measles: pathogenesis and control

Current Topics in Microbiology and Immunology

Series Editors

Richard W. CompansEmory University School of Medicine, Department of Microbiology and Immunology, 3001 Rollins Research Center, Atlanta, GA 30322, USA

Max D. CooperDepartment of Pathology and Laboratory Medicine, Georgia Research Alliance, Emory University, 1462 Clifton Road, Atlanta, GA 30322, USA

Tasuku HonjoDepartment of Medical Chemistry, Kyoto University, Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan

Hilary KoprowskiThomas Jefferson University, Department of Cancer Biology, Biotechnology Foundation Laboratories, 1020 Locust Street, Suite M85 JAH, Philadelphia, PA 19107-6799, USA

Fritz MelchersBiozentrum, Department of Cell Biology, University of Basel, Klingelbergstr. 50–70, 4056 Basel Switzerland

Michael B.A. Oldstone

Sjur Olsnes

Radium Hospital, Montebello 0310 Oslo, Norway

Peter K. VogtThe Scripps Research Institute, Dept. of Molecular & Exp. Medicine, Division of Oncovirology, 10550 N. Torrey Pines. BCC-239, La Jolla, CA 92037, USA

The Scripps Research Institute, Department of Immunology and Microbial Science, 10550 N. Torrey Pines, La Jolla, CA 92037, USA

Department of Biochemistry, Institute for Cancer Research, The Norwegian

Volume 330

• MEditorsDi nea E. Griffin ichael B.A. Oldstone

Measles

Pathogenesis and Control

Editors:

ISBN 978-3-540-70616-8 e-ISBN 978-3-540-70617-5

DOI 10.1007/978-3-540-70

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Diane E. GriffinJohns Hopkins UniversitySchool of Hygiene and Public HealthDepartment of Molecular Microbiology

USA

Michael B.A. OldstoneScripps Research Institute

617-5

931704

[email protected]@jhsph.edu

[email protected]

615 N. Wolfe StreetBaltimore, MD 21205 La Jolla, CA 92037

Department of Immunology and

10550 N. Torrey Pines Road Microbial Science

Current Topics in Microbiology and Immunology ISSN 0070-217X

reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication

Contents

Introduction ...................................................................................................... 1

1 Making It to the Synapse: Measles Virus Spread in and Among Neurons ............................................................................ 3

V.A. Young and G.F. Rall

2 Modeling Subacute Sclerosing Panencephalitis in a Transgenic Mouse System: Uncoding Pathogenesis of Disease and Illuminating Components of Immune Control ............ 31

M.B.A. Oldstone

3 Measles Studies in the Macaque Model ................................................. 55 R.L. de Swart

4 Ferrets as a Model for Morbillivirus Pathogenesis, Complications, and Vaccines ................................................................... 73

S. Pillet, N. Svitek, and V. von Messling

5 Current Animal Models: Cotton Rat Animal Model ........................................................................................... 89

S. Niewiesk

6 Current Animal Models: Transgenic Animal Models for the Study of Measles Pathogenesis ................................................... 111

C.I. Sellin and B. Horvat

7 Molecular Epidemiology of Measles Virus ............................................ 129 P.A. Rota, D.A. Featherstone, and W. J. Bellini

8 Human Immunology of Measles Virus Infection .................................. 151 D. Naniche

v

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vi Contents

9 Measles Control and the Prospect of Eradication ................................. 173 W.J. Moss

10 Measles: Old Vaccines, New Vaccines .................................................... 191 D.E. Griffin and C.-H. Pan

11 Measles Virus for Cancer Therapy ........................................................ 213 S.J. Russell and K.W. Peng

12 Measles Virus-Induced Immunosuppression ........................................ 243 S. Schneider-Schaulies and J. Schneider-Schaulies

13 Hostile Communication of Measles Virus with Host Innate Immunity and Dendritic Cells ................................................................ 271

B. Hahm

Index .................................................................................................................. 289

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W.J. BelliniMeasles, Mumps, Rubella and Herpesvirus Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA

R.L. de SwartDepartment of Virology, Erasmus MC, University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands, [email protected]

D.A. FeatherstoneImmunization, Vaccines and Biologicals, World Health Organization, Geneva, Switzerland

D.E. GriffinDepartment of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe St. Rm E5132, Baltimore, MD 21205, USA, [email protected]

B. HahmDepartments of Surgery and Molecular Microbiology and Immunology, Center for Cellular and Molecular Immunology, University of Missouri-Columbia School of Medicine, One Hospital Dr., Columbia, MO 65212, USA, [email protected]

B. HorvatU758-ENS Lyon, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France, [email protected]

W.J. MossDepartment of Epidemiology and the W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore MD, USA, [email protected]

Contributors

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D. NanicheBarcelona Center for International Health Research (CRESIB), Hospital Clinic, Institut d’Investigacions Biomedicas August Pi i Sunyer (IDIBAPS), C/Rossello 132, 4 08036, Barcelona, Spain, [email protected]

S. NiewieskCollege of Veterinary Medicine, Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210, USA, [email protected]

M.B.A. OldstoneThe Scripps Research Institute, Department of Immunology and Microbial Science, La Jolla CA, USA, [email protected]

C.-H. PanDepartment of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe St. Rm E5132, Baltimore, MD 21205, USA

K.W. PengMayo Clinic, Department of Molecular Medicine, 200 1st Street SW, Rochester, MN 55905, USA

S. PilletINRS-Institut Armand-Frappier, University of Quebec, 531, boul. des Prairies, Laval, QC, H7V 1B7, Canada

P.A. RotaMeasles, Mumps, Rubella and Herpesvirus Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA, [email protected]

S.J. RussellMayo Clinic, Department of Molecular Medicine, 200 1st Street SW, Rochester, MN 55905, USA, [email protected]

J. Schneider-SchauliesInstitute for Virology and Immunobiology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany

S. Schneider-SchauliesInstitute for Virology and Immunobiology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany, [email protected]

viii Contributors

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S. SvitekINRS-Institut Armand-Frappier, University of Quebec, 531, boul. des Prairies, Laval, QC, H7V 1B7, Canada

C.I. SellinU758-ENS Lyon, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France

V. von MesslingINRS-Institut Armand-Frappier, University of Quebec, 531, boul. des Prairies, Laval, QC, H7V 1B7, Canada, [email protected]

V.A. YoungDivision of Basic Science, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA

Contributors ix

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D.E. Griffin and M.B.A. Oldstone (eds.) Measles – Pathogenesis and Control. 1© Springer-Verlag Berlin Heidelberg 2009

Introduction

Measles virus, one of the most contagious of all human viruses, has been largely contained by the development and use of a vaccine that was introduced 50 years ago. These two volumes were timed to honor the introduction of the vaccine and to record the enormous advancements made in understanding the molecular and cell biology, pathogenesis, and control of this infectious disease. Where vaccine has been effectively delivered, endemic measles virus transmission has been eliminated. However, difficulties in vaccine delivery, lack of health care support and objection to vaccination in some communities continue to result in nearly 40 million cases and over 300,000 deaths per year from measles.

By itself measles virus infection has and still provides some of the most interest-ing phenomena in biology. Following infection of dendritic cells, measles virus causes a profound suppression of the host’s immune response that lasts a number of months after apparent recovery from infection. Indeed, measles virus was the first virus to be associated with immunosuppression with many of the manifesta-tions to be observed one hundred years later with HIV infection. Measles is also associated with development of both post-infectious encephalomyelitis, an autoim-mune demyelinating disease, and subacute sclerosing panencephalitis, a slowly progressive neurodegenerative disorder. How measles virus infects cells, spreads to various tissues and causes disease, as well as the role of the immune response, gen-eration of new vaccines, and use as a vector for gene delivery are topics covered in these two volumes. A unique highlight for readers of this series and those interested in the history of a major and profound biomedical research accomplishment is the chapter written by one of the participants who worked on the initial discovery and use of the vaccine who records the events that occurred at that time.

Baltimore, MD Diane E. GriffinLa Jolla, CA Michael B.A. Oldstone

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Chapter 1 Making It to the Synapse: Measles Virus Spread in and Among Neurons

V. A. Young and G. F. Rall (*)

G. F. Rall Division of Basic Science , Fox Chase Cancer Center , 333 Cottman Avenue, Philadelphia , PA 19111 , USA, e-mail: [email protected]

Abstract Measles virus (MV) is one of the most transmissible microorganisms known, continuing to result in extensive morbidity and mortality worldwide. While rare, MV can infect the human central nervous system, triggering fatal CNS diseases weeks to years after exposure. The advent of crucial laboratory tools to dissect MV neuropathogenesis, including permissive transgenic mouse models, the capacity to manipulate the viral genome using reverse genetics, and cell biology advances in understanding the processes that govern intracellular

Contents

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Measles Virus: Genome and Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Measles Virus Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Understanding MV CNS Complications: Results from Brains of Infected Individuals and Persistently Infected Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8The Big Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Tools. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Transgenic Mouse Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Other Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11In Vitro Culture Systems and Techniques in Cellular Transport Studies. . . . . . . . . . . . . . . 12Reverse Genetics for MV. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Access to the CNS: Lessons from CDV, PV, and WNV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14MV Spread and Transport in Non-neuronal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

MV Spread in Non-neuronal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Use of Microtubules and Their Associated Motor Proteins to Achieve Viral Transport Within an Infected Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

MV Spread in Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19MV Movement Within and Among Neurons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Effects of Immune Responses on MV Spread in Neurons. . . . . . . . . . . . . . . . . . . . . . . . . . 21

Remaining Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

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4 V.A. Young, G.F. Rall

trafficking of viral components, have substantially clarified how MV infects, spreads, and persists in this unique cell population. This review highlights some of these technical advances, followed by a discussion of our present understand-ing of MV neuronal infection and transport. Because some of these processes may be shared among diverse viruses, comparisons are made to parallel studies with other neurotropic viruses. While a crystallized view of how the unique environ-ment of the neuron affects MV replication, spread, and, ultimately, neuropatho-genesis is not fully realized, the tools and ideas are in place for exciting advances in the coming years.

Abbreviations

BBB Blood–brain barrier CNS Central nervous system CSF Cerebrospinal fluid EGFP Enhanced green fluorescence protein F MV fusion FIP Fusion inhibitory peptide H MV hemagglutinin HSV Herpes simplex virus IC Intracerebral IFNAR Interferon alpha receptor IL Interleukin IN Intranasal IV Intravenous KO Knockout L MV large (viral polymerase) M MV matrix MAP-2 Microtubule associated protein 2 MIBE Myelin inclusion body encephalitis MV Measles virus N MV nucleoprotein NK Neurokinin P MV phosphoprotein PIE Postinfectious encephalomyelitis PV Poliovirus PrV Pseudorabies virus RAG Recombinase activating gene RNP Ribonucleoprotein SLAM Signaling lymphocyte activation molecule SSPE Subacute sclerosing panencephalitis WNV West Nile virus YAC Yeast artificial chromosome

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1 Making It to the Synapse: Measles Virus Spread in and Among Neurons 5

Introduction

The basic steps of the infectious cycle of any pathogen’s entry into, spread within, and exit from the host offer crucial insights into how pathogens cause disease. As a result, a full understanding of the stages of a microorganism’s life cycle may inform the rational design of therapeutics to prevent or ameliorate consequent disease.

Measles virus (MV) is one of the most transmissible microorganisms known; it continues to result in hundreds of thousands of infections annually throughout the world, many of which have serious pathogenic consequences that can result in death. Despite the tremendous progress that has been made in deciphering the basis of MV pathogenesis and in the creation of effective attenuated vaccines, how MV causes disease, including rare, but serious, central nervous system (CNS) complications, remains poorly understood. It is our view that defining the pathogenesis of MV in the CNS necessitates an understanding of the interaction of the virus with the host in trafficking to and spreading within the CNS. This is the focus of this review.

Measles Virus: Genome and Proteins

MV is a member of the Paramyxoviridae , within the Morbillivirus genus. Its genome consists of approximately 16,000 bases of nonsegmented, single-stranded negative-sense RNA, meaning that the viral genome is transcribed immediately upon entry into the cell. Virions are spherical and enveloped, and the envelope is derived from the host cell as the viral particle buds from the plasma membrane. The viral genome encodes eight proteins, the function of which are briefly noted here. Inserted into the envelope of the virion are the two MV glycoproteins, hemagglutinin (H) and fusion (F). These proteins mediate attachment to cellular receptors and fusion of the virion with the tar-get cell or fusion of an infected cell with an adjacent uninfected cell. The matrix pro-tein (M) lies immediately underneath the virion envelope and serves in virus assembly and budding. The two polymerase proteins, large (L) and phosphoprotein (P), are closely associated with the genome, which is encapsidated by the nucleocapsid (N) protein. The nonstructural proteins V and C, encoded within the P cistron, are also packaged within the virion; these proteins have recently been shown to play a role in counteracting host antiviral immune responses (reviewed in Griffin 2001).

Measles Virus Pathogenesis

MV is a human-restricted pathogen that spreads among individuals by release of aerosol droplets. An infected individual will undergo a latent period of 10–14 days followed by a few days of fever, cough, coryza, and rash. Primary infection occurs in the upper respiratory tract, but MV will secondarily infect lymphoid cells (reviewed in Griffin 2001; Rall 2003; Schneider-Schaulies et al. 2003; Sips et al.

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2007), though a contrasting hypothesis has recently been proposed that lymphoid cells are a primary traget for MV replication, followed then by engagement of an as yet unidentified receptor on the basolateral membrance of lung epithelia (de Swart et al., 2007; Leonard et al., 2008). Although the immunological response made by most infected, immunocompetent individuals is sufficient to clear the virus and provide life-long protection, a period of transient immunosuppression is a notorious characteristic of MV infection and is likely the basis of most of the complications and the subsequent fatalities following acute infection (reviewed in Dhib-Jalbut and Johnson 1994; Griffin et al. 2008; Rall 2003). Additional consequences of acute infection include diarrhea pneumonia, and encephalitis.

Immunosuppression

Immunosuppression following MV infection can last for weeks, extending beyond the classical MV symptoms. The chief risk of immunosuppression is that it renders infected individuals more susceptible to secondary infections, though precisely how this occurs is not fully understood. In humans, MV-induced immunosuppression is characterized by a loss of delayed type hypersensitivity responses to recall antigens (e.g., tuberculin; Tamashiro et al. 1987), a limited response of lymphocytes to mitogens when cultured ex vivo (Hirsch et al. 1984), and impaired responses to new antigens (Coovadia et al. 1978). To date, a number of mechanisms have been pro-posed based on animal and cell culture studies, all of which could be pertinent in the natural infection. For example, Griffin and colleagues showed that MV infec-tion of antigen-presenting cells suppresses interleukin-12 (IL-12) production, which then in turn skews the CD4 + T cell response toward a Th2 profile (Karp et al. 1996). This altered CD4 + T cell response leads to inappropriate priming of T cells and a failure of T cells to proliferate following interaction with MV-infected den-dritic cells (Fugier-Vivier et al. 1997; Servet-Delprat et al. 2000). These studies correlate well with serum profiles in MV-infected macaques and humans that also show a skewing toward Th2-like cytokines (Atabani et al. 2001; Moss et al. 2002; Polack et al. 2000). In addition to influencing the Th1/Th2 balance, acute MV infection may precipitate immunosuppression by causing an overall lymphopenia, due to effects on T cell proliferation and progression through the cell cycle (Naniche et al. 1999; Niewiesk et al. 1999, 2000; Schnorr et al. 1997), as well as specifically inhibiting immune function via the production of unidentified, immu-nosuppressive molecules from infected T cells (Sun et al. 1998). A detailed discus-sion of MV-induced immunosuppression can be found in other chapters within this volume.

CNS Complications Following Acute MV Infection

Approximately 1 in 100,000 acutely infected individuals later go on to develop CNS complications. These diseases differ in terms of immune status of the affected

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host, onset of symptoms, presence of MV within the CNS, host survival rate, and neuropathological findings. These are briefly discussed below.

Subacute Sclerosing Panencephalitis

Subacute sclerosing panencephalitis (SSPE) is a slow, progressive disease that is invariably fatal, and can occur from 1 to 15 years following acute MV infection (Dubois-Dalcq et al. 1974). Children are far more likely to develop this compli-cation than adults (reviewed in Johnson 1998). The disease initially manifests as subtle cognitive losses, progressing to more overt cognitive dysfunction, fol-lowed by motor loss, seizures, and eventual organ failure in virtually all affected individuals. The rate of SSPE occurrence ranges from 1 in 10,000–300,000 acute MV infections (reviewed in Rima and Duprex 2005; Takasu et al. 2003). Neurons are predominantly infected, though at late times of infection, oligodendrocytes, astrocytes, and endothelial cells may also be involved (reviewed in Rima and Duprex 2005). SSPE affects both gray and white matter and is histologically characterized by the presence of cellular inclusion bodies, inflammation, glial activation, loss of blood–brain barrier (BBB) integrity, and neuronal loss (reviewed in Dhib-Jalbut and Johnson 1994; Rall 2003). A serologic hallmark of SSPE, as compared to the other CNS complications, is the elevation of measles-specific antibodies in the blood and cerebrospinal fluid (CSF) (Dubois-Dalcq et al. 1974).

Importantly, evidence from brain biopsies of SSPE patients indicates that infected neurons do not release budding virus (Paula-Barbosa and Cruz 1981). Based on extensive sequencing studies of MV from these specimens and from cells persistently infected with MV isolates from SSPE patients, it has been proposed that the failure of infected neurons to produce complete extracellular virus may be due to defects in protein expression caused by extensive point mutations in the envelope-associated genes, H, F, and M (Cattaneo et al. 1988; reviewed in Dhib-Jalbut and Johnson 1994; Rima and Duprex 2005), though what role these viral proteins play in neuronal spread of MV and how mutations may affect MV biology in infected neurons are not known.

Postinfectious Encephalomyelitis

Postinfectious encephalomyelitis (PIE) occurs more frequently than SSPE, affecting approximately 1 in 1,000 infected individuals. Symptoms of PIE nor-mally appear 5–14 days after the characteristic MV rash but can predate the rash (reviewed in Johnson 1998). This complication is thought to be an autoimmune reaction, perhaps to myelin basic protein (Johnson et al. 1984). MV antigen and nucleic acids have not been detected in PIE brain biopsies by immunohisto-chemistry or in situ hybridization (Johnson et al. 1984; reviewed in Dhib-Jalbut and Johnson 1994; Norrby and Kristensson 1997), supporting the notion that this

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is an autoimmune disease. Additional hallmarks of PIE include perivascular inflammation and demyelination (reviewed in Norrby and Kristensson 1997). Unlike SSPE, intrathecal production of MV antibodies has only been found in a few cases of PIE. Affected individuals present with seizures, deafness, ataxia, and movement disorders. There is an approximate 25% mortality rate associ-ated with PIE, and survivors are likely to suffer from frequent neurologic sequelae.

Measles Inclusion Body Encephalitis

Measles inclusion body encephalitis (MIBE), a rare CNS complication following acute MV infection, has been described in children and adults receiving immuno-suppressive drugs and therefore is thought to chiefly affect immunocompromised hosts. The neurologic disease appears 3–6 months after the acute MV rash (reviewed in Dhib-Jalbut and Johnson 1994; Johnson 1998). As the name suggests, MIBE is characterized by inclusion bodies in both neurons and glia, with accom-panying neuronal loss but an overall lack of inflammation (reviewed in Dhib-Jalbut and Johnson 1994; Johnson 1998; Norrby and Kristensson 1997; Rall 2003). Measles antigen is present in the brain, and virus has been isolated directly from the brains of affected individuals (Johnson 1998). MIBE differs from SSPE in the absence of elevated serum and cerebrospinal fluid neutralizing antibodies (reviewed in Dhib-Jalbut and Johnson 1994; Rima and Duprex 2005). The disease course is relatively short, lasting from days to weeks, causing seizures, motor deficits, and stupor, often leading to coma and death (reviewed in Johnson 1998).

Importantly, even though only a small percentage of acute MV infections will go on to develop CNS complications, a few studies have detected MV RNA in vari-ous organs, including brain, upon autopsy of elderly individuals who died of non-viral and non-CNS causes (Katayama et al. 1995, 1998).

These findings suggest that MV may persist in the brains (and other organs) of healthy individuals, and that the frequency with which MV invades the CNS cannot be determined by summing the occurrence of the above-described CNS complications.

Understanding MV CNS Complications: Results from Brains of Infected Individuals and Persistently Infected Cell Lines

The development of a robust live attenuated vaccine has profoundly decreased the infection rate in vaccinated populations. Vaccination has not been associated with SSPE, and only wild-type virus sequences have been isolated from SSPE tissues. However, vaccine strains have been isolated from the CNS of immunocompromised patients with MIBE (Bitnun et al. 1999; reviewed in Rima and Duprex 2005), implying that the attenuated vaccine strain can traffic to the brain under conditions of poor immune surveillance.

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Both SSPE and MIBE are characterized by mutations that apparently render the virus defective, though spread in neurons may still occur. As noted above, it has long been hypothesized that these mutations are the reason why infectious virus is not released from infected brain cells, especially since the mutations map to the envelope-associated proteins M, F, and H (Baczko et al. 1988; Billeter et al. 1994; reviewed in Johnson 1998). In SSPE, M protein expression is reduced, likely through one of two mechanisms: either a steeper gradient of transcription (Cattaneo et al. 1987a, 1987b) or increased read-through across the P–M junction of the MV genome (reviewed in Rima and Duprex 2005). In support of these data, hyperim-mune antibody responses in SSPE are directed to all MV proteins with the key exception of M (reviewed in Rima and Duprex 2005).

Mutations in F have been described for both SSPE and MIBE cases (Baczko et al. 1988; Billeter et al. 1994; reviewed in Johnson 1998). Interestingly, in all SSPE cases, mutations in F characteristically consist of the loss of the C-terminal pentadecapeptide, a sequence that is strictly conserved among morbilliviruses and is therefore thought to play an essential role in F protein function. This region contains the basolateral sorting signal of F (Maisner et al. 1998; reviewed in Rima and Duprex 2005), suggesting that missorting of F may contribute to altered MV spread and lack of complete MV assembly in neurons of SSPE patients. Interestingly, a study of ten SSPE cases by immunohistochemistry and in situ hybridization suggests that MV likely spreads transneuronally, an observation subsequently validated by in vitro model studies (Lawrence et al. 2000; Oldstone et al. 1999). The analysis of these infected brains revealed that MV infection in neuronal processes was predominantly dendritic, though there were signs of occasional axonal involvement as well (Allen et al. 1996). Whether mutations in viral proteins influence how MV is transported within neurons is currently under investigation.

The Big Questions

Years of research on MV CNS complications have provided key insights into MV neuronal spread and have identified future areas of study concerning the relation-ship between viral spread and pathogenesis. For example: how does MV move from the site of entry to the perinuclear space and then again to the site of viral egress? Once present at the synaptic membrane, what cellular and viral proteins mediate MV transsynaptic spread? Is the mechanism of interneuronal spread related to the pathogenesis of MV within the brain? The establishment of permissive animal and cell culture models, coupled with advances in manipulating the viral genome, and the advent of cell biology resources to explore intracellular trafficking have been essential for key developments over the past few years. A discussion of these recent observations serves as the basis for the remainder of this review. We first describe some of these important technical advances and then discuss how they have been applied to MV neuronal spread.

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Tools

Transgenic Mouse Models

To date, two human receptors for MV have been identified: CD46 (Dorig et al. 1993; Naniche et al. 1993) and signaling lymphocyte activation molecule (SLAM; Erlenhoefer et al. 2001; Hsu et al. 2001; Tatsuo et al. 2000). While this is still an evolving field, and many believe that other receptors will be identified in the future, the general consensus is that CD46 is principally a receptor for vaccine strains, such as Edmonston, whereas SLAM, though restricted to hematogenous cells, permits entry of wild-type MV. As the mouse and rat homologs of CD46 and SLAM do not confer susceptibility to MV infection (Dorig et al. 1993; Manchester et al. 1994; Naniche et al. 1993; Ono et al. 2001b), transgenic mice expressing human CD46 or SLAM were established, with the hypothesis that expression of these human pro-teins would overcome the initial barrier to viral entry (Mrkic et al. 1998; Oldstone et al. 1999; Rall et al. 1997; Sellin et al. 2006; reviewed in Manchester and Rall 2001). Importantly for neuron-focused studies, these transgenic mice also provide a source of primary neurons for ex vivo experiments to complement observations made in vivo (Lawrence et al. 2000; Makhortova et al. 2007). A brief introduction to some of the transgenic model systems follows.

NSE-CD46 mice, developed in the laboratory of Michael Oldstone, were engi-neered to express the BC1 isoform of human CD46 under the control of the neuron-specific enolase (NSE) promoter, restricting CD46 expression to CNS neurons. These mice can be infected intranasally (IN) or intracranially (IC) with a vaccine strain of MV (e.g., Edmonston); as predicted, only CD46-expressing neurons are initially permissive for infection. Adult immunocompetent mice mount an aggres-sive T cell response and survive infection, whereas NSE-CD46 neonatal mice, or adults on an immunodeficient background succumb to CNS disease. CD46 + pri-mary hippocampal neurons can be cultured from transgenic embryos, providing a parallel in vitro culture system to corroborate and extend the in vivo observations (Lawrence et al. 1999, 2000; Makhortova et al. 2007; Patterson et al. 2002, 2003; Rall et al. 1997).

Another CD46 transgenic model that was developed in Oldstone’s lab is the YAC-CD46 mouse, in which CD46 is expressed from its own promoter, more closely mirroring expression in humans. Indeed, CD46 distribution is found throughout the mouse, and this model has been used for both CNS and immunosup-pression studies. Importantly, in this model, all four major isoforms of CD46 are expressed to levels and in locations similar to those seen in humans. Furthermore, overall expression of CD46 was shown to be greater than that previously reported for other transgenic mouse models, including the NSE-CD46 mice (Oldstone et al. 1999; Patterson et al. 2001).

Mrkic et al. engineered mice to express CD46 on a mouse background lacking the interferon-α receptor [IFNAR –/– (Duprex et al. 2000; Ludlow et al. 2007; Mrkic et al. 1998)]. IFNAR –/– mice can be infected intranasally with MV, though

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replication of MV is limited. Engineering mice to express the MV receptor CD46 on the IFNAR –/– background resulted in higher levels of MV infection in the respiratory tract with subsequent inflammation, providing a more relevant model for the acute MV infection seen in humans.

A perplexing observation from the establishment of a number of other CD46 transgenic mice was that, despite CD46 expression and ability to infect such cells when cultured ex vivo, little if any infection was observed in vivo. For example, Blixenkrone-Moller et al. generated a CD46 transgenic mouse that expressed a genomic copy of CD46 on a Bl/6 × SJL mouse background (Blixenkrone-Moller et al. 1998). Despite apparently robust CD46 expression, MV replication could not be detected following intraperitoneal (IP) or IN inoculation, though kidney and lung cell cultures from these mice were permissive. An important observation made by these authors was that, in these cultures, MV did not reach full replication levels as compared to MV-infected Vero cells. Furthermore, while IC challenge of CD46 transgenic mice did result in limited CNS infection, infection was also observed in nontransgenic mice, suggesting that factors other than CD46 play a role in mediating MV uptake into murine cells, and that key intracellular factors are needed to achieve optimal levels of MV replication. This work was supported by the findings of Horvat et al. and Evlashev et al. who found that cells from CD46 transgenic mice showed cell-type specific susceptibility to MV infection, indicating a role for host factors other than CD46 in mediating productive MV infection (Evlashev et al. 2001; Horvat et al. 1996).

Thus far, four different SLAM-expressing transgenic mouse models have been created (Ohno et al. 2007; Sellin et al. 2006; Shingai et al. 2005; Welstead et al. 2005). However, to date, only one group has used these mice to look at MV CNS infections. Sellin et al. engineered mice to ubiquitously express SLAM. Wild-type and vaccine strains of MV could infect transgenic mice, either by IC or IN routes, but vaccine strains were less virulent (Sellin et al. 2006).

Other Animal Models

Other investigators have focused on rodent-adapted MVs to infect nontransgenic (i.e., wild-type) mice, rats, or hamsters (Castro et al. 1972; Duprex et al. 1999a; Griffin et al. 1974; Moeller-Ehrlich et al. 2007; Schubert et al. 2006; Johnson and Swoveland 1977; Johnson and Norrby 1974; Roos et al. 1978). Such studies have been ongoing since the 1970s and have allowed researchers to study MV in a small animal model prior to the identification of the human MV receptors. Importantly, despite the mutations that occurred in adapting MV to rodents, the pathogenesis of such strains is remarkably similar to that seen following MV infection of humans or transgenic mouse models.

Efforts to create animal models to study MV infection and pathogenesis have not been limited only to mice. For example, MV-infected cotton rats recapitulate the immunosuppression seen in MV-infected humans (Niewiesk 1999; Niewiesk et al.

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1999; Wyde et al. 1992), even with wild-type (clinical) isolates (Wyde et al. 1999). Furthermore, studies on rat brain slices grown in culture and infected with MV have revealed important clues about how MV spreads through an infected brain (Ehrengruber et al. 2002). Interestingly, the generation of a transgenic CD46-expressing rat demonstrated that although MV was taken up by CD46-expressing cells, subsequent intracellular blocks in MV replication prevented robust infection of the animal (Niewiesk et al. 1997). Ferret models have also been used by some researchers as an SSPE model of MV infection in the CNS (Brown et al. 1985, 1987; Mehta and Thormar 1979; Thormar et al. 1983).

Finally, while rhesus macaques have been chiefly used to study immune responses to MV, immune suppression induced by MV and efficacy of possible vaccines against MV (de Swart et al. 2007; Pan et al. 2005; Pasetti et al. 2007; Polack et al. 2000), some investigators have used tissues collected from infected monkeys to model human CNS diseases (Albrecht et al. 1977; Steele et al. 1982).

How results from all of these model systems have been used to advance our understanding of MV interneuronal spread and neuropathogenesis will be discussed in the final section of the chapter.

In Vitro Culture Systems and Techniques in Cellular Transport Studies

Primary Neuron Cultures and Neuron-Like Cell Lines

The ability to culture primary neurons from various CNS substructures (e.g., hip-pocampus, cortex, dorsal root ganglia, cerebellum) of transgenic mice has been a powerful tool in dissecting aspects of intra- and interneuronal MV transport. These cultures are validated as neurons in their expression of characteristic neuronal markers such as MAP-2 and NeuN, their ability to form synapses in culture, and the fact that, once plated, these cells are mitotically inactive. While in general quite pure neuron cultures can be obtained (90%–95%), contaminating glial cells, cul-ture-to-culture variability, difficulty in establishing these cultures and a fairly short lifespan in culture (10–14 days, typically), are some of the disadvantages.

Cells lines such as NT2 (human teratocarcinoma cells that can be terminally differentiated into neuronal cells by retinoic acid treatment), human astrocytoma cells, and mouse neuroblastoma cells offer an alternative that is free of the com-plexities and challenges of primary neuron cultures (Duprex et al. 1999b, 2000; Lawrence et al. 2000; Ludlow et al. 2005; McQuaid et al. 1998). The ease of using these established lines of CNS cells is balanced by concerns about whether these cells accurately reflect primary CNS cell biology.

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Slice Cultures

The introduction of organotypic monolayer cultures of nervous tissue has added an intermediate technique to the neurologist’s toolbox, falling between the complexity of a whole animal model and the culturing of cell lines or pure primary cultures that do not recapitulate the cellular heterogeneity of the CNS. Advantages to organo-typic brain slice cultures are the ability to take cells from a defined region of the brain, the ability to use phase microscopy on the monolayer of resulting brain cells, the relevance of culturing primary cells without performing single cell dissociation, and the power of studying heterogeneous cell populations and how such diverse cells impact viral spread (Gahwiler 1981). This approach has been used to assess the spread of rMV-EGFP through neurons of cultured rat brain slices (Ehrengruber et al. 2002) as well as the spread of SSPE isolates through hamster cerebellum slices (Sheppard et al. 1975).

Reagents to Study the Role of Motor Proteins in Viral Spread

Progress in our understanding of the cellular cytoskeleton and how organelles, sig-naling complexes, and proteins are transported within cells has greatly advanced our understanding of how viruses are transported within cells (Jouvenet et al. 2004; reviewed in Leopold and Pfister 2006; Mackenzie et al. 2006; reviewed in Ploubidou and Way 2001; Rietdorf et al. 2001; Ward and Moss 2004). For example, the devel-opment of dominant negatives to genetically manipulate the cellular transport sys-tem, as well as the increased availability of genetically altered mice with defined motor protein deficiencies, have enabled a dissection of which cellular proteins are utilized by viruses to enable transport. Although pharmaceutical approaches to ablate specific microtubule motors or microtubules themselves are broadly used (e.g., vanadate, colchicine, cytochalasin, nocodazole, etc.), the harsh and somewhat nonspecific consequences of using these cytotoxic reagents was always a limitation that can now be obviated with more precise and less caustic genetic strategies.

For example, the generation and characterization of mice via ENU-mutagenesis containing a single point mutation in dynein heavy chain has helped to define the role of dynein in the spread of mouse-adapted scrapie prions from the periphery to the CNS of infected mice (Hafezparast et al. 2004). These loa (legs at odd angles) mice are viable as heterozygotes, and are currently being used by a number of neu-rovirology labs to assess the role of dynein in virus transport (Ahmad-Annuar et al. 2003; Hafezparast et al. 2003, 2004).

Another tool to define viral–cytoskeletal interactions are dominant negative constructs. For example, overexpression of p50 dynamitin, a member of the multi-protein complex dynactin, which acts as an accessory factor to dynein, specifically disrupts dynein function (Ahmad et al. 1998; Burkhardt et al. 1997; Echeverri et al. 1996). This approach was used by Dohner et al. to show that herpes simplex virus type 1 (HSV-1) utilizes dynein following its entry into a cell to travel to the nucleus (Dohner et al. 2002). Similar reagents have been established for kinesins,

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motors that govern intracellular retrograde transport (Verhey et al. 1998; Verhey et al. 2001).

Reverse Genetics for MV

The establishment of a reverse genetics system for vaccine strains of MV has also furthered our understanding of MV replication in neurons, and in MV intra- and interneuronal spread (Devaux et al. 2007; Radecke et al. 1995; Schneider et al. 1997). Future studies using this approach will be key to identifying what roles each of the viral proteins play in neuronal infection, and will define whether some viral proteins may be dispensable for neuronal infection. Moreover, given the specula-tion that point mutations found in SSPE isolates might make these viruses more neuropathogenic, reverse genetics offers an opportunity to directly test these hypotheses in a controlled setting. Already, MV reverse genetics has been used to address the role of the H protein of rodent brain-adapted MV in conferring neuro-virulence to MV Edmonston in nontransgenic mice (Duprex et al. 1999a). Moreover, Maisner’s group engineered viruses with altered basolateral sorting signals in F and H and showed that these domains are important for MV propaga-tion through lymphoid cells, in addition to their previously described role in MV spread through epithelial cells (Runkler et al. 2008). Furthermore, the ability of SSPE-associated viral genes to confer certain phenotypes on wild-type or vaccine strains of MV has been tested through reverse genetics approaches. For example, a recombinant MV expressing the M gene of an SSPE isolate was found to repli-cate less efficiently and led to a CNS infection in YAC-CD46 transgenic mice with a longer course than that seen following infection with MV Edmonston. This MV-expressing SSPE M also showed defects in viral assembly and subsequent budding of progeny virus from infected cells (Patterson et al. 2001). Finally, the power of reverse genetics has provided some experimental convenience in that we can now molecularly tag MV with marker proteins such as GFP to more easily follow MV infections in vivo, in brain slices, and in cell culture (de Swart et al. 2007; Duprex et al. 1999b, 2000; Ehrengruber et al. 2002; Ludlow et al. 2007; Plumb et al. 2002; Schubert et al. 2006).

Access to the CNS: Lessons from CDV, PV, and WNV

Despite this broad palette of tools that are available to study MV–neuron interac-tions, many of the reagents and strategies have been developed only recently, and as a result we have more questions than answers about how MV gets to the CNS, spreads within CNS cells, and mediates neurological disease. How MV gains access to the CNS from the periphery is not known, though it has been previously proposed that MV spreads into the brain by infecting endothelial cells during secondary viremia (Esolen et al. 1995). Alternatively, there is speculation that MV may infect

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the CNS by infiltration of infected lymphoid cells, e.g., Alternatively there is specu-lation that MV may infect the CNS by infiltration of infected lymphoid cells, e.g., infiltrating macrophages. Despite a lack of certainty regarding MV neuroinvasion, clues about how MV spreads to the CNS can be gained by looking at the spread of other neurotropic viruses from the periphery to the CNS.

Two routes of CNS infection have been proposed for a related morbillivirus (CDV): the hematogenous route and anterograde trafficking via the olfactory nerve. Like MV, CDV can infect lymphocytes during the systemic phase of infection, thus providing an opportunity for infected lymphocytes to traffic across the BBB and release infectious virus to initiate infection within the brain parenchyma. Importantly, the work by Rudd et al. showed that entry of CDV into the CNS can also occur via the olfactory bulb and suggested that this may be a common mechanism of entry for neurotropic paramyxoviruses (Rudd et al. 2006).

Similar portals of entry have been proposed and tested for both polio virus (PV) and West Nile virus (WNV). One hypothesis is that these viruses cross the BBB; the second is that PV or WNV is transmitted via peripheral nerves (Ohka et al. 1998; Samuel et al. 2007). Yang et al. demonstrated that circulating PV crosses the BBB at a high rate that is independent of polio virus receptor (PVR) expression (Yang et al. 1997). More recently, studies in transgenic PVR-expressing mice have shown that PV is transported through the sciatic nerve by fast retrograde axonal transport. Furthermore, PV accesses the sciatic nerve from its intramuscular inoculation site by a process dependent on PVR (Ohka et al. 1998), and the mechanism by which PV is transported retrogradely along the sciatic nerve is thought to be an interaction of the PVR cytoplasmic domain with dynein light chain Tctex1 (Ohka et al. 2004). As with polio, axonal transport of WNV can also occur, mediating entry into the CNS. However, WNV can spread to the CNS even when the sciatic nerve has been transected, suggesting that WNV likely uses multiple routes to access the brain (Samuel et al. 2007). One of these routes of WNV entry has been shown to be dependent on Toll-like receptor 3 (TLR3)-mediated inflammation and subsequent opening of the BBB (Wang et al. 2004). Thus, as for CDV (Rudd et al. 2006), mul-tiple non-mutually exclusive routes of spread are available for PV and WNV entry into the CNS. How MV penetrates into the CNS, and perhaps more importantly, how often this occurs in a human population, remain to be determined.

MV Spread and Transport in Non-neuronal Cells

MV Spread in Non-neuronal Cells

MV infection is initiated when H binds to one of its cellular receptors, CD46 or SLAM (Dorig et al. 1993; Naniche et al. 1993; Tatsuo et al. 2000). The receptor usage correlates with the source of the MV H protein: wild-type isolates typically use SLAM (Erlenhoefer et al. 2001; Ono et al. 2001a, 2001b; Tatsuo et al. 2000), whereas attenuated vaccine strains, such as Edmonston and the strains derived from

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it, preferentially use CD46. Receptor selection is not exclusive, however: SLAM has been shown to be an effective receptor for MV entry for vaccine strains and conversely a few wild-type strains have the ability to bind to CD46 (Erlenhofer et al. 2002). It has been suggested that the receptor usage of a given MV isolate may depend on the cell line used to isolate and amplify the virus rather than the wild-type or vaccine strain status of the MV isolate (Manchester et al. 2000). Furthermore, genetic and structural studies of MV H proteins have illustrated that the H binding domains to CD46 and SLAM are spatially distinct (Colf et al. 2007; Erlenhofer et al. 2002), providing support for the idea that H can interact with both CD46 and SLAM, albeit with different affinities for each. In any case, given that SLAM expression is limited to lymphoid cells (McQuaid and Cosby 2002), it is likely that other MV receptors await discovery.

In non-neuronal cells, MV buds from the apical surface, resulting in the release of free virus or in cell fusion (reviewed in Griffin 2001; Fig. 1. 1 A). Transport of the viral nucleocapsid to the plasma membrane is dependent on levels of M protein and its accumulation at the cell surface (Runkler et al. 2007). Apical budding occurs despite the preferential sorting of MV glycoproteins F and H to the basolateral membrane through a tyrosine-based sorting signal (Blau and Compans 1995; Maisner et al. 1998; Moll et al. 2001). Appropriate budding is achieved by restricted expression of M at the apical surface, which retargets some of F and H in polarized cells (Naim et al. 2000; Riedl et al. 2002). The interaction of the cytoplasmic tails of F and H with M mediates virus assembly (Cathomen et al. 1998a, 1998b). However, the predominant presence of the glycoproteins F and H at the basolateral membrane has been hypothesized to play a key role in MV spread through an infected individual. The tyrosine-based sorting signals in F and H are not only required for basolateral sorting, but are also required for the fusogenicity of the F/H complex in polarized epithelial cells (Maisner et al. 1998; Moll et al. 2001). Consequently, it has been proposed that the basolaterally expressed F/H complex promotes cell–cell fusion, thus allowing the virus to spread to underlying tissues

Fig. 1.1 A, B Immunohistochemistry for MV antigen with hematoxylin counterstain. A MV-infected Vero cells form syncytia, or multinucleated giant cells. B MV-infected primary hippocampal neurons from NSE-CD46 mice do not form syncytia

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and facilitating systemic MV dissemination (Moll et al. 2004). This hypothesis emphasizes that apically released budding virus and basolaterally mediated cell–cell fusion are both important components of MV systemic dissemination.

It should be noted that the classical MV spread in non-neuronal cells (extracellu-lar progeny virus budding and cell–cell fusion) is dependent on MV glycoproteins. Thus, MV H interaction with its receptor likely triggers F protein fusogenic activity, initiating viral infection (Griffin 2001; Lamb and Kolakofsky 2001). As we point out later, these events may be substantially altered upon MV infection of neurons.

Use of Microtubules and Their Associated Motor Proteins to Achieve Viral Transport Within an Infected Cell

Introduction to Microtubules

How are the glycoproteins and RNP complexes shuttled through a cell to achieve appropriate assembly and egress? Microtubules are part of the cellular cytoskeleton and play a critical role in mediating movement of cellular proteins and organelles to their proper destinations. In neurons, their role is even more critical, as neurons rely on microtubules for neurite extension, synaptic vesicle trafficking, and synapse formation (Zhai and Bellen 2004). Microtubules are long, hollow cylindrical poly-mers of tubulin that assemble with a head-to-tail orientation (reviewed in Henry et al. 2006). The plus-end of microtubules typically extends to the plasma mem-brane and away from the nucleus, or toward the axon or dendrite and away from the cell body in neurons. In non-neuronal cells, microtubule minus-ends are bound and stabilized at the microtubule organizing center (MTOC) near the nucleus, whereas in neurons the minus-ends are oriented to the cell body, but lack an MTOC. The protein motors that move cargo along these microtubules are grouped into two families: the predominately plus-end-directed kinesin family of motors, which move cargo away from the cell body (anterograde transport), and the minus-end directed dynein family of motors, which move cargo to the cell body (retrograde transport), often as part of the lysosomal pathway. Cytoplasmic kinesin (or conven-tional kinesin) consists of two heavy chains and two light chains. The heavy chains contain the motor domains, which allow for the generation of force and subsequent movement along the microtubule. Furthermore, the heavy chains facilitate dimeri-zation and cargo binding. The kinesin light chains may also facilitate binding to cargo (reviewed in Mandelkow and Mandelkow 2002). Cytoplasmic dynein binds to microtubules via its two heavy chains, but other components of this complex (including six light chains, two light intermediate chains, and two intermediate chains) mediate cargo binding and contribute to the specificity of the cargo that is transported. Furthermore, cytoplasmic dynein associates with another protein complex dynactin, which acts as a co-factor to facilitate cargo binding and processivity along the microtubule (reviewed in Leopold and Pfister 2006; Radtke et al. 2006).

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Viruses of many families utilize the microtubule pathway during infection. Some viruses associate with microtubules and retrograde motors to move to the nucleus, including murine polyomavirus, adeno-associated virus, adenovirus, her-pes simplex virus 1, and human immunodeficiency virus (reviewed in Greber and Way 2006; Radtke et al. 2006). Other viruses are associated with anterograde trans-port, as has been shown for both Vaccinia virus and African swine fever virus transport to the plasma membrane through an interaction of viral cargo with kinesin-1 (Jouvenet et al. 2004; Rietdorf et al. 2001). Presumably, for these viruses, interaction with anterograde motors facilitates viral egress. Moreover, recent work with the alphaherpesvirus pseudorabies, a large DNA virus, illustrated fast axonal transport through neurons, a process mediated by microtubule motors (Smith et al. 2001). However, little is known about how neurotropic RNA viruses, including MV, interact with molecular motors in neurons.

MV and the Cytoskeleton

It has been known for some time that actin plays a key role in MV trafficking within cells. Actin is packaged within MV virions (Tyrrell and Norrby 1978), and treatment of infected cells with the actin-disrupting agent cytochalasin B resulted in disruption of MV virion formation (Stallcup et al. 1983). Electron microscopy studies of cytoskeletal preparations of MV-infected cells showed that the growing end of actin filaments protruded into budding virions. The authors suggested that the vectorial action of actin promotes MV budding and may also be involved in the transport of nucleocapsids to the cell surface (Bohn et al. 1986). Furthermore, actin was associated with transcriptionally silent MV nucleocapsids in vitro, supporting a role of actin in the budding of mature (i.e., not transcriptionally active) MV nucleocapsids. The same study showed that tubulin promoted MV RNA synthesis in in vitro RNA synthesis assays and could be co-immunoprecipitated with MV L, implying a further requirement for tubu-lin in MV replication (Moyer et al. 1990). These points, in addition to the large body of work illustrating the essential role of microtubules in facilitating long-distance transport within cells (reviewed in Greber and Way 2006), strongly support the notion that MV engages the microtubule network for transport within infected neurons. Ongoing work in a number of MV-focused laboratories is addressing this hypothesis, recognizing that it is hard to imagine a neurotropic virus achieving transport from one end of a neuron to the other without hijacking the cell’s railroad, the microtubule system.

Transport of Other Viruses in Neurons

The best-studied viruses, with regard to neuronal transport and spread, are the alphaherpesviruses, including herpes simplex virus 1 (HSV1) and pseudorabies virus (PRV). Cell imaging studies with both viruses have revealed by time-lapse

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fluorescence microscopy that these viruses travel retrogradely down the dendrite to the cell body (Bearer et al. 2000; Feierbach et al. 2007). This intracellular traffick-ing of the virus along microtubules can be disrupted by inhibitors of microtubule assembly, such as colchicine, vinblastine, or nocodazole (Sodeik et al. 1997; Topp et al. 1994). HSV-1 capsids entering a cell associate with the dynein complex, and overexpression of the dynactin component dynamitin (p50) blocks the transport of HSV1 to the nucleus (Dohner et al. 2002). Two somewhat contradictory models have emerged to explain virus transport away from the nucleus and subsequent viral egress. In the first model, herpesviral capsids and glycoproteins are transported separately, and assembly takes place somewhere along the axon shaft or at the axon terminus. The second model proposes that fully assembled enveloped virions are transported down the axon in vesicles (reviewed in Diefenbach et al. 2008; Lyman et al. 2007). As of last count, five different laboratories, three for HSV, two for PRV, have demonstrated contradictory findings as to which model is correct, and the controversy continues as the differences between variables, viruses, types of neu-rons, kinetics, and technical differences are sorted out (reviewed in Diefenbach et al. 2008). In either case, these two models provide the RNA virologist with an impetus for thought as to how smaller enveloped RNA viruses achieve egress from an infected neuron.

MV Spread in Neurons

MV Movement Within and Among Neurons

MV-infected CD46-expressing mice illustrated that, in vivo, neurons are the primary cell of the CNS infected by MV, even in animals where the expression of CD46 was not neuronally restricted (Oldstone et al. 1999). Similarly, neurons are the main tar-get of MV infection in the brain of infected SLAM transgenic mice (Sellin et al. 2006), as is the case for infected human CNS tissues (Allen et al. 1996). The Edmonston strain of MV engineered to express EGFP (rMV-EGFP) spread through brains of infected CD46 + /IFNAR –/– neonatal mice by neuronal processes. Fur-thermore, the authors detected EGFP-positive cells whose morphology was not neu-ronal and that the authors believed to be ependymal cells and neuroblasts (Duprex et al. 2000). This finding is of significance since it widens the population of cells sus-ceptible to MV in the CNS of this animal model, and the result differs from the pre-dominant neuronal infection described in other CD46-expressing animal models. Interestingly, the first report describing the generation and characterization of CD46 + /IFNAR –/– mice indicated that IC infection with MV Edmonston also resulted in infection of ependymal cells, oligodendrocytes, and neurons (Mrkic et al. 1998). One key difference among these studies and those of other CD46 transgenic mouse models and of infected humans is the absence of an intact type I interferon system, which may allow the virus the opportunity to access other cell populations.

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MV does not bud from infected neurons and MV-infected primary neurons do not form syncytia (Fig. 1.1B). The presence of nucleocapsids in the axons and at the presynaptic membranes of infected neurons suggests a contact-dependent, trans-synaptic spread of MV (Lawrence et al. 2000; Oldstone et al. 1999). This finding is consistent with data from autopsy specimens of SSPE cases, where MV budding is not apparent (Paula-Barbosa and Cruz 1981). Moreover, spread of MV-Edmonston in a neuronal population is independent of the receptor CD46 (Lawrence et al. 2000); thus, in this model, while CD46 was needed for viral entry, it was dispensable for neuron-to-neuron transmission.

MV spread via neuron–neuron contact has been confirmed in studies using rat organotypic hippocampal slices, as well as in tissue culture studies using differen-tiated human NT2 neurons (Lawrence et al. 2000; Ludlow et al. 2005; McQuaid et al. 1998). The spread of rMV-EGFP through hippocampal slices followed neu-ronal tracts, and no release of extracellular virus particles was observed. In this study, the interneuronal spread of rMV-EGFP was determined to be retrograde based on the spread of MV-EGFP from CA1 to CA3 pyramidal cells to granule cells within the slices and the known synaptic connectivity of these CNS substruc-tures. Furthermore, the MV envelope proteins (F, H, and M) and P proteins were detected in dendrites of infected neurons (Ehrengruber et al. 2002).

The mutations that accumulate upon MV adaptation to rodents (Rima et al. 1997; Vanchiere et al. 1995) do not seem to affect neuronal spread, as these rodent-adapted MVs have yielded data consistent with observations in humans and data from MV-infected CD46-expressing mice and primary neurons cultured from such mice (Duprex et al. 1999a; Mrkic et al. 1998; Schubert et al. 2006). For example, the hamster-neurotropic strain (HNT) of MV was used to infect Balb/c mice, and the resulting infection was limited to neurons and was not cytolytic. No virus assembly was detected (Van et al. 1979). Other work with HNT supports the lack of virus assembly following infection of weanling or adult mice (Griffin et al. 1974). The infection of weanling hamsters with HBS, another rodent-adapted MV isolated from an SSPE case, also revealed no budding virions in infected brains (Johnson and Swoveland 1977).

As mentioned earlier, MV neuronal spread can occur independent of the expression of CD46, suggesting that H may not be required for MV trans-synaptic spread. Recently, Makhortova et al. indirectly investigated the role of MV F in trans-synaptic spread by using FIP, fusion inhibitory peptide (Makhortova et al. 2007). FIP, a synthetic tripeptide (z-D-Phe-L-Phe-Gly), prevents fusion by multi-ple viruses, though its strongest activity is against MV (Norrby 1971; Richardson et al. 1980; Richardson and Choppin 1983). FIP reduced MV infection and sub-sequent spread through primary CD46-expressing neuron cultures, implicating a role for F in trans-neuronal transport. Furthermore, the neurotransmitter sub-stance P, whose active site is identical to FIP, also blocked MV neuronal spread. Genetic deletion of the substance P receptor, neurokinin-1 (NK-1), or pharmaco-logical inhibition of NK-1 decreased MV replication and subsequent disease in MV-infected mice. Overall, these data suggest that MV F may interact with NK-1 at the synapse to mediate trans-synaptic spread of MV (Makhortova et al. 2007). The idea that MV engages a cellular receptor other than SLAM or CD46 is not

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new (Andres et al. 2003; Blixenkrone-Moller et al. 1998; Takeda et al. 2007), though the potential that such a receptor may be bound by MV F, rather than H, broadens this original hypothesis. As a result of this work, the current model for MV trans-synaptic spread is a microfusion event at the synapse, illustrated in Fig. 1. 2 . In this model, at least the MV RNP and F glycoprotein are actively trans-ported to the synapse. F engagement of NK-1 may then allow a membrane fusion event to permit movement of the RNP from one neuron to the next. The neuro-transmitter receptors neurokinin-2 and -3 (NK-2 and NK-3) bind to neurotrans-mitters of the same family as substance P and therefore may also play a role in MV trans-synaptic spread.

Effects of Immune Responses on MV Spread in Neurons

It has long been known that multiple host factors contribute to the ability of a virus to replicate in a given cell. The literature contains several examples in which the elicitation or removal of a host immune response can alter MV replica-tion and subsequent spread. While most of these studies have measured the con-sequences of ablation of a particular immune cell type (e.g., recombinase-activating gene (RAG) knockout (KO) mice that lack functional T and B cells), intrinsic responses of neurons can also influence the outcome of infection. For example, expression of the heat shock protein hsp72, which would be induced during a febrile illness, increased MV gene expression following infection of neonatal hsp72 transgenic mice. The increased MV RNA levels correlated with a higher mortality rate (Carsillo et al. 2006), providing an example wherein the cellular response to viral infection increases MV pathogenesis. Conversely, the removal of the adaptive immune response protein TAP1 (the transporter associated with antigen presentation) led to enhanced spread of MV into the brains of infected mice. The HNT strain of MV trafficked from the olfactory bulb into the limbic

Fig. 2.2 Model for MV-trans-synaptic spread. RNPs and F are transported to the synapse. The interaction of F with neurokinin-1 triggers a microfusion event, allowing the RNP to cross the synapse and infect the synaptically connected neuron. Neurokinin-2 and -3 may act as receptors for MV F, given their homology to neurokinin-1

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system of wild-type and TAP1 –/– mice but showed a greater dissemination into the brains of TAP1 –/– mice, as compared to that seen in wild-type mice (Urbanska et al. 1997).

Finally, an association was found between promoter polymorphisms of the innate immunity protein MxA and the occurrence of SSPE in Japan. The most fre-quent polymorphisms caused the MxA promoter to be more responsive to type I interferon than the wild-type promoter sequence. Therefore, the increased activity of MxA in response to type I interferon positively correlated with SSPE occurrence, suggesting that MxA may play a role in the establishment of persistent MV infec-tions in neurons (Torisu et al. 2004), though these studies have been somewhat controversial (Pipo-Deveza et al. 2006). The analysis of SSPE lesions of affected individuals revealed that the cells staining positive for MxA were mainly astrocytes located in a belt around the region of MV antigen-positive cells in affected areas of the brain. Similar to the conclusions drawn from the Japanese cases of SSPE, the authors suggested that MxA plays an important role in slowing down viral spread in SSPE and therefore may contribute to the persistent nature of this MV CNS infection (Ogata et al. 2004; Torisu et al. 2004).

Remaining Questions

As this review has highlighted, the molecular mechanisms that govern MV trans-port in neurons are slowly coming into focus. Ehrengruber’s study on the spread of rMV-EGFP in cultured rat hippocampal slices found that MV spreads through the rat hippocampus in a retrograde direction, with the MV F, H, M, and P proteins localizing to the dendrite of infected neurons (Ehrengruber et al. 2002). Lawrence et al. demonstrated by electron microscopy the presence of MV nucleocapsids at the presynaptic membrane of infected primary hippocampal neurons, suggesting that MV may, in fact, spread in an anterograde direction, or from the axon of an infected neuron to the dendrite of a neighboring neuron (Lawrence et al. 2000). However, these studies could not distinguish between nucleocapsids that were exit-ing or nucleocapsids that were entering the neuron. Furthermore, there have been reports in support of both anterograde and retrograde trafficking of MV in neurons (McQuaid et al. 1998; Urbanska et al. 1997), which may not be unexpected as the virus must travel to the cell body to replicate and then back to the periphery of the neuron for egress, regardless of which neuronal process it uses (the dendrite or the axon) for entry and for exit. While a comprehensive view of how the unique environment of the neuron affects MV replication, spread and, ultimately, neu-ropathogenesis awaits further study, the tools and ideas are in place for exciting advances in the coming years.

Acknowledgements We gratefully acknowledge the assistance of Christine Matullo and Lauren O’Donnell in the preparation of this review. V.A. Young is supported by T32 NS007180-25 from the University of Pennsylvania. G. R. is supported by NIH grants NS40500, MH56951, and CA006927, as well as generous support from the F.M. Kirby Foundation.

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References

Ahmad FJ, Echeverri CJ, Vallee RB, Baas PW (1998) Cytoplasmic dynein and dynactin are required for the transport of microtubules into the axon. J Cell Biol 140:391–401

Ahmad-Annuar A, Shah P, Hafezparast M, Hummerich H, Witherden AS, Morrison KE, Shaw PJ, Kirby J, Warner TT, Crosby A, Proukakis C, Wilkinson P, Orrell RW, Bradley L, Martin JE, Fisher EMC (2003) No association with common Caucasian genotypes in exons 8, 13 and 14 of the human cytoplasmic dynein heavy chain gene (DNCHC1) and familial motor neuron disorders. Amyotrophic Lateral Scler Other Motor Neuron Disord 4:150–157

Albrecht P, Burnstein T, Klutch MJ, Hicks HT, Ennis FA (1977) Subacute sclerosing panencepha-litis: experimental infection in primates. Science 195:64–66

Allen IV, McQuaid S, McMahon J, Kirk J, McConnell R (1996) The significance of measles virus antigen and genome distribution in the CNS in SSPE for mechanisms of viral spread and demyelination. J Neuropathol Exp Neurol 55:471–480

Andres O, Obojes K, Kim KS, ter Meulen V, Schneider-Schaulies J (2003) CD46- and CD150-independent endothelial cell infection with wild-type measles viruses. J Gen Virol 84:1189–1197

Atabani S, Byrnes A, Jaye A, Kidd IM, Magnusen A, Whittle H, Karp C (2001) Natural measles causes prolonged suppression of Interleukin-12 production. J Infect Dis 184:1–9

Baczko K, Liebert UG, Cattaneo R, Billeter MA, Roos RP, ter Meulen, V (1988) Restriction of measles virus gene expression in measles inclusion body encephalitis. J Infect Dis 158:144–150

Bearer EL, Breakefield XO, Schuback D, Reese TS, LaVail JH (2000) Retrograde axonal transport of herpes simplex virus: evidence for a single mechanism and a role for tegument. Proc Natl Acad Sci U S A 97:8146–8150

Billeter MA, Cattaneo R, Spielhofer P, Kaelin K, Huber M, Schmid A, Baczko K, ter Meulen V (1994) Generation and properties of measles virus mutations typically associated with suba-cute sclerosing panencephalitis. Ann N Y Acad Sci 724:367–377

Bitnun A, Shannon P, Durward A, Rota PA, Bellini WJ, Graham C, Wang E, Ford-Jones EL, Cox P, Becker L, Fearon M, Petric M, Tellier R (1999) Measles inclusion-body encephalitis caused by the vaccine strain of measles virus. Clin Infect Dis 29:855–861

Blau DM, Compans RW (1995) Entry and release of measles virus are polarized in epithelial cells. Virology 210:91–99

Blixenkrone-Moller M, Bernard A, Bencsik A, Sixt N, Diamond LE, Logan JS, Wild TF (1998) Role of CD46 in measles virus infection in CD46 transgenic mice. Virology 249:238–248

Bohn W, Rutter G, Hohenberg H, Mannweiler K, Nobis P (1986) Involvement of actin fila-ments in budding of measles virus: studies on cytoskeletons of infected cells. Virology 149:91–106

Bonami F, Rudd PA, von Messling V (2007) Disease duration determines canine distemper virus neurovirulence. J Virol 81:12066–12070

Brown HR, Thormar H, Barshatzky M, Wisniewski HM (1985) Localization of measles virus antigens in subacute sclerosing panencephalitis in ferrets. Lab Anim Sci 35:233–237

Brown HR, Goller NL, Thormar H, Rudelli R, Tourtellotte WW, Shapshak P, Boostanfar R, Wisniewski HM (1987) Measles virus matrix protein gene expression in a subacute sclerosing panencephalitis patient brain and virus isolate demonstrated by cDNA hybridization and immunocytochemistry. Acta Neuropathol 75:123–130

Burkhardt JK, Echeverri CJ, Nilsson T, Vallee RB (1997) Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution. J Cell Biol 139:469–484

Carsillo T, Traylor Z, Choi C, Niewiesk S, Oglesbee M (2006) hsp72, a host determinant of mea-sles virus neurovirulence. J Virol 80:11031–11039

Castro AE, Burnstein T, Byington DP (1972) Properties in cell culture of a hamster brain-adapted subacute sclerosing panencephalitis virus. J Gen Virol 16:413–417

b12325337-0001ztc.indd 23 10/3/2008 10:29:37 AM


Cathomen T, Mrkic B, Spehner D, Drillien R, Naef R, Pavlovic J, Aguzzi A, Billeter MA, Cattaneo R (1998a) A matrix-less measles virus is infectious and elicits extensive cell fusion: consequences for propagation in the brain. EMBO J 17:3899–3908

Cathomen T, Naim HY, Cattaneo R (1998b) Measles viruses with altered envelope protein cyto-plasmic tails gain cell fusion competence. J Virol 72:1224–1234

Cattaneo R, Rebmann G, Baczko K, ter Meulen V, Billeter MA (1987a) Altered ratios of measles virus transcripts in diseased human brains. Virology 160:523–526

Cattaneo R, Rebmann G, Schmid A, Baczko K, ter Meulen V, Billeter MA (1987b) Altered tran-scription of a defective measles virus genome derived from a diseased human brain. EMBO J 6:681–688

Cattaneo R, Schmid A, Eschle D, Baczko K, ter Meulen V, Billeter MA (1988) Biased hypermuta-tion and other genetic changes in defective measles viruses in human brain infections. Cell 55:255–265

Colf LA, Juo ZS, Garcia KC (2007) Structure of the measles virus hemagglutinin. Nat Struct Mol Biol 14:1227–1228

Coovadia HM, Wesley A, Henderson LG, Brain P, Vos GH, Hallett AF (1978) Alterations in immune responsiveness in acute measles and chronic post-measles chest disease. Int Arch Allergy Appl Immunol 56:14–23

de Swart RL, Ludlow M, deWitte L, Yanagi Y, van Amerongen G, McQuaid S, Yuksel S, Geijtenbeek TBH, Duprex WP, Osterhaus ADME (2007) Predominant infection of CD150 (+) lymphocytes and dendritic cells during measles virus infection of macaques. PLoS Pathog 3:l771

Devaux P, von Messling V, Songsungthong W, Springfeld C, Cattaneo R (2007) Tyrosine 110 in the measles virus phosphoprotein is required to block STAT1 phosphorylation. Virology 360:72–83

Dhib-Jalbut S, Johnson KP (1994) Measles virus diseases. In: McKendall RR, Stroop WG (eds) Handbook of neurovirology. Marcel Dekker, New York, pp 539–554

Diefenbach RJ, Miranda-Saksena M, Douglas MW, Cunningham AL (2008) Transport and egress of herpes simplex virus in neurons. Rev Med Virol 18:35–51

Dohner K, Wolfstein A, Prank U, Echeverri C, Dujardin D, Vallee R, Sodeik B (2002) Function of dynein and dynactin in herpes simplex virus capsid transport. Mol Biol Cell 13:2795–2809

Dorig RE, Marcil A, Chopra A, Richardson CD (1993) The human CD46 molecule is a receptor for measles virus (Edmonston strain). Cell 75:295–305

Dubois-Dalcq M, Coblentz JM, Pleet AB (1974) Subacute sclerosing panencephalitis. Unusual nuclear inclusions and lengthy clinical course. Arch Neurol 31:355–363

Duprex WP, Duffy I, McQuaid S, Hamill L, Cosby SL, Billeter MA, Schneider-Schaulies J, ter Meulen V, Rima BK (1999a) The H gene of rodent brain-adapted measles virus confers neu-rovirulence to the Edmonston vaccine strain. J Virol 73:6916–6922

Duprex WP, McQuaid S, Hangartner L, Billeter MA, Rima BK (1999b) Observation of measles virus cell-to-cell spread in astrocytoma cells by using a green fluorescent protein-expressing recombinant virus. J Virol 73:9568–9575

Duprex WP, McQuaid S, Roscic-Mrkic B, Cattaneo R, McCallister C, Rima BK (2000) In vitro and in vivo infection of neural cells by a recombinant measles virus expressing enhanced green fluorescent protein. J Virol 74:7972–7979

Echeverri CJ, Paschal BM, Vaughan KT, Vallee RB (1996) Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J Cell Biol 132:617–633

Ehrengruber MU, Ehler E, Billeter MA, Naim HY (2002) Measles virus spreads in rat hippocam-pal neurons by cell-to-cell contact and in a polarized fashion. J Virol 76:5720–5728

Erlenhoefer C, Wurzer WJ, Loffler S, Schneider-Schaulies S, ter Meulen V, Schneider-Schaulies J (2001) CD150 (SLAM) is a receptor for measles virus but is not involved in viral contact-mediated proliferation inhibition. J Virol 75:4499–4505

Erlenhofer C, Duprex WP, Rima BK, ter Meulen V, Schneider-Schaulies J (2002) Analysis of receptor (CD46, CD150) usage by measles virus. J Gen Virol 83:1431–1436

b12325337-0001ztc.indd 24 10/3/2008 10:29:37 AM


Esolen LM, Takahashi K, Johnson RT, Vaisberg A, Moench TR, Wesselingh SL, Griffin DE (1995) Brain endothelial cell infection in children with acute fatal measles. J Clin Invest 96:2478–2481

Evlashev A, Moyse E, Valentin H, Azocar O, Trescol-Biemont MC, Marie JC, Rabourdin-Combe C, Horvat B (2000) Productive measles virus brain infection and apoptosis in CD46 transgenic mice. J Virol 74:1373–1382

Evlashev A, Valentin H, Rivailler P, Azocar O, Rabourdin-Combe C, Horvat B (2001) Differential permissivity to measles virus infection of human and CD46-transgenic murine lymphocytes. J Gen Virol 82:2125–2129

Feierbach B, Bisher M, Goodhouse J, Enquist LW (2007) In vitro analysis of transneuronal spread of an alphaherpesvirus infection in peripheral nervous system neurons. J Virol 81:6846–6857

Firsching R, Buchholz CJ, Schneider U, Cattaneo R, ter Meulen V, Schneider-Schaulies J (1999) Measles virus spread by cell-cell contacts: uncoupling of contact-mediated receptor (CD46) downregulation from virus uptake. J Virol 73:5265–5273

Fugier-Vivier I, Servet-Delprat C, Rivailler P, Rissoan MC, Liu YJ, Rabourdin-Combe C (1997) Measles virus suppresses cell-mediated immunity by interfering with the survival and func-tions of dendritic and T cells. J Exp Med 186:813–823

Gahwiler BH (1981) Organotypic monolayer cultures of nervous tissue. J Neurosci Methods 4:329–342

Greber UF, Way M (2006) A superhighway to virus infection. Cell 124:741–754 Griffin DE (2001) Measles virus. In: Knipe DM, Howley PM (eds) Fields virology. Lippincott

Williams and Wilkins, Philadelphia, pp 1401–1441 Griffin DE, Pan CH, Moss WJ (2008) Measles vaccines. Front Biosci 13:1352–1370 Griffin DE, Mullinix J, Narayan O, Johnson RT (1974) Age dependence of viral expression: com-

parative pathogenesis of two rodent-adapted strains of measles virus in mice. Infect Immun 9:690–695

Hafezparast M, Klocke R, Ruhrberg C, Marquardt A, Ahmad-Annuar A, Bowen S, Lalli G, Witherden AS, Hummerich H, Nicholson S, Morgan PJ, Oozageer R, Priestley JV, Averill S, King VR, Ball S, Peters J, Toda T, Yamamoto A, Hiraoka Y, Augustin M, Korthaus D, Wattler S, Wabnitz P, Dickneite C, Lampel S, Boehme F, Peraus G, Popp A, Rudelius M, Schlegel J, Fuchs H, de Angelis MH, Schiavo G, Shima DT, Russ AP, Stumm G, Martin JE, Fisher EMC (2003) Mutations in dynein link motor neuron degeneration to defects in retrograde transport. Science 300:808–812

Hafezparast M, Brandner S, Linehan J, Martin JE, Collinge J, Fisher EMC (2004) Prion disease incubation time is not affected in mice heterozygous for a dynein mutation. Biochem Biophys Res Comm 326:18–22

Henry T, Gorvel JP, Meresse S (2006) Molecular motors hijacking by intracellular pathogens. Cell Microbiol 8:23–32

Hirsch RL, Griffin DE, Johnson RT, Cooper SJ, Lindo de Soriano I, Roedenbeck S, Vaisberg A (1984) Cellular immune responses during complicated and uncomplicated measles virus infec-tions of man. Clin Immunol Immunopathol 31:1–12

Horvat B, Rivailler P, Varior-Krishnan G, Cardoso A, Gerlier D, Rabourdin-Combe C (1996) Transgenic mice expressing human measles virus (MV) receptor CD46 provide cells exhibit-ing different permissivities to MV infections. J Virol 70:6673–6681

Hsu EC, Iorio C, Sarangi F, Khine AA, Richardson CD (2001) CDw150(SLAM) is a receptor for a lymphotropic strain of measles virus and may account for the immunosuppressive properties of this virus. Virology 279:9–21

Johnson KP, Norrby E (1974) Subacute sclerosing panencephalitis (SSPE) agent in hamsters. III. Induction of defective measles infection in hamster brain. Exp Mol Pathol 21:166–178

Johnson KP, Swoveland P (1977) Measles antigen distribution in brains of chronically infected hamsters. An immunoperoxidase study of experimental subacute sclerosing panencephalitis. Lab Invest 37:459–465

Johnson RT (1998) Viral infections of the nervous system. Lippincott-Raven, Philadelphia.

b12325337-0001ztc.indd 25 10/3/2008 10:29:37 AM


Jouvenet N, Monaghan P, Way M, Wileman T (2004) Transport of African swine fever virus from assembly sites to the plasma membrane is dependent on microtubules and conventional kinesin. J Virol 78:7990–8001

Karp CL, Wysocka M, Wahl LM, Ahearn JM, Cuomo PJ, Sherry B, Trinchieri G, Griffin DE (1996) Mechanism of suppression of cell-mediated immunity by measles virus. Science 273:228–231

Katayama Y, Hotta H, Nishimura A, Tatsuno Y, Homma M (1995) Detection of measles virus nucleoprotein mRNA in autopsied brain tissues. J Gen Virol 76:3201–3204

Katayama Y, Kohso K, Nishimura A, Tatsuno Y, Homma M, Hotta H (1998) Detection of measles virus mRNA from autopsied human tissues. J Clin Microbiol 36:299–301

Lamb RA, Kolakofsky D (2001) Paramyxoviridae: the viruses and their replication. In: Knipe DM, Howley PM (eds) Fields virology. Lippincott Williams and Wilkins, Philadelphia, pp 1305–1340

Lawrence DM, Vaughn MM, Belman AR, Cole JS, Rall GF (1999) Immune response-mediated protection of adult but not neonatal mice from neuron-restricted measles virus infection and central nervous system disease. J Virol 73:1795–1801

Lawrence DM, Patterson CE, Gales TL, D’Orazio JL, Vaughn MM, Rall GF (2000) Measles virus spread between neurons requires cell contact but not CD46 expression, syncytium formation, or extracellular virus production. J Virol 74:1908–1918

Leonard VHJ, Sinn PL, Hodge G, Miest T, Devaux P, Oezguen N, Braun W, McCray PB, McChesney MB, Cattaneo R (2008) Measles virus blind to its epithelial cell receptor remains virulent in rhesus monkeys but cannot cross the airway epithelium and is not shed. J Clin Invest 118:2448

Leopold PL, Pfister KK (2006) Viral strategies for intracellular trafficking: motors and microtubules. Traffic 7:516–523

Ludlow M, McQuaid S, Cosby SL, Cattaneo R, Rima BK, Duprex WP (2005) Measles virus superinfection immunity and receptor redistribution in persistently infected NT2 cells. J Gen Virol 86:2291–2303

Ludlow M, Duprex WP, Cosby SL, Allen IV, McQuaid S (2007) Advantages of using recombinant measles viruses expressing a fluorescent reporter gene with vibratome slice technology in experimental measles neuropathogenesis. Neuropathol Appl Neurobiol doi: 10.1111/j.1365–2990.2007.00900.x

Lyman MG, Feierbach B, Curanovic D, Bisher M, Enquist LW (2007) Pseudorabies virus Us9 directs axonal sorting of viral capsids. J Virol 81:11363–11371

Mackenzie GG, Keen CL, Oteiza PI (2006) Microtubules are required for NF-kappaB nuclear translocation in neuroblastoma IMR-32 cells: modulation by zinc. J Neurochem 99:402–415

Maisner A, Klenk HD, Herrler G (1998) Polarized budding of measles virus is not determined by viral surface glycoproteins. J Virol 72:5276–5278

Makhortova NR, Askovich P, Patterson CE, Gechman LA, Gerard NP, Rall GF (2007) Neurokinin-1 enables measles virus trans-synaptic spread in neurons. Virology 362:235–244

Manchester M, Rall GF (2001) Model systems: transgenic mouse models for measles pathogene-sis. Trends Microbiol 9:19–23

Manchester M, Eto DS, Oldstone MBA (1999) Characterization of the inflammatory response during acute measles encephalitis in NSE-CD46 transgenic mice. J Neuroimmunol 96:207–217

Manchester M, Eto DS, Valsamakis A, Liton PB, Fernandez-Munoz R, Rota PA, Bellini WJ, Forthal DN, Oldstone MBA (2000) Clinical isolates of measles virus use CD46 as a cellular receptor. J Virol 74:3967–3974

Mandelkow E, Mandelkow EM (2002) Kinesin motors and disease. Trends Cell Biol 12:585–591

McQuaid S, Cosby SL (2002) An immunohistochemical study of the distribution of the measles virus receptors, CD46 and SLAM, in normal human tissues and subacute sclerosing panen-cephalitis. Lab Invest 82:403–409

b12325337-0001ztc.indd 26 10/3/2008 10:29:37 AM


McQuaid S, Campbell S, Wallace IJ, Kirk J, Cosby SL (1998) Measles virus infection and replica-tion in undifferentiated and differentiated human neuronal cells in culture. J Virol 72:5245–5250

Mehta PD, Thormar H (1979) Immunological studies of subacute measles encephalitis in ferrets: similarities to human subacute sclerosing panencephalitis. J Clin Microbiol 9:601–604

Moeller-Ehrlich K, Ludlow M, Beschorner R, Meyermann R, Rima BK, Duprex WP, Niewiesk S, Schneider-Schaulies J (2007) Two functionally linked amino acids in the stem 2 region of measles virus haemagglutinin determine infectivity and virulence in the rodent central nervous system. J Gen Virol 88:3112–3120

Moll M, Klenk HD, Herrler G, Maisner A (2001) A single amino acid change in the cytoplasmic domains of measles virus glycoproteins H and F alters targeting, endocytosis, and cell fusion in polarized Madin-Darby canine kidney cells. J Biol Chem 276:17887–17894

Moll M, Pfeuffer J, Klenk HD, Niewiesk S, Maisner A (2004) Polarized glycoprotein targeting affects the spread of measles virus in vitro and in vivo. J Gen Virol 85:1019–1027

Moss W, Ryon J, Monze M, Griffin D (2002) Differential regulation of interleukin-4 (IL-4), IL-5, and IL-10 during measles in Zambian children. J Infect Dis 186:879–887

Moyer SA, Baker SC, Horikami SM (1990) Host cell proteins required for measles virus repro-duction. J Gen Virol 71:775–783

Mrkic B, Pavlovic J, Rulicke T, Volpe P, Buchholz CJ, Hourcade D, Atkinson JP, Aguzzi A, Cattaneo R (1998) Measles virus spread and pathogenesis in genetically modified mice. J Virol 72:7420–7427

Naim HY, Ehler E, Billeter MA (2000) Measles virus matrix protein specifies apical virus release and glycoprotein sorting in epithelial cells. EMBO J 19:3576–3585

Naniche D, Varior-Krishnan G, Cervoni F, Wild TF, Rossi B, Rabourdin-Combe C, Gerlier D (1993) Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus. J Virol 67:6025–6032

Naniche D, Reed SI, Oldstone MB (1999) Cell cycle arrest during measles virus infection: a G0-like block leads to suppression of retinoblastoma protein expression. J Virol 73:1894–1901

Niewiesk S (1999) Cotton rats ( Sigmodon hispidus ): an animal model to study the pathogenesis of measles virus infection. Immunol Lett 65:47–50

Niewiesk S, Schneider-Schaulies J, Ohnimus H, Jassoy C, Schneider-Schaulies S, Diamond L, Logan JS, ter Meulen V (1997) CD46 expression does not overcome the intracellular block of measles virus replication in transgenic rats. J Virol 71:7969–7973

Niewiesk S, Ohnimus H, Schnorr JJ, Gotzelmann M, Schneider-Schaulies S, Jassoy C, ter Meulen V (1999) Measles virus-induced immunosuppression in cotton rats is associated with cell cycle retardation in uninfected lymphocytes. J Gen Virol 80:2023–2029

Niewiesk S, Gotzelmann M, ter Meulen V (2000) Selective in vivo suppression of T lym p-hocyte responses in experimental measles virus infection. Proc Natl Acad Sci U S A 97:4251–4255

Norrby E (1971) The effect of a carbobenzoxy tripeptide on the biological activities of measles virus. Virology 44:599–608

Norrby E, Kristensson K (1997) Measles virus in the brain. Brain Res Bull 44:213–220 Ogata S, Ogata A, Schneider-Schaulies S, Schneider-Schaulies J (2004) Expression of the inter-

feron-alpha/beta-inducible MxA protein in brain lesions of subacute sclerosing panencephali-tis. J Neurol Sci 223:113–119

Ohka S, Yang WX, Terada E, Iwasaki K, Nomoto A (1998) Retrograde transport of intact polio-virus through the axon via the fast transport system. Virology 250:67–75

Ohka S, Matsuda N, Tohyama K, Oda T, Morikawa M, Kuge S, Nomoto A (2004) Receptor (CD155)-dependent endocytosis of poliovirus and retrograde axonal transport of the endo-some. J Virol 78:7186–7198

Ohno S, Ono N, Seki F, Takeda M, Kura S, Tsuzuki T, Yanagi Y (2007) Measles virus infection of SLAM (CD150) knockin mice reproduces tropism and immunosuppression in human infec-tion. J Virol 81:1650–1659

b12325337-0001ztc.indd 27 10/3/2008 10:29:38 AM


Oldstone MBA, Lewicki H, Thomas D, Tishon A, Dales S, Patterson J, Manchester M, Homann D, Naniche D, Holz A (1999) Measles virus infection in a transgenic model: virus-induced immunosuppression and central nervous system disease. Cell 98:629–640

Ono N, Tatsuo H, Hidaka Y, Aoki T, Minagawa H, Yanagi Y (2001a) Measles viruses on throat swabs from measles patients use signaling lymphocytic activation molecule (CDw150) but not CD46 as a cellular receptor. J Virol 75:4399–4401

Ono N, Tatsuo H, Tanaka K, Minagawa H, Yanagi Y (2001b) V domain of human SLAM (CDw150) is essential for its function as a measles virus receptor. J Virol 75:1594–1600

Pan CH, Valsamakis A, Colella T, Nair N, Adams RJ, Polack FP, Greer CE, Perri S, Polo JM, Griffin DE (2005) Inaugural article: modulation of disease, T cell responses, and measles virus clearance in monkeys vaccinated with H-encoding alphavirus replicon particles. Proc Natl Acad Sci U S A 102:11581–11588

Pasetti MF, Resendiz-Albor A, Ramirez K, Stout R, Papania M, Adams RJ, Polack FP, Ward BJ, Burt D, Chabot S, Ulmer J, Barry EM, Levine MM (2007) Heterologous prime-boost strategy to immunize very young infants against measles: pre-clinical studies in rhesus macaques. Clin Pharmacol Ther 82:672–685

Patterson JB, Cornu TI, Redwine J, Dales S, Lewicki H, Holz A, Thomas D, Billeter MA, Oldstone MBA (2001) Evidence that the hypermutated M protein of a subacute sclerosing panencephalitis measles virus actively contributes to the chronic progressive CNS disease. Virology 291:215–225

Patterson CE, Lawrence DM, Echols LA, Rall GF (2002) Immune-mediated protection from measles virus-induced central nervous system disease is noncytolytic and gamma interferon dependent. J Virol 76:4497–4506

Patterson CE, Daley JK, Echols LA, Lane TE, Rall GF (2003) Measles virus infection induces chemokine synthesis by neurons. J Immunol 171:3102–3109

Paula-Barbosa MM, Cruz C (1981) Nerve cell fusion in a case of subacute sclerosing panencephalitis. Ann Neurol 9:400–403

Pipo-Deveza JR, Kusuhara K, Silao CLT, Lukban MB, Salonga AM, Sanchez BC, Kira R, Takemoto M, Torisu H, Hara T (2006) Analysis of MxA, IL-4, and IRF-1 genes in Filipino patients with subacute sclerosing panencephalitis. Neuropediatrics 37:222–228

Ploubidou A, Way M (2001) Viral transport and the cytoskeleton. Curr Opin Cell Biol 13:97–105

Plumb J, Duprex WP, Cameron CH, Richter-Landsberg C, Talbot P, McQuaid S (2002) Infection of human oligodendroglioma cells by a recombinant measles virus expressing enhanced green fluorescent protein. J Neurovirol 8:24–34

Polack FP, Lee SH, Permar S, Manyara E, Nousari HG, Jeng Y, Mustafa F, Valsamakis A, Adams RJ, Robinson HL, Griffin DE (2000) Successful DNA immunization against measles: neutral-izing antibody against either the hemagglutinin or fusion glycoprotein protects rhesus macaques without evidence of atypical measles. Nat Med 6:776–781

Radecke F, Spielhofer P, Schneider H, Kaelin K, Huber M, Dotsch C, Christiansen G, Billeter MA (1995) Rescue of measles viruses from cloned DNA. EMBO J 14:5773–5784

Radtke K, Dohner K, Sodeik B (2006) Viral interactions with the cytoskeleton: a hitchhiker’s guide to the cell. Cell Microbiol 8:387–400

Rall GF (2003) Measles virus 1998–2002: progress and controversy. Annu Rev Microbiol 57:343–367

Rall GF, Manchester M, Daniels LR, Callahan EM, Belman AR, Oldstone MB (1997) A trans-genic mouse model for measles virus infection of the brain. Proc Natl Acad Sci U S A 94:4659–4663

Richardson CD, Choppin PW (1983) Oligopeptides that specifically inhibit membrane fusion by paramyxoviruses: studies on the site of action. Virology 131:518–532

Richardson CD, Scheid A, Choppin PW (1980) Specific inhibition of paramyxovirus and myxo-virus replication by oligopeptides with amino acid sequences similar to those at the N-termini of the F1 or HA2 viral polypeptides. Virology 105:205–222

Riedl P, Moll M, Klenk HD, Maisner A (2002) Measles virus matrix protein is not cotransported with the viral glycoproteins but requires virus infection for efficient surface targeting. Virus Res 83:1–12

b12325337-0001ztc.indd 28 10/3/2008 10:29:38 AM


Rietdorf J, Ploubidou A, Reckmann I, Holmstrom A, Frischknecht F, Zettl M, Zimmermann T, Way M (2001) Kinesin-dependent movement on microtubules precedes actin-based motility of vaccinia virus. Nat Cell Biol 3:992–1000

Rima BK, Duprex WP (2005) Molecular mechanisms of measles virus persistence. Virus Res 111:132–147

Rima BK, Earle JA, Baczko K, ter Meulen V, Liebert UG, Carstens C, Carabana J, Caballero M, Celma ML, Fernandez-Munoz R (1997) Sequence divergence of measles virus haemagglutinin during natural evolution and adaptation to cell culture. J Gen Virol 78:97–106

Roos RP, Griffin DE, Johnson RT (1978) Determinants of measles virus (hamster neurotropic strain) replication in mouse brain. J Infect Dis 137:722–727

Rudd PA, Cattaneo R, von Messling V (2006) Canine distemper virus uses both the anterograde and the hematogenous pathway for neuroinvasion. J Virol 80:9361–9370

Runkler N, Pohl C, Schneider-Schaulies S, Klenk HD, Maisner A (2007) Measles virus nucleo-capsid transport to the plasma membrane requires stable expression and surface accumulation of the viral matrix protein. Cell Microbiol 9:1203–1214

Runkler N, Dietzel E, Moll M, Klenk HD, Maisner A (2008) Glycoprotein targeting signals influ-ence the distribution of measles virus envelope proteins and virus spread in lymphocytes. J Gen Virol 89:687–696

Samuel MA, Wang H, Siddharthan V, Morrey JD, Diamond MS (2007) Axonal transport mediates West Nile virus entry into the central nervous system and induces acute flaccid paralysis. Proc Natl Acad Sci U S A 104:17140–17145

Schneider H, Spielhofer P, Kaelin K, Dotsch C, Radecke F, Sutter G, Billeter MA (1997) Rescue of measles virus using a replication-deficient vaccinia-T7 vector. J Virol Methods 64:57–64

Schneider-Schaulies J, ter Meulen V, Schneider-Schaulies S (2003) Measles infection of the central nervous system. J Neurovirol 9:247–252

Schnorr JJ, Seufert M, Schlender J, Borst J, Johnston IC, ter Meulen V, Schneider-Schaulies S (1997) Cell cycle arrest rather than apoptosis is associated with measles virus contact-medi-ated immunosuppression in vitro. J Gen Virol 78:3217–3226

Schubert S, Moller-Ehrlich K, Singethan K, Wiese S, Duprex WP, Rima BK, Niewiesk S, Schneider-Schaulies J (2006) A mouse model of persistent brain infection with recombinant measles virus. J Gen Virol 87:2011–2019

Sellin CI, Davoust N, Guillaume V, Baas D, Belin MF, Buckland R, Wild TF, Horvat B (2006) High pathogenicity of wild-type measles virus infection in CD150 (SLAM) transgenic mice. J Virol 80:6420–6429

Servet-Delprat C, Vidalain PO, Bausinger H, Manie S, Le Deist F, Azocar O, Hanau D, Fischer A, Rabourdin-Combe C (2000) Measles virus induces abnormal differentiation of CD40 lig-and-activated human dendritic cells. J Immunol 164:1753–1760

Sheppard RD, Raine CS, Burnstein T, Bornstein MB, Feldman LA (1975) Cell-associated suba-cute sclerosing panencephalitis agent studied in organotypic central nervous system cultures: viral rescue attempts and morphology. Infect Immun 12:891–900

Shingai M, Inoue N, Okuno T, Okabe M, Akazawa T, Miyamoto Y, Ayata M, Honda K, Kurita-Taniguchi M, Matsumoto M, Ogura H, Taniguchi T, Seya T (2005) Wild-type measles virus infection in human CD46/CD150-transgenic mice: CD11c-positive dendritic cells establish systemic viral infection. J Immunol 175:3252–3261

Sips GJ, Chesik D, Glazenburg L, Wilschut J, De Keyser J, Wilczak N (2007) Involvement of morbilliviruses in the pathogenesis of demyelinating disease. Rev Med Virol 17:223–244

Smith GA, Gross SP, Enquist LW (2001) Herpesviruses use bidirectional fast-axonal transport to spread in sensory neurons. Proc Natl Acad Sci U S A 98:3466–3470

Sodeik B, Ebersold MW, Helenius A (1997) Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus. J Cell Biol 136:1007–1021

Stallcup KC, Raine CS, Fields BN (1983) Cytochalasin B inhibits the maturation of measles virus. Virology 124:59–74

Steele MD, Giddens WE Jr, Valerio M, Sumi SM, Stetzer ER (1982) Spontaneous paramyxoviral encephalitis in nonhuman primates ( Macaca mulatta and M. nemestrina ). Vet Pathol 19:132–139

Sun X, Burns JB, Howell JM, Fujinami RS (1998) Suppression of antigen-specific T cell prolifer-ation by measles virus infection: role of a soluble factor in suppression. Virology 246:24–33

b12325337-0001ztc.indd 29 10/3/2008 10:29:38 AM


Takasu T, Mgone JM, Mgone CS, Miki K, Komase K, Namae H, Saito Y, Kokubun Y, Nishimura T, Kawanishi R, Mizutani T, Markus TJ, Kono J, Asuo PG, Alpers MP (2003) A continuing high incidence of subacute sclerosing panencephalitis (SSPE) in the Eastern Highlands of Papua New Guinea. Epidemiol Infect 131:887–898

Takeda M, Tahara M, Hashiguchi T, Sato TA, Jinnouchi F, Ueki S, Ohno S, Yanagi Y (2007) A human lung carcinoma cell line supports efficient measles virus growth and syncytium forma-tion via a SLAM- and CD46-independent mechanism. J Virol 81:12091–12096

Tamashiro VG, Perez HH, Griffin DE (1987) Prospective study of the magnitude and duration of changes in tuberculin reactivity during uncomplicated and complicated measles. Pediatr Infect Dis J 6:451–454

Tatsuo H, Ono N, Tanaka K, Yanagi Y (2000) SLAM (CDw150) is a cellular receptor for measles virus. Nature 406:893–897

Thormar H, Mehta PD, Lin FH, Brown HR, Wisniewski HM (1983) Presence of oligoclonal immunoglobulin G bands and lack of matrix protein antibodies in cerebrospinal fluids and sera of ferrets with measles virus encephalitis. Infect Immun 41:1205–1211

Topp KS, Meade LB, LaVail JH (1994) Microtubule polarity in the peripheral processes of trigeminal ganglion cells: relevance for the retrograde transport of herpes simplex virus. J Neurosci 14:318–325

Torisu H, Kusuhara K, Kira R, Bassuny WM, Sakai Y, Sanefuji M, Takemoto M, Hara T (2004) Functional MxA promoter polymorphism associated with subacute sclerosing panencephalitis. Neurology 62:457–460

Tyrrell DLJ, Norrby E (1978) Structural polypeptides of measles virus. J Gen Virol 39:219–229 Urbanska EM, Chambers BJ, Ljunggren HG, Norrby E, Kristensson K (1997) Spread of measles

virus through axonal pathways into limbic structures in the brain of TAP1 –/– mice. J Med Virol 52:362–369

Van PC, Rammohan KW, McFarland HF, Dubois-Dalcq M (1979) Selective neuronal, dendritic, and postsynaptic localization of viral antigen in measles-infected mice. Lab Invest 40:99–108

Vanchiere JA, Bellini WJ, Moyer SA (1995) Hypermutation of the phosphoprotein and altered mRNA editing in the hamster neurotropic strain of measles virus. Virology 207:555–561

Verhey KJ, Lizotte DL, Abramson T, Barenboim L, Schnapp BJ, Rapoport TA (1998) Light chain-dependent regulation of kinesin’s interaction with microtubules. J Cell Biol 143:1053–1066

Verhey KJ, Meyer D, Deehan R, Blenis J, Schnapp BJ, Rapoport TA, Margolis B (2001) Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules. J Cell Biol 152:959–970

Wang T, Town T, Alexopoulou L, Anderson JF, Fikrig E, Flavell RA (2004) Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis. Nat Med 10:1366–1373

Ward BM, Moss B (2004) Vaccinia virus A36R membrane protein provides a direct link between intracellular enveloped virions and the microtubule motor kinesin. J Virol 78:2486–2493

Welstead GG, Iorio C, Draker R, Bayani J, Squire J, Vongpunsawad S, Cattaneo R, Richardson CD (2005) Measles virus replication in lymphatic cells and organs of CD150 (SLAM) transgenic mice. Proc Natl Acad Sci U S A 102:16415–16420

Wyde PR, Ambrose MW, Voss TG, Meyer HL, Gilbert BE (1992) Measles virus replication in lungs of hispid cotton rats after intranasal inoculation. Proc Soc Exp Biol Med 201:80–87

Wyde PR, Moore-Poveda DK, Daley NJ, Oshitani H (1999) Replication of clinical measles virus strains in hispid cotton rats. Proc Soc Exp Biol Med 221:53–62

Yang WX, Terasaki T, Shiroki K, Ohka S, Aoki J, Tanabe S, Nomura T, Terada E, Sugiyama Y, Nomoto A (1997) Efficient delivery of circulating poliovirus to the central nervous system independently of poliovirus receptor. Virology 229:421–428

Zhai RG, Bellen HJ (2004) Hauling t-SNAREs on the microtubule highway. Nat Cell Biol 6:918–919

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Chapter 2 Modeling Subacute Sclerosing Panencephalitis in a Transgenic Mouse System: Uncoding Pathogenesis of Disease and Illuminating Components of Immune Control

M. B. A. Oldstone

Abstract Subacute sclerosing panencephalitis (SSPE) is a chronic neurodegenera-tive disease of the central nervous system (CNS) that afflicts eight to 20 individuals per one million of those who become infected with measles virus (MV). The six cardinal elements of SSPE are: (1) progressive fatal CNS disease developing sev-eral years after MV infection begins; (2) replication of MV in neurons; (3) defective nonreplicating MV in the CNS that is recoverable by co-cultivation with permissive tissue culture cells; (4) biased hypermutation of the MV recovered from the CNS with massive A to G (U to C) base changes primarily in the M gene of the virus; (5) high titers of antibody to MV ; and (6) infiltration of B and T cells into the CNS. All these parameters can be mimicked in a transgenic (tg) mouse model that expresses the MV receptor , thus enabling infection of a usually uninfectable mouse in which the immune system is or is not manipulated. Utilization and analysis of


M.B.A. Oldstone Viral-Immunobiology Laboratory, Department of Immunology and Microbial Science , The Scripps Research Institute , La Jolla CA , USA , e-mail: [email protected]

Contents

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Introduction: Measles Virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Historical Background: Subacute Sclerosing Panencephalitis. . . . . . . . . . . . . . . . . . . . . . . . . 33Transgenic Mouse Model of SSPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35Contribution of the Hypermutated M Protein to the Chronic Progressive CNS Disease . . . . 38Hypothesis to Explain the Initiation and Pathogenesis of SSPE . . . . . . . . . . . . . . . . . . . . . . . 40

Contribution of Biased Hypermutation Predominantly in the M Gene of Measles Virus to the Pathogenesis of SSPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41Contribution of Antibody-Induced Modulation of Measles Virus Antigens to the Pathogenesis of SSPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Dual-Hit Hypothesis: Acute Immunosuppressive Event Preceding Measles Virus Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Identification of the Components of Measles Virus-Specific Immune Response Required for Clearing Measles Virus from the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46Future: Studies to Further Dissect the Molecular Pathogenesis of SSPE . . . . . . . . . . . . . . . . 48References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

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32 M.B.A. Oldstone

such mice have illuminated how chronic measles virus infection of neurons can be initiated and maintained, leading to the SSPE phenotype. Further, an active role in prolonging MV replication while inhibiting its spread in the CNS can be mapped to a direct affect of the biased hypermutations (A to G changes) of the MV M gene in vivo.

Abbreviations

SSPE Subacute sclerosing panencephalitis CNS Central nervous system MV Measles virus

Introduction: Measles Virus

Although diseases caused by viruses, including measles, were known and described in antiquity, viruses per se were not recognized as separate infectious agents until the late 1890s, over 110 years ago. Their recognition followed the pioneering work in bacteriology by Louis Pasteur and Robert Koch and their associates in the mid-1800s. During that interval, the laboratory culturing process was developed, so it became possible to grow bacteria in culture for eventual placement on glass slides, staining, and observation under the microscope. Importantly, bacteria placed on Pasteur-Chamberland filters of varying pore sizes were retained upon vacuum pres-sure enabling the identification of specific bacteria, which when re-introduced into the appropriate host recaused the specific illness, thus linking the infectious agent with a particular disease state. It was on this framework that the first viruses were uncovered in 1898 (Loeffler and Frosch 1898; Beijerinck 1899; Ivanovski 1899). In contrast to the retention of bacteria on the Pasteur-Chamberland filters, viruses were characterized by their passage through the filters, their invisibility under light microscopy and their inability to grow in bacterial (cell-free) cultures.

Thirteen years later, in 1911, Goldberger and Anderson (1911) showed that res-piratory tract secretions from measles virus-infected patients were not retained but passed through the Pasteur-Chamberland-like filter, were invisible to light micros-copy, and were unable to replicate in cell-free bacterial cultures. Moreover, macaque monkeys inoculated with material that passed through the filter developed the features of a measles virus-like disease. Another 43 years passed before MV was isolated and adapted to grow in cell cultures (Enders and Peebles 1954). This feat was accomplished by John Enders , to whom this volume is dedicated. His work formed the basis of subsequent extensive biological research on the pathogenesis of MV, methods for diagnosing MV, and eventually the development of the measles virus vaccine, again the achievement of Enders and his laboratory associates (Katz and Enders 1959; Enders et al. 1960; Katz 1965). Sam Katz, a protégé of Enders who was instrumental in the development and testing of the vaccine has recorded some of his personal observations with Enders in chapter 1 of volume 329.

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2 Modeling Subacute Sclerosing Panencephalitis in a Transgenic Mouse System 33

In addition to the usual transient CNS symptoms and signs that accompany acute measles virus infection, two serious and debilitating manifestations of CNS injury, although uncommon, are consequences of acute MV infection: postinfectious encephalitis and subacute sclerosing panencephalitis (SSPE).

Measles virus infection can occasionally be complicated by sudden onset of encephalitis. The incidence of MV postinfection encephalitis is 1 per 1000 cases (Miller 1964; Scott 1967) and the clinical picture includes high fever, headache, vomiting, and convulsions, often leading to coma in an individual who has begun to recover from the initial acute infection. This manifestation usually occurs at 5 days, but in some instances 25 days after the measles virus rash appears. Mortality is approximately 10% with one-quarter of those who survive showing a permanent neurologic defect. Rarely is MV recovered from the patient’s cerebral spinal fluid or brain, and the pathogenesis of this disease is immunologic in nature. Infection by other viruses such as mumps, varicella, rubella, smallpox, and vaccinia may cause a similar postinfection encephalitis. In contrast to post-MV encephalitis, SSPE does not appear for several years after the primary MV infection, and no fever or other signs of acute CNS injury are present. SSPE presents with behavioral changes and mental deterioration followed by neurodegeneration of the pyramidal, extrapyramidal, and cerebellar systems, invariably leading to death. In this situa-tion, MV genetic material is easily identified in the CNS. Despite its rarity, the lethal sequence of SSPE and its long period of latency have motivated the author’s investigation of this disease. This chapter discusses SSPE and recent observations from the author’s laboratory detailing the molecular pathogenesis of the disease.

Historical Background: Subacute Sclerosing Panencephalitis

As early as the 1930s, the clinical picture of SSPE was suggestive of a potential infectious etiology. Neuropathologic evaluation (Dawson 1933; van Bogaert 1945) showed acidophilic intranuclear and cytoplasmic inclusion bodies in neurons and glia cells of the CNS. However, although infection was the suspected cause, attempts to implicate any microorganism failed. Indeed, evidence for a viral etiol-ogy required an additional 30 years. Not until the mid-1960s did Bouteille and his associates (1965) view by electron microscopy structures resembling nucleocapsids of a paramyxovirus within neurons from the brain material of a patient with SSPE. Two years later, Connolly and colleagues (1967) demonstrated that patients with SSPE had excessively high titers of antibodies to measles virus in their sera and cerebral spinal fluid. Further, by immunohistochemistry measles virus antigens were found in neurons and glia. The last and essential piece of evidence establish-ing an association with MV came independently from two laboratories, one at the NIH from Horta-Barbosa and colleagues (1969) and the second from a team at the University of Michigan lead by Francis Payne (Payne et al. 1969). Their work veri-fied that the MV was defective in replication and that the recovery of virus required the assistance of a co-cultivation assay. Thus, viable brain tissue from SSPE

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34 M.B.A. Oldstone

patients when co-cultivated in tissue culture with cells permissive for MV lead to recovery of the virus. These results have now been confirmed multiple times and extended by direct cloning of measles virus from the brains of SSPE patients, thus firmly establishing the relationship between SSPE chronic progressive neurologic disease and MV infection.

Thereafter, a plethora of papers appeared concerning measles virus and SSPE and occasionally also implicating mumps virus with SSPE (see PubMed). The fact that SSPE cases have decreased in parallel with the implementation of vaccination against MV clearly indicates that MV vaccination does not cause SSPE (Campbell et al. 2007). Noteworthy and seminal was the report concerning SSPE from the laborato-ries of Cattaneo, ter Meulen, and Billeter (Cattaneo et al. 1988), who performed direct cloning and molecular analysis of the MV genome recovered from brains of SSPE patients. These investigators noted biased hypermutation in the MV genome, most often affecting the matrix (M) gene, with conversion of U to C and A to G bases. The debate that followed centered on the question could such a defective MV cause or result from the persistent infection? The evidence on hand given later in this review indicates that the defective virus does not initiate infection but is a consequence of it. Further, the defective virus contributes to the ongoing pathogenesis of the disease.

Currently, there is no reliable treatment that delays or aborts this chronic fatal neu-rodegenerative disease. Thus, investigation has centered first on establishing animal model(s) to mimic this disorder and second, on utilizing such models to understand how measles virus can establish, under rare conditions, a persistent infection and why the host cannot clear the infection. With the availability of such models, the pathogen-esis of this disease can be decoded and therapeutic approaches designed and tested.

An animal model that mimics SSPE must exhibit six cardinal elements:

1. Progressive fatal CNS disease; 2. Replication of measles virus in neurons; 3. Defective (nonreplicating) virus that can be recovered by co-cultivation with

permissive tissue culture cells; 4. Dramatic biased hypermutation in the measles virus genome primarily involving

one of the eight measles virus genes, the M gene, with conversion of U to C and A to G bases;

5. High titers of antibodies to measles virus; 6. Infiltration of B cells and T cells into the CNS.

Several animal models discussed in this current volume of CTMI or the CTMI vol-ume 191 on this subject satisfy one or more of these demands. However, the one described below fulfills all six criteria.

The tg mouse model we use in which the MV receptor CD46 is transcriptionally expressed by its authentic CD46 promoter (YAC-CD46 tgs) , placed on the C57Bl/6 background and then crossed onto the Rag1 genetically deleted (Rag1 −/− ) back-ground provides all of the six necessary fingerprints cited above. First, such tg mice express CD46 on neurons as well as all nucleated cells. Crossing these tg mice on to the Rag1 −/− background results in offspring whose B and T lymphocytes have been deleted. This maneuver removes the adaptive immune response and allows

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MV infection to persist. This approach was first successfully employed in the Rall laboratory where Rag −/− mice (Lawrence et al. 1999) were crossed with previously constructed tg mice that expressed the CD46 molecule exclusively on neurons by use of the neuron-specific enolase (NSE) promoter (Rall et al. 1997). A further benefit of using either the YAC-CD46 or NSE-CD46 mice is that reconstitution of purified and specific components of the host’s immune system can be adaptively transferred to the measles virus-infected host deleted of immune cells (YAC-CD46 × Rag −/− or NSE-CD46 × Rag −/− tg mice), thereby allowing insight into which the missing component(s) of the immune system is essential for the control and purging of measles virus from the CNS.

Transgenic Mouse Model of SSPE

The primary host for measles virus is humans. Measles virus can infect monkeys but as far as we know, that occurs by transmission from humans to monkeys either by experimental inoculation or by air droplet spread to susceptible monkeys from humans infected with the virus. Earlier investigators utilized forced adaptation of measles virus by serial passage through brains of a variety of animals. Eventually, use of this method produced rodent adapted MV capable of infecting in the rodent host in which the virus had been passed. Although useful data were obtained from such experimental systems, the models were limited primarily by the disease phe-notype produced, the age of the host required for infection, and the forced passage production of a mutated measles virus genome that was very distant from the sequence of the measles viruses isolated from humans and used for adaptation.

Once the receptors (CD46, SLAM) for measles virus in humans were discov ered, this limitation was overcome by expressing the authentic human virus receptor in mice using tg protocols. The transcriptional expression of CD46 or SLAM in mice, especially in C57Bl/6 (B6) mice, provided an easily manipulatable small-animal model in which the genetics were known and abundant markers were present to dissect the immunologic response to the virus by deletion and reconstitution of unique cells of the immune system. However, a limitation remained. Although the mouse and human genomes are over 98% identical, not all the host factors required for optimal measles virus replication and control of infection may be present or optimally functional in the rodent. Nevertheless, either tissue-specific promoters to transcriptionally express MV in cells of the immune system (e.g., CD11c for dendritic cells, LCK for T cells) in neurons in NSE for neurons or promoters to express receptors more generally have been used by a number of laboratories (Rall et al. 1997; Thorley et al. 1997; Blixenkrone-Moller et al. 1998; Mrkic et al. 1998; Oldstone et al. 1999; Hahm et al. 2003, 2004; Carsillo et al. 2006; Kurihara et al. 2006; Sellin et al. 2006; Ohno et al. 2007).

Our tack and the tg model we utilized were the CD46 molecule expressed under control of its own CD46 promoter. This strategy allowed us to infect cells of the immune system and neurons in the brain. Since the CD46 genome was obtained from a yeast artificial chromosome (YAC) library (Yannoutsos et al. 1996), the

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36 M.B.A. Oldstone

resultant tg mice were termed YAC-CD46. Infection of YAC-CD46 mice led to virus replication in and recovery from cells of the animal immune system. This process was associated with suppression of primary and secondary humoral and cell-mediated immune responses (Oldstone et al. 1999). Further, the use of YAC-CD46 tg mice, measles virus infection, and challenge with a secondary bacterial infection, Listeria monocytogenes , demonstrated that measles virus suppressed both the innate and adoptive immune responses required to control the Listeria infection. Further, the molecules and immune cells involved in the process were mapped (Slifka et al. 2003).

Pertinent for this chapter, infectious virus also replicated in and was recovered from neurons in the CNS. However, replication of MV in the CNS was markedly enhanced and prolonged when YAC-CD46 mice were placed on a Rag1 −/− back-ground. Rag1 −/− mice have a deletion in B and T immune cells and thus cannot gener-ate an immune response when challenged with an antigen. Thus, by avoiding a host-generated immune response to MV, adult YAC-CD46 × Rag1 −/− mice, replicated and spread MV infections in their neurons. This allowed the infection to persist in the CNS for months. Figure 2. 1 shows neurons of YAC-CD46 × Rag1 −/− tg mice 60 days after the initiation of MV infection in adult mice. However, no MV could be

Fig. 2.1 Infection of neurons and accumulative mortality of YAC-CD46 × Rag1 −/− tg mice infected with MV (Edmonston strain)

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Fig. 2.2 Electron micrographs of brains from YAC-CD46 × Rag1 −/− tg mice infected with MV. Note presence of viral nucleocapsids in neurons. EM courtesy of Sam Dales

recovered from brains of infected YAC-CD46 × Rag1 −/− tg mice unless viable brain tissue was co-cultured with viable permissive tissue culture cells such as Vero cells. Electron microscopy performed by Samuel Dales, currently in Gunter Blobel’s labo-ratory at the Rockefeller University in New York, showed a picture in which struc-tures of measles virus nucleocapsids were clearly present in the cytoplasm and nucleus of hippocampal neurons in MV-infected YAC-CD46 × Rag1 −/− tg mice (Fig. 2. 2 ). Further, direct cloning of the MV genome from brains of such tg mice by Lee Martin, when a postdoctoral fellow in my laboratory, revealed biased hypermu-tations in the M gene. Figure 2. 3 displays the geography of A to G hypermutation in the M protein from one such mouse, #1919, in which a remarkable 35%–40% of the gene is mutated. Also listed in Fig. 2.3 is the A to G hypermutations in four other YAC-CD46 × Rag1 −/− mice. Their clinical state at the time of sacrifice and the number of A to G mutations found are displayed. Hypermutation of the M gene commonly knocked out a unique Alu1 restriction site. This allowed Martin to devise a scheme for enrichment of additional hyperimmune M gene sequences (see Oldstone et al. 2005 for details). Cattaneo and Billeter (1988, 1992) had previously reported similar mutations and hypermutations in MV persisting in brain material from patients with SSPE. Thus the YAC-CD46 × Rag1 −/− tg mouse infected with measles virus (Edmonston strain) mimicked the SSPE disease in humans by the fol-lowing criteria. The first is a progressive fatal CNS disease with measles virus repli-cation in neurons. Second, measles virus in the CNS was defective in that it was recoverable only by co-cultivation on permissive cells in culture. Third, analysis of the measles virus genome revealed biased hypermutation primarily in the M gene

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38 M.B.A. Oldstone

with A to G (U to C) changes. Further, classic SSPE-like nucleocapsid structures were seen by electron microscopic examination of infected neurons. In addition, when such YAC-CD46 × Rag1 −/− tg mice were reconstituted with splenic lym-phocytes and infected with measles virus, high titers of measles virus antibodies were recorded in the sera, presumably in response to repetitive stimulation of the immune system by measles virus (Oldstone et al. 2005; Tishon et al. 2006). The heightened anti-measles virus antibody response was accompanied by a modest infiltration of B and T cells in the CNS (Oldstone et al. 2005; Tishon et al. 2006).

Contribution of the Hypermutated M Protein to the Chronic Progressive CNS Disease

Now that a reproducible small-animal model of SSPE was available, we could answer the question of whether or not the hypermutated A to G bases in the M gene of measles virus directly contributed to the chronic progressive CNS disease. This was accomplished by John Patterson, a former postdoctoral fellow in my labora-tory, who used the reverse genetics system for MV developed by Martin Billeter and his colleagues in Zurich (Radecke et al. 1995). Patterson replaced the M gene

Fig. 2.3 Biased hypermutations (A to G) in the M gene of MV from brains of YAC-CD46 × Rag1 −/− tg mice persistently infected with MV. Each dot records A to G mutation

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of Edmonston strain measles virus with the M gene taken from Biken strain SSPE MV (Patterson et al. 2001a). The newly constructed recombinant virus was then injected into adult YAC-CD46 × Rag1 −/− tg mice. Comparison of Edmonston mea-sles virus (contains non-A to G mutated M gene) with recombinant Edmonston/M genome Biken (A to G mutated M gene) indicated that the Edmonston/M gene Biken recombinant was infectious and produced a protracted CNS disease in the range of 1–3 months longer than Edmonston alone. Both viruses caused death. Further, the recombinant virus appeared predominantly in clusters so that the infec-tion of neurons was limited. That is, samples of multiple levels of brain tissues from tg mice infected with the recombinant Edmonston/Biken SSPE virus when com-pared to those infected with Edmonston virus showed less spread of the Edmonston/Biken SSPE virus in neurons. The best interpretation of these results is that the biased hypermutations of the M gene most likely slowed the migration of the virus and thereby prolonged the infection. These data noted in vivo supported the earlier observations in vitro of Cathomen et al. (1998) that a functioning M gene is not required for measles virus replication or transcription. The other conclusion from this work is that mutations in the M gene do not harm the virus, as do some muta-tions to other MV genes required for replication, transcription, or viral assembly. Those detrimental mutations in other MV genes likely result in the virus’ elimina-tion, whereas mutations in M are well tolerated and have little if any affect on virus survival.

Fig. 2.4 Evidence of a pathogenic role for the biased hypermutated M gene in the pathogenesis of SSPE. The M gene of Edmonston strain of MV was replaced by the hypermutated M gene from human SSPE MV (Biken isolate). The resultant recombinant MV when inoculated into YAC-CD46 × Rag1 −/− tg mice significantly prolonged the time of the progressive CNS degenerative disease ( left panel ). Further, MV-infected neurons appeared as clumps, suggesting impairment of viral spread in vivo (compare infected neurons in Fig. 1 and Fig. 5, Edmonston MV infection) with neurons in right panels (recombinant Edmonston/Biken M gene MV). For details see Cattaneo et al. 1988; Wong et al. 1989; Patterson et al. 2001a

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40 M.B.A. Oldstone

Figure 2. 4 illustrates the accumulated mortality curves of both groups and reveals the enhanced survival time of YAC-CD46 × Rag1 −/− mice infected with the recombinant measles virus bearing the A to G hypermutated M gene. The insert shows clumping of MV-infected neurons as detected by immunochemistry.

Hypothesis to Explain the Initiation and Pathogenesis of SSPE

Hypotheses regarding the initiation and pathogenesis of SSPE revolve around three clinical/pathological findings. The first concerns the issue of whether biased hyper-mutations in the M gene of measles virus contribute to the development of SSPE or if such mutations per se are not involved in either the initiation or maintenance of pathogenesis, but simply accumulate because the M gene is not necessary for the virus’s life cycle during CNS infection. For instance, it has been suggested that the measles virus causing SSPE may be a unique strain or recombinant virus. However, this explanation is unlikely because epidemiologic analysis of identical twins infected simultaneously, i.e., with the same measles virus, show discordance in that only one twin developed SSPE (Houff et al. 1979; Cianchetti et al. 1983; Dhib-Jalbut and Haddad 1984). Thus, although it is unlikely that a unique strain of mea-sles virus (carrying the biased hypermutation primarily of the M gene) is responsible for initiation of measles virus infection leading to SSPE, it is possible that such biased hypermutated viruses in the CNS, once formed, contribute to ongoing SSPE disease. Further, a suspected enzyme may be responsible for the U to C (A to G) M hypermutations and could be triggered by virus-induced cytokines in the CNS. Such cytokines would enhance the interferon-inducible double-stranded RNA ade-nosine deaminase (ADAR1). ADAR1 is presumed to propagate the biased hyper-mutations by adenosine to inosine conversion in double-stranded RNA intermediates (Cattaneo et al. 1988; Bass and Weintraub 1988; Bass et al. 1989; Baczko et al. 1993; Patterson and Samuel 1995; Patterson et al. 2001a, 2001b). Further, MVs, as they continue their infection of neurons, may alter these cells’ expression of selected genes and function without killing the infected cell. ADAR is associated with Ca ++ flux and glutamate receptor editing.

The second hypothesis focuses on the role of excessively high titers of antibody to measles virus in the blood and cerebral spinal fluid of patients with SSPE. This theory questions whether such antibody could participate in modulating MV gene products and thus alter the virus’s life cycle, favoring its ability to persist in infected cells. The basic idea here is that MV per se is not cytotoxic for cells but causes their injury by cell-to-cell fusions and syncytial formation, which depends on the viral glycoproteins (Joseph and Oldstone 1975; Oldstone and Tishon 1978; Fujinami and Oldstone 1979, 1980, 1983; Oldstone et al. 1980). Fusion/syncytial formation is the sine qua non of measles virus infection in tissue culture and during acute infection, i.e., syncytia of lymphoid cells in lymphoid organs, but is surprisingly absent or minimally present in neurons of the CNS in SSPE patients (Fenner 1974; Adams et al. 1984).

The third hypothesis centers on the profound suppression of the immune system associated with measles virus infection. Indeed, among viruses, measles was the

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first to be recognized as causing a profound immunosuppression (Osler 1904; von Pirquet 1908; Wagner 1968), which was discovered several years before the virus was isolated (Goldberger and Anderson 1911). Measles virus-induced immunosup-pression led to the activation of a latent tuberculosis, changing it to a miliary or systemic spread of tuberculin disease (von Pirquet 1908). Further, the strength of immune system suppression by MV is profound and, in fact, measles was used by physicians therapeutically before the discovery of corticosteroids to treat aggressive autoimmune nephritis (Blumberg and Cassady 1947). The activation of secondary microbial infections and other infectious agents was linked with MV well before MV was isolated and parallels the later observations with the human immunodefi-ciency (HIV, AIDS) virus (reviewed in McChesney and Oldstone 1989).

The foundation of this hypothesis rests on three observations. The first is the study of identical twins infected simultaneously with MV (presumably the same strain) show a discordance because SSPE develops in only one twin (Houff et al. 1979; Cianchetti et al. 1983; Dhib-Jalbut and Haddad 1984). This observation sug-gests that an environmental effect, not a genetic predisposition, is important in initi-ating SSPE. This observation also disputes a component of the first hypothesis that a unique measles virus strain or altered measles virus is responsible for initiation of SSPE. The second arm of the immunosuppressive hypothesis is the clinical docu-mentation that unimmunized patients who receive intensive immunosuppressive therapy are more vulnerable to SSPE than immunocompetent individuals infected with measles virus (Coulter et al. 1979; Fukuya et al. 1992). The third arm is that the majority of SSPE cases occur in children infected before the age of 2 years (Cattaneo et al. 1988; Griffin 2007) when the immune system is still immature.

In the next section follows the experimental support for these various hypothe-ses, along with the possibility or realization that components of each and not neces-sarily only one may be involved in the initiation and pathogenesis of SSPE.

Contribution of Biased Hypermutation Predominantly in the M Gene of Measles Virus to the Pathogenesis of SSPE

To determine whether a biased hypermutation of the M gene directly participates in SSPE pathogenesis we used reverse genetics to construct and rescue a recombinant measles virus containing the hypermutated M gene. The Edmonston strain of MV backbone was modified to contain the biased hypermutated A to G M gene of a human SSPE isolate Biken strain (Cattaneo et al. 1988; Wong et al. 1989). Once CNS infection was initiated in CD46 MV receptor expressing tg mice, this recom-binant virus was found to be defective in budding from the plasma membranes of neurons and was recoverable only by co-cultivation of infected brains with tissue culture cells permissive for measles virus. The recombinant virus was expressed in neurons and the time course for the persistent infection was dramatically prolonged by months when compared to similar tg mice persistently infected with Edmonston virus alone. Further, recombinant Edmonston/Biken SSPE biased hypermutated

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42 M.B.A. Oldstone

M gene measles virus appeared predominantly in clusters and did not spread as widely through the CNS as did the Edmonston measles virus. Together these obser-vations indicated that the biased hypermutations in the M gene likely slow the migration of the virus through the CNS and prolong the infectious cycle in the host. This outcome in an experimental animal model documented clearly and for the first time that the biased hypermutated M gene of SSPE virus per se contributes to the chronicity of SSPE disease. Probably, however, the M protein is not necessary for MV replication and transcription (Cathomen et al. 1998). Hence, mutations in the M gene are not lethal for the virus. Yet, the hypermutated M gene still allows the virus to replicate and spread. This conclusion amplifies that of Baczko et al. (1993) who recorded clonal expansion of a different hypermutated MV variant.

Similarly, our use of Edmonston measles virus infection in YAC-CD46 × Rag1 −/− tg mice resulted in long stretches of A to G hypermutations in the M gene and the COOH − -terminal end of the F gene (Oldstone et al. 2005). The preference for A to G and U to C mutations was dramatic accounting for over 95% of muta-tions in M and 73% for F genes, respectively. Further, all five mice studied showed clonal expansion of M genes with biased hypermutations. Each of the mice had Alu1 hypermutated M genes , thus mimicking the end stage found in brains of humans with SSPE (Cattaneo and Billeter 1992; Baczko et al. 1993; Radecke et al. 1995). Further, the high percentage of A to G and U to C mutations suggests the possible superimposition of mutations induced by a host enzyme such as adenosine deaminase (ADAR) (Cattaneo et al. 1988; Bass et al. 1989; Patterson and Samuel 1995). Accordingly, the hypothetical scenario is that cytokines made in the CNS during persistent MV infection enhances the inter-feron-inducible ADAR1 enzyme. ADAR1 would then propagate the biased hyper-mutation by adenosine to inosine conversion in viral double-stranded intermediates. Since the measles virus M gene is not essential to virus replication or transcrip-tion, it could absorb multiple mutations, whereas, for example, the virus polymer-ase–replication complex could not, resulting in the virus’s survival. Further, by bearing an increase of mutations in M, the infecting virus survives longer, thereby establishing a near continuous feedback loop cycle over a number of years in SSPE patients to favor persistence and continuous stimulation for the production of antibody to MV.

Contribution of Antibody-Induced Modulation of Measles Virus Antigens to the Pathogenesis of SSPE

Patients with SSPE possess logarithmically higher titers of antibodies to measles virus than found in individuals following either acute measles virus infection or vaccination against measles. Presumably the cause is continuous immunogenic stimulation from the persisting MV. Over three decades ago, investigators recog-nized that MV-infected cells in culture bathed in antibodies to MV resulted in a redistribution of MV antigens on the cell surface and the suppression of both

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immune mediate injury and, more importantly, the infected cells continued to survive and switch into a state of persistent infection (Joseph and Oldstone 1974, 1975; Oldstone et al. 1980). Impressively, by removing (capping off) the MV glycoproteins the hemagglutinin (HA) and the fusion (F) protein, antibody to MV prevented cell-to-cell fusion and giant cell syncytial formation, essential components for MV-induced death. In addition, by antibody modulation therapy a time occurred when restoration of HA and F proteins (by removal of anti-mea-sles virus antibody) no longer occurred on the surface of infected cells and, uniquely, measles virus nucleocapsids similar to those in SSPE patients appeared in the nucleus and cytoplasm of infected cells. Of interest here is that the preven-tion of cell-to-cell fusion by another means recapitulates these events. When MV-infected cells are actively maintained in suspension to prevent cell–cell con-tact, the cell lysis does not occur and persistent infection follows. Thus the abnormal nucleocapsid formation seen in patients with SSPE is also observed in either antibody-treated cells or in tg mice expressing the CD46 measles virus receptor and whose neurons are persistently infected with measles. At the molec-ular level, MV antibody-induced modulation occurs after using monoclonal anti-bodies to specific epitopes on the measles virus HA and results in a signal delivered to the plasma membrane of infected cells that alters the measles virus transcriptional complex, especially the measles virus phosphoprotein inside the cell (Fujinami and Oldstone 1979, 1980; Oldstone et al. 1980; Fujinami et al. 1984). These results from our experiments with antibodies to MV used to treat measles-infected HeLa cells were subsequently confirmed in ter Meulen’s labo-ratory in mouse neuroblastoma cells or rat glioma cells infected with MV (Schneider-Schaulies et al. 1992). Further, we noted that this antibody induced modulation of measles-infected cells was specific since antibodies to HeLa cell-surface antigens as well as antibodies to other viruses, i.e., influenza, failed to modulate MV-infected cells. Conversely, the antibodies to MV failed to modulate off-surface antigens of influenza or lymphocytic choriomeningitis virus on infected cells (Fujinami and Oldstone 1979, 1980; Oldstone et al. 1980; Fujinami et al. 1984; Go et al. 2006). Recently, we obtained RNA profiles and performed mass spectrometry on the antibody-modulated cells in order to identify the host genes or proteins either up- or downregulated. These gene products are still being characterized (Go et al. 2006). One gene product of interest is ADAR1.

The role played by MV antibody-induced antigen modulation in vivo has been more difficult to define, although two observations are noteworthy. Wear and Rapp (1971) demonstrated that only MV (adapted to the hamster brain) infected newborn hamster pups suckled to mothers having antibodies to measles developed a persist-ent infection in their CNS. In contrast, similarly infected pups suckled to nonim-mune mothers died of acute encephalitis. Later, working with rats and rat-adapted measles virus, ter Meulen’s laboratory (Liebert et al. 1990) found that passive trans-fer of neutralizing antibodies directed against selected MV HA determinants were able to modulate MV gene expression at the level of transcription and switched an expected acute necrotizing encephalitis in either Lewis rats or brown Norway rats into a persistent CNS infection.

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Dual-Hit Hypothesis: Acute Immunosuppressive Event Preceding Measles Virus Infection

Clinical reports that SSPE was associated with measles virus infection occurring after drug-induced immunosuppression (Coulter et al. 1979; Fukuya et al. 1992) usually used in treatment of acute childhood leukemia, coupled with the discordance of SSPE in identical twins infected at the same time with measles virus, raised the possibility that an immunosuppressive event narrowly preceding acute MV infection might allow the virus to initially escape the host’s immune response that would oth-erwise terminate the acute virus infection. Persistent virus infection would then fol-low. Further, the timing of both events would be critical and thus account for the low frequency of SSPE. To test this hypothesis, we initially infected tg mice expressing the MV receptor with a virus, lymphocytic choriomeningitis virus (LCMV) Clone 13 (Cl 13), known to transiently suppress the mouse immune system yet itself be noncytopathic (Oldstone 2002). Infection with measles virus 10 days later, a time of maximal immunosuppression by LCMV Cl 13 in its natural murine host (Sevilla et al. 2000), resulted in persistent measles virus infection of neurons. Such mice then developed a chronic CNS disease leading to death. Analysis of their brains showed the biased A to G (U to C) hypermutations in the measles virus M gene. Further, the other cardinal manifestations of SSPE occurred in this dual-hit model: (1) generation of a defective measles virus in the CNS that could be recovered by co-cultivation with tissue culture cells permissive for measles virus; (2) high titers of antibodies to measles, and (3) infiltration of B and T cells into the CNS.

The timing between initiation of the immunosuppressive event and contracting measles virus infection was crucial. LCMV Cl 13 infection of mice causes a gener-alized suppression of both T (cell-mediated) and B cell (humoral antibody) arms of the adoptive immune system (Sevilla et al. 2000; Oldstone 2002). Thus, antigen-specific T cell responses to several RNA and DNA viruses as well as antibody responses to soluble and particulate antigens are significantly dampened. The decreased immune response peaks at day 10–14 following initiation of infection, wanes after day 20, and by 60–90 days after LCMV Cl 13 infection, the virus is usually cleared from the mouse (except from neurons and glomeruli in which viral clearance is delayed until days 110–120) (see Oldstone et al. 1986; Tishon et al. 1993). Further, LCMV Cl 13, by virtue of its tropism for dendritic cells, also inter-feres with or suppresses the early innate immune response (Zuniga et al. 2008) as well as the later adoptive immune response (Sevilla et al. 2000).

Thus, when MV infection occurred 3 days after LCMV Cl 13 infection, a time when there is no LCMV-induced immunosuppression, none of the inoculated mice developed persistent infection of neurons. When MV was administered 30 days after the initiation of LCMV Cl 13 infection, a time when immunosuppression has begun to wane, only a minority of mice had evidence of persistent MV infection in their neurons.

In contrast, when LCMV Cl 13 was given initially followed by MV 10 days later, the time of maximal LCMV-induced immunosuppression, all mice developed

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a persistent measles virus infection of neurons (see Fig. 2. 5 ). Cloning of virus from their brains revealed A to G (U to C) biased hypermutations prevalent in the M gene and electron microscopy revealed viral nucleocapsids in the cytoplasm and nuclei of infected neurons (Oldstone et al. 2005). Sera harvested from these mice had antibody titers that were 20- to 30-fold higher than observed in immunocompetent mice immunized with measles virus. Further, like human patients with SSPE, such mice displayed infiltration of predominantly CD4 T cells and B cells as well as CD8 T cells in their brains.

Fig. 2.5 Experimental evidence for dual-hit strategy to explain the initiation of persistent MV infection in CNS neurons. Lymphocytic choriomeningitis virus (LCMV) Clone (Cl) 13 induces a generalized immunosuppression in mice due to its replication in dendritic cells (DCs) and inter-feres with DC activation of T and B cells (see Sevilla et al. 2000 and Oldstone 2002 for details). Judged by results of mixed leukocyte reaction (MLR), there is negligible inhibition on MLR by day 3 but maximal inhibition at day 10 with modest inhibition at day 30 post-LCMV Cl 13 infec-tion. The SSPE phenotype in YAC-CD46 mice infected first with LCMV and then with measles refers to: (1) persistent MV infection of neurons with chronic progressive neurologic disease; (2) presence of nucleocapsids in disarray in the cytoplasm and nuclei of infected neurons; (3) biased hypermutation in MV M genome cloned from CNS; (4) recovery of defective MV from the CNS only by co-cultivation of viable brains with MV permissive cultured cells; (5) markedly elevated amounts of antibodies to MV in the sera of infected mice (significantly higher than antibody titers in acutely infected or vaccinated mice); and (6) infiltration of T and B cells into the brains of infected mice. See Oldstone et al. 2005 for details

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46 M.B.A. Oldstone

Patients with clinical manifestations of SSPE when their disease emerges, sev-eral years after the initial MV infection, are not severely immunosuppressed. Our early studies with Rag1 −/− - mice in which T and B cells are deleted, crossed to YAC-CD46 recapitulated all the findings of the LCMV Cl 13/measles virus dual-hit model (10-day interval before the second infection).

The longer-lasting persistent MV infection observed with the dual-hit model then likely results from two separate factors. One is the ability of antibody to MV to prolong the lifespan of MV-infected cells (Joseph and Oldstone 1975; Oldstone et al. 1980). This observation and its consequence are discussed above in Sect. 5.2. The second factor is the enhanced A to G (U to C) mutation in the M gene in patients with SSPE (Cattaneo et al. 1988) and in the murine model outlined here (Patterson et al. 2001a; Oldstone et al. 2005). Indeed, mice given recombinant Edmonston/Biken M SSPE measles virus in which the M gene of the Biken strain from an SSPE patient replaced the normal nonmutated M gene in Edmonston measles virus (Patterson et al. 2001a) have a persistent infection lasting several months longer than mice given the Edmonston virus alone. Two essential points emerge from this model. First, a system is available to further dissect the molecu-lar pathogenesis of SSPE and, second, an animal model of SSPE is available for testing and evaluating various protocols and therapies for treatment of this chronic neurologic disease. At present, therapy for SSPE remains controversial and overall has not worked. Drugs such as steroids, ribavirin, type I and II interferons, have been tried singularly or in combination but with either conflicting or negative results (reviewed in Gascon 2003).

Identification of the Components of Measles Virus-Specific Immune Response Required for Clearing Measles Virus from the CNS

Measles virus infection is most often an acute self-limiting disease. Both humoral (antibody) and cell-mediated (T cell) immune responses act to restrict infection, although T cells appear to be the major players (Burnet 1968; Whitton and Oldstone 2001; Griffin 2007). However, clearance of persisting virus from the CNS may require a unique combination of immune cells. For example, CD8 T cells alone can overcome an acute LCMV infection, but the mix of CD4 T cells along with CD8 T cells is an absolute requirement to purge this virus from persistently infected neu-rons (Tishon et al. 1995). To determine the essential prerequisite for clearance of MV from neurons during a persistent MV infection, we took advantage of the YAC-CD46 tg mouse model and the technical ability to purify or delete individual cell constituents of the immune response and adaptively transfer them into the YAC-CD46 × Rag1 −/− tg mice so infected. Measles virus fails to establish a persistent infection of neurons in adult YAC-CD46 tg mice because such mice have a compe-tent and mature immune system. However, when the immune system is voided by crossing YAC-CD46 mice onto the Rag1 −/− background, infection with MV then causes a persistent neuronal infection. Adaptive transfer of 5 × 10 7 or 1 × 10 7 splenic

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colleagues, we have received NOD-SCID mice implanted when 1-day-old with human neuronal stem cells. Accordingly, 1 × 10 5 stem cells were inoculated into six different CNS locations. These cells express the CD46 molecule and are permissive to MV infection in vivo. When the mice were 8 weeks old, they were inoculated with MV, which subsequently induced a persistent infection in the animals’ acquired human neurons. Since the MV was engineered to express GFP, infected neurons were easily identified. This technique allowed analysis of the MV genome biased mutations of A to G (U to C) and use of gene display to profile RNA expres-sion of human neuronal genes during persistent MV infection. The MV-infected neurons in this stem cell model, as well as those in the YAC-CD46 × Rag1 −/− model, mirror the situation of most infected neurons in brains of SSPE patients in that none of these neurons engage in fusion. Additionally, since MV per se is not cytotoxic for cells, neuronal apoptosis was not an issue. The hypothesis being tested is that, in SSPE, persistent MV infection of neurons affects the differentiation (luxury) function of neurons without killing them. As a result, the altered homeostasis of neuronal function leads to disease. This mechanism of disease, e.g., disturbed cell function in the absence of cell cytotoxicity, was first uncovered during study of persistent LCMV infection of several differentiated cell types in vivo – e.g., neu-rons, endocrine cells, immune cells – and has been extended to a variety of RNA and DNA viruses (reviewed in Oldstone 2002).

A second ongoing study tests the hypothesis that cytokines released in the brain during persistent MV infection induce the dsRNA adenosine deaminase enzyme responsible for A to G (U to C) changes in the virus’s M gene (see Fig. 2. 8 ). The resultant behavioral/neurodegenerative phenotype may likely be caused by faulty glutamate receptor editing in neurons.

Fig. 2.8 Illustrated hypothesis for the induction of cytokines inducing ADAR1 enzyme and its conversion of A to G base changes in the MV genome

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50 M.B.A. Oldstone

ADAR is an interesting candidate to evaluate for four reasons. First, the presence of ADAR could account for the molecular hallmark of prolonged MV infection of the CNS, the biased conversion of A to G (U to C) found in humans with SSPE (Cattaneo et al. 1988; Bass and Weintraub 1988; Bass et al. 1989; Baczko et al. 1993; Patterson and Samuel 1995; Patterson et al. 2001a, 2001b) and in the tg ani-mal model of SSPE (Patterson et al. 2001a; Oldstone et al. 2005). Second, we found elevations of ADAR in brains of some measles virus-infected YAC-CD46 mice compared to uninfected YAC-CD46 controls. Third, ADAR is involved in neuronal glutamate receptor editing, defects that can cause neuronal dysfunction due to excess Ca ++ flux (Seeburg et al. 1998; Liu and Samuel 1999). Fourth, ADAR is an interferon-inducible protein (Patterson and Samuel 1995; Patterson et al. 1995) and amounts of this cytokine are elevated in MV-infected brains.

To explore the role of ADAR in the pathogenesis of SSPE disease, Bumsuk Hahm and Lee Martin, former postdoctoral fellows in my laboratory, in collabora-tion with Charles Samuel and Cyril George at the University of California, Santa Barbara, constructed an ADAR1a plasmid with a neo-insert for the purpose of pro-ducing ADAR1a knockout mice. At present, several potential ADAR1a knockout founders have been identified and are currently being bred to determine germline transmission of the gene. Once ADAR1a knockout lines have been confirmed, these mice will be used to generate triple tg mice: ADAR1a −/− × YAC-CD46 × Rag1 −/− . Such mice, after infection with MV, will provide a valuable resource to determine the roles of ADAR in the generation of biased hypermutations A to G (U to C) and the role, if any, of glutamate receptor editing in the pathogenesis of chronic measles virus infection of neurons.

Acknowledgements This is Pub. No. 19363 from the Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA. This work was supported in part by NIH grant AI036222 and NIH postdoctoral training fellowships to Glenn Rall, Lee Martin, and John Patterson. Bumsuk Hahm was supported by NIH grants AI036222 and AI074564.

References

Adams JA, Corsellis JAN, Duchen LW (eds) Greenfield’s neuropathology, 4th edn. (1984) John Wiley and Son’s, New York

Amanna IJ, Carlson NE, Slifka MK (2007) Duration of humoral immunity to common viral and vaccine antigens. N Engl J Med 357:1903–1915

Baczko K, Lampe J, Liebert UG, Brinckmann U, ter Meulen V, Pardowitz I, Budka H, Crosby SL, Isserte S, Rima BK (1993) Clonal expansion of hypermutated measles virus in a SSPE brain. Virology 197:188–195

Bass BL, Weintraub H (1988) An unwinding activity that covalently modifies its double-stranded RNA substrate. Cell 55:1089–1098

Bass BL, Weintraub H, Cattaneo R, Billeter MA (1989) Biased hypermutation of viral RNA genomes could be due to unwinding/modification of double-stranded RNA. Cell 56:331

Beijerinck MW (1899) Bemerkung zu dem Aufsatz fon Herrn Iwanowsky uber die Mosaikkrankheit der Tabakspflanze. Zentbl Bakt ParasitKde Abt I 5:310–311


b12325337-0002ztc.indd 50 10/3/2008 10:32:20 AM


Blumberg RW, Cassady HA (1947) Effect of measles on nephrotic syndrome. Am J Dis Child 63:151

Bouteille M, Fontaine C, Vedrenne CL, Delarue J (1965) Sur un cas d’encéphalite subaigüe a inclusions. Étude anatomoclinique et ultrastructurale. Rev Neurol 113:454–458

Burnet FM (1968) Measles as an index of immunologic function. Lancet ii:610 Campbell H, Andrews N, Brown KE, Miller E (2007) Review of the effect of measles vaccination

on the epidemiology of SSPE. Int J Epidemiol 36:1334–1348 Carsillo T, Traylor Z, Choi C, Niewiesk S, Oglesbee M (2006) hsp72, a host determinant of mea-

sles virus neurovirulence. J Virol 80:11031–11039 Cathomen T, Mrkic B, Spehner D, Drillien R, Naef R, Pavlovic J, Aguzzi A, Billeter MA,

Cattaneo R (1998) A matrix-less measles virus is infectious and elicits extensive cell fusion: consequences for propagation in the brain. EMBO J 17:3899–3908

Cattaneo R, Billeter MA (1992) Mutations and A/I hypermutations in measles virus persistent infections. Curr Topics Microbiol Immunol 176:63–74


Cianchetti C, Marrosu MG, Manconi PE, Loi M, Cao A (1983) Subacute sclerosing panencepha-litis in only one of identical twins. Case report with study of cell-mediated immunity. Eur Neurol 22:428–432

Connolly JH, Allen IV, Hurwitz LJ, Millar JHD (1967) Measles-virus antibody and antigen in subacute sclerosing panencephalitis. Lancet 1:542–544

Coulter JB, Balch N, Best PV (1979) Subacute sclerosing panencephalitis after drug-induced immunosuppression. Arch Dis Child 54:640–642

Dawson JR Jr (1933) Cellular inclusions in cerebral lesions of lethargic encephalitis. Am J Pathol 9:7–15

Dhib-Jalbut S, Haddad FS (1984) Subacute sclerosing panencephalitis in one member of identical twins. Neuropediatrics 15:49–51

Enders JF, Peebles TC (1954) Propagation in tissue culture of cytopathogenic agents from patients with measles. Proc Soc Exp Biol Med 86:277–286

Enders JF, Katz SL, Milovanovic MV, Holloway A (1960) Studies on an attenuated measles-virus vaccine. 1. Development and preparation of the vaccine: techniques for assay of effects of vaccination. N Engl J Med 263:153–159

Fenner F (1974) The biology of animal viruses. Academic Press, New York Fujinami RS, Oldstone MB (1979) Antiviral antibody reacting on the plasma membrane alters

measles virus expression inside the cell. Nature 279:529–530 Fujinami RS, Oldstone MB (1980) Alterations in expression of measles virus polypeptides

by antibody: molecular events in antibody-induced antigenic modulation. J Immunol 125:78–85

Fujinami RS, Oldstone MB (1983) Antigenic modulation: a mechanism of viral persistence. Prog Brain Res 59:105–111

Fujinami RS, Norrby E, Oldstone MB (1984) Antigenic modulation induced by monoclonal anti-bodies: antibodies to measles virus hemagglutinin alters expression of other viral polypeptides in infected cells. J Immunol 132:2618–2621

Fukuya H, Ohfu M, Tomoda Y, Nibu K, Ichiki S, Midsudome A (1992) A case of subacute scleros-ing panencephalitis developing 8 years after immunosuppressive treatment for acute lym-phocytic leukemia. No To Hattatsu 24:60–64

Gascon GG (2003) Randomized treatment study of inosiplex versus combined inosiplex and intraventricular interferon-alpha in subacute sclerosing panencephalitis (SSPE): international multicenter study. J Child Neurol 18:819–827

Go EP, Wikoff WR, Shen Z, O’Maille G, Morita H, Conrads TP, Nordstrom A, Trauger SA, Uritboonthai W, Lucas DA, Chan KC, Veenstra TD, Lewicki H, Oldstone MB, Schneemann A, Siuzdak G (2006) Mass spectrometry reveals specific and global molecular transformations during viral infection. J Proteome Res 5:2405–2416

b12325337-0002ztc.indd 51 10/3/2008 10:32:20 AM

52 M.B.A. Oldstone

Goldberger J, Anderson JF (1911) An experimental demonstration of the presence of the virus of measles in the mixed buccal and nasal secretions. J Am Med Assoc 57:476–478

Griffin DE (2007) Measles Virus. In: Knipe DM, Howley PM (eds) Fields virology, 5th edn. Lippincott Williams Wilkins, Philadelphia, pp 1551–1585

Hahm B, Arbour N, Naniche D, Homann D, Manchester M, Oldstone MB (2003) Measles virus infects and suppresses proliferation of T lymphocytes from transgenic mice bearing human signaling lymphocyte activation molecule. J Virol 77:3505–3515

Hahm B, Arbour N, Oldstone MB (2004) Measles virus interacts with human SLAM receptor on dendritic cells to cause immunosuppression. Virology 323:292–302

Homann D, Teyton L, Oldstone MBA (2001) Differential regulation of antiviral T-cell immunity results in stable CD8 + but declining CD4 + T-cell memory. Nature Med 7:913–919

Horta-Barbosa L, Fuccillo DA, Sever JL, Zeman W (1969) Subacute sclerosing panencephalitis: isolation of measles virus from a brain biopsy. Nature 221:974

Houff SA, Madden DL, Sever JL (1979) Subacute sclerosing panencephalitis in only one of identi-cal twins. A seven-year follow-up. Arch Neurol 36:854–856

Ivanovski DI (1899) Ueber die Mosaikkrankheit der Tabakspflanze. Zentbl Bakt ParasitKde Abt II 5:250–254

Joseph BS, Oldstone MB (1974) Antibody-induced redistribution of measles virus antigens on the cell surface. J Immunol 113:1205–1209

Joseph BS, Oldstone MB (1975) Immunologic injury in measles virus infection. II. Suppression of immune injury through antigenic modulation. J Exp Med 142:864–876

Katz SL (1965) Immunization of children with live attenuated measles vaccines: five years of experience. Arch Gesamte Virusforsch 16:222–230

Katz SL, Enders JF (1959) Immunization of children with live attenuated measles vaccine. Am J Dis Child 85:605–607

Kurihara N, Zhou H, Reddy SV, Garcia-Palacios V, Subler MA, Dempster DW, Windle JJ, Roodman GD (2006) Experimental models of Paget’s disease. J Bone Miner Res Suppl 2:P55–P57

Lawrence DMP, Vaughn MM, Belman AR, Cole JS, Rall GF (1999) Immune-mediated protection of adult but not neonatal mice from neuron-restricted measles virus infection and CNS disease. J Virol 73:1795–1801

Liebert UG, Schneider-Schaulies S, Baczko K, ter Meulen V (1990) Antibody-induced restriction of viral gene expression in measles encephalitis in rats. J Virol 64:706–713

Liu Y, Samuel CE (1999) Editing of glutamate receptor subunit B pre-mRNA by splice-site vari-ants of interferon-inducible double-stranded RNA-specific adenosine deaminase ADAR1. J Biol Chem 274:5070–5077

Loeffler F, Frosch P (1898) Berichte der Kommission zur Erforschung der Maul und Klauenseuche bei dem Institut fur Infektionskrankheiten in Berlin. Zentbl Bakt ParasitKde Abt I 23:371–391

McChesney MB, Oldstone MBA (1989) Virus-induced immunosuppression: infections with mea-sles virus and human immunodeficiency virus. Adv Immunol 45:335–380

Miller DL (1964) Frequency of complications of measles, 1963. Br Med J 2:75–78 Mrkic B, Pavlovic J, Rulicke T, Volpe P, Buchholz CJ, Hourcade D, Atkinson JP, Aguzzi A,

Cattaneo R (1998) Measles virus spread and pathogenesis in genetically modified mice. J Virol 72:7420–7427

Naniche D, Garenne M, Rae C, Manchester M, Buchta R, Brodine SK, Oldstone MBA (2004) Decrease in measles virus-specific CD4 T cell memory in vaccinated subjects. J Infect Dis 109:1387–1395


Oldstone MBA (2002) Biology and pathogenesis of lymphocytic choriomeningitis virus infection. Curr Topics Microbiol Immunol 262:83–117

b12325337-0002ztc.indd 52 10/3/2008 10:32:20 AM


Oldstone MB, Tishon A (1978) Immunologic injury in measles virus infection. IV. Antigenic modulation and abrogation of lymphocyte lysis of virus-infected cells. Clin Immunol Immunopathol 9:55–62

Oldstone MB, Fujinami RS, Lampert PW (1980) Membrane and cytoplasmic changes in virus-infected cells induced by interactions of antiviral antibody with surface viral antigen. Prog Med Virol 26:45–93

Oldstone MBA, Blount P, Southern PJ, Lampert PW (1986) Cytoimmunotherapy for persistent virus infection: unique clearance pattern from the central nervous system. Nature 321:239–243

Oldstone MBA, Lewicki H, Thomas D, Tishon A, Dales S, Patterson J, Manchester M, Homann D, Naniche D, Holz A (1999) Measles virus infection in a transgenic model: virus-induced central nervous system disease and immunosuppression. Cell 98:629–640

Oldstone MBA, Dales S, Tishon A, Lewicki H, Martin L (2005) A role for dual viral hits in causa-tion of subacute sclerosing panencephalitis. J Exp Med 202:1185–1190

Osler W (1904) The principles and practice of medicine. New York Patterson JB, Samuel CE (1995) Expression and regulation by interferon of a double-stranded

RNA-specific adenosine deaminase from human cells: evidence for two forms of the deami-nase. Mol Cell Biol 15:5376–5388

Patterson JB, Cornu TI, Redwine J, Dales S, Lewicki H, Holz A, Thomas D, Billeter MA, Oldstone MB (2001a) Evidence that the hypermutated M protein of a subacute sclerosing panencephalitis measles virus actively contributes to the chronic progressive CNS disease. Virology 291:215–225

Patterson JB, Manchester M, Oldstone MBA (2001b) Disease model: dissecting the pathogenesis of the measles virus. Trends Mol Med 7:85–88

Patterson JB, Thomis DC, Hans SL, Samuel CE (1995) Mechanism of interferon action: double-stranded RNA-specific adenosine deaminase from human cells is inducible by alpha and gamma interferons. Virology 210:508–511

Payne FE, Baublis JV, Habashi HH (1969) Isolation of measles virus from cell cultures of brain from a patient with subacute sclerosing panencephalitis. N Engl J Med 281:585–589

Radecke F, Spielhofer P, Schneider H, Kaelin K, Huber M, Dotsch C, Christiansen G, Billeter MA (1995) Rescue of measles viruses from cloned DNA. EMBO J 14:5773–5784

Rall GF, Manchester M, Daniels LR, Callahan EM, Belman AR, Oldstone MB (1997) A trans-genic mouse model for measles virus infection of the brain. Proc Natl Acad Sci U S A 94:4659–4663

Schneider-Schaulies S, Liebert UG, Segev Y, Rager-Zisman B, Wolfson M, ter Meulen V (1992) Antibody-dependent transcriptional regulation of measles virus in persistently infected neural cells. J Virol 66:5534–5541

Scott T (1967) Postinfectious and vaccinial encephalitis. Med Clinics North Am 51:701–707 Seeburg PH, Higuchi M, Sprengel R (1998) RNA editing of brain glutamate receptor channels:

mechanism and physiology. Brain Res Rev 26:217–229 Sellin CI, Davoust N, Guillaume V, Baas D, Belin MF, Buckland R, Wild TF, Horvat B (2006)

High pathogenicity of wild-type measles virus infection in CD150 (SLAM) transgenic mice. J Virol 80:6420–6429

Sevilla N, Kunz S, Holz A, Lewicki H, Homann D, Yamada H, Campbell KP, de la Torre JC, Oldstone MBA (2000) Immunosuppression and resultant viral persistence by specific viral targeting of dendritic cells. J Exp Med 192:1249–1260

Slifka MK, Homann D, Tishon A, Pagarigan R, Oldstone MB (2003) Measles virus infection results in suppression of both innate and adaptive immune responses to secondary bacterial infection. J Clin Invest 111:805–810

Thorley BR, Milland J, Christiansen D, Lanteri MB, McInnes B, Moeller I, Rivailler P, Horvat B, Rabourdin-Combe C, Gerlier D, McKenzie IF, Loveland BE (1997) Transgenic expression of a CD46 (membrane cofactor protein) minigene: studies of xenotransplantation and measles virus infection. Eur J Immunol 27:726–734

b12325337-0002ztc.indd 53 10/3/2008 10:32:20 AM

54 M.B.A. Oldstone

Tishon A, Eddleston M, de la Torre JC, Oldstone MB (1993) Cytotoxic T lymphocytes cleanse viral gene products from individually infected neurons and lymphocytes in mice persistently infected with lymphocytic choriomeningitis virus. Virology 197:463–467

Tishon A, Lewicki H, Rall G, von Herrath M, Oldstone MB (1995) An essential role for type 1 interferon-gamma in terminating persistent viral infection. Virology 212:244–250

Tishon A, Lewicki H, Andaya A, McGavern D, Martin L, Oldstone MBA (2006) CD4 T cell con-trol primary measles virus infection of the CNS: regulation is dependent on combined activity with either CD8 T cells or with B cells: CD4, CD8 or B cells alone are ineffective. Virology 347:234–245

van Bogaert L (1945) Une leucoencéphalite sclérosante subaigüe. J Neurol, Neurosurg, Psych 8:101–120

von Pirquet C (1908) Das Verhalten del kutanen Tuberkulin-Reaktion Wahrend der Masern. Dtsch Med Wochenschr 34:1297

Wagner R (1968) Clements von Pirquet: his life and work. Baltimore, MD Wear DJ, Rapp F (1971) Latent measles virus infection of the hamster central nervous system. J

Immunol 107:1593–1598 Whitton JL, Oldstone MBA (2001) The immune response to viruses. In: Knipe DM, Howley PM

(eds) Fields virology, 4th edn. Lippincott Williams Wilkins, Philadelphia, pp 285–320 Wong TC, Ayata M, Hirano A, Yoshikawa Y, Tsuruoka H, Yamanouchi K (1989) Generalized and

localized biased hypermutation affecting the matrix gene of a measles virus strain that causes subacute sclerosing panencephalitis. J Virol 63:5464–5468

Yannoutsos N, Ijzermans JN, Harkes C, Bonthuis F, Zhou CY, White D, Marquet RL, Grosveld F (1996) A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection. Genes Cells 1:409–419

Zuniga EI, Liou LY, Mack L, Oldstone MBA (2008) In vivo virus infection inhibits type 1 inter-feron production by plasmacytoid dendritic cells thereby facilitating opportunistic infections. Cell Host Microbe (in press)

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Chapter 3 Measles Studies in the Macaque Model

R. L. de Swart

Abstract Much of our current understanding of measles has come from experi-ments in non-human primates. In 1911, Goldberger and Anderson showed that macaques inoculated with filtered secretions from measles patients developed measles, thus demonstrating that the causative agent of this disease was a virus. Since then, different monkey species have been used for experimental measles virus infections. Moreover, infection studies in macaques demonstrated that serial passage of the virus in vivo and in vitro resulted in virus attenuation, providing the basis for all current live-attenuated measles vaccines. This chapter will review the macaque model for measles, with a focus on vaccination and immunopathogenesis studies conducted over the last 15 years. In addition, recent data are highlighted demonstrating that the application of a recombinant measles virus strain expressing enhanced green fluorescent protein dramatically increased the sensitivity of virus

R. L. de Swart Department of Virology, Erasmus MC , University Medical Center , P.O. Box 2040 , 3000 CA Rotterdam , The Netherlands , e-mail: [email protected]

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56Vaccination Studies in Macaques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Atypical Measles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57Live-Attenuated Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59New-Generation Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59Alternative Routes of Administration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Pathogenesis Studies in Macaques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Cell Lines and Virus Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Differences Between Macaque Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62Immunity, Protection, and Immunosuppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64Infections with MV Expressing EGFP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

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detection, both in living and sacrificed animals, allowing new approaches to old questions on measles vaccination and pathogenesis.

Introduction

At the beginning of the twentieth century, it was demonstrated that measles virus (MV) could be transmitted from humans to non-human primates. One to 2 weeks after inoculation with blood collected from measles patients, macaques developed fever and skin rash (Anderson and Goldberger 1911). Disease transmission could also be achieved using filtered respiratory secretions, demonstrating that the causa-tive agent of measles was a virus (Goldberger and Anderson 1911). A decade later, Blake and Trask followed up on these studies by experimentally infecting macaques with nasopharyngeal washings of measles patients, thereby successfully inducing measles in 16 animals (Blake and Trask 1921a). The virus could be passaged from monkey to monkey by intratracheal inoculation of tissue homogenates or by intra-venous injection of citrated blood (Blake and Trask 1921a). The authors carefully documented incubation time, fever, leukopenia and pathology of enanthem and exanthem (Blake and Trask 1921b). In addition, they demonstrated that experimen-tal MV infection resulted in complete immunity against reinfection (Blake and Trask 1921c). In the 1950s, it was demonstrated that experimental infections with MV isolated in cell culture also caused measles in macaques (Peebles et al. 1957). The authors were able to reisolate the virus and detect MV-specific serum antibody responses, thus bringing the model close to its current state.

Although experimental MV infection of macaques had been demonstrated suc-cessfully, a number of other studies yielded negative results. Retrospectively, it can be concluded that in many of these cases animals were immune to measles due to prior exposure to the virus. Peebles et al. detected MV-specific serum antibodies in 22 out of 24 macaques tested from US laboratories, whereas animals screened immediately after capture all proved to be serum antibody-negative (Peebles et al. 1957). This illustrates that measles is a disease of humans and normally does not affect non-human primates unless they are brought in close contact with humans. Monkeys used as experimental animals were at that time in most cases wild-caught animals. Before and during transport they were housed under crowed conditions, and contacts with MV-infected humans could result in virus transmission (Meyer et al. 1962). On several occasions, measles outbreaks occurred at animal facilities after arrival of new animals, in some cases associated with significant morbidity (Potkay et al. 1966; Hime and Keymer 1975; Scott and Keymer 1975; Remfry 1976; MacArthur et al. 1979; Welshman 1989). Thus monkeys intended to be used for experimental MV infections needed to be transported using special precautions (Tauraso 1973). At present, all macaques used for experimental measles virus infec-tions are purpose-bred animals, but prescreening for the absence of MV-specific antibodies remains imperative.

The high susceptibility of non-human primates to MV infection provided the first animal model for measles. The two species used most often are the rhesus

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3 Measles Studies in the Macaque Model 57

macaque ( Macaca mulatta ) and the cynomolgus macaque ( Macaca fascicularis ) (El Mubarak et al. 2007). As a result of the close similarity of clinical, virologi-cal, immunological and pathological parameters to those associated with mea-sles in humans, the model continues to be used almost a century later. Although several rodent species and other small laboratory animals have been inoculated with MV, most of these do not or poorly replicate the virus (Stittelaar et al. 2002a; Pütz et al. 2003). Studies in cotton rats or transgenic animal species do reproduce certain aspects of measles, but none of these show the same level of similarity with the pathogenesis of measles in humans as the macaque model (see chapters 5 and 6).

New world monkeys proved to be even more susceptible to MV infection than old world monkeys, but developed a disease with a different pathogenesis than that of measles in humans, which was associated with high mortality (Levy and Mirkovic 1971; Albrecht et al. 1980). Due to their high susceptibility, marmosets ( Saguinus mystax ) were later used for encephalogenicity studies, as reviewed elsewhere (Van Binnendijk et al. 1995). The current review will address macaque models of measles, with a focus on studies published after 1995.

Vaccination Studies in Macaques

The first isolation of MV in cell culture (Enders and Peebles 1954) was immedi-ately followed by attempts to develop a vaccine against measles. Two different strategies were pursued in parallel: inactivated vaccines and live-attenuated vac-cines. Whereas the first category of vaccines proved to predispose for enhanced disease, the second was highly successful. In the 1990s studies were initiated to develop new-generation vaccines as potential successors of the current live-attenu-ated vaccines, some of which showed promise in preclinical studies in macaques. However, none of these candidate new measles vaccines have been pushed forward towards licensing for human use. Studies in the 1980s had already demonstrated that administration of the current MV vaccine by aerosol held great promise (Sabin et al. 1982). However, regulatory authorities consider a vaccine and its route of administration as one entity, which means that before the aerosol route for measles vaccination can be implemented licensure will be required, for which preclinical studies were conducted in macaques.

Atypical Measles

In the 1960s, classical inactivated measles vaccines were manufactured by formalin inactivation of whole MV preparations, which were subsequently precipitated with aluminum salts (referred to as FI-MV). Initially, vaccination with FI-MV was shown to induce MV-specific serum antibody responses (Hilleman et al. 1962;

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Norrby et al. 1964; Foege et al. 1965), and the vaccine was used in large-scale clinical trials in humans. However, after a number of years it turned out that vaccination predisposed for enhanced disease upon natural MV infection, which was referred to as atypical measles (Fulginiti et al. 1967) (see also chapter 10). During the same period, vaccination trials with formalin-inactivated respiratory syncytial virus (FI-RSV), another member of the family Paramyxoviridae , also proved to predispose for enhanced disease upon natural infection (Fulginiti et al. 1969; Kapikian et al. 1969; Kim et al. 1969). Whereas availability of small animal models for RSV allowed extensive studies on the pathogenesis of this vaccine-mediated enhanced disease, this remained difficult for measles. Because the patho-genesis remained subject to speculation, the risk of inducing atypical measles continued to form a major stumbling block for the development of new generation nonreplicating MV vaccines.

In 1999, Polack and Griffin were successful in reproducing atypical measles in macaques (Polack et al. 1999), thus providing an opportunity to study the patho-genesis of the disease as well as a model to test candidate new MV vaccines for predisposition of similar aberrant responses upon challenge infection. They immunized macaques with the original FI-MV preparation that had been manufac-tured in the 1960s. When challenged with pathogenic wild-type MV, two out of five rhesus macaques vaccinated with FI-MV developed atypical measles, charac-terized by a petechial rash and pneumonitis associated with the presence of eosi-nophils and immune complexes in their lungs. The authors concluded that atypical measles “results from previous priming for a nonprotective type 2 CD4 T cell response rather than from lack of functional antibody against the fusion protein” (Polack et al. 1999).

In follow-up studies, they further characterized immune responses in these ani-mals, demonstrating in vivo impairment of interleukin (IL)-12 production and increased production of IL-4 by peripheral blood mononuclear cells (Polack et al. 2002). When characterizing the antibody responses, they found that these were not only transient but also lacked avidity maturation. Challenge infection resulted in anamnestic production of low avidity antibodies, which could explain the immune complex deposition in FI-MV-primed animals (Polack et al. 2003a). Before repro-duction of atypical measles in the macaque model, it was speculated that inactiva-tion with formalin had resulted in destruction of critical B cell epitopes on the fusion protein (Norrby et al. 1975). However, this hypothesis could be rejected on the basis of two macaque experiments: animals primed with FI-MV developed atypical measles in the presence of fusion-inhibiting antibodies (Polack et al. 1999), and macaques primed with a DNA vaccine encoding for the haemagglutinin gene that developed H- but not F-specific antibodies did not develop any of the clinical signs associated with atypical measles (Polack et al. 2000). Interestingly, vaccination of macaques with formalin-inactivated respiratory syncytial virus (RSV) or human metapneumovirus (hMPV) preparations also predisposed for hypersensitivity to challenge infection with the respective viruses, suggesting that these phenomena are characteristic for all members of the family Paramyxoviridae (De Swart et al. 2002, 2007c).

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Live-Attenuated Vaccines

As mentioned above, the MV strain Edmonston isolated by Enders and colleagues in 1954 induced measles-like clinical signs in macaques (Peebles et al. 1957). Serial passage of this virus in vivo (in chicken embryos) and in vitro (in chicken embryo fibroblast [CEF] cells) resulted in virus attenuation: upon experimental infection of macaques, the virus still induced MV-specific immune responses but virtually no clinical signs (Enders et al. 1960). More than 30 years later, direct comparison of MV isolated in lymphoid cells (strain Bilthoven) with MV passaged in human and monkey kidney cells (strain Edmonston wild type) or live-attenuated MV passaged in CEF cells (strain Schwarz) demonstrated that these strains dis-played high, intermediate and low pathogenicity in macaques, respectively (Van Binnendijk et al. 1994). Virus loads in peripheral blood mononuclear cells and in bronchoalveolar lavage cells were several log values lower in animals infected with MV strains of reduced pathogenicity. Whereas levels of MV-specific serum IgM antibodies seemed directly related to the magnitude of virus loads, levels of specific serum IgG and virus neutralizing (VN) antibodies induced by the three virus strains were on the same order of magnitude (Van Binnendijk et al. 1994).

Macaques and other non-human primate species have been used to assess the levels of attenuation of different candidate vaccine virus strains. In most cases, this was done by studying pathological lesions induced by intracerebral MV infection (Buynak et al. 1962; Nii et al. 1964b; Albrecht et al. 1981; Sharova et al. 1984). At present, vaccine manufacturers still use this method to assess appropriate attenu-ation of new MV vaccine virus seed stocks.

New-Generation Vaccines

Despite its documented safety and efficacy, live-attenuated MV vaccines also have drawbacks, most importantly their dependence on cold chain maintenance and their ineffectiveness in the presence of maternal antibodies (Stittelaar et al. 2002a; Pütz et al. 2003). Live-attenuated MV vaccines are ineffective when used before the age of 9 months, resulting in a window of susceptibility in young infants between waning of maternal immunity and acquisition of vaccine-induced immunity. Since the 1980s, new-generation candidate MV vaccines have been developed to address these issues, including subunit vaccines, vectored vaccines and nucleic acid vac-cines (see chapter 10). In the macaque model, passive transfer of MV-specific VN antibodies inhibited effectiveness of the live-attenuated MV vaccine, thus providing a model to evaluate the potential of candidate new vaccines in the presence of maternal antibodies (Van Binnendijk et al. 1997). The scientific steering committee of the World Health Organization (WHO) adopted the macaque model for pre-clinical comparison of the different candidates. A strategy was developed in which vaccine candidates were first used to immunize juvenile animals, either in the absence or presence of passively transferred MV-specific antibodies. This would

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allow assessment of immunogenicity and longevity of the induced specific immune responses. Approximately 1 year after vaccination, animals were challenged with a pathogenic wild-type MV strain to assess levels of protection. Vaccine candidates that performed well in this evaluation were subsequently tested in infant macaques, to assess their potential effectiveness at an early age in the presence of true mater-nally derived VN antibodies. This strategy resulted in the identification of a number of promising candidate MV vaccines. However, further preclinical and clinical evaluation of a new MV vaccine will require huge financial investments. Moreover, improved coverage of the live-attenuated MV vaccine in recent years in combina-tion with the implementation of a two-dose strategy has been highly successful in reducing measles mortality. As a result, it remains uncertain if any of the candidate new-generation MV vaccines will ever be licensed for human use.

Alternative Routes of Administration

For regulatory purposes, a vaccine and the device used for administration form one integral entity: the current live-attenuated MV vaccines are licensed for injection only. However, alternative routes of administration have extensively been studied in humans. Whereas intradermal, conjunctival, oral or intranasal administration were not particularly successful, aerosol administration of nebulized MV vaccine proved highly effective (Cutts et al. 1997). The aerosol route closely mimics the natural route of MV infection and may result in both mucosal and systemic immunity (Valdespino-Gomez et al. 2006).

The WHO, in partnership with the American Red Cross and the Centers for Disease Control and Prevention and with funding from the Bill and Melinda Gates Foundation, has initiated a Product Development Group (PDG) for measles aerosol vaccination. The ultimate objective of the PDG is to achieve licensure of a combi-nation of measles vaccine and nebulizer, which is equally safe, effective and cheap as the currently licensed measles vaccines administered by injection, but is easier to administer, less invasive, and could be administered by nonmedical personnel. The PDG evaluated two alternative strategies for measles aerosol vaccination: neb-ulization of a reconstituted vaccine (Dilraj et al. 2000) or inhalation of a dry powder aerosol (LiCalsi et al. 2001).

Both vaccination strategies were evaluated in immunocompetent and immuno-compromised macaques and proved equally safe. However, vaccination with a nebulized aerosol proved more effective than inhalation of a dry powder vaccine (De Swart et al. 2006, 2007a). The animals used in these studies had body weights of 1.8–4.5 kg and consequently had much smaller tidal volumes than those of children. To mimic vaccination using a similar dose to that inhaled by a child in 30 s, the exposure time therefore had to be prolonged (De Swart et al. 2006). Before proceeding to clinical trials, a toxicology study was conducted using larger study groups than those used in the exploratory studies. These studies were also

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conducted in macaques, to allow MV vaccine replication in all exposed tissues. This study revealed no adverse events, and measles aerosol vaccination is currently under investigation in clinical trials. The PDG aims to achieve licensure for this vaccination route in 2009.

Pathogenesis Studies in Macaques

Experimental MV infections of macaques have been crucial for our understanding of the pathogenesis of measles. Infections with MV isolated in cell culture demon-strated the importance of passage history of the virus: MV strains exclusively cul-tured in lymphoid cells retained pathogenicity in macaques, whereas passage in other cell lines often resulted in virus attenuation. However, nonattenuated MV strains were in some cases also associated with subclinical infections, which seemed to be related to species differences between rhesus and cynomolgus macaques rather than to virus differences. A large number of studies have focused on the development of MV-specific immune responses, and the role of different arms of the immune system in protection from measles. Measles is usually not recognized before onset of rash, approximately 2 weeks after MV infection, making studies on the early events fol-lowing virus transmission in humans difficult to perform. Pathological studies of experimentally infected macaques have provided important insights in the tissue dis-tribution and cell tropism of the virus. Recently, infections with recombinant MV expressing enhanced green fluorescent protein (EGFP) resulted in improved sensitiv-ity of virus detection, and provided new insights in the role of different target cells. This new approach to an old animal model provides alternative possibilities to further unravel the pathogenesis of measles and measles-associated immunosuppression.

Cell Lines and Virus Strains

The introduction of Epstein-Barr virus (EBV)-transformed marmoset B cell line B95a as a substrate for isolation of MV (Kobune et al. 1990) made it possible to isolate non-culture-adapted wild-type MV strains. Whereas isolation of wild-type MV from patient samples in Vero cells usually takes at least 2 weeks (and in many cases requires serial blind passage), virus isolation in B95a cells can be as rapid as 2–4 days (WHO 2007). Experimental infections with the Edmonston wild-type strain did not always result in detectable clinical signs (Enders et al. 1960; Hicks et al. 1977; Van Binnendijk et al. 1994), whereas non-cell culture-passaged virus usually induced more fulminant infections (Yamanouchi et al. 1973; Sakaguchi et al. 1986; McChesney et al. 1989). Kobune and colleagues demonstrated that MV iso-lated in B95a cells retained its pathogenicity in macaques, resulting in development of viremia, lymphopenia and rash (Kobune et al. 1996).

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Van Binnendijk et al. isolated wild-type MV in human EBV-transformed B-lymphoblastic cell lines (BLCL) and demonstrated that this virus also retained pathogenicity (Van Binnendijk et al. 1994). These authors applied the infectious center assay to the model, thus quantifying the frequency of MV-infected cells dur-ing viremia. Intratracheal inoculation of macaques with a single infectious unit of wild-type MV strain Bilthoven could spark infection associated with similar viral load kinetics as infection with 10 4 infectious units, with the only difference that the peak of viremia shifted slightly backwards in time with decreasing infectious dosage (Van Binnendijk et al. 1994).

McChesney and colleagues developed a measles model in macaques using a MV strain isolated during an outbreak of measles in a primate facility (McChesney et al. 1997). The virus was isolated in Raji cells, a human BLCL isolated from a patient with Burkitt’s lymphoma, a disease resulting from in vivo transformation of B lym-phocytes by EBV infection. Subsequently, the virus was passaged in vivo in macaques, after which a challenge stock was produced in macaque mononuclear cells (McChesney et al. 1997). This virus was also fully pathogenic in macaques, as demonstrated by induction of clinical and pathological changes typical for measles (McChesney et al. 1997) and MV-specific immune responses (Zhu et al. 1997). Auwaerter and colleagues compared the pathogenicity in macaques of six different MV strains, demonstrating that the nonadapted Bilthoven strain was fully patho-genic while the other cell culture-adapted strains were not (Auwaerter et al. 1999).

Interestingly, a recent study addressing genetic changes that affect the virulence of MV in macaques demonstrated that wild-type MV isolated in Vero cells express-ing CD150 also retained pathogenicity in macaques and induced skin rash (Bankamp et al. 2008). In contrast, the same MV isolated and passaged in normal Vero cells or in CEF cells did not induce rash. This suggests that expression of CD150 on the cells used for virus isolation is crucial, and that the cells are not required to be of lymphoid origin. Cell culture adaptation does not necessarily result in adaptation to the use of CD46 as a receptor, as the adapted MV isolates in the above-mentioned study did not infect Chinese Hamster Ovary cells expressing CD46 (Bankamp et al. 2008).

Differences Between Macaque Species

Although experimental MV infections have been conducted both in rhesus and cynomolgus macaques, clinical signs such as rash and conjunctivitis were espe-cially reported in rhesus macaques (Blake and Trask 1921b; McChesney et al. 1997; Auwaerter et al. 1999). Although skin rash has also been reported in cynomolgus macaques (Kobune et al. 1996), this symptom seemed to be less prominent in this species. The first direct comparison of MV infection in these two macaque species by experimental infection with two different non-culture-adapted wild-type MV strains indeed seemed to confirm this assumption: although animals of both species displayed similar virus replication curves in peripheral blood and broncho-alveolar

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lavage cells as well as similar MV-specific immune responses, the appearance of skin rash was more prominent in rhesus macaques (El Mubarak et al. 2007).

Infection studies with a recombinant MV strain expressing EGFP conducted in parallel in rhesus and cynomolgus macaques showed that in both animal species MV-infected cells were detected in many different tissues, including the skin (De Swart et al. 2007b). Also with respect to other virological and immunological parameters, including infection of specific lymphocyte subsets, MV infection followed a virtually identical course in both animal species, suggesting that both macaque species can be used for measles pathogenesis studies.

Immunity, Protection, and Immunosuppression

Both in humans and macaques recovery from measles is accompanied by lifelong immunity (Blake and Trask 1921c). MV infection induces strong specific humoral and cellular immune responses, and many different assays have been developed to characterize these ex vivo in macaques. The most important serological parameter is the detection of VN antibodies in serum, of which levels above 0.1–0.2 IU/ml have been identified as a correlate of protection from measles in infants (Chen et al. 1990; Samb et al. 1995). In macaques, similar levels of passively transferred VN antibodies were shown to interfere with MV vaccination (Van Binnendijk et al. 1997). Measurement of specific cell-mediated immunity (CMI) is less well stand-ardized, but several techniques including assessment of lymphoproliferation (Van Binnendijk et al. 1997; Pan et al. 2005), cytotoxicity (Van Binnendijk et al. 1997; Zhu et al. 1997, 2000), cytokine production (Auwaerter et al. 1999; Polack et al. 2002, 2003b; Stittelaar et al. 2002b; Premenko-Lanier et al. 2003; Pan et al. 2005; Pasetti et al. 2007) or flow cytometry-based stimulation assays (Stittelaar et al. 2000; Pahar et al. 2005; De Swart et al. 2006) have been employed as correlates of CMI.

VN antibodies can confer complete protection from measles, as also illustrated by the fact that infants born of a mother with adequate MV-specific antibody titers are protected from measles during their first months of life. In contrast, clearance of an established MV infection is largely dependent on CMI. Agammaglobulinemic patients recover normally from measles, while individuals with impaired CMI may succumb to MV infection (Burnet 1968). The role of specific lymphocyte popula-tions in clearance of MV was addressed in the macaque model by depleting single or multiple populations using monoclonal antibodies to CD20 and/or CD8. Macaques depleted of all T lymphocytes or of CD8 + T lymphocytes only at the moment of MV infection exhibited a more extensive rash, increased viral loads and delayed viral clearance (Hicks et al. 1977; Permar et al. 2003). In contrast, deple-tion of CD20 + B lymphocytes did not result in alterations of clinical signs or kinet-ics of MV clearance (Permar et al. 2004). These studies demonstrate that CMI (and more specifically CD8 + T lymphocyte responses), but not humoral immunity, plays a crucial role in MV clearance. These data also highlight a major strength of the

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64 R.L. de Swart

macaque model, correlating specific immune responses to protection from or clear-ance of experimental MV infection.

Measles is not only associated with the induction of strong MV-specific immune responses but also with a transient immunosuppression, leading to enhanced susceptibility to opportunistic infections (see also chapter 12). Many putative mechanisms have been suggested, but in vivo assessment of the relative importance of these hypotheses has remained difficult. MV infects lymphocytes in vitro and in vivo (Yamanouchi et al. 1973; McChesney et al. 1989), but may also alter function-ality of noninfected lymphocytes (Okada et al. 2000) or antigen-presenting cells (Schneider-Schaulies et al. 2003). Furthermore, MV infection interferes with pro-duction of specific cytokines, thus changing the host response to invading pathogens (Griffin et al. 1994; Moss et al. 2002). Paradoxically, MV inhibits proliferation of lymphocytes in vitro (Hirsch et al. 1984), but recovery from measles is associated with extensive lymphocyte expansion in vivo (Mongkolsapaya et al. 1999).

Due to difficulties in standardization of CMI assays, it has been difficult to eval-uate mechanisms of immunosuppression in macaques. Lymphopenia and reduced responses to mitogen stimulation have been described extensively (McChesney et al. 1989; Kobune et al. 1996; Zhu et al. 1997; Auwaerter et al. 1999). MV infection of macaques specifically alters the production of IL-10 and IL-12, thus affecting the balance between phenotypically different T lymphocyte populations (Polack et al. 2002; Hoffman et al. 2003a, 2003b). Perhaps the most functional assessment of immunocompetence of macaques during measles is the assessment of responses to immunization with other antigens, e.g., tetanus toxoid, which has been applied both to assess recall of previously primed immune responses (Bankamp et al. 2008) or as primary immunization during measles (Premenko-Lanier et al. 2004). Flow cytometry studies on lymphocytes of macaques infected with MV-EGFP suggest that infection of specific memory T lymphocyte subsets may also play an important role in measles-associated immunosuppression.

Pathology

Early studies on the pathology of measles in macaques demonstrated the remarka-ble similarity to measles in humans (Blake and Trask 1921b). However, experimen-tal infections in an animal model offer the possibility to evaluate pathological changes during different phases of the pathogenesis, whereas human tissue samples collected during the prodromal phase of measles are relatively rare. The classical Warthin-Finkeldey-type syncytial cells observed in human lymphoid tissues during the prodromal phase (Warthin 1931; Finkeldey 1931) were also observed in macaque tissues (Nii et al. 1964a; Yamanouchi et al. 1970, 1973; Hall et al. 1971; Sakaguchi et al. 1986; McChesney et al. 1997).

The major tissues affected by MV infection of macaques are the upper and lower respiratory tract, the gastrointestinal tract, the lymphoid system and the skin (Blake

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and Trask 1921b; Sergiev et al. 1960; Nii et al. 1964a; Hall et al. 1971; McChesney et al. 1997). However, the cellular origin of the lesions has been debated for dec-ades (Nii et al. 1964a; De Swart et al. 2007b). Both in human and macaque tissues, MV antigen is commonly detected in association with epithelial tissues. In addi-tion, culture-adapted MV strains grow very well in epithelial cells, which has led to the assumption that respiratory epithelial cells were a primary target for MV infection. However, epithelial cells do not express CD150 and are not easily infected with non-culture-adapted wild-type MV strains in vitro (Takeuchi et al. 2003). These observations warrant a reevaluation of the pathogenesis of measles, which may be facilitated by the combination of modern immunohistochemistry and flow cytometry techniques for characterizing cell subsets with experimental infec-tions of macaques with MV strains expressing EGFP.

Infections with MV Expressing EGFP

The development of reverse genetics techniques for non-culture-adapted MV strains resulted in the rescue of a MV strain from cloned cDNA that retained patho-genicity in macaques (Takeda et al. 2000). This molecular clone was used as a backbone for insertion of the gene encoding EGFP as an additional transcription unit upstream of the MV N gene. The resulting recombinant virus displayed similar in vitro replication characteristics as its parental strain, and infected cells produced high amounts of EGFP (Hashimoto et al. 2002). The MV-EGFP strain still proved to be virulent in macaques, and EGFP could be visualized macroscopically in both living and sacrificed animals, and microscopically by confocal microscopy and flow cytometry (De Swart et al. 2007b). As illustrated in Fig. 3.1 , EGFP fluores-cence was detected in skin, respiratory tract and digestive tract, but most intensely in lymphoid tissues. B and T lymphocytes expressing CD150 were the major target cells for MV infection. Highest percentages (up to more than 30%) of infected lymphocytes were detected in lymphoid tissues. In peripheral tissues, large num-bers of MV-infected CD11c + MHC class-II + myeloid dendritic cells (DCs) were detected in conjunction with infected T lymphocytes, suggesting transmission of MV between these cell types. In the respiratory tract, the majority of MV-infected cells was detected in subepithelial tissues, but infected ciliated epithelial cells were also observed. In the T lymphocyte compartment, MV preferentially infected CD45RA − cells, which have a memory phenotype. This observation, which is in good accordance with recent data from MV infections in a human tonsillar explant model (Condack et al. 2007), pointed to a possible role for depletion of specific lymphocyte subsets in the transient disappearance of recall responses, as described in the early twentieth century (Von Pirquet 1908). By using cell-sorting techniques, the model allows both identification and functional assessment of MV-infected cell populations. The macaque model using the pathogenic autofluorescent wild-type MV strain IC323/EGFP clearly opens new possibilities for measles pathogenesis

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66 R.L. de Swart

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Fig. 3.1 A–M Imaging of MV-EGFP infection in macaques. A–G Macroscopic EGFP fluorescence in tissues of a cynomolgus macaque 9 days after experimental infection with MV-IC323-EGFP (close to the peak of virus replication): EGFP fluorescence in skin ( A ), gingiva and buccal mucosa ( B ), tongue and tonsils ( C ), inguinal lymph nodes ( D ), lungs with tracheobronchial lymph nodes

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studies, including identification of the first target cells infected upon transmission of MV to a naive host.

Concluding Remarks

In conclusion, the macaque model has proven to be of crucial importance for devel-opment of measles vaccines, as well as for our understanding of measles pathogen-esis. Vaccination and challenge studies in macaques have provided information on immunogenicity and protective capacity of both new vaccines and old vaccines given via alternative routes of administration. Modeling atypical measles has pro-vided a tool for safety assessment of nonreplicating candidate measles vaccines. This important lesson from the past should help us avoid making similar mistakes in the future with other viral vaccines intended for priming immunity to respiratory viruses (Marshall and Enserink 2004; Ruat et al. 2008). In those cases, preclinical studies should not only evaluate acute toxicity responses to the vaccination, but also potential immunopathological responses to challenge infection.

Further employment of the potential of reverse genetics holds the promise of new insights in measles pathogenesis in the coming years. Infection studies in macaques using pathogenic MV strains expressing EGFP may provide new insights in the pathogenesis of measles and measles-associated immunosuppression. In addition, by comparing pathogenic and vaccine strains expressing EGFP we may be able to learn more on the in vivo tropism of vaccine strains that can use either CD150 or CD46 as a receptor in vitro. Finally, manipulation of viruses and viral genes will enable direct assessment of the role of the different viral proteins in these processes.

Fig. 3.1 (Continued) ( E ), stomach ( left ), spleen ( upper left ) and large intestine with gut-associated lymphoid tissue (GALT) ( F ), spleen ( right ) and large intestine with GALT ( G ). H, I cynomolgus macaque 13 days after MV-IC323-EGFP infection (late stage of the infection): skin rash shown under normal light ( H ) or by EGFP fluorescence ( I ). J Flow cytometric detection of EGFP + cells in peripheral blood mononuclear cell (PBMC) subpopulations of a cynomolgus macaque at differ-ent time points after infection. Freshly isolated PBMCs were stained with monoclonal antibodies, and analyzed in a FACScalibur measuring approximately 500,000 events per sample to allow detection of low-frequent MV-infected cell populations. Results are shown as dot plots, with EGFP expression on the y-axis and CD150 expression on the x-axis. EGFP expression in CD3 + CD4 + T lymphocytes is shown in red ; in CD3 + CD8 + T lymphocytes in green and in MHC class-II + CD20 + B lymphocytes in blue . K Confocal scanning laser microscopical image of EGFP + cells in lymphoid follicles of the spleen of a rhesus macaque 9 days after infection with MV-IC323-EGFP. MV infection was visualized in paraformaldehyde-fixed vibratome-cut tissue sec-tions (100 µm) by direct detection of EGFP fluorescence. Propidium iodide ( red ) was used as a structural counter stain. L, M Confocal scanning laser microscopical image of EGFP + cells in the trachea of a rhesus macaque 9 days after infection with MV-IC323-EGFP. MV infection was visualized in formalin-fixed microtome-cut tissue sections (6 µm) by staining with anti-EGFP antibodies. Propidium iodide ( red ) was used as a structural counter stain. A single MV-infected ciliated epithelial cell can be seen in the mucociliary epithelium ( L ), but the majority of infected cells is present in the lamina propria and submucosa of the epithelium. These include cells with the phenotype of lymphocytes and of dendritic cells. (From de Swart et al. 2007b)

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68 R.L. de Swart

Acknowledgements I thank Albert Osterhaus, Rob van Binnendijk, Teunis Geijtenbeek and Lot de Witte for critical comments on the manuscript.

References

Albrecht P, Lorenz D, Klutch MJ, Vickers JH, Ennis FA (1980) Fatal measles infection in marmo-sets pathogenesis and prophylaxis. Infect Immun 27:969–978

Albrecht P, Lorenz D, Klutch MJ (1981) Encephalitogenicity of measles virus in marmosets. Infect Immun 34:581–587

Anderson JF, Goldberger J (1911) Experimental measles in the monkey: a supplemental note. Public Health Reports 26:887–895

Auwaerter PG, Rota PA, Elkins WR, Adams RJ, DeLozier T, Shi Y, Bellini WJ, Murphy BR, Griffin DE (1999) Measles virus infection in rhesus macaques: altered immune responses and comparison of the virulence of six different virus strains. J Infect Dis 180:950–958

Bankamp B, Hodge G, McChesney MB, Bellini WJ, Rota PA (2008) Genetic changes that affect the virulence of measles virus in a rhesus macaque model. Virology 373:39–50

Blake FG, Trask Jr JD (1921a) Studies on measles. I. Susceptibility of monkeys to the virus of measles. J Exp Med 33:385–412

Blake FG, Trask Jr JD (1921b) Studies on measles. II. Symptomatology and pathology in mon-keys experimentally infected. J Exp Med 33:413–422

Blake FG, Trask Jr JD (1921c) Studies on measles. III. Acquired immunity following experimen-tal measles. J Exp Med 33:621–626

Burnet FM (1968) Measles as an index of immunological function. Lancet 292:610–613 Buynak EB, Peck HM, Creamer AA, Goldner H, Hilleman MR (1962) Differentiation of virulent

from avirulent measles strains. Am J Dis Child 103:290–303 Chen RT, Markowitz LE, Albrecht P, Stewart JA, Mofenson LM, Preblud SR, Orenstein WA

(1990) Measles antibody: reevaluation of protective titers. J Infect Dis 162:1036–1042 Condack C, Grivel J-C, Devaux P, Margolis L, Cattaneo R (2007) Measles virus vaccine attenua-

tion: suboptimal infection of lymphatic tissue and tropism alteration. J Infect Dis 196:541–549

Cutts FT, Clements CJ, Bennett JV (1997) Alternative routes of measles immunization: a review. Biologicals 25:323–338

De Swart RL, Kuiken T, Timmerman HH, Van Amerongen G, van den Hoogen BG, Vos HW, Neijens HJ, Andeweg AC, Osterhaus ADME (2002) Immunization of macaques with forma-lin-inactivated respiratory syncytial virus (RSV) induces interleukin-13-associated hypersen-sitivity to subsequent RSV infection. J Virol 76:11561–11569

De Swart RL, Kuiken T, Fernandez de Castro J, Papania MJ, Valdespino JL, Minor P, Witham C, Yüksel S, Vos HW, Van Amerongen G, Osterhaus ADME (2006) Aerosol measles vaccination in macaques: preclinical studies of immune responses and safety. Vaccine 24:6424–6436

De Swart RL, LiCalsi C, Quirk AV, Van Amerongen G, Nodelman V, Alcock R, Yüksel S, Ward GH, Hardy JG, Vos HW, Witham CL, Grainger CI, Kuiken T, Greenspan BJ, Gard TG, Osterhaus ADME (2007a) Measles vaccination of macaques by dry powder inhalation. Vaccine 25:1183–1190

De Swart RL, Ludlow M, De Witte L, Yanagi Y, Van Amerongen G, McQuaid S, Yüksel S, Geijtenbeek TBH, Duprex WP, Osterhaus ADME (2007b) Predominant infection of CD150 + lymphocytes and dendritic cells during measles virus infection of macaques. PLoS Pathog 3:e178

De Swart RL, van den Hoogen BG, Kuiken T, Herfst S, Van Amerongen G, Yüksel S, Sprong L, Osterhaus ADME (2007c) Immunization of macaques with formalin-inactivated human metapneumovirus induces hypersensitivity to hMPV infection. Vaccine 25:8518–8528

b12325337-0003ztc.indd 68 10/3/2008 10:40:51 AM


Dilraj A, Cutts FT, Fernandez de Castro J, Wheeler JG, Brown D, Roth C, Coovadia HM, Bennett JV (2000) Response to different measles vaccine strains given by aerosol and subcutaneous routes to schoolchildren: a randomised trial. Lancet 355:798–803

El Mubarak HS, Yüksel S, Van Amerongen G, Mukhtar MM, De Swart RL, Osterhaus ADME (2007) Infection of cynomolgus macaques ( Macaca fascicularis ) and rhesus macaques ( Macaca mulatta ) with different wild-type measles viruses. J Gen Virol 88:2028–2034

Enders JF, Peebles TC (1954) Propagation in tissue cultures of cytophatic agents from patients with measles. Proc Soc Exp Biol Med 86:277–286

Enders JF, Katz SL, Milovanovic MV, Holloway A (1960) Studies on an attenuated measles-virus vaccine I. Development and preparation of the vaccine: techniques for assay of effects of vac-cination. N Engl J Med 263:153–159

Finkeldey W (1931) Über Riesenzellbefunde in den Gaumenmandeln, zugleich ein Beitrag zur Histopathologie der Mandelveränderungen im Maserninkubationsstadium. Virchows Arch 281:323–329

Foege WH, Leland OS, Mollohan CS, Fulginiti VA, Henderson DA, Kempe CH (1965) Inactivated measles-virus vaccine. Public Health Reports 80:60–64

Fulginiti VA, Eller JJ, Downte AW, Kempe CH (1967) Altered reactivity to measles virus. Atypical measles in children previously immunized with inactivated measles virus vaccines. JAMA 202:1075–1080

Fulginiti VA, Eller JJ, Sieber OF, Joyner JW, Minamitani M, Meiklejohn G (1969) Respiratory virus immunization. I. A field trial of two inactivated respiratory virus vaccines; an aqueous trivalent parainfluenza virus vaccine and an alum-precipitated respiratory syncytial virus vac-cine. Am J Epidemiol 89:435–448

Goldberger J, Anderson JF (1911) An experimental demonstration of the presence of the virus of measles in the mixed buccal and nasal secretions. J Am Med Assoc 57:476–478

Griffin DE, Ward BJ, Esolen LM (1994) Pathogenesis of measles virus infection: an hypothesis for altered immune responses. J Infect Dis 170 [Suppl. 1]:S24–S31

Hall WC, Kovatch RM, Herman PH, Fox JG (1971) Pathology of measles in rhesus monkeys. Vet Pathol 8:307–319

Hashimoto K, Ono N, Tatsuo H, Minagawa H, Takeda M, Takeuchi K, Yanagi Y (2002) SLAM (CD150)-independent measles virus entry as revealed by recombinant virus expressing green fluorescent protein. J Virol 76:6743–6749

Hicks JT, Sullivan JL, Albrecht P (1977) Immune responses during measles infection in immuno-suppressed rhesus monkeys. J Immunol 119:1452–1456

Hilleman MR, Stokes J, Buynak EB, Reilly CM, Hampil B (1962) Immunogenic response to killed measles-virus vaccine. Am J Dis Child 103:274–281

Hime JM, Keymer IF (1975) Measles in recently imported colobus monkeys ( Colobus guereza ). Vet Rec 97:392

Hirsch RL, Griffin DE, Johnson RT, Cooper SJ, Lindo de Soriano I, Roedenbeck S, Vaisberg A (1984) Cellular immune responses during complicated and uncomplicated measles virus infec-tions of man. Clin Immunol Immunopathol 31:1–12

Hoffman SJ, Polack FP, Hauer DA, Griffin DE (2003a) Measles virus infection of rhesus macaques affects neutrophil expression of IL-12 and IL-10. Viral Immunol 16:369–379

Hoffman SJ, Polack FP, Hauer DA, Singh M, Billeter MA, Adams RJ, Griffin DE (2003b) Vaccination of rhesus macaques with a recombinant measles virus expressing interleukin-12 alters humoral and cellular immune responses. J Infect Dis 188:1553–1561

Kapikian AZ, Mitchell RH, Chanock RM, Shvedoff RA, Stewart CE (1969) An epidemiologic study of altered clinical reactivity to respiratory syncytial (RS) virus infection in children previously vaccinated with an inactivated RS virus vaccine. Am J Epidemiol 89:405–421

Kim HW, Canchola JG, Brandt CD, Pyles G, Chanock RM, Jensen K, Parrott RH (1969) Respiratory syncytial virus disease in infants despite prior administration of antigenic inacti-vated vaccine. Am J Epidemiol 89:422–434

b12325337-0003ztc.indd 69 10/3/2008 10:40:51 AM

70 R.L. de Swart

Kobune F, Sakata H, Sugiura A (1990) Marmoset lymphoblastoid cells as a sensitive host for iso-lation of measles virus. J Virol 64:700–705

Kobune F, Takahashi H, Terao K, Ohkawa T, Ami Y, Suzaki Y, Nagata N, Sakata H, Yamanouchi K, Kai C (1996) Nonhuman primate models of measles. Lab Anim Sci 46:315–320

Levy BM, Mirkovic RR (1971) An epizootic of measles in a marmoset colony. Lab Anim Sci 21:33–39

LiCalsi C, Maniaci MJ, Christensen T, Phillips E, Ward GH, Witham C (2001) A powder formula-tion of measles vaccine for aerosol delivery. Vaccine 19:2629–2636

MacArthur JA, Mann PG, Oreffo V, Scott GBD (1979) Measles in monkeys: an epidemiological study. J Hyg 83:207–212

Marshall E, Enserink M (2004) Caution urged on SARS vaccines. Science 303:944–946 McChesney MB, Fujinami RS, Lerche NW, Marx PA, Oldstone MBA (1989) Virus-induced

immunosuppression: infection of peripheral blood mononuclear cells and suppression of immunoglobulin synthesis during natural measles virus infection of rhesus monkeys. J Infect Dis 159:757–760

McChesney MB, Miller CJ, Rota PA, Zhu Y, Antipa L, Lerche NW, Ahmed R, Bellini WJ (1997) Experimental measles I. Pathogenesis in the normal and the immunized host. Virology 233:74–84

Meyer HM, Brooks BE, Douglas RD, Rogers NG (1962) Ecology in measles in monkeys. Am J Dis Child 103:138–143

Mongkolsapaya J, Jaye A, Callan MFC, Magnusen AF, McMichael AJ, Whittle HC (1999) Antigen-specific expansion of cytotoxic T lymphocytes in acute measles virus infection. J Virol 73:67–71

Moss WJ, Ryon JJ, Monze M, Griffin DE (2002) Differential regulation of interleukin (IL)-4, IL-5, and IL-10 during measles in Zambian children. J Infect Dis 186:879–887

Nii S, Kamahora J, Mori Y, Takahashi M, Nishimura S, Okuno Y (1964a) Experimental pathology of measles in monkeys. Biken J 6:271–297

Nii S, Kamahora J, Okuno Y, Nishimura S, Kato S, Aoyama Y (1964b) Studies on the biological effects of live attenuated measles vaccines in monkeys. Biken J 7:65–69

Norrby E, Carlstrom G, Lagercrantz R, Gard S (1964) Measles vaccination II. Antibody titers 8 to 9 months after vaccination with an inactivated vaccine. Acta Paediat 53:533–540

Norrby E, Enders-Ruckle G, Ter Meulen V (1975) Differences in the appearance of antibodies to structural components of measles virus after immunization with inactivated and live virus. J Infect Dis 132:262–269

Okada H, Kobune F, Sato TA, Kohama T, Takeuchi Y, Abe T, Takayama N, Tsuchiya T, Tashiro M (2000) Extensive lymphopenia due to apoptosis of uninfected lymphocytes in acute measles patients. Arch Virol 145:905–920

Pahar B, Li J, McChesney MB (2005) Detection of T cell memory to measles virus in experimen-tally infected rhesus macaques by cytokine flow cytometry. J Immunol Methods 304:174–183

Pan CH, Valsamakis A, Colella T, Nair N, Adams RJ, Polack FP, Greer CE, Perri S, Polo JM, Griffin DE (2005) Modulation of disease, T cell responses, and measles virus clearance in monkeys vaccinated with H-encoding alphavirus replicon particles. Proc Natl Acad Sci U S A 102:11581–11588


Peebles TC, McCarthy K, Enders JF, Holloway A (1957) Behavior of monkeys after inoculation of virus derived from patients with measles and propagated in tissue culture together with observations on spontaneous infections of these animals by an agent exhibiting similar anti-genic properties. J Immunol 78:63–74

Permar SR, Klumpp SA, Mansfield KG, Kim WK, Gorgone DA, Lifton MA, Williams KC, Schmitz JE, Reimann KA, Axthelm MK, Polack FP, Griffin DE, Letvin NL (2003) Role of CD8 + lymphocytes in control and clearance of measles virus infection of rhesus monkeys. J Virol 77:4396–4400

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Permar SR, Klumpp SA, Mansfield KG, Carville AA, Gorgone DA, Lifton MA, Schmitz JE, Reimann KA, Polack FP, Griffin DE, Letvin NL (2004) Limited contribution of humoral immunity to the clearance of measles viremia in rhesus monkeys. J Infect Dis 190:998–1005

Polack FP, Auwaerter PG, Lee SH, Nousari HC, Valsamakis A, Leiferman KM, Diwan A, Adams RJ, Griffin DE (1999) Production of atypical measles in rhesus macaques: evidence for disease mediated by immune complex formation and eosinophils in the presence of fusion-inhibiting antibody. Nat Med 5:629–634

Polack FP, Lee SH, Permar S, Manyara E, Nousari HC, Jeng Y, Mustafa F, Valsamakis A, Adams RJ, Robinson HL, Griffin D (2000) Successful DNA immunization against measles: neutraliz-ing antibody against either the hemagglutinin or fusion glycoprotein protects rhesus macaques without evidence of atypical measles. Nat Med 6:776–781

Polack FP, Hoffman SJ, Moss WJ, Griffin DE (2002) Altered synthesis of interleukin-12 and type 1 and type 2 cytokines in rhesus macaques during measles and atypical measles. J Infect Dis 185:13–19

Polack FP, Hoffman SJ, Crujeiras G, Griffin DE (2003a) A role for nonprotective complement-fixing antibodies with low avidity for measles virus in atypical measles. Nat Med 9:1209–1213

Polack FP, Hoffman SJ, Moss WJ, Griffin DE (2003b) Differential effects of priming with DNA vaccines encoding the hemagglutinin and/or fusion proteins on cytokine responses after mea-sles virus challenge. J Infect Dis 187:1794–1800

Potkay S, Ganaway JR, Rogers NG, Kinard R (1966) An epizootic of measles in a colony of rhe-sus monkeys ( Macaca mulatta ). Am J Vet Res 27:331–334

Premenko-Lanier M, Rota PA, Rhodes G, Verhoeven D, Barouch DH, Lerche NW, Letvin NL, Bellini WJ, McChesney MB (2003) DNA vaccination of infants in the presence of maternal antibody: a measles model in the primate. Virology 307:67–75

Premenko-Lanier M, Rota PA, Rhodes GH, Bellini WJ, McChesney MB (2004) Protection against challenge with measles virus (MV) in infant macaques by an MV DNA vaccine administered in the presence of neutralizing antibody. J Infect Dis 189:2064–2071

Pütz MM, Bouche FB, De Swart RL, Muller CP (2003) Experimental vaccines against measles in a world of changing epidemiology. Int J Parasitol 33:525–545

Remfry J (1976) A measles epizootic with 5 deaths in newly-imported rhesus monkeys ( Macaca mulatta ). Lab Anim 10:49–57

Ruat C, Caillet C, Bidaut A, Simon J, Osterhaus ADME (2008) Vaccination of macaques with adjuvanted formalin-inactivated influenza A (H5N1) vaccines: protection against H5N1 chal-lenge without disease enhancement. J Virol 82:2565–2569

Sabin AB, Fernandez de Castro J, Flores Arechiga A, Sever JL, Madden DL, Shekarchi I (1982) Clinical trials of inhaled aerosol of human diploid and chick embryo measles vaccine. Lancet 2:604

Sakaguchi M, Yoshikawa Y, Yamanouchi K, Sata T, Nagashima K, Takeda K (1986) Growth of measles virus in epithelial and lymphoid tissues of cynomolgus monkeys. Microbiol Immunol 30:1067–1073

Samb B, Aaby P, Whittle HC, Coll-Seck AM, Rahman S, Bennett J, Markowitz L, Simondon F (1995) Serologic status and measles attack rates among vaccinated and unvaccinated children in rural Senegal. Pediatr Infect Dis J 14:203–209

Schneider-Schaulies S, Klagge IM, Ter Meulen V (2003) Dendritic cells and measles virus infec-tion. Curr Top Microbiol Immunol 276:77–101

Scott GBD, Keymer IF (1975) The pathology of measles in Abyssinian colobus monkeys ( Colobus guereza ): a description of an outbreak. J Pathol 117:229–233

Sergiev PG, Ryazantseva NE, Shroit IG (1960) The dynamics of pathological processes in experi-mental measles in monkeys. Acta Virol 4:265–273

Sharova OK, Rozina EE, Gordienko NM, Shteinberg LSH (1984) Effect of immunosuppression on morphological changes in CNS of monkeys infected with different measles virus vaccine strains. Acta Virol 28:144–147

Stittelaar KJ, Wyatt LS, De Swart RL, Vos HW, Groen J, Van Amerongen G, Van Binnendijk RS, Rozenblatt S, Moss B, Osterhaus ADME (2000) Protective immunity in macaques vaccinated

b12325337-0003ztc.indd 71 10/3/2008 10:40:51 AM

72 R.L. de Swart

with a modified vaccinia virus Ankara-based measles virus vaccine in the presence of pas-sively acquired antibodies. J Virol 74:4236–4243

Stittelaar KJ, De Swart RL, Osterhaus ADME (2002a) Vaccination against measles: a neverending story. Expert Rev Vaccines 1:151–159

Stittelaar KJ, Vos HW, Van Amerongen G, Kersten GFA, Osterhaus ADME, De Swart RL (2002b) Longevity of neutralizing antibody levels in macaques vaccinated with Quil A-adjuvanted measles vaccine candidates. Vaccine 21:155–157

Takeda M, Takeuchi K, Miyajima N, Kobune F, Ami Y, Nagata N, Suzaki Y, Nagai Y, Tashiro M (2000) Recovery of pathogenic measles virus from cloned cDNA. J Virol 74:6643–6647

Takeuchi K, Miyajima N, Nagata N, Takeda M, Tashiro M (2003) Wild-type measles virus induces large syncytium formation in primary human small airway epithelial cells by a SLAM(CD150)-independent mechanism. Virus Res 94:11–16

Tauraso NM (1973) Review of recent epizootics in nonhuman primate colonies and their relation to man. Lab Anim Sci 23:201–210

Valdespino-Gomez JL, De Lourdes Garcia-Garcia M, Fernandez de Castro J, Henao-Restrepo AM, Bennett J, Sepulveda-Amor J (2006) Measles aerosol vaccination. Curr Top Microbiol Immunol 304:165–193

Van Binnendijk RS, van der Heijden RWJ, Van Amerongen G, UytdeHaag FGCM, Osterhaus ADME (1994) Viral replication and development of specific immunity in macaques after infection with different measles virus strains. J Infect Dis 170:443–448

Van Binnendijk RS, van der Heijden RWJ, Osterhaus ADME (1995) Monkeys in measles research. Curr Top Microbiol Immunol 191:135–148

Van Binnendijk RS, Poelen MCM, Van Amerongen G, De Vries P, Osterhaus ADME (1997) Protective immunity in macaques vaccinated with live attenuated, recombinant, and subunit measles vaccines in the presence of passively acquired antibodies. J Infect Dis 175:524–532

Von Pirquet CE (1908) Das Verhalten der kutanen Tuberkulin-reaktion während der Masern. Dtsch Med Wochenschr 34:1297–1300

Warthin AS (1931) Occurrence of numerous large giant cells in the tonsils and pharyngeal mucosa in the prodromal stage of measles. Arch Pathol 11:864–874

Welshman MD (1989) Measles in the cynomolgus monkey ( Macaca fascicularis ). Vet Rec 124:184–186

WHO (2007) Manual for the laboratory diagnosis of measles and rubella virus infection. WHO/IVB/07.01, Geneva

Yamanouchi K, Egashira Y, Uchida N, Kodama H, Kobune F, Hayami M, Fukuda A, Shishido A (1970) Giant cell formation in lymphoid tissues of monkeys inoculated with various strains of measles virus. J Med Sci Biol 23:131–145

Yamanouchi K, Chino F, Kobune F, Kodama H, Tsuruhara T (1973) Growth of measles virus in the lymphoid tissues of monkeys. J Infect Dis 128:795–799

Zhu Y, Heath J, Collins J, Greene T, Antipa L, Rota PA, Bellini W, McChesney MB (1997) Experimental measles II. Infection and immunity in the rhesus macaque. Virology 233:85–92

Zhu Y-D, Rota P, Wyatt LS, Tamin A, Rozenblatt S, Lerche N, Moss B, Bellini W, McChesney M (2000) Evaluation of recombinant vaccinia virus – measles vaccines in infant rhesus macaques with preexisting measles antibody. Virology 276:202–213

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Chapter 4 Ferrets as a Model for Morbillivirus Pathogenesis, Complications, and Vaccines

S. Pillet , N. Svitek , and V. von Messling (*)

Abstract The ferret is a standard laboratory animal that can be accommodated in most animal facilities. While not susceptible to measles, ferrets are a natural host of canine distemper virus (CDV), the closely related carnivore morbillivirus. CDV infection in ferrets reproduces all clinical signs associated with measles in humans, including the typical rash, fever, general immunosuppression, gastrointestinal and respiratory involvement, and neurological complications. Due to this similarity, experimental CDV infection of ferrets is frequently used to assess the efficacy of novel vaccines, and to characterize pathogenesis mechanisms. In addition, direct intracranial inoculation of measles isolates from subacute sclerosing panencephali-tis (SSPE) patients results in an SSPE-like disease in animals that survive the acute phase. Since the advent of reverse genetics systems that allow the targeted manipu-lation of viral genomes, the model has been used to evaluate the contribution of the accessory proteins C and V, and signalling lymphocyte activation molecule (SLAM)-binding to immunosuppression and overall pathogenesis. Similarly produced green

V. von Messling INRS-Institut Armand-Frappier , University of Quebec , 531, boul. des Prairies, Laval , QC, H7V 1B7 , Canada , e-mail: [email protected]

Contents

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74Ferret Husbandry and Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75Availability and Development of Ferret-Specific Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . 75The Ferret as a Model for Measles Vaccine Development . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

The Role of Maternal Antibodies for Measles Vaccination Strategies . . . . . . . . . . . . . . . . 76Vaccination Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

The Ferret as Model for Measles-Induced Immunosuppression . . . . . . . . . . . . . . . . . . . . . . . 79The Ferret as Model for Measles-Associated Neurological Complications . . . . . . . . . . . . . . 80

Overview of Morbillivirus-Associated Central Nervous System Complications . . . . . . . . 80Subacute Sclerosing Panencephalitis in Ferrets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81Morbillivirus Neuroinvasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

Conclusion and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83


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fluorescent protein-expressing derivatives that maintain parental virulence have been instrumental in the direct visualization of systemic dissemination and neuroinvasion. As more immunological tools become available for this model, its contribution to our understanding of morbillivirus–host interactions is expected to increase.

Abbreviations

ADEM Acute disseminated encephalomyelitis CAV2 Canine adenovirus type 2 CDV Canine distemper virus CNS Central nervous system eGFP Enhanced green fluorescent protein F Fusion H Hemagglutinin IFN Interferon Ig Immunoglobulin IL Interleukin MV Measles virus MHC Major histocompatibility complex MIBE Inclusion body encephalitis N Nucleocapsid P Phosphoprotein SLAM Signalling lymphocyte activation molecule SSPE Subacute sclerosing panencephalitis

Introduction

Domestic ferrets ( Mustela putorius furo ) belong to the family Mustelidae , which also includes weasels, minks, martens, sables, skunks, otters, and badgers, in the order Carnivora (McKenna and Bell 1997). Due to their relatively small size and natural susceptibility to a broad range of respiratory viruses, ferrets are an attractive animal model for pathogenesis research and the development of new vaccines and treatment approaches (Hsu et al. 1994; Maher and DeStefano 2004; Osterhaus et al. 2004). While they do not develop signs of disease upon intranasal infection with measles virus (MV), intracranial inoculation of certain subacute sclerosing panen-cephalitis (SSPE) isolates results in a histopathologically similar subacute encepha-litis (Thormar et al. 1985). In addition, ferrets are a natural host for canine distemper virus (CDV), a close relative of MV that infects a broad range of carnivores. They develop all signs of disease seen in MV-infected humans, including the characteris-tic rash, but usually succumb to the infection (von Messling et al. 2003). This clini-cal similarity combined with the high sensitivity make the study of CDV in ferrets an ideal model to characterize MV pathogenesis mechanisms.

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4 Ferrets as a Model for Morbillivirus Pathogenesis, Complications, and Vaccines 75

Ferret Husbandry and Physiology

Ferrets have long tubular bodies with short legs, and they are very flexible. While most rabbit and cat cages correspond to regulatory requirements for the housing of ferrets in experimental settings, they require adaptation to prevent the animals’ escape through feeders or other small openings. Ferrets are generally friendly and display a broad range of behaviors. Whenever possible, they should be housed in groups and appropriate toys should be provided to avoid boredom. They can be fed ad libitum with commercially available cat food, and food should not be withheld for more than 6 h because of their rapid metabolism (Fox 1998; Moody et al. 1985) .

Ferrets reach sexual maturity around 6–8 months of age, with the weight of adult males reaching up to 2 kg, while females will weigh between 0.5 and 1 kg. Meas-urement of the rectal temperature and subcutaneous and intramuscular injections require only scruffing of the loose skin on the back of the neck, while intravenous injections and venipunctures are greatly facilitated by general anesthesia. This can be either achieved by intramuscular injection of a combination of ketamine and either midazolam, xylazine, or acepromazine, or by inhalation of isoflurane. Ferrets have a total blood volume of approximately 60 ml/kg, and 10% can be safely with-drawn once every 2 weeks. The most commonly used site for venipuncture is the jugular vein, but the anterior vena cava can also be used. For smaller volumes, the lateral saphenous or the cephalic vein and the tail artery are an alternative option. A detailed description of the techniques involved can be found in Quesenberry (1996).

Availability and Development of Ferret-Specific Reagents

The limited availability of ferret-specific immunological reagents has been a major drawback of this animal model. Aside from secondary antisera to ferret IgG, IgM, IgA, an increasing number of cross-reacting antibodies against various epitopes have become commercially available (Table 4. 1 ). In addition, the susceptibility of ferrets to influenza and SARS (Maher and DeStefano 2004; Osterhaus et al. 2004) has led to an increased effort to develop ferret-specific reagents. Several ferret cytokine and chemokine sequences have been published in GenBank, and PCR-based assays have been developed to assess gene expression (Danesh et al. 2008; Senchak et al. 2007; Svitek and von Messling 2007). While ferret-specific protein-based assays are still rare (Ochi et al. 2008), the potential applicability of commer-cial canine or feline tests can now be evaluated based on sequence homology comparisons. The recent addition of the ferret genome to the sequencing targets approved by the NIH National Human Genome Research Institute will further accelerate the development of this model.

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Table 4.1 Noncomprehensive list of cross-reactive antibodies commercially available for ferrets

Antibody Clone Company

Mouse monoclonal anti-CD79A HM47 Santa Cruz Biotechnology, CA

Mouse monoclonal anti-CD79α cy HM57 DakoCytomation, CAMouse monoclonal anti-CD3 PC3/188A Santa Cruz

Biotechnology, CAMouse monoclonal anti-CD3 F7.2.38 DakoCytomation, CAMouse monoclonal anti-CD14 CAM36A VMRD, WAMouse monoclonal anti-CD172a DH59B VMRD, WAMouse monoclonal anti-CD18 BAQ30A VMRD, WAMouse monoclonal anti-CD21-like F46A VMRD, WAMouse monoclonal anti-CD41/46 CL2A VMRD, WAMouse monoclonal anti-CD44 BAG40A VMRD, WAMouse monoclonal anti-MHC I H58A VMRD, WAMouse monoclonal anti-MHC II CAT82A VMRD, WAMouse monoclonal anti-MHC II

(HLA-DP)H425A VMRD, WA

Mouse monoclonal anti-MHC II (HLA-DQ)

TH81A5 VMRD, WA

Mouse monoclonal anti-MHC II (HLA-DRa)

TH14B VMRD, WA

Mouse monoclonal anti-MHC II (HLA-DR)

H34A VMRD, WA

Mouse monoclonal anti-keratin Pan Ab-3

Lu-5 NeoMarkers, CA

Rabbit polyclonal anti-glial fibrillary acidic protein

– DakoCytomation, CA

Rabbit polyclonal anti-Von Willebrand Factor

– DakoCytomation, CA

Goat polyclonal anti-ferret IgG H and l chains

– Bethyl Laboratories, TX

Goat polyclonal anti-ferret IgAa – Rockland Immunochemicals, PA

Goat polyclonal anti-ferret IgM μ – Rockland Immunochemicals, PA

The Ferret as a Model for Measles Vaccine Development

The Role of Maternal Antibodies for Measles Vaccination Strategies

In addition to the immaturity of the infant’s immune system, the poor response to MV vaccination observed in infants under 1 year of age has also been linked to the presence of maternal antibodies (Gans et al. 1998; Schluederberg et al. 1973; Wilkins and Wehrle 1979). Thus, maternal antibodies are an important consideration for MV vaccination development and for the ongoing eradication

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campaign. Ferrets and dogs have been extensively used to characterize the inter-actions of live-attenuated morbillivirus vaccines with maternal antibodies. Since both species are highly susceptible to CDV, annual vaccination with a live-atten-uated vaccine similar to that used in humans is recommended. Consequently, ferret kits and puppies are born with maternal antibodies that diminish as the animal ages. In ferret kits born to mothers vaccinated with a modified-live CDV vaccine of chicken tissue culture origin, virus-specific maternal antibodies have a half-life of approximately 9 days and reach levels below the detection limit after 12 weeks (Appel and Harris 1988), levels comparable to maternal canine parvovirus antibodies transferred to puppies (Pollock and Carmichael 1982). Despite this shorter half-life of maternal antibodies, ferrets and dogs thus reca-pitulate the overall aspects of maternally acquired anti-MV antibodies in human infants (Gans et al. 1998) .

In a study using a recombinant canarypox virus expressing the CDV fusion (F) and hemagglutinin (H) proteins, only 14% survival was seen in ferret kits with preexisting maternal immunity after mucosal or parenteral immunization with the recombinant candidate vaccines, despite high levels of neutralizing antibod-ies at the time of challenge. However, the percentage of survival increased to 33% when animals were inoculated by the combined parenteral/mucosal route (Welter et al. 2000), suggesting that multiple routes of inoculation may be advan-tageous. In puppies, maternal antibody interference can be efficiently overcome by the use of the same canarypox-based vaccine, a similarly constructed adeno-viral vector, or DNA vaccination with a mixture of plasmids coding for the CDV nucleocapsid (N), phosphoprotein (P), and H genes (Fischer et al. 2002, 2003; Griot et al. 2004; Pardo et al. 2007). In all cases, the vaccination elicited an increase in neutralizing antibodies, and all animals were protected from subse-quent challenge. This difference in vaccine efficacy observed between dogs and ferrets is likely due to differences in experimental setup and readout, but may also reflect the higher sensitivity of the latter to CDV infection. Taken together, these studies provide valuable insights for the development of MV vaccination strate-gies that retain the effectiveness in the presence of maternal antibodies.

Vaccination Studies

Modified-Live and Inactivated Vaccines

Ferrets and dogs have also been used to assess the efficacy of new CDV candidate vaccines and novel vaccination strategies. Early studies demonstrated the efficacy of the live-attenuated CDV vaccines upon subcutaneous and intramuscular inocu-lation, but also raised concerns because of the residual immunosuppressive prop-erties and the occasional anaphylactic reactions observed (Cabasso et al. 1953; Gorham et al. 1954; Greenacre 2003; Hoover et al. 1989; Morris et al. 1954; Rockborn et al. 1965; Shen et al. 1984; Stephensen et al. 1997; Williams et al. 1996; Wimsatt et al. 2001). To circumvent this problem, the cross-protective

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78 S. Pillet et al.

properties of the live-attenuated MV vaccine were evaluated in puppies that had no maternal antibodies. While none of the adverse effects associated with the CDV vaccine were observed, the level of protection conferred was variable (Appel et al. 1984; Chalmers and Baxendale 1994; Strating 1975). A comparative study of a beta-propiolactone-inactivated virus and the live-attenuated CDV vac-cine revealed that both vaccines conferred protective immunity, even though neu-tralizing antibody levels were lower in the group receiving the inactivated product. However, animals receiving the live-attenuated vaccine developed a transient lymphopenia after vaccination, demonstrating again that the live-attenuated vac-cine virus had retained some immunosuppressive properties (Williams et al. 1996). This residual virulence of the commercially available live-attenuated vac-cines is particularly pertinent for the development of vaccination strategies for CDV-susceptible wildlife species in zoos or in conservation programs. Many of these species may develop severe disease or even die from inoculation with these vaccines. The near extinction of black-footed ferrets caused by a vaccination campaign is the best known example (Carpenter et al. 1976; Pearson 1977), but there are similar reports for several other species, including domestic ferrets (Bush et al. 1976; Carpenter et al. 1976; Ek-Kommonen et al. 2003; Gill et al. 1988; Greenacre 2003). It was initially recommended to use either an inactivated vaccine or the live-attenuated MV vaccine for highly sensitive or endangered spe-cies, possibly followed by inoculation with the live-attenuated CDV vaccine after verification of antibody titers (Loeffler et al. 2007; Qin et al. 2007; Wimsatt et al. 2003). However, since its approval in 1997 (Pardo et al. 1997), the recombinant canarypox vaccine expressing the CDV envelope glycoproteins has replaced these approaches. In addition to providing insight into the risks and limitations of live-attenuated vaccines, these studies constitute a framework for the development of vaccination strategies for immunosuppressed individuals.

Novel Vaccine Candidates

The main disadvantages of the currently commercially available live-attenuated morbillivirus vaccines lie in their temperature sensitivity and the need for intramus-cular injection. Among the novel vaccine candidates brought forward to address these issues, recombinant poxviruses expressing the CDV envelope glycoproteins have been evaluated in ferrets (Stephensen et al. 1997). While the traditionally vac-cinated group still experienced weight loss and developed erythematous rash and leukopenia, animals receiving the recombinant vaccine mounted a strong humoral immune response and were completely protected from clinical disease. This study provided the first proof-of-principle that recombinant poxviruses expressing mor-billivirus F and H proteins elicit protective immunity. The same group subsequently demonstrated that these poxviral candidate vaccines were similarly efficacious upon intranasal inoculation while intraduodenal infection conferred only partial protection (Welter et al. 1999). In ferrets less than 12 weeks of age, vaccine efficacy was dependent on the absence of maternal antibodies (Welter et al. 2000). A similar

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vaccination strategy using two replication-competent canine adenovirus type 2 (CAV2) that expressed the CDV F and H proteins, respectively, was equally effi-cient in protecting CAV2 seronegative dogs from lethal challenge after intranasal inoculation (Fischer et al. 2002). In the presence of anti-CAV2 antibodies, this vac-cine was only effective when inoculated via the subcutaneous route.

DNA-based vaccines have not yet been evaluated in ferrets; however, studies conducted in dogs and minks (Dahl et al. 2004) have provided the proof-of-concept for this type of vaccination. Dogs immunized with a combination of plasmids expressing the CDV N, F, H proteins mounted a neutralizing immune response and were protected from challenge (Cherpillod et al. 2000). However, antibody titers markedly increased only after the third DNA immunization and reached high neu-tralizing values only after challenge. The formulation of plasmids encoding the CDV F and H proteins with the cationic lipid DMRIE-DOPE resulted in significant seroconversion and protection from lethal intracranial challenge after the second immunization (Fischer et al. 2003). These studies demonstrate that several alterna-tives to the currently used live-attenuated vaccines are available and suggest that similar approaches may be applicable to MV.

The Ferret as Model for Measles-Induced Immunosuppression

Since the host range of MV is limited to humans and certain non-human primates, animal models using other morbilliviruses have been developed to characterize the mechanisms underlying the long-lasting immunosuppression associated with MV infection. The clinical and immunological similarities of the diseases caused by MV and CDV in their respective hosts, the fact that ferrets and dogs are common laboratory animals, and the lack of regulatory limitations restricting the work with ungulate morbilliviruses have resulted in preferential use of CDV-based models (Kauffman et al. 1982; Krakowka 1982; Krakowka et al. 1987; McCullough et al. 1974; von Messling et al. 2003, 2004). Ferrets or dogs infected with wild-type CDV strains develop the characteristic clinical signs of morbillivirus disease, including the typical rash, fever, and gastrointestinal and respiratory involvement. In addition, severe leukopenia, inhibition of lymphocyte proliferation upon nonspecific stimula-tion, and reduction or loss of delayed-type hypersensitivity reactions are observed within 1 week after infection (Griffin 2007; Kauffman et al. 1982; Krakowka et al. 1975; von Messling et al. 2003). This immunosuppression is sustained throughout the acute phase of the disease and may persist in dogs for weeks after virus clear-ance (McCullough et al. 1974; Schobesberger et al. 2005) .

The ability to generate genetically modified wild-type viruses by reverse genet-ics and the introduction of an additional transcriptional unit encoding the enhanced green fluorescent protein (eGFP) in the viral genome have proven an invaluable tool for the characterization of virus–host interactions on the macroscopic and microscopic level (von Messling et al. 2004). Time course studies showed that the in-fection is initially limited to lymphatic tissues, where T and B cells are the primary targets. Spread to epithelia occurs only after massive infection of the immune

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system and coincided with the onset of clinical signs, followed by gradual neuroinvasion (Rudd et al. 2006). The extent of the lymphotropism observed, where over 70% of T and B cells were eGFP-positive within the 1st week of a wild type infection, illustrated the direct impact of the infection on the immune system (von Messling et al. 2004). The importance of immune cell infection for viral pathogenesis was further demonstrated by the inability of a signalling lymphocyte activation molecule (SLAM)-blind virus to spread and cause disease (von Messling et al. 2006) .

Since ferrets usually succumb to the infection with wild-type CDV strains, the model is well suited for the characterization of genetic factors of immune evasion. A comparative study involving viruses that lacked the accessory proteins V and C either individually or in combination demonstrated the essential role of V, while indicating that C had a more subtle function (von Messling et al. 2006). Confirming findings from in vitro and murine studies with the corresponding recombinant MVs, a V-mediated block in cytokine induction was observed (Devaux et al. 2007; Fontana et al. 2008; Ohno et al. 2004; Palosaari et al. 2003). The subsequent analy-sis of cytokine profiles from animals that survived the infection revealed a response similar to that seen in patients with naturally acquired MV infection (Atabani et al. 2001; Moss et al. 2002; Svitek and von Messling 2007; Tetteh et al. 2003; Zilliox et al. 2007). The response was characterized by a strong interleukin (IL)-10 response as early as 3 days after infection, and sustained induction of cytokines of the proinflamatory or the TH1 and TH2 pathways such as IL-6, IL-2, IL-12p40, IL-4 and interferon (IFN)-gamma (Svitek and von Messling 2007). Taken together, these studies demonstrate the value of CDV-based models for the characterization of morbillivirus immunosuppression and highlight the complementarities of different experimental systems.

The Ferret as Model for Measles-Associated Neurological Complications

Overview of Morbillivirus-Associated Central Nervous System Complications

MV can result in early or late central nervous system (CNS) involvement. Based on onset, virus presence in the brain, and mitigating host factors, three distinct forms are recognized: acute disseminated encephalomyelitis (ADEM), which is thought to be mainly immune-mediated and occurs within 2–4 weeks after infec-tion; inclusion body encephalitis (MIBE), a rare complication prevalent in immunocompromised patients, which develops within weeks after recovery and is associated with high amounts of replicating virus in the CNS; and SSPE, which manifests months to years after the initial infection and is characterized by the development and subsequent persistence of a highly cell-associated nonproductive form of the virus (Griffin 2007; Sips et al. 2007) .

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CDV is associated with one of the highest incidences of CNS complications among morbilliviruses. Up to 30% of dogs exhibit signs of neurologic involvement during or after CDV infection, and most wild carnivores that succumb to CDV display some evidence of CNS involvement (Appel and Summers 1995; Confer et al. 1975; Summers et al. 1984; van Moll et al. 1995). Similarly to MV, CDV-induced CNS complications can be categorized into acute encephalopathy, inclu-sion body polioencephalitis, and subacute to chronic demyelinating encephalitis (Krakowka et al. 1985). The Snyder Hill strain, which was selected for increased neurovirulence by multiple passages in dogs via intracerebral inoculation, induces acute encephalitis with high mortality that mimics the MV encephalitis seen in patients with acquired immune deficiency syndrome (Gillespie and Rickard 1956; McQuaid et al. 1998; Summers et al. 1984). In contrast, typical wild-type strains mainly induce demyelinating SSPE-like lesions in dogs. These lesions are first observed around 3 weeks postinfection and can progress even after the virus has been cleared from the periphery and the animal has recovered from the acute dis-ease (Vandevelde et al. 1982; Vandevelde and Zurbriggen 2005). This chronic demyelinating encephalitis is characterized by perivascular cuffing, intracerebral infiltra tion of immune cells, MHC II upregulation, and increased levels of anti-CDV antibodies in the cerebrospinal fluid, all hallmarks of SSPE (Alldinger et al. 1996; Dorries and Ter Meulen 1984; Nagano et al. 1991; Schneider-Schaulies et al. 1999; Vandevelde et al. 1986).

Subacute Sclerosing Panencephalitis in Ferrets

Forty years ago, Katz et al. (1968) inoculated ferrets intracerebrally with brain cell cultures from SSPE patients, producing acute encephalitis in these animals. In con-trast to typical cell-associated nonproductive SSPE strains, wild-type MV strains and the efficiently replicating SSPE isolates did not cause detectable encephalitis in young ferrets. Subsequent studies showed that the course of disease depends on the respective cell-associated SSPE strain and the type of cell culture used for virus propagation (Thormar et al. 1985). However, despite severe clinical signs and large amounts of virus in the brain, only minor lesions and no humoral immune response or inflammatory changes were observed in the CNS, in contrast to human SSPE tissues (Brown et al. 1985) .

In an attempt to reproduce SSPE more accurately, a nonproductive SSPE strain was inoculated into ferrets previously immunized with the live-attenuated MV vaccine. Among the 50% of survivors, some developed subacute encephalitis weeks or months after inoculation (Mehta and Thormar 1979). The neurological signs observed in these ferrets had certain similarities to those seen in SSPE patients, leading to coma and death. Cell-associated nonproductive MV was always present in the brain and sometimes in the spinal cord, and widespread inflammatory lesions were observed in both the white and gray matter. Ferrets developing suba-cute encephalitis furthermore exhibited increased IgG concentrations in the cere-brospinal fluid, another hallmark of SSPE (Mehta and Thormar 1979; Thormar

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82 S. Pillet et al.

et al. 1983). The lack of antibodies against the viral matrix protein despite high titers against other structural proteins was also observed (Thormar et al. 1985). In a follow-up study, cell-associated nonproductive SSPE viruses were inoculated intracardially to elucidate the route of CNS invasion. Although infectious virus was not detectable in the blood of ferrets tested daily following inoculation, clinical signs and inflammatory lesions as well as the presence of viral antigens in the brain demonstrate the ability of the virus to spread to the CNS and cause encephalitis within 5–7 days (Thormar et al. 1988) .

By reproducing key features of the disease, inoculation of ferrets with human isolates provides unique insights in SSPE pathogenesis. The findings emphasize the importance of the host’s immune response, especially the humoral response, in the establishment of a persistent morbillivirus infection in the CNS. Even though the intracerebral route of inoculation constitutes a major drawback of this model, it merits further development as more ferret-specific reagents for the assessment of the immune response become available.

Morbillivirus Neuroinvasion

Due to the high incidence of CNS involvement associated with CDV infections, morbillivirus neuroinvasion has been extensively studied in dogs and more recently in ferrets (Axthelm and Krakowka 1987; Bonami et al. 2007; Rudd et al. 2006; Vandevelde and Zurbriggen 2005). In dogs experimentally infected with a neuro-virulent strain, the outcome is determined by the individual’s ability to mount a rapid and specific immune response. Compared to complete recovery without CNS involvement, persistent CNS infection was associated with a delayed and reduced immune response, while animals that died during the acute disease phase were unable to mount any response (Appel et al. 1982). The regular presence of extravas-cular infected lymphocytes in the white matter and the choroid plexus of CDV-infected dogs during the acute disease phase was indicative of neurovasion via the hematogenous route (Summers et al. 1979), which may be facilitated by alterations of the blood–brain barrier resulting from direct infection of endothelial cells (Axthelm and Krakowka 1987) .

Using an eGFP-expressing derivative of A75/17 that retained parental virulence and tropism in the ferret model, it was shown that neuroinvasion occurred not only hematogenously through infected circulating lymphocytes, but also anterogradely via the olfactory signaling route (Rudd et al. 2006). In fact, the earliest macroscopically detectable infected foci in the brain were located in the olfactory bulb, emphasizing the importance of this route of entry for CNS dissemination. CNS invasion only occurred after extensive infection of immune and epithelial tissues, when the animal was already highly immunosuppressed, indicating that morbillivirus neuroinvasion is a late event and requires an inefficient or absent immune response. In a follow-up study, it was shown that the duration of the infection in the absence of an immune response, and not differences in attachment protein-mediated tropism, determines the ability of a CDV strain to invade the CNS (Bonami et al. 2007) .

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Conclusion and Perspectives

The recognition that CDV causes several pathologies in its natural hosts that are similar to MV-associated disease syndromes in humans has promoted the study of host–CDV interactions as a model for measles. Compared to non-human primates or even dogs, ferrets are relatively inexpensive small animals that do not require costly facilities and are easy to house and manipulate. CDV in ferrets recapitulates the clinical signs of measles in humans including the typical rash, fever, severe leu-kopenia, and general immunosuppression as well as gastrointestinal and respiratory involvement. The study of CDV infection in ferrets is therefore increasingly used as a model for pathogenesis studies and vaccine development. The ongoing effort to develop ferret-specific immunological tools and the advances in the genetic manipulation of morbilliviruses will further increase the attractivity of this model for morbillivirus research.

References

Alldinger S, Wunschmann A, Baumgartner W, Voss C, Kremmer E (1996) Up-regulation of major histocompatibility complex class II antigen expression in the central nervous system of dogs with spontaneous canine distemper virus encephalitis. Acta Neuropathol 92:273–280

Appel MJ, Harris WV (1988) Antibody titers in domestic ferret jills and their kits to canine dis-temper virus vaccine. J Am Vet Med Assoc 193:332–333

Appel MJ, Shek WR, Summers BA (1982) Lymphocyte-mediated immune cytotoxicity in dogs infected with virulent canine distemper virus. Infect Immun 37:592–600

Appel MJ, Shek WR, Shesberadaran H, Norrby E (1984) Measles virus and inactivated canine distemper virus induce incomplete immunity to canine distemper. Arch Virol 82:73–82

Appel MJ, Summers BA (1995) Pathogenicity of morbilliviruses for terrestrial carnivores. Vet Microbiol 44:187–191

Atabani SF, Byrnes AA, Jaye A, Kidd IM, Magnusen AF, Whittle H, Karp CL (2001) Natural measles causes prolonged suppression of interleukin-12 production. J Infect Dis 184:1–9

Axthelm MK, Krakowka S (1987) Canine distemper virus: the early blood-brain barrier lesion. Acta Neuropathol 75:27–33

Bonami F, Rudd PA, von Messling V (2007) Disease duration determines canine distemper virus neurovirulence. J Virol 81:12066–12070

Brown HR, Thormar H, Barshatzky M, Wisniewski HM (1985) Localization of measles virus antigens in subacute sclerosing panencephalitis in ferrets. Lab Anim Sci 35:233–237

Bush M, Montali RJ, Brownstein D, James AE Jr, Appel MJ (1976) Vaccine-induced canine dis-temper in a lesser panda. J Am Vet Med Assoc 169:959–960

Cabasso VJ, Stebbins MR, Cox HR (1953) Active immunization of ferrets by simultaneous injec-tions of avianized canine distemper vaccine and anticanine distemper hyperimmune serum. Cornell Vet 43:179–183

Carpenter JW, Appel MJ, Erickson RC, Novilla MN (1976) Fatal vaccine-induced canine distem-per virus infection in black-footed ferrets. J Am Vet Med Assoc 169:961–964

Chalmers WS, Baxendale W (1994) A comparison of canine distemper vaccine and measles vac-cine for the prevention of canine distemper in young puppies. Vet Rec 135:349–353

Cherpillod P, Tipold A, Griot-Wenk M, Cardozo C, Schmid I, Fatzer R, Schobesberger M, Zurbriggen R, Bruckner L, Roch F et al (2000) DNA vaccine encoding nucleocapsid and sur-face proteins of wild type canine distemper virus protects its natural host against distemper. Vaccine 18:2927–2936

b12325337-0004ztc.indd 83 10/3/2008 10:50:42 AM

84 S. Pillet et al.

Confer AW, Kahn DE, Koestner A, Krakowka S (1975) Biological properties of a canine distem-per virus isolate associated with demyelinating encephalomyelitis. Infect Immun 11:835–844

Dahl L, Jensen TH, Gottschalck E, Karlskov-Mortensen P, Jensen TD, Nielsen L, Andersen MK, Buckland R, Wild TF, Blixenkrone-Moller M (2004) Immunization with plasmid DNA encod-ing the hemagglutinin and the nucleoprotein confers robust protection against a lethal canine distemper virus challenge. Vaccine 22:3642–3648

Danesh A, Seneviratne C, Cameron CM, Banner D, Devries ME, Kelvin AA, Xu L, Ran L, Bosinger SE, Rowe T et al (2008) Cloning, expression and characterization of ferret CXCL10. Mol Immunol 45:1288–1297


Dorries R, Ter Meulen V (1984) Detection and identification of virus-specific, oligoclonal IgG in unconcentrated cerebrospinal fluid by immunoblot technique. J Neuroimmunol 7:77–89

Ek-Kommonen C, Rudback E, Anttila M, Aho M, Huovilainen A (2003) Canine distemper of vac-cine origin in European mink, Mustela lutreola – a case report. Vet Microbiol 92:289–293

Fischer L, Tronel JP, Pardo-David C, Tanner P, Colombet G, Minke J, Audonnet JC (2002) Vaccination of puppies born to immune dams with a canine adenovirus-based vaccine protects against a canine distemper virus challenge. Vaccine 20:3485–3497

Fischer L, Tronel JP, Minke J, Barzu S, Baudu P, Audonnet JC (2003) Vaccination of puppies with a lipid-formulated plasmid vaccine protects against a severe canine distemper virus challenge. Vaccine 21:1099–1102

Fontana JM, Bankamp B, Bellini WJ, Rota PA (2008) Regulation of interferon signaling by the C and V proteins from attenuated and wild-type strains of measles virus. Virology 374:71–81

Fox JG (1998) Biology and diseases of the ferret. Wiley-Blackwell, New York Gans HA, Arvin AM, Galinus J, Logan L, DeHovitz R, Maldonado Y (1998) Deficiency of the

humoral immune response to measles vaccine in infants immunized at age 6 months. JAMA 280:527–532

Gill JM, Hartley WJ, Hodgkinson NL (1988) An outbreak of post-vaccinal suspected distemper-like encephalitis in farmed ferrets ( Mustela putorius furo ) NZ Vet J 36:173–176

Gillespie JH, Rickard CG (1956) Encephalitis in dogs produced by distemper virus. Am J Vet Res 17:103–108

Gorham JR, Leader RW, Gutierrez JC (1954) Distemper immunization of ferrets by nebulization with egg adapted virus. Science 119:125–126

Greenacre CB (2003) Incidence of adverse events in ferrets vaccinated with distemper or rabies vaccine: 143 cases (1995–2001) J Am Vet Med Assoc 223:663–665 virology, 5 th edn. Philadelphia, Lippincott Williams Wilkins, pp 1550–1585

Griot C, Moser C, Cherpillod P, Bruckner L, Wittek R, Zurbriggen A, Zurbriggen R (2004) Early DNA vaccination of puppies against canine distemper in the presence of maternally derived immunity. Vaccine 22:650–654

Hoover JP, Baldwin CA, Rupprecht CE (1989) Serologic response of domestic ferrets ( Mustela puto-rius furo ) to canine distemper and rabies virus vaccines. J Am Vet Med Assoc 194:234–238

Hsu KH, Lubeck MD, Bhat BM, Bhat RA, Kostek B, Selling BH, Mizutani S, Davis AR, Hung PP (1994) Efficacy of adenovirus-vectored respiratory syncytial virus vaccines in a new ferret model. Vaccine 12:607–612

Katz M, Rorke LB, Masland WS, Koprowski H, Tucker SH (1968) Transmission of an encepha-litogenic agent from brains of patients with subacute sclerosing panencephalitis to ferrets. Preliminary report. N Engl J Med 279:793–798

Kauffman CA, Bergman AG, O’Connor RP (1982) Distemper virus infection in ferrets: an animal model of measles-induced immunosuppression. Clin Exp Immunol 47:617–625

Krakowka S (1982) Mechanisms of in vitro immunosuppression in canine distemper virus infec-tion. J Clin Lab Immunol 8:187–196

Krakowka S, Cockerell G, Koestner A (1975) Effects of canine distemper virus infection on lym-phoid function in vitro and in vivo. Infect Immun 11:1069–1078

b12325337-0004ztc.indd 84 10/3/2008 10:50:42 AM


Krakowka S, Axthelm MK, Johnsen GC (1985) Canine distemper virus. In: Olsen RG, Krakowka S, Blakeslee JR (eds) Comparative pathobiology of viral diseases. Boca Raton, FL, CRC, pp 137–164

Krakowka S, Ringler SS, Lewis M, Olsen RG, Axthelm MK (1987) Immunosuppression by canine distemper virus: modulation of in vitro immunoglobulin synthesis, interleukin release and prostaglandin E2 production. Vet Immunol Immunopathol 15:181–201

Loeffler IK, Howard J, Montali RJ, Hayek LA, Dubovi E, Zhang Z, Yan Q, Guo W, Wildt DE (2007) Serosurvey of ex situ giant pandas ( Ailuropoda melanoleuca ) and red pandas ( Ailurus fulgens ) in China with implications for species conservation. J Zoo Wildl Med 38:559–566

Maher JA, DeStefano J (2004) The ferret: an animal model to study influenza virus. Lab Anim (NY) 33:50–53

McCullough B, Krakowka S, Koestner A (1974) Experimental canine distemper virus-induced lymphoid depletion. Am J Pathol 74:155–170

McKenna MC, Bell SK (1997) Classification of mammals above the species level. Columbia University Press, New York

McQuaid S, Cosby SL, Koffi K, Honde M, Kirk J, Lucas SB (1998) Distribution of measles virus in the central nervous system of HIV-seropositive children. Acta Neuropathol 96:637–642

Mehta PD, Thormar H (1979) Immunological studies of subacute measles encephalitis in ferrets: similarities to human subacute sclerosing panencephalitis. J Clin Microbiol 9:601–604

Moody KD, Bowman TA, Lang CM (1985) Laboratory management of the ferret for biomedical research. Lab Anim Sci 35:272–279

Morris JA, Coburn DR, O’Connor JR (1954) Rapid protection of ferrets against fully virulent dis-temper virus with nebulized attenuated distemper virus. Cornell Vet 44:198–207


Nagano I, Nakamura S, Yoshioka M, Kogure K (1991) Immunocytochemical analysis of the cellu-lar infiltrate in brain lesions in subacute sclerosing panencephalitis. Neurology 41:1639–1642

Ochi A, Danesh A, Seneviratne C, Banner D, Devries ME, Rowe T, Xu L, Ran L, Czub M, Bosinger SE et al (2008) Cloning, expression and immunoassay detection of ferret IFN-gamma. Dev Comp Immunol 32:890–897

Ohno S, Ono N, Takeda M, Takeuchi K, Yanagi Y (2004) Dissection of measles virus V protein in relation to its ability to block alpha/beta interferon signal transduction. J Gen Virol 85:2991–2999

Osterhaus AD, Fouchier RA, Kuiken T (2004) The aetiology of SARS: Koch’s postulates fulfilled. Philos Trans R Soc Lond B Biol Sci 359:1081–1082

Palosaari H, Parisien JP, Rodriguez JJ, Ulane CM, Horvath CM (2003) STAT protein interference and suppression of cytokine signal transduction by measles virus V protein. J Virol 77:7635–7644

Pardo MC, Bauman JE, Mackowiak M (1997) Protection of dogs against canine distemper by vaccination with a canarypox virus recombinant expressing canine distemper virus fusion and hemagglutinin glycoproteins. Am J Vet Res 58:833–836

Pardo MC, Tanner P, Bauman J, Silver K, Fischer L (2007) Immunization of puppies in the pres-ence of maternally derived antibodies against canine distemper virus. J Comp Pathol 137 Suppl 1:S72–S75

Pearson GL (1977) Vaccine-induced canine distemper virus in black-footed ferrets. J Am Vet Med Assoc 170:103, 106, 109

Pollock RV, Carmichael LE (1982) Maternally derived immunity to canine parvovirus infection: transfer, decline, and interference with vaccination. J Am Vet Med Assoc 180:37–42

Qin Q, Wei F, Li M, Dubovi EJ, Loeffler IK (2007) Serosurvey of infectious disease agents of carnivores in captive red pandas ( Ailurus fulgens ) in China. J Zoo Wildl Med 38:42–50

Quesenberry KE (1996) Basic approach to veterinary care. In: Hillyer EV, Quesenberry KE (eds) Ferrets, rabbits, and rodents: clinical medicine and surgery. W.B. Saunders, Philadelphia, pp 14–25

Rockborn G, Norrby E, Lannek N (1965) Comparison between the immunizing effect in dogs and ferrets of living distemper vaccines, attenuated in dog tissue cultures and embryonated eggs. Res Vet Sci 6:423–427

b12325337-0004ztc.indd 85 10/3/2008 10:50:42 AM

86 S. Pillet et al.

Rudd PA, Cattaneo R, von Messling V (2006) Canine distemper virus uses both the anterograde and the hematogenous pathway for neuroinvasion. J Virol 80:9361–9370

Schluederberg A, Lamm SH, Landrigan PJ, Black FL (1973) Measles immunity in children vac-cinated before one year of age. Am J Epidemiol 97:402–409

Schneider-Schaulies J, Niewiesk S, Schneider-Schaulies S, ter Meulen V (1999) Measles virus in the CNS: the role of viral and host factors for the establishment and maintenance of a persistent infection. J Neurovirol 5:613–622

Schobesberger M, Summerfield A, Doherr MG, Zurbriggen A, Griot C (2005) Canine distemper virus-induced depletion of uninfected lymphocytes is associated with apoptosis. Vet Immunol Immunopathol 104:33–44

Senchak AJ, Sato AK, Vazquez R, Keller CE, Cable BB (2007) Characterization of transforming growth factors beta1 and 2 in ferrets ( Mustela putorius furo ) Comp Med 57:594–596

Shen DT, Gorham JR, Evermann JF, McKeirnan AJ (1984) Comparison of subcutaneous and intramuscular administration of a live attenuated distemper virus vaccine in ferrets. Vet Rec 114:42–43

Sips GJ, Chesik D, Glazenburg L, Wilschut J, De Keyser J, Wilczak N (2007) Involvement of morbilliviruses in the pathogenesis of demyelinating disease. Rev Med Virol 17:223–244

Stephensen CB, Welter J, Thaker SR, Taylor J, Tartaglia J, Paoletti E (1997) Canine distemper virus (CDV) infection of ferrets as a model for testing Morbillivirus vaccine strategies: NYVAC- and ALVAC-based CDV recombinants protect against symptomatic infection. J Virol 71:1506–1513

Strating A (1975) Measles vaccine in dogs: efficacy against aerosol challenge with virulent canine distemper virus. J Am Vet Med Assoc 167:59–62

Summers BA, Greisen HA, Appel MJ (1979) Early events in canine distemper demyelinating encephalomyelitis. Acta Neuropathol 46:1–10

Summers BA, Greisen HA, Appel MJ (1984) Canine distemper encephalomyelitis: variation with virus strain. J Comp Pathol 94:65–75

Svitek N, von Messling V (2007) Early cytokine mRNA expression profiles predict Morbillivirus disease outcome in ferrets. Virology 362:404–410

Tetteh JK, Addae MM, Ishiwada N, Yempewu SM, Yamaguchi S, Ofori-Adjei D, Kamiya H, Komada Y, Akanmori BD (2003) Plasma levels of Th1 and Th2 cytokines in Ghanaian chil-dren with vaccine-modified measles. Eur Cytokine Netw 14:109–113

Thormar H, Mehta PD, Lin FH, Brown HR, Wisniewski HM (1983) Presence of oligoclonal immunoglobulin G bands and lack of matrix protein antibodies in cerebrospinal fluids and sera of ferrets with measles virus encephalitis. Infect Immun 41:1205–1211

Thormar H, Mehta PD, Barshatzky MR, Brown HR (1985) Measles virus encephalitis in ferrets as a model for subacute sclerosing panencephalitis. Lab Anim Sci 35:229–232

Thormar H, Brown HR, Goller NL, Barshatzky MR, Wisniewski HM (1988) Transmission of measles virus encephalitis to ferrets by intracardiac inoculation of a cell-associated SSPE virus strain. APMIS 96:1125–1128

van Moll P, Alldinger S, Baumgartner W, Adami M (1995) Distemper in wild carnivores: an epidemiological, histological and immunocytochemical study. Vet Microbiol 44:193–199

Vandevelde M, Zurbriggen A (2005) Demyelination in canine distemper virus infection: a review. Acta Neuropathol 109:56–68

Vandevelde M, Kristensen F, Kristensen B, Steck AJ, Kihm U (1982) Immunological and patho-logical findings in demyelinating encephalitis associated with canine distemper virus infec-tion. Acta Neuropathol 56:1–8

Vandevelde M, Zurbriggen A, Steck A, Bichsel P (1986) Studies on the intrathecal humoral immune response in canine distemper encephalitis. J Neuroimmunol 11:41–51

von Messling V, Springfeld C, Devaux P, Cattaneo R (2003) A ferret model of canine distemper virus virulence and immunosuppression. J Virol 77:12579–12591

von Messling V, Milosevic D, Cattaneo R (2004) Tropism illuminated: lymphocyte-based path-ways blazed by lethal morbillivirus through the host immune system. Proc Natl Acad Sci U S A 101:14216–14221

b12325337-0004ztc.indd 86 10/3/2008 10:50:42 AM


von Messling V, Svitek N, Cattaneo R (2006) Receptor (SLAM [CD150]) recognition and the V protein sustain swift lymphocyte-based invasion of mucosal tissue and lymphatic organs by a morbillivirus. J Virol 80:6084–6092

Welter J, Taylor J, Tartaglia J, Paoletti E, Stephensen CB (1999) Mucosal vaccination with recom-binant poxvirus vaccines protects ferrets against symptomatic CDV infection. Vaccine 17:308–318

Welter J, Taylor J, Tartaglia J, Paoletti E, Stephensen CB (2000) Vaccination against canine dis-temper virus infection in infant ferrets with and without maternal antibody protection, using recombinant attenuated poxvirus vaccines. J Virol 74:6358–6367

Wilkins J, Wehrle PF (1979) Additional evidence against measles vaccine administration to infants less than 12 months of age: altered immune response following active/passive immuni-zation. J Pediatr 94:865–869

Williams ES, Anderson SL, Cavender J, Lynn C, List K, Hearn C, Appel MJ (1996) Vaccination of black-footed ferret ( Mustela nigripes ) x Siberian polecat ( M. eversmanni ) hybrids and domestic ferrets ( M. putorius furo )against canine distemper. J Wildl Dis 32:417–423

Wimsatt J, Jay MT, Innes KE, Jessen M, Collins JK (2001) Serologic evaluation, efficacy, and safety of a commercial modified-live canine distemper vaccine in domestic ferrets. Am J Vet Res 62:736–740

Wimsatt J, Biggins D, Innes K, Taylor B, Garell D (2003) Evaluation of oral and subcutaneous delivery of an experimental canarypox recombinant canine distemper vaccine in the Siberian polecat ( Mustela eversmanni ) J Zoo Wildl Med 34:25–35

Zilliox MJ, Moss WJ, Griffin DE (2007) Gene expression changes in peripheral blood mononu-clear cells during measles virus infection. Clin Vaccine Immunol 14:918–923

b12325337-0004ztc.indd 87 10/3/2008 10:50:42 AM

Chapter 5 Current Animal Models: Cotton Rat Animal Model

Cotton Rat

S. Niewiesk

S. Niewiesk College of Veterinary Medicine, Department of Veterinary Biosciences , The Ohio State University , 1925 Coffey Road , Columbus , OH 43210 , USA, e-mail: [email protected]

Abstract The cotton rat ( Sigmodon hispidus ) model has proven to be a suitable small animal model for measles virus pathogenesis to fill the niche between tissue culture and studies in macaques. Similar to mice, inbred cotton rats are available in a microbiologically defined quality with an ever-increasing arsenal of reagents and

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91Reagents and Methods for the Analysis of Infectious Diseases in Cotton Rats . . . . . . . . . . . 91

Growth Behavior of Measles Wild Type, Vaccine, and Recombinant Viruses in Cotton Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91Viral Replication and Spread of Measles Virus in Cotton Rats . . . . . . . . . . . . . . . . . . . . . . 93Role of Cytoplasmic Tails of MV Glycoproteins in Cell-to-Cell Transfer . . . . . . . . . . . . . 94Role of Measles Virus V Protein for Virus Replication. . . . . . . . . . . . . . . . . . . . . . . . . . . . 94Interaction of Heat Shock Protein 72 with Measles Virus Nucleocapsid . . . . . . . . . . . . . . 95Vector Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

Measles Virus-Induced Immune Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96MV Inhibits T Cell Rather than B Cell Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96MV Suppresses the CD8 T Cell Response In Vivo Using the DTH Model of Epicutaneous DNFB Application. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97MV Glycoproteins Are Sufficient and Necessary to Suppress Proliferation. . . . . . . . . . . . . . 98MV Induces Cell Cycle Retardation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Testing of Antiviral Substances and Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99Evaluating Antivirals in Cotton Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100Immune Response Against Measles Virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100Testing of Measles Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100Inhibition of Seroconversion in the Presence of Maternal Antibodies . . . . . . . . . . . . . . . . 102Experimental Vector Systems Do Not Induce Protection in the Presence of MV-Specific (Maternal) Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . 104Failure of Experimental Vaccines to Immunize in the Presence of Maternal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Summary and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105


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90 S. Niewiesk

methods available for the study of infectious diseases. Cotton rats replicate measles virus in the respiratory tract and (depending on virus strain) in lymphoid organs. They can be infected with vaccine, wild-type, and recombinant measles viruses and have been used to study viruses with genetic modifications. Other areas of study include efficacy testing of antivirals and vaccines. The cotton rat also has been an informative animal model to investigate measles virus-induced immune suppres-sion and suppression of vaccination by maternal antibodies. In addition, the cotton rat promises to be a useful model for the study of polymicrobial disease (interaction between measles virus and secondary pathogens).

Introduction

The value of the cotton rat ( Sigmodon hispidus ) for the investigation of measles vaccination and pathogenesis is its natural susceptibility toward measles virus (MV) infection after intranasal inoculation. Virus replicates mostly in the respira-tory tract, causing interstitial pneumonia without overt clinical signs of disease. The cotton rat is semipermissive for measles virus infection, which means that the amount of virus obtained from the animal is proportionate to the amount of virus inoculated. In contrast, in a fully permissive animal, only viral kinetics and not the level of virus replication are determined by the viral inoculum. The first report of the use of cotton rats as laboratory animals stems from 1939 when it was found that poliovirus induced paralysis in cotton rats (Armstrong 1939). Since then, it has been shown that cotton rats can be infected with a variety of human viral, bacterial, and parasitic pathogens: human immunodeficiency virus (Rytik et al. 1995; Langley et al. 1998); herpes simplex virus type 1 (Lewandowski et al. 2002); herpes simplex virus type 2 (Yim et al. 2005); adenovirus (Pacini et al. 1984); measles virus (Wyde et al. 1992, 1999); respiratory syncytial virus (Dreizin et al. 1971; Murphy et al. 1990); human metapneumovirus (Wyde et al. 2005; Steinbach and Duca 1940; Williams et al. 2005; Hamelin et al. 2005); parainfluenza virus (Murphy et al. 1981); influenza A virus (Brydak 1990; Ottolini et al. 2003); Helicobacter pylori (Mähler et al. 2002); Staphylococcus aureus (Weidenmaier et al. 2004); Mycobacterium tuberculosis (Elwood et al. 2007); Borrelia burgdorferi (Burgdorfer and Gage 1987; Oliver et al. 1995); Francisella tularensis (Lowery 1981); Toxoplasma gondii (Clark 1984); Leishmania donovani (Azazy et al. 1994); and Echinococcus multilocularis (Kroeze and Tanner 1985); Dipetalonema vita e (Bayer and Wenk 1988). The high susceptibility to productive infection might be linked to similarities between cotton rats and humans in their antipathogen response, which differs from mice (Mestas and Hughes 2004). Examples of these similarities are the low production of nitric oxide by macrophages (Carsillo et al. 2008) and the presence of func-tional Mx proteins in cotton rats (Stertz et al. 2007). The additional value of the cotton rat is its well-defined genetics and an increasing pool of reagents, thus providing a small animal model for measles virus research (for review Niewiesk and Prince 2002). The cotton rat has been used as a pathogenesis model, a tool

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5 Current Animal Models: Cotton Rat Animal Model 91

to investigate the effect of modifications in measles virus on in vivo replication and a model for screening purposes to test antivirals and vaccines.

Genetics

Cotton rats are new world rodents that have their natural habitat in the south of the United States of America, Central America, and northern regions of South America. In contrast to the more often used members of the Muridae family, mice ( Mus musculus ) and rats ( Rattus norwegicus ), cotton rats belong to the Sigmodontinae family. Originally, wild hispid cotton rats were captured to set up an outbred colony at the National Institutes of Health (NIH), Bethesda, MD, USA, Veterinary Resources Program. By caesarean section and weaning onto rat ( Rattus norvegicus ) foster mothers, a specific pathogen-free colony was reder-ived. By serial brother–sister mating, an inbred strain was established (SIG/N). Offspring from these animals are marketed worldwide by various commercial breeders in a microbiologically defined quality. Currently, some 300 genes of cotton rats have been sequenced, and sequence comparisons demonstrate a 75%–95% homology to mice and rats and approximately 50% homology to humans.

Reagents and Methods for the Analysis of Infectious Diseases in Cotton Rats

The use of cotton rats has been supported by the availability of inbred animals and the development of reagents and methods for the analysis of immune responses against infectious diseases in cotton rats. These include standard immunological methods for antibody detection such as ELISA and neutralization assays, and methods for T cell analysis such as T cell proliferation and cytotoxic T cell assays. In addition, antibodies against cell surface markers have been produced and detec-tion systems for various chemokines and cytokines have been established (for details see Tables 5.1 and 5.2 ). For cotton rats, permanent cell lines are available and tissue culture methods for primary cell culture have been established (Niewiesk and Prince 2002; Blanco et al. 2004).

Growth Behavior of Measles Wild Type, Vaccine, and Recombinant Viruses in Cotton Rats

The kinetics and extent of measles virus replication has been defined for vaccine and wild-type viruses. Based on this knowledge, recombinant viruses with specific mutations have been used to address the question of tropism and to compare in vitro with in vivo consequences of these mutants.

92 S. Niewiesk

Chemokine/cytokine Gene sequence Assay system Reference

Interferon-alpha AF421386 ELISA, PCR Blanco et al. 2002Interferon-gamma AF167349 ELISA, PCR Blanco et al. 2002Tumor necrosis factor alpha AF421388 ELISA, PCR Blanco et al. 2002Interleukin-1-alpha AF398548 ELISA -Interleukin-1-beta AF421387 ELISA, PCR Blanco et al. 2002Interleukin-2 AF398549 ELISA, PCR Boukhvalova et al. 2006Interleukin-4 AF421390 ELISA, PCR Ottolini et al. 2005Interleukin-5 AF148211 PCR Boukhvalova et al. 2006Interleukin-6 AF421389 ELISA, PCR Blanco et al. 2002Interleukin-9 AY327895 PCR Boukhvalova et al. 2006Interleukin-10 AF398550 ELISA, PCR Blanco et al. 2002Interleukin-12 p35 AF421396 Real-time PCR Carsillo et al. 2008Interleukin-12/23 p40 AF421395 PCR Ottolini et al. 2005Interleukin-13 AY327896 PCR Hassantoufighi et al. 2007Interleukin-18 AY059406 Real-time PCR Carsillo et al. 2008CCL2/MCP-1 AY165953 ELISA, PCR Boukhvalova et al. 2006CCL3/MIP1-alpha AY059407 ELISA CCL4/MIP1-beta AF421392 NT, WB, PCR Blanco et al. 2002CCL5/RANTES AF421391 ELISA, PCR Blanco et al. 2002MIP2 AY059408 PCR CXCL10/IP-10/CRG-2 AF421394 ELISA, PCR Blanco et al. 2002Growth-regulated protein AF421393 ELISA, PCR Blanco et al. 2002GM-CSF AY065645 TGF beta AF480858 PCR Carsillo et al. 2008Lymphotactin DQ136045 PCR Boukhvalova et al. 2006

Published gene sequences and detection systems are listed for cotton rat chemokines and cytokines. Antibody-based detection systems are commercially available from R&D Systems

Target molecule Antibody Reference

CD3 Polyclonal Elwood et al. 2007CD4 mAB Richter et al. 2005CD8alpha mAB Streif et al. 2004CD18 Polyclonal Carsillo et al. 2008CD57 mAB (mouse, clone NK1.1) Eichelberger et al. 2006CD79a mAB (human, clone HM57) Elwood et al. 2007MHC I mAB (human, clone W6/32) Carsillo et al. 2008MHC II mAB (mouse, clone 13/4) Pueschel et al. 2007CD68 mAB (rat, clone ED1) Schachtner et al. 1995IgA mAB Schlereth et al. 2003IgG Polyclonal Schlereth et al. 2003Mx 1 and 2 mAB (human, clone M143) Pletneva et al. 2008

Various published antibodies specific for cotton rat antigens are listed in the table. If the antibodies are cross-reactive with cotton rats, the original species and the clone name are given in brackets. mAB monoclonal antibody

Table 5.1 Antibodies for the analysis of immune responses in cotton rats

Table 5.2 Chemokine/cytokine detection systems in the cotton rat model


Viral Replication and Spread of Measles Virus in Cotton Rats

Philip Wyde made the seminal observation that cotton rats can be infected with both vaccine and wild-type measles virus without adaptation (Wyde et al. 1992, 1999). In subsequent studies, it was determined that recombinant MV viruses also repli-cate well in cotton rat lung tissue (Tober et al. 1998; Moll et al. 2004). After infec-tion, vaccine viruses replicate in the upper and lower respiratory tract but rarely are found in the lung-draining lymph node (Pfeuffer et al. 2003). In contrast, wild-type viruses spread to the mediastinal lymph nodes and (depending on viral strain) also to the spleen (Pfeuffer et al. 2003; Wyde et al. 1999). In tissue culture, tracheal epi-thelial cells can be infected, and in cotton rats MV infection was detected in lung epithelial cells (Moll et al. 2004). In addition, macrophages isolated by bronchoal-veolar lavage from intranasally infected animals carried infectious virus (Wyde et al. 1999; Pfeuffer et al. 2003). After intraperitoneal inoculation of virus, infected macrophages could be obtained by lavage from the peritoneal cavity (Wyde et al. 1999). When virus was injected intramuscularly into the limb muscle of cotton rats, virus replication was detected in inguinal lymph nodes by GFP-expressing recom-binant MV (Haga et al. 2008). Currently, the most likely carrier of virus seems to be the macrophage. However, dendritic cells are another potential candidate as car-rier cells (de Swart et al. 2007) and need to be analyzed. An interesting finding is the fact that peripheral blood lymphocytes are positive for MV RNA by PCR, but no infectious virus could be obtained by co-cultivation (Niewiesk et al. 1997). The differences in virus spread between vaccine and wild-type strains are independent of the strain of virus used (Pfeuffer et al. 2003). This might be related to differences in receptor usage between vaccine and wild-type measles viruses (see the chapters by Y. Yanagi et al. and C. Kemper and J.P. Atkinson, This Volume). Whereas wild-type viruses use human CD150, vaccine viruses are able to use both human CD46 and CD150 as receptor molecules. CD150 is expressed on activated lymphoid cells. Since vaccine viruses were developed by passage on fibroblast cell lines (no expres-sion of CD150 but expression of CD46), they are thought to have mutated their receptor usage in adaptation to the fibroblast as growth substrate. Interestingly, MV vaccine virus also replicates in CCRT, a cotton rat osteosarcoma cell line, whereas wild-type virus does not (S. Niewiesk, unpublished data). To investigate the rele-vance of receptor usage in cotton rats, recombinant viruses (on the vaccine strain Edmonston backbone) expressing the receptor-binding protein hemagglutinin (H) from wild-type virus WTF (uses human CD150) or vaccine strain Edmonston (uses both human CD150 and CD46) were generated. In cotton rats, recombinant viruses expressing the hemagglutinin (H) of wild-type strain WTF spread to mediastinal lymph nodes (MDLN) in similar fashion to wild-type strain WTF (Pfeuffer et al. 2003). In contrast, viruses expressing Edmonston-H did not spread to MDLN. These results were independent of the fusion (F) protein expressed by the respective virus. If a point mutation at amino acid 481 [asparagine (N) to tyrosine (Y)] was introduced in the H of WTF, the receptor usage switched to both human CD46 and CD150 (like vaccine virus) and virus replication was restricted to the respiratory

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tract. These data indicate that measles virus also uses two receptors in cotton rats that presumably are homologous to the human molecules.

Role of Cytoplasmic Tails of MV Glycoproteins in Cell-to-Cell Transfer

In tissue culture, single tyrosine residues in the cytoplasmic tail of the measles virus glycoproteins H and F are critical for basolateral targeting of these proteins in polarized epithelial cells (Moll et al. 2001). After infection with virus mutants in which the basal lateral targeting signal of either one or both glycoproteins was destroyed due to substitutions of the tyrosine residues, H and F were expressed predominantly on the apical membrane of polarized Madin-Darby canine kidney cells. In contrast to parental measles virus, none of these virus mutants was able to spread by syncytia formation in polarized cells demonstrating that the presence of both glycoproteins at the basolateral cell surface is required for cell-to-cell fusion in vitro (Moll et al. 2004). To test the pathogenicity of these mutants in vivo, cotton rats were infected intranasally. After 4 days, virus titers in lung lav-age cells and lung tissue of infected cotton rats were clearly lower than those found in animals infected with the parental virus. Thus, measles virus mutants encoding glycoproteins with altered basolateral targeting signals clearly showed reduced replication in the cotton rat respiratory tract. When animals infected with mutant virus were compared with animals infected with the parental virus by his-tology, in animals infected with the parental virus, small foci of infected epithelial cells were found in the nose and lung. In contrast, in animals infected with mutant virus, only individually infected epithelial cells were found in nose and lung. These data indicate that basolateral expression of MV glycoproteins is required for cell-to-cell spread in vivo.

Role of Measles Virus V Protein for Virus Replication

The cotton rat model also has been used to evaluate the importance of MV V pro-tein for replication in vivo. The V protein is translated from an edited P protein mRNA. V protein is not associated with intracellular or released viral particles. Recombinant MV strains genetically engineered to be deficient for V protein (ED-V − ) or to overexpress V protein (ED-V + ) do not replicate as well as the parental strain (ED-tag) in tissue culture (Tober et al. 1998). In the absence of V protein, both measles virus-specific protein and RNA accumulated to levels higher than those in the parental measles virus derived from the molecular clone. Furthermore, infection resulted in similar viral titers 24 h after tissue culture infection and slightly reduced titers (0.5 log10) after 48 h. After overexpression of the V pro-tein, measles virus gene expression, as well as viral titers, were strongly reduced

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in tissue culture (reduction of 1–1.5 log10). In cotton rats, ED-V - replication was reduced by 1 log

10 TCID

50 in lung tissue days 4 and 5 after infection (Tober et al.

1998). By comparison, ED-V + replication was reduced by 2.5 log 10

TCID 50

in lung tissue on day 4 and no virus was found on day 5. This study indicated that replica-tion characteristics of measles virus in tissue culture are recapitulated (and even more pronounced) in cotton rats thus making it an excellent model to study ques-tions of how viral replication is controlled in vivo.

Interaction of Heat Shock Protein 72 with Measles Virus Nucleocapsid

Heat shock protein 72 is a cellular factor that increases transcription of measles vaccine virus. This is achieved by complex formation with the nucleocapsid (N) of MV vaccine virus. The carboxy-terminus of N (N-tail) has two contact sites for HSP 72, the low-affinity binding site Box 2 and the high-affinity binding site Box 3. In contrast, wild-type viruses in their overwhelming majority interact with HSP72 through Box 2 only and do not change replication characteristics in response to heat shock. To test the relative merit of one versus two contact sites, the molecular MV Edmonston clone (which has two contact sites) was mutated at amino acid position 522 from asparagine (N) to aspartic acid (D). This change abolishes the HSP72 interaction with Box 3. When both viruses were compared in vitro they replicated to comparable titers. After infection of cotton rats, no difference in viral titers was detected after 5 days. Since the N522D mutation occurs in nearly all wild-type viruses, it was hypothesized that this mutant should be able to outcompete the virus derived from the vaccine-based parental clone. However, after co-infection of cot-ton rats with both viruses, the parental clone Edmonston clearly had a growth advantage over N522D. This in vivo experiment indicated that the difference in interaction with HSP72 between wild-type and vaccine viruses is not simply dependent on one amino acid change but might depend on the overall protein context.

Vector Development

For the studies discussed so far, the molecular clone of the Edmonston B strain (ED-tag) (and derivatives thereof) that was produced by Martin Billeter’s group was used (Radecke et al. 1995). Based on the availability of molecularly cloned measles virus, it has been proposed to use recombinant measles viruses expressing addi-tional genes either as vaccines (see the chapter by M.A. Billeter et al., This volume) and tools for oncolytic cancer therapy (see the chapter by S.J. Russell and K.W. Peng, this volume). For this purpose, a molecular clone of the temperature sensitive AIK-C virus, a vaccine derivative from Edmonston strain, was produced. When

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cotton rats were infected with this virus, it did not replicate in lung tissue but induced an immune response after intramuscular inoculation (Haga et al. 2008). This would indicate that AIK-C could be used as a very safe and immunogenic vector system. Other established molecular clones of measles virus include one of the Schwarz vaccine strains (Combredet et al. 2003) and the IBC wild-type strain (Takeda et al. 2000). Both molecularly cloned viruses have not been tested in cotton rats but the parental viruses replicated well in cotton rats (Pfeuffer et al. 2003; S. Niewiesk, unpublished data).

Measles Virus Induced Immune Suppression

Children with acute measles often contract secondary infections that can be lethal. The increased susceptibility to opportunistic infections has been thought to be due to severe suppression of the immune system by measles virus infection itself (see the chapter by S. Schneider-Schaulies and J. Schneider-Schaulies, this volume). In contrast to infection with the wild-type virus, vaccination with the live-attenu-ated vaccine at the current dosage does not lead to clinical symptoms of immune suppression. However, the use of high titer vaccines (10 5 pfu) has been correlated with increased mortality, presumably related to immune suppression, which led to discontinuation of this immunization regimen (Aaby et al. 1988; Halsey 1993). Laboratory-based methods to determine immune suppression have included the measurement of the suppression of antigen-specific T cell proliferation (which measures CD4 T cells) and of mitogen-stimulated B cell and T cell responses. In tissue culture, the suppressive effect of measles virus on proliferation of periph-eral blood lymphocytes could be demonstrated by infection with live virus or incubation with UV-inactivated virus. In the cotton rat model, it also was found that measles virus induces immune suppression. After infection, suppression of proliferation of keyhole limpet hemocyanin (KLH)-specific T cells and mitogen-stimulated (Concanavalin A; ConA) proliferation of spleen cells was detected ex vivo (Niewiesk et al. 1997, 2000). Vaccine and wild-type viruses differ in their suppressive effect. Whereas wild-type virus suppresses immune responses at all titers tested (Pfeuffer et al. 2003), only high titers of vaccine virus (≥10 6 pfu) are suppressive (Niewiesk et al. 1997). After inoculation with 10 5 TCID

50 wild-type

virus (strain WTF), the ConA-stimulated proliferation of cotton rat spleen cells was suppressed for up to 20 days, whereas inoculation with 10 6 pfu vaccine strain (strain Edmonston B) suppressed proliferation for up to 10 days.

MV Inhibits T Cell Rather than B Cell Responses

In order to investigate whether B cells or T cells were inhibited by MV infection, spleen cells from infected animals were stimulated with B cell and/or T cell mitogens. Ex vivo, both B cell and T cell proliferation were inhibited by MV

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(Niewiesk et al. 2000). This is consistent with findings in patients where infection with wild-type measles virus leads to reduction of B cell and T cell stimulation by mitogens ex vivo. To further investigate how the antigen-specific B cell and T cell response are affected by MV infection, cotton rats were injected with various anti-gens and immune responses measured ex vivo. After application of keyhole limpet hemocyanin coupled to dinitrophenyl groups (KLH-DNP), the antibody response measured from serum was impaired in infected animals after 2 weeks but was back to normal 1 week later (Niewiesk et al. 2000). During this time, there was no sig-nificant difference in the number of antigen-specific B cells (measured by Elispot) secreting either IgM or IgG specific for KLH-DNP so that the overall effect of MV infection on the B cell response was small. In contrast, T cell responses were severely affected. Antigen-specific T cell proliferation was reduced by MV infec-tion in a mixed lymphocyte reaction after primary and secondary stimulation and in a KLH-specific proliferation assay. Similar to proliferation of T helper cells, the number of cytotoxic vaccinia virus-specific T cells generated ex vivo was markedly reduced in spleen cells from MV-infected animals. However, on a per cell basis, cytotoxicity was not reduced. Overall, the proliferation of T cells was severely inhibited, whereas B cell help and cytotoxicity did not seem to be affected. With only semiquantitative bioassays available at the time, the reduced secretion of IL2 and interferon gamma by antigen-specific T cells was not statistically significant. The difference in suppression of the B cell and T cell response measured in vivo and ex vivo might be due to differences in activation of these cells. Ex vivo, both cell populations were driven to proliferate for 3 days by mitogen. In vivo, the gen-eration of antigen-specific T cell responses also occurred within a few days in a so-called clonal burst (Hou et al. 1996); immune suppression resulted in a reduced expansion and subsequently lower numbers of memory T cells. In contrast, B cells proliferated in vivo more slowly over weeks and therefore might have been less susceptible. In contrast, T cells might not only be affected by the above-mentioned cell cycle arrest, but also by a defect in antigen presentation.

MV Suppresses the CD8 T Cell Response In Vivo Using the DTH Model of Epicutaneous DNFB Application

In order to test the suppression of both CD4 and CD8 T cells directly in vivo by MV, a delayed-type hypersensitivity model of epicutaneous sensitization (ECS) by 2,4 dinitrofluorobenzene (DNFB) to the ear was used (Niewiesk et al. 2000). After application of DNFB, dendritic cells (DCs) in the skin take up the antigen and transport it to draining mandibular lymph nodes to stimulate T cells (Bour et al. 1995). These lymph node cells can be analyzed by flow cytometry and will incor-porate 3 H-thymidine after overnight culture, thus giving a measure of induction and proliferation of T cells. After DNFB application to the ear of cotton rats, the number of lymphocytes was markedly increased (fivefold). The ratio of CD4 T cells to CD8 T cells changed from 1.5:1 in nonactivated lymph nodes to 1:1.7 in activated lymph nodes, indicating a strong stimulation of CD8 T cells (similar to

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98 S. Niewiesk

the mouse model; Bour et al. 1995). T cell proliferation of lymph node cells from MV-infected animals was strongly reduced in comparison to control animals (Streif et al. 2004), whereas the CD4/CD8 ratio remained unchanged (as seen in humans).

If DNFB is applied to the flank of mice, ear painting 10 days later will result in ear swelling due to the recall CD8 T cell response. In mice, CD8 cytotoxic T cells are the effector cells in ear swelling (Bour et al. 1995), whereas CD4 T cells can (down)regulate the activity of CD8 T cells (Dubois et al. 2003). To investigate which T cell subset is the effector subset in cotton rats, monoclonal antibodies specific for cotton rat CD4 and CD8 were used. When CD8 T cells were depleted, a marked reduction in ear swelling was observed, indicating that CD8 T cells are the main effector T cells in this model (Streif et al. 2004). In contrast, the deple-tion of CD4 T cells did not significantly influence ear swelling. These data suggest that in cotton rats CD4 T cells do not regulate the DNFB response and that the reduction in proliferation of DNFB-specific CD8 T cells is not due to regulatory CD4 T cells.

MV Glycoproteins are Sufficient and Necessary to Suppress Proliferation

In vitro, a cotton rat T cell line infected with either vaccine or wild-type virus was UV-inactivated and tested for expression of equal amounts of MV glycopro-teins. After contact with these cells, mitogen-dependent proliferation of spleen cells from naive animals was reduced (Pfeuffer et al. 2003). However, in contrast to in vivo findings and similar to in vitro data with human cells, no difference was seen between vaccine and wild-type virus. The only viral proteins to contact spleen cells in this assay are the MV glycoproteins fusion protein (F) and hemag-glutinin (H). Therefore, it was investigated whether this contact alone is suffi-cient to suppress proliferation. 293T fibroblast cells transfected with both H and F suppressed proliferation of lymphocytes in vitro (Niewiesk et al. 1997). In addition, 293T cells transfected with both H and F suppressed proliferation of spleen cells 4 days after intraperitoneal injection. In contrast, a recombinant MV, where H and F were replaced by the G protein of vesicular stomatitis virus (MGV), did not suppress proliferation in cotton rats. In order to investigate the effect of the different glycoproteins in the context of measles virus in vivo, recombinant viruses (based on the molecular Edmonston clone) expressing either alone or in combination the H and the F of wild-type virus WTF and/or vaccine strain Edmonston were used. In cotton rats, recombinant viruses express-ing the WTF-H spread to mediastinal lymph nodes (MDLN) and induced prolif-eration inhibition like wild-type WTF (Pfeuffer et al. 2003). In contrast, viruses expressing Edmonston-H did not spread to MDLN and did not suppress immu-nity at titers of up to 10 5 pfu. These results were independent of the F protein expressed by the respective viruses. In summary, these data show that both H and F proteins are necessary and sufficient to suppress proliferation. In the context

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of the whole virus and in vivo, however, H seems to be more important in explaining differences seen between vaccine and wild-type virus. In aggregate with the results that implicate that the dual receptor usage by MV in human cells is reflected in cotton rats, these data suggest that receptor usage is important for viral spread and immune suppression.

MV Induces Cell Cycle Retardation

In vitro, suppression of lymphocyte proliferation has been investigated using live virus or inactivated virus. It was found that mechanisms such as apoptosis, fusion of cells, and unresponsiveness to IL-2 do not contribute to suppress proliferation (McChesney et al. 1987, 1988; Naniche et al. 1999; Schnorr et al. 1997) but that contact with MV is able to downregulate AKT, which results in cell cycle arrest (see the chapter by S. Schneider-Schaulies and J. Schneider-Schaulies, this vol-ume). These results were confirmed in cotton rats in that only the retardation of cell cycle progression was found to be responsible for suppression (Niewiesk et al. 1999). Using fluorescent dyes (CFSE), it was found that all lymphocytes progress through the cell cycle, but preferentially gather in the G1/S phase. In agreement, the cell cycle-dependent kinases (CDK) 2 and 4, which propel pro-gression through the cell cycle, were reduced in MV-infected animals (S. Niewiesk, unpublished data). Furthermore, the activity of AKT was severely reduced (Avota et al. 2001). When the kinetics of AKT regulation and T cell pro-liferation were investigated in the DNFB model, it was found that the reduction of AKT activity preceded a reduction of DNFB-specific T cell proliferation by 4 days, which is consistent with the notion that a change in signal transduction is responsible for reduced proliferation.

These data indicate that the suppression of T cell proliferation is a major part of the immune suppression found after measles virus infection and that B cells are not affected. However, B cells and T cells are not the only effector cells of the immune system. Additional studies will be needed to investigate the effect on innate immune responses (see the chapter by B. Hahm, this volume) and the effect of infection on antigen-presenting cells like macrophages and dendritic cells and their role in T cell suppression.

Testing of Antiviral Substances and Vaccines

The safety and efficacy of antivirals and vaccines against measles virus can be tested in Rhesus macaques, which develop a disease very similar to acute measles in children (see the chapter by R.L. de Swart, this volume). However, it is difficult and costly to use these animals to determine a number of basic questions such as evaluation of routes of administration, drug and vaccine doses, schedules, and regi-mens. For this reason, the use of cotton rats as a screen can be useful for primary evaluation of vaccines and drugs.

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100 S. Niewiesk

Evaluating Antivirals in Cotton Rats

Early during the drug screening process, antiviral substances found in tissue culture must be tested in a small animal model. In an example of how cotton rats can be utilized for drug testing of anti-measles virus substances, three compounds shown to be active against measles virus were tested in vivo (Wyde et al. 2000a). These compounds were ribavirin, 5-ethyl-1-β-D-ribo-furanosylimidazole-4-carboamide (EICAR) and poly(acryl-amidomethyl propanesulfonate) (PAMPS). When the drugs were administered twice daily i.p. and virus titers analyzed 4 days after infec-tion, ribavarin (360 mg/kg/day) and EICAR (120 mg/kg/day) were shown to reduce virus titers by 1 log

10 TCID

50 /g lung tissue. In contrast, PAMPS inhibited virus

infection only when directly applied intranasally after virus infection. Later appli-cation of PAMPS was not effective, indicating that PAMPS acts extracellularly during viral binding and uptake. These data indicate that cotton rats may be a useful rodent model for testing antiviral substances against measles.

Immune Response Against Measles Virus

In cotton rats, measles virus infection is overcome within 10 days. The decline in virus titers correlates with the development of measles virus-specific CD4 T cells. The T cell response is first detectable on day 5, reaches its peak on days 7–8 and remains detectable for at least 3 months (Pueschel et al. 2007). CD4 T cells recog-nize two epitopes (amino acid 261–285 and 348–372) on the nucleocapsid protein, one epitope on the hemagglutinin (amino acid 553–567) and one on the fusion pro-tein (amino acid 347–372) (Pueschel et al. 2007). However, these CD4 T cells do not control viral replication since neither immunization with the hemagglutinin epitope nor depletion of CD4 T cells influences viral titers. In contrast to T cells, antibodies specific for measles develop later during infection. By ELISA, antibod-ies can be detected 6 days after infection and by neutralization assay antibodies can be found 12 days after infection (Niewiesk and Germann 2000). Although neutral-izing antibodies develop too late during primary infection to protect against chal-lenge, they protect against secondary infection. As in humans, passive transfer of MV neutralizing antibodies protects against infection (Schlereth et al. 2000b).

Testing of Measles Vaccines

Acute measles can be prevented by immunization of seronegative children with the live attenuated measles virus vaccine (see the chapter by D.E. Griffin and C.-H. Pan, this volume). Protection against the acute disease is correlated with the titer of neutralizing antibodies (Chen et al. 1990). Although CD8 and CD4 T cells are activated during the acute disease in humans (van Binnendijk et al. 1990)

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and CD8 T cells seem to play a role in clearance in the late phase of infection in monkeys (Permar et al. 2003, 2004), stimulation of the T cell response by experi-mental vaccine vectors only confers limited protection (Fennelly et al. 1995; Schlereth et al. 2000a). Furthermore, a case report describing persistent MV infection in T cell-deficient patients supports the notion that T cells are required to clear infection but do not provide protection (Nahmias et al. 1967). This is in agreement with data from animal models (cotton rat, rhesus macaque) (see Schlereth et al. 2003 and references therein), where the only immune correlate consistently linked to protection against measles virus infection in animal models is the titer of neutralizing antibodies.

To compare the efficacy of different routes of immunization with MV vaccine virus (Edmonston strain), cotton rats were immunized subcutaneously, intrana-sally, or intraperitoneally with different doses of virus. Similar to humans (Wong-Chew et al. 2004), immunization efficiency was twice as high after s.q. immunization than after i.n. immunization (studies in humans with aerosols show superior results to intranasal droplet immunization but have not been repeated in cotton rats). Immunization efficiency after i.p. immunization, which is not used in humans, was fourfold higher than after s.q. immunization (see Table 5. 3 ). As in humans, cotton rats were effectively protected against chal-lenge after vaccination. The only difference from humans is that for successful immunization of cotton rats, roughly tenfold more virus is necessary (10 5 pfu instead of 10 4 pfu s.q.).

Based on the principle that the induction of neutralizing antibodies is protec-tive, a number of alternate vaccine vectors have been produced that express the hemagglutinin and/or the fusion protein of measles virus, since these proteins are

Table 5.3 Measles virus immunization in the presence and absence of passively transferred (maternal) antibodies

Route of Dose of Amount of passively NT titer (±SD)immunization immunization (pfu) transferred antibodies (NT titer)

i.n. 10 4 0 <10 5 × 10 5 0 30 + 20 320 <10 2 × 10 7 320 <10s.q. 10 4 0 20 + 10 320 <10 5 × 10 5 0 60 + 20 320 <10i.p. 10 4 0 85 ± 60 320 <10 10 5 0 185 + 60 320 0

Cotton rats were immunized either intranasally (i.n.), subcutaneously (s.q.), or intraperitoneally (i.p.) with different doses of Edmonston vaccine virus in the absence or presence (1 mL antiserum of NT titer 320) of maternal antibodies. Neutralizing antibodies were determined 6 weeks after immunization.

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102 S. Niewiesk

the target of neutralizing antibodies. The advantage of the cotton rat model is that both markers of successful immunization (level of neutralizing antibodies as well as CD4 T cell responses) and markers of protection (level of neutralizing antibod-ies, reduction of viral titers after challenge infection and histological changes in lung tissue) can be measured and experiments can be conducted in group sizes large enough for statistical analysis in a relatively cost-efficient manner. For the measurement of neutralizing antibodies, the plaque reduction neutralization assay (PRNT) and the neutralization assay (NT) are utilized, which differ tenfold in titer (PRNT titer of 100 correlates with NT titer of 10). It is also easy to compare neu-tralizing antibody titers from animal experiments with studies in humans. In cot-ton rats, the titer of the virus inoculum determines whether it is possible to obtain sterile immunity (complete virus clearance) after immunization. If a low titer inoculum (≤10 4 pfu or TCID

50 ) is used, virus usually is cleared completely. If

higher inocula are used, it is not possible to achieve sterile immunity by day 5. A number of vector systems that have been shown to induce good immune

responses in other laboratory animals have been used in cotton rats expressing the H, F, and/or N protein. Basically, all vectors were successful in inducing protective immunity, with the exception of plasmids expressing the N protein. This particular immunization induced good T cell and non-neutralizing antibody responses but no protective neutralizing antibodies (Schlereth et al. 2000a). After repeated immuni-zation, all vector systems induced protective levels of neutralizing antibodies: plas-mid immunization (Schlereth et al. 2000a), Salmonella and Shigella -mediated plasmid transfer (Pasetti et al. 2003), modified virus Ankara (MVA) (Weidinger et al. 2001) and canarypox virus vectors (ALVAC) (Wyde et al. 2000b), adenovirus (Fooks et al. 1998), immune stimulating complexes (ISCOM) (Wyde et al. 2000b) and TLR-2 specific adjuvants with measles vaccine virus (Luhrman et al. 2005). Probably the most remarkable results were obtained with recombinant vesicular stomatitis virus, which induced high titers of neutralizing antibodies after a single intranasal immunization. The reasons for this efficient induction are not understood fully but it might be due to increased immunogenicity of the rigid highly repetitive envelope of VSV-H (see Schlereth et al. 2003 and references therein) or to the interaction and subsequent stimulation of immune responses by VSV with TLR-7 (Lund et al. 2004).

Inhibition of Seroconversion in the Presence of Maternal Antibodies

Although vaccination of seronegative children is hugely successful with the live-attenuated measles virus, vaccination of children with maternal antibodies is noto-riously difficult. Maternal antibodies protect children after birth but are metabolized over time. Eventually, low antibody titers no longer protect but still are able to interfere with immunization, which leads to the phenomenon that vaccination in the presence of maternal antibodies does not induce seroconversion. To test the

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inhibitory activity of maternal antibodies in young cotton rats, dams were immu-nized with measles virus before they gave birth and the level of MV-specific anti-bodies transmitted was measured in pups after birth (Schlereth et al. 2000b). Neutralizing antibodies declined with an approximate half-life of 7 days. After i.p. immunization with the MV vaccine strain, maternal antibodies inhibited vaccina-tion in cotton rats in the same way as in humans. However, the amount of maternal antibody transferred to individual pups varied (probably according to their pecking order). To better standardize the experimental system, human MV-specific antibod-ies were transferred into cotton rats (Schlereth et al. 2000b). By injection of various amounts of human MV-specific antibodies, it is possible to quantify the level of passively transferred MV-specific antibodies. After vaccination, passively trans-ferred human MV-specific antibodies can be distinguished from actively induced cotton rat antibodies using ELISA. In cotton rats, the half-life of human MV-spe-cific antibodies is reduced twofold in comparison to cotton rat antibodies, thus reducing the time required per experiment. Immunization 1 day after serum trans-fer with 10 5 pfu vaccine virus i.p. failed to induce neutralizing antibody responses or protection against intranasal challenge with MV. It also has been tested whether different immunization routes, doses, or repeated administration could induce neu-tralizing antibodies in the presence of MV-specific antibodies. The results from the

MV in the presence of MV-specific antibodies was never successful. In these studies, a human serum sample was used as a source of human MV-

specific antibodies that contained 16 IU/ml as measured by ELISA against a refer-ence serum and had a neutralization titer (NT) of 320. After injection of 1 ml of human serum (NT 320), antibodies declined with a half-life of roughly 3.5 days and neutralizing antibodies were detectable for 3 weeks (Schlereth et al. 2000b). Similar to humans, the presence of neutralizing antibodies was predictive of pro-tection against infection. In children, neutralization titers of 120 or more measured in a plaque reduction neutralization (PRNT) assay were shown to protect against reinfection (Chen et al. 1990). In cotton rats, the same titers were protective [if tenfold less sensitive neutralization (NT) assay was used, this correlates with titers ≥12]). In the presence of neutralizing antibodies after serum transfer, virus titers in lung tissue 5 days after challenge were reduced by 1.5–2 log

10 (Schlereth et al.

2000b). This level of reduction is comparable to that seen after s.q. immunization of cotton rats.

To evaluate how strongly B cell and T cell responses were affected and how much MV-specific (maternal) antibody was needed to suppress immune responses, cotton rats were inoculated with the equivalent of 1 ml of human MV-specific antiserum with neutralization titers of 320, 160, and 80, respectively (Pueschel et al. 2007). One day later, this transfer resulted in neutralization titers in serum of 60, 30, and 15/ml blood, respectively. At this point, cotton rats were immunized i.p. with 10 5 pfu MV vaccine virus. After 9 weeks, animals were challenged with MV intranasally and T cell proliferation, neutralizing antibodies and virus titers in lung tissue were measured. Immunization in the presence of high levels of maternal antibodies (NT 320 and NT 160) reduced (although did not abolish) the T cell

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various immunization regimens are summarized in Table 5.3. Immunization with

104 S. Niewiesk

response, whereas low levels had no effect. In contrast, the neutralizing antibody response was blocked completely after immunization in the presence of NT 320 and NT 160, and only a low level NT titer of 12.5 was generated after immunization in the presence of NT 80. The lack of NT antibody generation correlated with a lack of protection after viral challenge (after immunization in the presence of NT 320 and NT 160). Generation of a low NT response resulted in partial protection (after immunization in the presence of NT 80) (Pueschel et al. 2007).

These data demonstrated that the generation of neutralizing antibodies is much more sensitive than T cell responses to the presence of passively transferred antibody at the time of immunization. They also showed that no protection was achieved in the absence of neutralizing antibodies, although T cell responses were detectable.

Experimental Vector Systems Do Not Induce Protection in the Presence of MV-Specific (Maternal) Antibodies

The lack of protection by MV-specific T cells also was observed in our vaccina-tion studies using plasmids (Schlereth et al. 2000a). To find potential alternative vaccine vector systems that might induce protection in the presence of maternal antibodies, plasmids expressing the MV nucleocapsid, the fusion protein and hemagglutinin were tested in seronegative cotton rats. Immunization with the nucleocapsid induced high antibody titers (which did not neutralize!) and T cell responses. However, no protection was conferred by T cells in the absence of neutralizing antibodies. In contrast, plasmids expressing the fusion protein and hemagglutinin induced both neutralizing antibodies and effector T cells and suc-cessfully protected against infection (Schlereth et al. 2000a). However, in the presence of passively transferred antibodies, immunization with both plasmids failed to induce neutralizing antibodies and did not protect against infection. The same was true for a modified vaccinia virus Ankara (MVA-H) expressing MV hemagglutinin (Weidinger et al. 2001). In contrast, a vesicular stomatitis virus vector expressing MV-H (VSV-H) was partially effective, where intranasal immu-nization with a single dose of VSV-H led to the generation of neutralizing anti-bodies and protection against viral challenge (Schlereth et al. 2000b, 2003). After immunization with VSV-H in the presence of passively transferred antibodies, MV neutralizing antibody titers were reduced tenfold in comparison to immuniza-tion in the absence of passively transferred antibodies (NT titers 60 instead of 600). However, neutralizing antibody titers against VSV remained the same, indi-cating a specific suppression of MV-H-specific B cells. Comparison of all three vector systems indicated that the route of immunization does not seem to be deci-sive because in contrast to VSV-H, both plasmid and MVA-H (and measles vac-cine virus) did not induce protection after intranasal administration. Even though the hemagglutinin was a passenger protein not required for vector replication in all three systems, B cell responses were inhibited, indicating that neutralization by passively transferred antibodies is not important for inhibition.

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Failure of Experimental Vaccines to Immunize in the Presence of Maternal Antibodies

In the cotton rat and the rhesus/cynomolgous macaque models of measles virus infection, two vaccine candidates (plasmid immunization and modified vaccinia virus Ankara (MVA), which are used in clinical trials for HIV) have been tested in the presence of maternal antibodies (Schlereth et al. 2000a; Weidinger et al. 2001; van Binnendijk et al. 1997; Stittelaar et al. 2000; Premenko-Lanier et al. 2003; Zhu et al. 2000). Although the results, at first glance, seem to be contradic-tory in nature, a common theme emerges when taking into account the level of maternal antibodies present at the time of immunization, the number of immuni-zations, induction of immune responses (T cell or B cell response), and level of protection (reduced viral titers or reduced histologically detectable tissue dam-age). High antibody titers inhibit vaccination more strongly than low titers and repeated immunizations improve vaccination success in the face of waning anti-body titers. It is easier to detect T cell proliferation than neutralizing antibodies and to find reduced tissue damage by histology than reduction of virus titers. Given these caveats, neither plasmid nor MVA immunization have proven to be effective by strict standards (i.e., single immunization in the presence of high tit-ers of maternal antibody, induction of neutralizing antibodies, and reduction of viral titers as measure of protection).

Summary and Outlook

The cotton rat has proven that it can fill the void left by the lack of a mouse model for measles virus pathogenesis research as a small rodent model. By now, this ani-mal model is well defined because of the commercial availability of microbiologi-cally defined inbred cotton rats. The on-going development of genetic sequences, reagents, and methods increasingly allows investigators to address mechanistic questions in this animal model. An added advantage is the susceptibility of cotton rats to other human pathogens, potentially making it a good animal model to study polymicrobial disease (e.g., tuberculosis and measles virus infection).

References

Aaby P, Jensen T, Hansen H (1988) Trial of high dose Edmonston-Zagreb measles vaccine in Guinea-Bissau: protective efficacy. Lancet 2:809–811

Armstrong C (1939) The experimental transmission of poliomyelitis to the eastern cotton rat Sigmodon hispidus hispidus . Public Health Rep 54:1719–1721

Avota E, Avots A, Niewiesk S, Kane LP, Bommhardt U, ter Meulen V, Schneider-Sschaulies S (2001) Disruption of Akt kinase activation is important for immunosuppression induced by measles virus. Nat Med 6:725–731

b12325337-0005ztc.indd 105 10/3/2008 10:52:56 AM

106 S. Niewiesk

Azazy AA, Devaney E, Chance ML (1994) A PEG-ELISA for the detection of Leishmania dono-vani antigen in circulating immune complexes. Trans Royal Soc Trop Med Hyg 88:62–66

Bayer M, Wenk P (1988) Homologous and crossreacting immune response of the jird and cotton rat against microfilariae of Dipetalomena vitae and Litosomoides carinii (Nematoda Filaroidea). Trop Med Parasit 39:304–308

Blanco JC, Richardson JY, Darnell ME, Rowzee A, Pletneva L, Porter DD, Prince GA (2002) Cytokine and chemokine gene expression after primary and secondary respiratory syncytial virus infection in cotton rats. J Infect Dis 185:1780–1785

Blanco JC, Pletneva L, Boukhvalova M, Richardson JY, Harris KA, Prince G (2004) The cotton rat: an underutilized animal model for human infectious diseases can now be exploited using specific reagents to cytokines, chemokines, and interferons. J Interferon Cytokine Res 24:21–28

Boukhvalova MS, Prince GA, Soroush L, Harrigan DC, Vogel SN, Blanco JC (2006) The TLR4 agonist, monophosphoryl lipid A, attenuates the cytokine storm associated with respiratory syncytial virus vaccine-enhanced disease. Vaccine 24:5027–5035

Bour H, Peyron E, Gaucherand M, Garrigue JL, Desvignes C, Kaiserlian D, Revillard JP, Nicolas JF (1995) Major histocompatibility complex class I-restricted CD8 + T cells and class II-restricted CD4 + T cells, respectively, mediate and regulate contact sensitivity to dinitrofluor-obenzene. Eur J Immunol 25:3006–3010

Brydak L (1990) Studies on adaptation of influenza virus replicated at low temperature. IV. Sensitivity of neuraminidase and hemagglutinin to some proteolytic enzymes, detergents and chemical agents. Acta Microbiol Pol 39:137–147

Burgdorfer W, Gage KL (1987) Susceptibility of the hispid cotton rat ( Sigmodon hispidus ) to the Lyme disease spirochete ( Borrelia burgdorferi ). Am J Trop Med Hyg 37:624–628

Chen RT, Markowitz LE, Albrecht P, Stewart JA, Mofenson LM, Preblud SR, Orenstein WA (1990) Measles antibody: reevalution of protective titers. J Inf Dis 162:1036–1042

Clark JD (1984) Biology and diseases of other rodents. In: Fox JG, Cohen BJ, Loew FM (eds) Laboratory animal medicine. Academic, Orlando, pp 183–206

Combredet C, Labrousse V, Mollet L, Lorin C, Delebecque F, Hurtrel B, McClure H, Feinberg MB, Brahic M, Tangy F (2003) A molecularly cloned Schwarz strain of measles virus vaccine induces strong immune responses in macaques and transgenic mice. J Virol 77:11546–11554

de Swart RL, Ludlow M, de Witte L, Yanagi Y, van Amerongen G, McQuaid S, Yuksel S, Geijtenbeek TB, Duprex WP, Osterhaus AD (2007) Predominant infection of CD150 + lymphocytes and dendritic cells during measles virus infection of macaques. PLoS Pathog 3:e178

Dreizin RS, Vyshnevetskaia LO, Bagdamian EE, Iankevich OD, Tarasova LB (1971) Experimental RS virus infection of cotton rats. A viral and immunofluorescent study(in Russian). Vopr Virusol 16:670–676

Dubois B, Chapat L, Goubier A, Papiernik M, Nicolas JF, Kaiserlian D (2003) Innate CD4 + CD25 + regulatory T cells are required for oral tolerance and inhibition of CD8 + T cells mediating skin inflammation. Blood 102:3295–3301

Eichelberger MC, Bauchiero S, Point D, Richter BW, Prince GA, Schuman R (2006) Distinct cel-lular immune responses following primary and secondary influenza virus challenge in cotton rats. Cell Immunol 243:67–74

Elwood RL, Wilson S, Blanco JC, Yim K, Pletneva L, Nikonenko B, Samala R, Joshi S, Hemming VG, Trucksis M (2007) The American cotton rat: a novel model for pulmonary tuberculosis. Tuberculosis 87:145–154

Fennelly GJ, Flynn JAL, ter Meulen V, Liebert UG, Bloom BR (1995) Recombinant Bacille Calmette Guérin priming against measles. J Infect Dis 172:698–705

Fooks AR, Jeevarajah D, Lee J, Warnes A, Niewiesk S, ter Meulen V, Stephensom JR, Clegg JCS (1998) Oral or parenteral administration of replication-deficient adenoviruses expressing the measles virus proteins: protective immune responses in rodents. J Gen Virol 79:1027–1031

Haga T, Murayama N, Shimizu Y, Saito A, Sakamoto T, Morita T, Komase K, Nakayama T, Uchida K, Katayama T, Shinohara A, Koshimoto C, Sato H, Miyata H, Katahira K, Goto Y

b12325337-0005ztc.indd 106 10/3/2008 10:52:56 AM


(2008) Analysis of antibody response by temperature-sensitive measles vaccine strain in the cotton rat model. Comp Immunol Microbiol Infect Dis (in press)

Halsey N (1993) Increased mortality following high titer measles vaccines: too much good thing. J Pediatr Infect Dis 12:462–465

Hamelin ME, Yim K, Kuhn KH, Cragin RP, Boukhvalova M, Blanco JC, Prince GA, Boivin G (2005) Pathogenesis of human metapneumovirus lung infection in BALB/c mice and cotton rats. J Virol 79:8894–8903

Hassantoufighi A, Oglesbee M, Richter BW, Prince GA, Hemming V, Niewiesk S, Eichelberger MC (2007) Respiratory syncytial virus replication is prolonged by a concomitant allergic response. Clin Exp Immunol 148:218–229

Hou S, Hyland L, Ryan KW, Portner A, Doherty PC (1996) Virus-specific CD8 + T-cell memory determined by clonal burst size. Nature 369:652–654

Kroeze WK, Tanner CE (1985) Echinococcus multilocularis : responses to infection in cotton rats ( Sigmodon hispidus ). Int. J Parasitol 15:233–238

Langley RJ, Prince GA, Ginsberg HS (1998) HIV type-1 infection of the cotton rat (Sigmodon fulviventer and S. hispidus ). Proc Natl Acad Sci U S A 95:14355–14360

Lewandowski G, Zimmerman MN, Denk LL, Porter DD, Prince GA (2002) Herpes simplex type 1 infects and establishes latency in the brain and trigeminal ganglia during primary infection of the lip in cotton rats and mice. Arch Virol 147:167–179

Lowery GH (1981) The mammals of Louisiana and its adjacent waters. Louisiana State University Press, Baton Rouge

Luhrman A, Tschernig T, Pabst R, Niewiesk S (2005) Improved intranasal immunization with live-attenuated measles virus after co-inoculation of the lipopeptide MALP-2. Vaccine 23:4721–4726

Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC, Gale NW, Iwasaki A, Flavell RA (2004) Recognition of single-stranded RNA viruses by Toll-like receptor 7. PNAS 101:5598–5603

Mähler M, Heidtmann W, Hedrich HJ, Beil W, Gruber A, Niewiesk S, Wagner S (2002) Helicobacter pylori infection induces chronic active gastritis in cotton rats. Gastroenterology 122:A532–A533

McChesney MB, Kehrl JH, Valsamakis A, Fauci AS, Oldstone MBA (1987) Measles virus infec-tion of B lymphocytes permits cellular activation but blocks progression through the cell cycle. J Virol 61:3441–3447

McChesney MB, Altmann A, Oldstone MBA (1988) Suppression of T lymphocyte function by measles virus is due to cell cycle arrest in G1. J Virol 140:1269–1273

Mestas J, Hughes CC (2004) Of mice and not men: differences between mouse and human immu-nology. J Immunol 172:2731–2738

Moll M, Klenk HD, Herrler G, Maisner A (2001) A single amino acid change in the cytoplasmic domains of measles virus glycoproteins H and F alters targeting, endocytosis, and cell fusion in polarized Madin-Darby canine kidney cells. J Biol Chem 276:17887–17894

Moll M, Pfeuffer J, Klenk H-D, Niewiesk S, Maisner A (2004) Polarized glycoprotein targeting affects spread of measles virus in vitro and in vivo. J Gen Virol 85:1019–1027

Murphy BR, Sotnikov AV, Lawrence LA, Banks SM, Prince GA (1990) Enhanced pulmonary histopathology is observed in cotton rats immunized with formalin-inactivated respiratory syncytial virus (RSV) or purified F glycoprotein and challenged with RSV 3–6 months after immunization. Vaccine 8:497–502

Murphy TF, Dubovi EJ, Clyde WA (1981) The common cotton rat as an experimental model of human parainfluenza virus type 3 disease. Exp Lung Res 2:97–109

Nahmias AJ, Griffith D, Salsbury C, Yoshida K (1967) Thymic aplasia with lymphopenia, plasma cells, and normal immunoglobulins. JAMA 201:729–734

Naniche D, Reed SI, Oldstone MBA (1999) Cell cycle arrest during measles virus infection: a G0-like block leads to suppression of retinoblastoma protein expression. J Virol 73:1894–1901

Niewiesk S, Germann P-G (2000) Development of neutralizing antibodies correlates with resolu-tion of interstitial pneumonia after measles virus infection in cotton rats. J Exp Anim Sci 40:201–210

b12325337-0005ztc.indd 107 10/3/2008 10:52:56 AM

108 S. Niewiesk

Niewiesk S, Prince GA (2002) Diversifying animal models: the use of hispid cotton rats ( Sigmodon hispidus ) in infectious diseases. Lab Anim 36:357–372

Niewiesk S, Eisenhuth I, Fooks A, Clegg JCS, Schnorr J-J, Schneider-Schaulies S, ter Meulen V (1997) Measles virus-induced immune suppression in the cotton rat ( Sigmodon hispidus ) model depends on viral glycoproteins. J Virol 71:7214–7219

Niewiesk S, Ohnimus H, Schnorr J-J, Götzelmann M, Schneider-Schaulies S, Jassoy C, ter Meulen V (1999) Measles virus-induced immunosuppression in cotton rats is associated with cell cycle retardation in uninfected lymphocytes. J Gen Virol 80:2023–2029

Niewiesk S, Götzelmann M, ter Meulen V (2000) Selective in vivo suppression of T lymphocyte responses in experimental measles virus infection. Proc Natl Acad Sci U S A 74:4652–4657

Oliver JH, Chandler FW, James AM, Sanders FH, Hutcheson HJ, Huey LO, McGuire BS, Lane RS (1995) Natural occurrence and characterization of the Lyme spirochete, Borrelia burgdor-feri , in cotton rats ( Sigmodon hispidus ) from Georgia and Florida. J Parasitol 81:30–36

Ottolini M, Blanco J, Porter D, Peterson L, Curtis S, Prince G (2003) Combination anti-inflam-matory and antiviral therapy of influenza in a cotton rat model. Pediatr Pulmonol 36:290–294

Ottolini MG, Blanco JC, Eichelberger MC, Porter DD, Pletneva L, Richardson JY, Prince GA (2005) The cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis. J Gen Virol 86:2823–2830

Pacini DL, Dubovi EJ, Clyde WA (1984) A new animal model for human respiratory tract disease due to adenovirus. J Infect Dis 150:92–97

Pasetti MF, Barry EM, Losonsky G, Singh M, Medina-Moreno SM, Polo JM, Ulmer J, Robinson H, Sztein MB, Levine MM (2003) Attenuated Salmonella enterica serovar Typhi and Shigella flexneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemagglutinin, inducing specific immune responses and protection in cotton rats. J Virol 77:5209–5217

Permar SR, Klumpp SA, Mansfield KG, Kim WK, Gorgone DA, Lifton MA, Williams KC, Schmitz JE, Reimann KA, Axthelm MK, Polack FP, Griffin DE, Letvin NL (2003) Role of CD8(+) lymphocytes in control and clearance of measles virus infection of rhesus monkeys. J Virol 77:4396–4400


Pfeuffer J, Püschel K, ter Meulen V, Schneider-Schaulies J, Niewiesk S (2003) Extent of measles virus spread and immune suppression differentiates between wildtype and vaccine strains in the cotton rat model ( Sigmodon hispidus ). J Virol 77:150–158

Pletneva LM, Haller O, Porter DD, Prince GA, Blanco JC (2008) Induction of type I interferons and interferon-inducible Mx genes during respiratory syncytial virus infection and reinfection in cotton rats. J Gen Virol 89:261–270

Premenko-Lanier M, Rota PA, Rhodes G, Verhoeven D, Barouch DH, Lerche NW, Letvin NL, Bellini WJ, McChesney MB (2003) DNA vaccination of infants in the presence of maternal antibody: a measles model in the primate. Virology 307:67–75

Pueschel K, Tietz A, Carsillo M, Steward M, Niewiesk S (2007) Measles virus-specific CD4 T-cell activity does not correlate with protection against lung infection or viral clearance. J Virol 81:8571–8578

Radecke F, Spielhofer P, Schneider H, Kaelin K, Huber M, Dötsch C, Christiansen G, Billeter MA (1995) Rescue of measles virus from cloned DNA. EMBO J 14:5773–5784

Richter BW, Onuska JM, Niewiesk S, Prince GA, Eichelberger MC (2005) Antigen-dependent proliferation and cytokine induction in respiratory syncytial virus-infected cotton rats reflect the presence of effector-memory T cells. Virology 337:102–110

Rytik PG, Kucherov II, Muller WE, Podol´skaia IA, Kruzo M, Duboiskaia GP, Poleshchuk NN (1995) The use of the polymerase chain reaction in modelling HIV infection in animals (in Russian). Zh Mikrobiol Epidemiol Immunobiol 8:86–89

Schachtner SK, Rome JJ, Hoyt RF Jr, Newman KD, Virmani R, Dichek DA (1995) In vivo adeno-virus-mediated gene transfer via the pulmonary artery of rats. Circ Res 76:701–709

b12325337-0005ztc.indd 108 10/3/2008 10:52:56 AM


Schlereth B, Germann P-G, ter Meulen V, Niewiesk S (2000a) DNA vaccination with the hemag-glutinin and the fusion proteins, but not the nucleocapsid protein protects against experimental measles virus infection. J Gen Virol 81:1321–1325

Schlereth B, Rose KJ, Buonocore L, ter Meulen V, Niewiesk S (2000b) Successful vaccine-induced seroconversion by single dose immunization in the presence of measles virus specific maternal antibodies. J Virol 74:4652–4657

Schlereth B, Buonocore L, Tietz A, ter Meulen V, Rose JK, Niewiesk S (2003) Successful mucosal immunization of cotton rats in the presence of measles virus-specific antibodies depends on degree of attenuation of vaccine vector and virus dose. J Gen Virol 84:2145–2151

Schnorr J-J, Seufert M, Schlender J, Borst J, Johnson ICD, ter Meulen V, Schneider-Schaulies S (1997) Cell cycle arrest rather than apoptosis is associated with measles virus contact-medi-ated immunosuppression in vitro. J Gen Virol 78:3217–3226

Steinbach MM, Duca CJ (1940) Experimental tuberculosis in the cotton rat ( Sigmodon hispidus littoralis ). Proc Soc Exp Biol Med 44:288–290

Stertz S, Dittmann J, Blanco JC, Pletneva LM, Haller O, Kochs G (2007) The antiviral potential of interferon-induced cotton rat mx proteins against orthomyxovirus (influenza), rhabdovirus, and bunyavirus. J Interferon Cytokine Res 27:847–855

Stittelaar K, Wyatt L, de Swart R, Vos H, Groen J, van Amerongen G, van Binnendijk R, Rozenblatt S, Moss B, Osterhaus A (2000) Protective immunity in macaques vaccinated with a modified vaccinia virus Ankara-based measles virus vaccine in the presence of passively acquired antibodies. J Virol 74:4236–4243

Streif S, Pueschel K, Tietz A, Blanco J, Meulen VT, Niewiesk S (2004) Effector CD8 + T cells are suppressed by measles virus infection during delayed type hypersensitivity reaction. Viral Immunol 17:604–608

Takeda M, Takeuchi K, Miyajima N, Kobune F, Ami Y, Nagata N, Suzaki Y, Nagai Y, Tashiro M (2000) Recovery of pathogenic measles virus from cloned cDNA. J Virol 74:6643–6647

Tober C, Seufert M, Schneider H, Billeter MA, Johnston ICD, Niewiesk S, ter Meulen V, Schneider-Schaulies S (1998) Expression of measles virus V protein is associated with tran-scriptional control and pathogenicity. J Virol 72:8124–8132

van Binnendijk RS, Poelen MCM, Kuijpers KC, Osterhaus ADME, Uytdehaag FGCM (1990) The predominance of CD8 T cells after infection with measles virus suggests a role for CD8 class I MHC-restricted cytotoxic T lymphocytes (CTL) in recovery from measles. J Immunol 144:2394–2399

van Binnendijk RS, Poelen MCM, van Amerongen G, de Vries P, Osterhaus ADME (1997) Protective immunity in macaques vaccinated with live attenuated, recombinant, and subunit measles vaccines in the presence of passively acquired antibodies. J Infect Dis 175:524–532

Weidenmaier C, Kokai-Kun JF, Kristian SA, Chanturiya T, Kalbacher H, Gross M, Nicholson G, Neumeister B, Mond JJ, Peschel A (2004) Role of teichoic acids in Staphylococcus aureus nasal colonization, a major risk factor in nosocomial infections. Nat Med 10:243–245

Weidinger G, Ohlmann M, Schlereth B, Sutter G, Niewiesk S (2001) Vaccination with recom-binant modified vaccinia virus Ankara protects against measles virus infection in the mouse and cotton rat model. Vaccine 19:2764–2768

Williams JV, Tollefson SJ, Johnson JE, Crowe JE Jr (2005) The cotton rat ( Sigmodon hispidus ) is a permissive small animal model of human metapneumovirus infection, pathogenesis, and protective immunity. J Virol 79:10944–10951

Wong-Chew RM, Islas-Romero R, Garcia-Garcia ML, Beeler JA, Audet S, Santos-Preciado JI, Gans H, Lew-Yasukawa L, Maldonado YA, Arvin AM, Valdespino-Gomez JL (2004) Induction of cellular and humoral immunity after aerosol or subcutaneous administration of Edmonston-Zagreb measles vaccine as a primary dose to 12-month-old children. J Infect Dis 189:254–257

Wyde PR, Ambrosi MW, Voss TG, Meyer HL, Gilbert BF (1992) Measles virus replication in lungs of hispid cotton rats after intranasal inoculation. Proc Soc Exp Biol Med 201:80–87

Wyde PR, Moore-Poveda DK, Daley NJ, Oshitani H (1999) Replication of clinical measles virus strains in hispid cotton rats. Proc Soc Exp Biol Med 221:53–62

b12325337-0005ztc.indd 109 10/3/2008 10:52:57 AM

110 S. Niewiesk

Wyde PR, Moore-Poveda DK, De Clercq E, Neyts J, Matsuda A, Minakawa N, Guzman E, Gilbert BE (2000a) Use of cotton rats to evaluate the efficacy of antivirals in treatment of measles virus infections. Antimicrob Agents Chemother 44:1146–1152

Wyde PR, Stittelaar KJ, Osterhaus ADME, Guzman E, Gilbert BE (2000b) Use of cotton rats for preclinical evaluation of measles vaccines. Vaccine 19:42–53

Wyde PR, Chetty SN, Jewell AM, Schoonover SL, Piedra PA (2005) Development of a cotton rat-human metapneumovirus (hMPV) model for identifying and evaluating potential hMPV antivirals and vaccines. Antiviral Res 2005:57–66

Yim KC, Carroll CJ, Tuyama A, Cheshenko N, Carlucci MJ, Porter DD, Prince GA, Herold BC (2005) The cotton rat provides a novel model to study genital herpes infection and to evaluate preventive strategies. J Virol 79:14632–14639

Zhu Y, Rota P, Wyatt L, Tamin A, Rozenblatt S, Lerche N, Moss B, Bellini W, McChesney M (2000) Evaluation of recombinant vaccinia virus—measles vaccines in infant rhesus macaques with preexisting measles antibody. Virology 276:202–213

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Chapter 6 Current Animal Models: Transgenic Animal Models for the Study of Measles Pathogenesis

C. I. Sellin and B. Horvat (*)

Abstract Animal models are highly important to understand the pathologic mecha-nisms of viral diseases. Therefore, the lack of a suitable animal model has greatly hindered the research into the pathogenesis of measles. Identification of two human receptors for measles virus, CD46 and CD150 (SLAM) has opened new perspec-tives in this field. During the last decade, numerous transgenic animal models have been developed in order to humanize mice and use them to study measles infection and virus–host interactions. Despite their limitations, these models have provided remarkable insights in different aspects of measles infection, providing a better understanding of virus-induced neuropathology, immunosuppression, mechanisms of virus virulence, and contribution of innate and adaptive immune response in viral clearance. They should certainly continue to help in studies of the host and viral fac-tors that are important in measles infection and in developing of new antiviral agents

B. Horvat U758-ENS Lyon , 21 Avenue Tony Garnier, 69365 Lyon Cedex 07 , France , e-mail: [email protected]


Contents

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112Mice Transgenic for CD46 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

HMGCR-CD46 Mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114NSE-CD46 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115Genomic CD46 Transgenic Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

CD150 (SLAM) Transgenic Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119Lck-CD150 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119CD11c-CD150 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121CD150Ge Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121Knockin CD150 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122HMGCR-CD150 Mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122CD46 × CD150 Mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

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112 C.I. Sellin, B. Horvat

and measles virus-based vaccines. In addition, as CD46 serves as a receptor for two other human viruses, some of these models may also find an important application in the study of adenovirus and herpesvirus 6 infection. In this review, we describe different CD46 and CD150 transgenic models and detail their utilization in the study of various aspects of measles pathogenesis.

Abbreviations

CNS Central nervous system DCs Dendritic cells HMGCR Hydroxymethylglutaryl coenzyme A reductase i.c. Intracranial IFN Interferon IFNAR Interferon alpha/beta receptor i.n. Intranasal i.p. Intraperitoneal i.v. Intravenous M Matrix MV Measles virus NSE Neuron-specific enolase RAG Recombinase-activating gene TLR Toll-like receptor SLAM Signaling lymphocytic activation molecule SSPE Subacute sclerosing encephalitis STAT Signal transducer and activator of transcription YAC Yeast artificial chromosome.

Introduction

Infectious diseases remain a major cause of mortality in the world today, particularly among children in developing countries, where they are the first cause of death (WHO 2002). The pathogenesis of many viral diseases that pose a significant pub-lic health problem cannot be easily studied because of the lack of an adequate small-animal model. Although monkeys could often be used to study infection by human viruses, more practical and less expensive animal models are highly desira-ble. As mice remain the most convenient and well-characterized animal model, a great effort has been made to find the best way to use them to study the pathogene-sis of different viral diseases, including measles. The natural resistance of mice to measles virus infection has forced different laboratories to search for a means to overcome this obstacle.

As viral tropism is determined by the pattern of expression of virus-specific cel-lular receptor(s): these molecules are the key player in infection. A lack of human cell receptors on small animals therefore presents a major barrier to successful

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6 Current Animal Models 113

infection. Development of transgenic technology has helped enormously in this field to generate animals expressing receptors for several human viruses. Therefore, the identification of two human cell receptors for measles virus (MV)—CD46 (Dorig et al. 1993; Naniche et al. 1993) and CD150 or SLAM (signaling lym-phocytic activation molecule) (Tatsuo et al. 2000) (Fig. 6. 1 )—has made it possible to create transgenic animals that express these receptors, with the aim of establish-ing mice that are susceptible to MV infection.

Since 1996, numerous transgenic animal lines have been engineered to study MV pathogenesis. The choice of the promoter, composition and integration site of the transgenic construct, differentially engineered by various laboratories, is responsible for the principal differences between the models obtained. These trans-genic lines express one or both human MV receptors on different cell types and some of them have been crossed in a particular genetic background, usually allow-ing a higher susceptibility to the MV infection. They have been intensively used during the last decade to study different aspects of MV infection and provide new insights into MV–host interaction. This review summarizes different characteristics of these transgenic models and their application to the study of MV pathogenesis.

Mice Transgenic for CD46

CD46 is a ubiquitously expressed human transmembrane protein, whose primary function is to regulate complement activation by binding and inactivating the C3b and C4b component of the complement (Fig. 6.1). During the last decade, CD46

CD46NH2 NH2

C4bC3b

STP

Ig-likedomains

Extracellulardomains

Intracellulardomains ITSM

ITSM

COOH

COOH

Cyt1 orCyt2

ShortConsensus

Repeat

MVMVV

1

2

3

4

BC

C

CD150

Fig. 6.1 Schematic representation of two identified human MV receptors CD46 and CD150 (SLAM) expressed in different transgenic animal lines. STP serine threonine and proline rich region, Cyt cytoplasmic tail, ITSM immuno-tyrosine based switch motif

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Table 6.1 Main characteristics of mouse lines transgenic for human CD46, used as models of measles

Name of the transgenic model

Pattern of expression and immunological status

Major clinical signs and pathology following MV infection

HMGCR-CD46(Horvat et al. 1996)

Ubiquitous expressionImmuno-competent

i.c. injection in neonates → neurological syndrome, death: age-dependent susceptibility

NSE-CD46(Rall et al. 1997)

Neuron-restrictedImmunocompetent

i.c. injection in neonates → neurological syndrome, death: age-dependent susceptibility

NSE-CD46X RAG–2(Lawrence et al. 1999)

Neuron-restrictedImmunodeficient

i.c. injection in neonates and adults → neurological syndrome, death

YAC-CD46(Oldstone et al. 1999)

Ubiquitous expression similar to humansImmunocompetent

i.c. injection in neonates → neurological syndrome, death/i.p. and i.v. injection on adults → replication in lymphoid cells + immunosuppression

YAC-CD46 Ubiquitous expression similar to humans

i.c. injection in adults → neurological syndrome, death

X RAG-1(Patterson et al. 2001)

Immunodeficient

CD46GeX IFNAR-KO(Mrkic et al. 2000)

Ubiquitous expression similar to humansImmunodeficient

i.c. and i.p. injection in adults → replication in PBMC and lymphoid organs + MV systemic propagation

has been also identified as a receptor for several different human viruses, includ-ing laboratory strains of measles virus (Dorig et al. 1993; Naniche et al. 1993), herpesvirus 6 (Santoro et al. 1999), and groups B and D of adenovirus (Gaggar et al. 2003). As mice express a CD46 murine counterpart only in testis, numerous transgenic mouse lines have been produced to obtain human CD46 expression in mice. These lines were generated using different promoters and genetic back-grounds and they showed differential susceptibility to MV infection (Table 6. 1 ).

HMGCR-CD46 Mice

The first CD46 transgenic mice were generated using the HMGCR promoter, which drives the expression of an enzyme, hydroxymethylglutaryl coenzyme A reductase, involved in cholesterol metabolism (Horvat et al. 1996; Evlashev et al. 2000). These mice thus express CD46 ubiquitously with one of two different cytoplasmic domains either Cyt1 or Cyt2. Permissibility of transgenic lymphocytes to MV infection in vitro is much lower than human lymphocytes, suggesting the existence

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of intracellular factors in murine cells, which limit MV replication (Evlashev et al. 2001). However, intracranial infection of newborn transgenic mice with laboratory strain of MV, Edmonston, induces a severe neurological disease, followed with a lack of mobility, weight loss, ataxia, and death (Evlashev et al. 2000). Measles virus replication in these mice is followed with the production of infectious virus parti-cles and associated with widely spread neuronal lesions and apoptosis. The pathol-ogy observed is similar to what is seen in progressive infectious measles encephalitis in immunocompromised patients.

In addition to being used to analyze MV-induced neuropathology, CD46 trans-genic mice expressing Cyt1 domain (Evlashev et al. 2000; Thorley et al. 1997) have been used to study the most important pathology associated with measles: immu-nosuppression. These studies demonstrated that MV proteins in the absence of viral replication could induce an inhibition of inflammatory responses known to be altered in measles patients (Marie et al. 2001). UV-inactivated MV injection in CD46 transgenic mice resulted in a decrease in hypersensitivity responses, associ-ated with an alteration in antigen-presenting cell functions, a decrease in the number of dendritic cells producing IL-12, and an abrogation of antigen specific proliferation of T cells. Two distinct mechanisms have been implicated: the interac-tion between CD46 and MV envelope glycoproteins and MV nucleoprotein binding to Fcγ receptor, both with the important role in the generation of MV-induced immunosuppression.

HMGCR transgenic mice wee used to analyze the role of CD46 in the genera-tion of anti-MV responses. It has been demonstrated that the interaction between MV hemagglutinin and CD46 increases the uptake of MV particles and leads to the enhanced MHC-class II presentation MV antigens in vivo (Rivailler et al. 1998). Finally, these mouse lines were of critical importance to demonstrate a differential effect CD46 cytoplasmic domains in the regulation of the immune response, fol-lowing the CD46 engagement by MV hemagglutinin (Marie et al. 2002). Mice expressing Cyt-1 generate decreased inflammatory responses, whereas mice expressing the Cyt-2 isoform develop increased inflammation following CD46 engagement. This differential effect was associated with the modulation of Vav phosphorylation in lymphocytes, thus affecting their cytotoxic activity, T cell pro-liferation, IL-2 and IL-10 production. These findings underline the importance of CD46 receptor in the regulation of the immune response, which could be subverted by pathogens capable of binding to this molecule.

NSE-CD46 Mice

To study pathogenesis of MV-induced persistent brain infection, another model was generated: a transgenic mouse line expressing human CD46 under the control of a neuron-specific promoter (NSE-CD46) (Rall et al. 1997), therefore restricting the expression of CD46 to the brain. An intracranial injection of MV Edmonston into NSE-CD46 neonates resulted in the development of clinical neurological signs of infection and consecutive death of infected animals. Similarly to what has been

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observed with HMGCR-CD46 mice, MV susceptibility depends on the age at the time of infection, as adult CD46 mice were resistant to infection. However, viral particles could not be isolated from injected mouse brains, resembling persistent MV infection in patients with subacute sclerosing encephalitis (SSPE).

Although NSE-CD46 mice succumb to MV infection, they developed a substan-tial inflammatory response during MV-induced encephalitis, similarly to what has been observed in human cases of SSPE. Infiltrates of T and B lymphocytes and mac-rophages were found in the brain, associated with upregulation of MHC class I and II expression, reactive astrocytosis and microgliosis, upregulation of RANTES, IP-10 (IFNγ-induced 10-kD protein), IL-6, TNFα and IL-1β, and an increase in neuronal apoptosis (Manchester et al. 1999). IP-10 and RANTES were shown to be secreted by infected neurons and to attract lymphocytes in the brain (Patterson et al. 2003).

To assess the role and the importance of the different immune cell subsets in the regulation of MV infection, NSE-CD46 mice have been crossed on RAG-KO immunodeficient background, lacking both B and T lymphocytes. In contrast to adult single transgenic mice, NSE-CD46 × RAG-2 adult mice are susceptible to MV brain infection (Lawrence et al. 1999). These results suggested that the adap-tive immune response had a protective role against MV infection, indicting that the major cause of MV-induced neurological complications is the viral replication rather then the antiviral response in infected brains. In addition, NSE-CD46 mice have then been crossed on other backgrounds in order to measure the role of differ-ent cell subsets, deficient in CD4, β2-microglobulin, perforin or IFNγ. Results obtained with these transgenic models suggested that IFNγ plays a crucial role in viral protection, but that this protection is multifactorial. Moreover, CD4 + and CD8 + T cells are implicated in the local antiviral response and are both required for com-plete antiviral protection. Finally, these models suggested that anti-MV immune response favors viral clearance without concomitant neuronal loss, probably via cytokine secretion (Patterson et al. 2002).

Primary neuronal cultures were obtained from NSE-CD46 mice to study the spread of MV between neurons (Lawrence et al. 2000). These experiments sug-gested that CD46 expression, syncytia formation or viral budding are not required for viral propagation in the brain. CD46 remains required for initial infection, but cellular contact is necessary for the virus to spread, suggesting an alternative viral propagation, potentially via synapses, specific of the neuronal environment. The mechanism of viral spread in the central nervous system (CNS) was further ana-lyzed by studying the role of MV fusion protein (Makhortova et al. 2007). Viral spread and infection were inhibited by a tri-peptide, known to have a strong anti-fusogenic activity, showing that fusion played a role in MV infection and propaga-tion in neurons. Substance P, a neurotransmitter, with the same active site as the tri-peptide, also blocked neuronal MV spread. Finally both genetic deletion and pharmacological inhibition of substance P receptor, neurokinin 1, reduced infection of susceptible mice. These data indicated a role for neurokinin 1 in MV CNS infec-tion and spread, perhaps serving as an MV fusion receptor or co-receptor in neu-rons. This study was conducted using MV Edmonston strain and should therefore be confirmed with wild-type MV strains.

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Genomic CD46 Transgenic Animals

In order to mimic the cellular distribution and amount of CD46 found in humans as closely as possible, several transgenic lines have been generated using genomic clones carrying the complete CD46 gene. However, the first transgenic models generated using this approach were not susceptible to MV infection in vivo. Both CD46 trans-genic rats (Niewiesk et al. 1997) and mice (Blixenkrone-Moller et al. 1998) expressed widely transgenic human CD46 receptor and murine transgenic cell cultures were permissive to MV infection i n vitro, although both models were resistant to the infec-tion in vivo. Nevertheless, the race to obtain better CD46 transgenic models continued. Utilization of the yeast artificial chromosome (YAC) libraries with a full-length CD46 genomic clone (>400 kb) allowed generation of several lines of CD46 transgenic mice expressing all major isoforms of CD46, with a pattern of expression that is very similar to humans, except a CD46 expression on mouse erythrocytes (Kemper et al. 2001). Two of them, YAC-CD46 and CD46ge, have been crossed in different genetic backgrounds and intensively characterized regarding MV infection

YAC-CD46 Mice

The first transgenic line that contained YAC with the complete human CD46-encoding gene, expressed comparably to what is observed in human tissues, was generated to study the role of CD46 in the regulation of complement activation in transplantation (Yannoutsos et al. 1996). This animal model was then successfully used to analyze different aspects of MV pathogenesis (Oldstone et al. 1999). Intracranial infection of YAC-CD46 newborns with MV Edmonston is lethal, asso-ciated with an intensive MV replication in neurons, where viral nucleocapsids accumulate without budding. MV is able to replicate in cells of the immune system as well, although at a low level, and viral RNA and proteins are detectable in lym-phoid cells from peripheral blood, spleen, and lymph nodes. Injection of MV into adult YAC-CD46 mice induced a suppression of humoral and cellular responses to secondary antigens, suggesting that this model may be used to study MV-induced immunopathology. Indeed, a few years later it was demonstrated that MV-infected YAC-CD46 mice are much more susceptible then noninfected mice to the secondary infection with Listeria monocytogenes , due to the inhibition of both innate and adaptive immunity by MV (Slifka et al. 2003).

This transgenic line has been further used to study the mechanisms initiating SSPE. YAC-CD46 adult mice were initially infected with lymphocytic choriomeningitis virus, which transiently suppresses the murine immune system. Ten days later, infec-tion by MV Edmonston resulted in persistent MV infection of neurons and infiltration of T and B cells in the brain, MV presenting numerous mutations in the M gene. Furthermore, similarly to what is observed in SSPE, mice produced a high titer of anti-MV antibodies (Oldstone et al. 2005). This interesting experimental setup made it possible to suggest the importance of the immunological status of patients with

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measles, where existence of a stage of immunosuppression before infection may allow the generation of the persistent brain infection afterwards, therefore shedding new light on the immunopathogenesis of SSPE.

YAC-CD46 mice were crossed into RAG-1-deficient background to obtain a model where adult mice are more susceptible to infection and then used to analyze MV brain infection. Using a MV reverse genetics system, an infectious MV virus was generated, in which the matrix (M) gene was replaced with the M gene of a SSPE MV strain (Patterson et al. 2001). As frequent mutations in the M gene, which are observed in viruses recovered from SSPE patients, could result in defective virus, the pathogenicity of this SSPE M-MV was tested in YAC-CD46 × RAG-1 adult mice. Results showed that this virus is infectious in vivo but produces a pro-tracted progressive infection with a death of animals delayed compared to Edmonston strain. Studies were extended to primary neurons and showed that SSPE M-MV initiated infection only through the CD46 receptor. However, after initial entry, viral spread among neurons did not appear to require CD46, similarly to what has been shown in another study using NSE-CD46 mice (Lawrence et al. 2000).

Finally, in order to measure the contribution of different immune cell subsets, YAC-CD46 mice were crossed on immunodeficient backgrounds, as this has been done with NSE-CD46 mice. Different cell subpopulations were tested with RAG-1-KO, CD4-KO, CD8-KO, lymphocyte B-KO, Perforin-KO, TNF-α KO and IFN-γ KO backgrounds (Tishon et al. 2006). This study confirmed that IFN-γ was neces-sary for viral clearance, and that its absence results in persistent infection. B, CD4 + T or CD8 + T cells by themselves are not sufficient to control the infection. However, a combination of B and CD4 + T cells or CD4 + and CD8 + cells leads to viral clear-ance. Although B cells did not play a major role in the resolution of primary infec-tion, it has been shown in HMGCR CD46 mice that immunized mothers can transfer immunity to their pups, probably via transplacental transfer of maternal antibodies (Evlashev et al. 2000). These different transgenic models help to obtain more insight into the complex interaction between MV and innate and adaptive responses and the role of different components of the immune system in viral clearance.

CD46Ge × IFNAR-KO Mice

Another line generated using a full-length CD46 genomic clone was back crossed on the IFNAR-KO background, lacking alpha/beta interferon receptor and therefore deficient in the innate immune response (Mrkic et al. 1998). In contrast to the other models described, this transgenic line, CD46Ge × IFNAR-KO, was susceptible to developing MV infection by natural route of MV infection: intranasal inoculation. The absence of type I IFN signaling allowed an enhanced MV spread to the lungs and more prominent inflammatory responses. Virus replication was also detected in peripheral blood mononuclear cells, the spleen, and the liver, showing a systemic viral propagation in this transgenic model. Cells of the monocyte/macrophage line-age appeared to be early MV targets but also contributed to protect other immune cells from infection (Mrkic et al. 2000; Roscic-Mrkic et al. 2001).

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This model has also been used to assess the pathogenicity of different recom-binant MV viruses. Viruses lacking nonstructural MV C or V proteins are less effec-tive in spreading through the lymphatic system and were not detected in the liver (Mrkic et al. 2000). After intracerebral inoculation, recombinant viruses caused lethal disease less often and induced distinctive patterns of gliosis and inflamma-tion. So V and C proteins seem to act as virulence factors, similarly to what has been demonstrated in parallel in YAC-CD46 mice (Patterson et al. 2000).

Two other recombinant viruses were generated to mimic SSPE variants: MV deficient in matrix protein M (MV-Δ M) and a virus bearing glycoproteins with shortened cytoplasmic tails (MV-Δ tails). Both lost acute pathogenicity in CD46Ge × IFNAR-KO mice but penetrated more deeply into the brain parenchyma than standard MV (Cathomen et al. 1998).

These different CD46 mouse models helped study the pathogenesis of infection with MV laboratory strains and reproduced many aspects of MV pathology, which were observed in monkeys or in naturally infected humans. In addition, they have an important application in the study of vaccination approaches, based on the recom-binant MV vaccines (Combredet et al. 2003; Despres et al. 2005; Lorin et al. 2004), in MV-based oncolytic therapy (Peng et al. 2002), as well as in testing new antiviral concepts (Christiansen et al. 2000). However, all CD46 transgenic mice were resist-ant to wild-type MV strains, emphasizing that any direct extrapolation of results to human physiopathology may be hazardous. Furthermore, the obligation to eliminate either the adaptive (RAG KO) or innate immune system (Interferon type 1 receptor KO) to obtain susceptibility to MV in adult mice limited their application in studies where these immune responses are desirable to obtain relevant conclusions.

CD150 (SLAM) Transgenic Mice

The identification of CD150 or SLAM as a receptor both for wild-type and labora-tory MV strains (Tatsuo et al. 2000) (Fig. 6.1) opened up new perspectives in the generation of transgenic mouse models for infection with clinical MV isolates. Therefore, the race to generate better models to study MV pathogenesis has contin-ued. As murine CD150 can not serve as a receptor for MV, over the last several years, numerous mouse lines that are transgenic for human CD150 have been devel-oped. They differ in the promoter used to drive CD150 expression and therefore in the cell type expressing it, as well as in susceptibility to MV infection (Table 6. 2 ).

Lck-CD150 Mice

The first reported model transgenic for CD150 was engineered using the lck pro-moter, specific for T lymphocytes (Hahm et al. 2003). CD150 was thus restricted to immature and mature lymphocytes, in spleen, thymus, and blood. As in human

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Table 6.2 Main characteristics of mouse lines transgenic for human CD150, used as models of measles

Name of the transgenic model

Pattern of expression and immunological status

Major clinical signs and pathology following MV infection

Lck-CD150 Expression restricted to T lymphocytes

i.p. injection in neonates → infection of thymocytes

(Hahm et al. 2003) Immunocompetent ex vivo infection → inhibition of T cell proliferation

CD11c-CD150

(Hahm et al. 2004)

Expression restricted to CD11c+ dendritic cellsImmunocompetent

i.v. injection in adults → infection of 2%–5% of dendritic cells/ex vivo infection of dendritic cells → immunosuppression

CD150Ge(Welstead et al. 2005)

Human-like expressionImmunocompetent

i.n. injection in adults → transient infection of nasal lymph nodes

CD150GeX STAT1-KO(Welstead et al. 2005)

Human-like expressionImmunodeficient

i.n. and i.p. injection in adults → infection of the thymus spleen and nasal mesenteric and femoral lymph nodes + splenomegaly

Knockin CD150 mouse C + human V (Ohno et al. 2007)

Human-like expressionImmunocompetent

ex vivo infection of splenocytes

Knockin CD150X IFNAR-KO(Ohno et al. 2007)


i.n. and i.p. injection in adults → infection of spleen and lymph nodes + immuno suppression

HMGCR-CD150(Sellin et al. 2006)

Ubiquitous expressionImmunocompetent

i.n. and i.c. injection in suckling mice → neurological syndrome, death

HMGCR-CD150X IFNAR-KO(Druelle et al. 2008)

Ubiquitous expressionImmunodeficient

i.n. and i.c. injection in suckling mice and adults → neurological syndrome, death

CD46GeX CD150GeX IFNAR-KO(Shingai et al. 2005)


i.n. Injection in adults → infection of dendritic cells in lymph nodes

T cells, splenic transgenic lymphocytes were susceptible to in vitro infection with wild-type and vaccine MV strains, resulting in an inhibition of cell proliferation. Following infection or contact with an infected cell, downregulation of CD150 cell surface expression was observed. In addition, in neonate animals, Lck CD150 thy-mocytes expressed MV antigens at the surface 2 days after i.p. injection of MV, suggesting either their infection or binding of the residual MV to the cell receptor. Although no clinical signs of infection are observed in this model, inhibited capac-ity of infected lymphocytes to proliferate suggested MV-induced immunosuppression in these mice.

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CD11c-CD150 Mice

Several in vitro studies have suggested that MV infection of human dendritic cells (DCs) may play an important role in MV-induced immunosuppression (Kerdiles et al. 2006). To test this hypothesis in vivo and analyze the mechanisms implicated, mice transgenic for CD150 were generated using a CD11c-specific promoter, thus allowing expression of MV receptor on splenic and bone-marrow-derived DCs (Hahm et al. 2004). Although in vitro infection of splenic DCs gave approximately 35% cells positive for MV, followed by a downregulation of co-stimulatory mole-cules (CD80, CD86, CD40) and MHC class I and II molecules, only 2%–5% of splenic DCs were infected upon i.v. MV injection. In vitro infected transgenic DCs fail to stimulate alloreactive responses and efficiently inhibit T cell proliferation, independently of IFN-α/β, TNF-α or lymphotoxin α or β. Results from these studies demonstrated that CD150 expression on T cells is not required for the inhibition of T cell functions by DCs and characterized the role of DCs in MV-induced immunosuppression.

The same transgenic model was used few years later to study the role of Toll-like receptors (TLRs) in MV-induced immunosuppression (Hahm et al. 2007). Transgenic bone-marrow-derived DCs were infected with a wild-type MV, then stimulated with different TLR ligands, and the production of several cytokines was measured. The results showed that transgenic infected DCs were defective in the selective synthesis of IL-12 in response to stimulation of TLR4 signaling, but not to engagements of TLR2, 3, 7, or 9. Nonstructural MV V and C proteins were not responsible for this inhibition, and interaction of MV hemagglutinin with CD150 facilitated the suppression. These results suggested that MV, by altering DC func-tion, renders them unresponsive to secondary pathogens via TLRR4.

As virus virulence is often related to the ability of viruses to interfere with the host interferon system, the role of IFN α/β in MV infection was studied using CD11c-CD150 mice (Hahm et al. 2005). It was shown that MV infection interfered with development and expansion of DC both in vivo and in vitro, through genera-tion of IFN α/β by DCs. Type I IFN acted via a signal transducer and activator of transcription 2 (STAT-2)-dependent but STAT-1-independent pathway, suggesting a new mechanism in the abrogating the function of DCs and generation of virus- induced immunosuppression.

CD150Ge Mice

In 2005, another mouse model that was transgenic for CD150 was created, mice expressing the complete human gene, under the control of endogenous promoter (Welstead et al. 2005). The pattern of CD150 expression was similar to what has been described in humans: activated B and T lymphocytes and activated DCs. In mice infected intranasally by recombinant wild-type MV, a transient infection of nasal lymph nodes was observed, although they did not show clinical signs of

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disease. To improve the efficiency of MV infection, these mice were crossed on a STAT-1-deficient background and the resulting mice showed higher susceptibility to the infection and produced more viral particles. After i.n. and i.p. inoculation, infection in the thymus, spleen, and lymph nodes was detected, with increased lymph node and spleen size. Abnormally large numbers of mature neutrophils and natural killer cells caused the splenomegaly. Therefore, although STAT-1 was not required for the DC development in CD11c-CD150 mice, in this model ablation of STAT-1 significantly increased susceptibility to MV infection.

Knockin CD150 Mice

In order to generate transgenic mice regulating the expression of human CD150 in the same way as murine CD150, and therefore to closely approach the immuno-physiological conditions of the host, a mouse model bearing a chimeric CD150 molecule was generated (Ohno et al. 2007). As the V domain is necessary and suf-ficient for MV binding to CD150 (Ono et al. 2001) (Fig. 6.1), a knockin approach was used to replace the murine V domain by the human V domain, using homologous recombination in embryonic stem cells. Generated knockin mice express chimeric CD150 molecule with an expected distribution and normal signal-ing function. Although cells from this transgenic model could be infected in vitro with wild-type MV, these mice had to be crossed on an IFNAR-KO background to obtain a productive MV infection in vivo. In knockin-CD150 × IFNAR-KO mice, MV propagation was detected in different lymphoid organs following i.n. and i.p. wild-type MV injection. Transgenic splenocytes showed suppression of prolifera-tive responses to concanavalin A. Thus MV infection of these mice reproduces lymphotropism and immunosuppression as in MV human infection and should serve as a useful small-animal model to study MV pathogenesis in conditions when production of interferon type I is not critical.

HMGCR-CD150 Mice

To further increase the susceptibility of mice to wild-type MV infection, another CD150 transgenic model was generated using HMGCR promoter (Sellin et al. 2006). These mice ubiquitously express CD150 and present the only model reported so far where suckling mice are highly susceptible to the natural route of MV infection. Following intranasal MV inoculation, mice develop a severe neuro-logic syndrome, characterized by lethargy, seizures, ataxia, weight loss, and death within 3 weeks, associated with MV propagation to different organs. The highest level of replication was observed in the brain, where virus infects neurons and oligodendrocytes, but not astrocytes, although an intensive astrogliosis is observed

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(Fig. 6. 2 ). Moreover, this model was shown to be useful to test anti-measles preven-tive and therapeutic approaches, as a soluble recombinant CD150 molecule suc-cessfully protected HMGCR-CD150 mice from MV infection. This transgenic model, by allowing a simple readout of the efficacy of an antiviral treatment, pro-vides a unique experimental tool to evaluate innovative anti-measles approaches.

This mouse line was then used to study the mechanism of attenuation of MV by repeated cellular passages (Druelle et al. 2008). Indeed, virulence of parental and attenuated MV strains could be distinguished in this transgenic model. Moreover, HMGCR-CD150 mice were crossed on an IFNAR-KO background, where attenu-ated virus did not show any higher virulence. This model demonstrated that attenuation of this particular MV strain was independent of type I IFN and therefore opens new perspectives to compare the level of neurovirulence of different MV strains and further dissect MV-induced neurological disease.

CD46 × CD150 Mice

Finally, to approach murine models as closely as possible to humans, a double trans-genic mice was generated, expressing both known human MV receptors, CD46 and CD150 (Shingai et al. 2005). These mice present the same pattern of expression of CD46 and CD150 as humans: CD46 expression is ubiquitous and CD150 is induci-ble on T lymphocytes and can be upregulated on B cells and mature DCs. However, expression of both receptors on murine cells turned out to be insufficient to render them more susceptible to infection. This was not completely surprising because intracellular factors were shown to limit MV replication in murine cells (Evlashev et al. 2001). Again, to obtain higher susceptibility to infection, these double trans-genic mice were crossed on an IFNAR-KO background. Consequently, following

Fig. 6.2 a–c Cellular targets of MV infection in the brain of HMGCR-CD150 mice. Suckling mice were infected intranasally with wild-type MV strain G954 and brains were dissected and analyzed immunohistochemically 10 days later by confocal microscopy using anti-MV nucleo-protein antibody ( red , a–c ) and neuron-specific anti-NF antibody ( green , a ); astrocyte-specific anti-GFAP antibody ( green , b ) and anti-GC oligodendrocytic antibody ( c ), as described previously (Sellin et al. 2006). Co-localization ( yellow ) is observed only in a and c , demonstrating that neu-rons and oligodendrocytes but not astrocytes are infected by MV

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i.p. MV inoculation, infected DCs were detectable in lymph nodes. This transgenic model therefore helped to demonstrate that mature DCs are the main vectors of viral spread in vivo, and because of the similarity in the expression of both MV receptors compared to humans, it may give new insights into MV pathogenesis.

Conclusions

There is a frequent feeling among scientists that an animal model must exactly mimic the human disease studied. However, this goal has only rarely been achieved. Many different transgenic mouse lines have been generated to study MV pathogenesis and we must admit that none of them reproduces all the aspects of MV infection in humans. Indeed, it seems clear now that if receptor expression is necessary for viral entry, additional obstacles during other steps of the viral cycle prevent expected viral pathogenesis in receptor humanized mice. In most cases, an immunodeficient background was necessary to obtain the susceptibility for MV infection in vivo. However, despite imperfections, these different trans-genic models have provided remarkable insights into diverse aspects of MV pathogenesis in a relatively short period of time. Real progress has been accom-plished particularly with CD150 transgenic models, since these lines are suscep-tible to wild-type MV strains and some of them allow MV to spread via the natural route of infection, the airways. These models are now available for the molecular dissection of the measles immunopathology, testing antiviral agents and new vaccines. In addition, since CD46 serves as a receptor for the other human viruses, these transgenic models have been recently used for adenovirus infection in vivo (DiPaolo et al. 2006) and therefore open new avenues to study other viral infections.

Finally, several studies have demonstrated the existence of other way(s) of entry into human cells, independently of CD46 and CD150 (Andres et al. 2003; Hashimoto et al. 2002; Kouomou and Wild 2002; Takeuchi et al. 2003). However, the nature of this third receptor remains elusive for the moment. It is certain that its identification in the future will inspire a generation of new transgenic models and help the race towards the development of improved and more perfect models of MV pathogenesis to continue.

Acknowledgements Studies cited from our laboratory were supported by institutional grants from INSERM and Fondation pour la Recherche Médicale (FRM); CIS was supported by FRM in 2006–2007.

References

Andres O, Obojes K, Kim KS, ter Meulen V, Schneider-Schaulies J (2003) CD46- and CD150-inde-pendent endothelial cell infection with wild-type measles viruses. J Gen Virol 84:1189–1197


b12325337-0006ztc.indd 124 10/3/2008 10:54:19 AM


Cathomen T, Mrkic B, Spehner D, Drillien R, Naef R, Pavlovic J, Aguzzi A, Billeter MA, Cattaneo R (1998) A matrix-less measles virus is infectious and elicits extensive cell fusion: consequences for propagation in the brain. EMBO J 17:3899–3908

Christiansen D, Devaux P, Reveil B, Evlashev A, Horvat B, Lamy J, Rabourdin-Combe C, Cohen JH, Gerlier D (2000) Octamerization enables soluble CD46 receptor to neutralize measles virus in vitro and in vivo. J Virol 74:4672–4678


Despres P, Combredet C, Frenkiel MP, Lorin C, Brahic M, Tangy F (2005) Live measles vaccine expressing the secreted form of the West Nile virus envelope glycoprotein protects against West Nile virus encephalitis. J Infect Dis 191:207–214

DiPaolo N, Ni S, Gaggar A, Strauss R, Tuve S, Li ZY, Stone D, Shayakhmetov D, Kiviat N, Toure P et al (2006) Evaluation of adenovirus vectors containing serotype 35 fibers for vaccination. Mol Ther 13:756–765


Druelle J, Sellin CI, Waku-Kouomou D, Horvat B, Wild FT (2008) Wild type measles virus atten-uation independent of type I IFN. Virol J 5:22

Evlashev A, Moyse E, Valentin H, Azocar O, Trescol-Biemont MC, Marie JC, Rabourdin-Combe C, Horvat B (2000) Productive measles virus brain infection and apoptosis in CD46 transgenic mice. J Virol 74:1373–1382

Evlashev A, Valentin H, Rivailler P, Azocar O, Rabourdin-Combe C, Horvat B (2001) Differential permissivity to measles virus infection of human and CD46-transgenic murine lymphocytes. J Gen Virol 82:2125–2129

Gaggar A, Shayakhmetov DM, Lieber A (2003) CD46 is a cellular receptor for group B adenovi-ruses. Nat Med 9:1408–1412

Hahm B, Arbour N, Naniche D, Homann D, Manchester M, Oldstone MB (2003) Measles virus infects and suppresses proliferation of T lymphocytes from transgenic mice bearing human signaling lymphocytic activation molecule. J Virol 77:3505–3515


Hahm B, Trifilo MJ, Zuniga EI, Oldstone MB (2005) Viruses evade the immune system through type I interferon-mediated STAT2-dependent but STAT1-independent signaling. Immunity 22:247–257

Hahm B, Cho JH, Oldstone MB (2007) Measles virus-dendritic cell interaction via SLAM inhibits innate immunity: selective signaling through TLR4 but not other TLRs mediates suppression of IL-12 synthesis. Virology 358:251–257

Hashimoto K, Ono N, Tatsuo H, Minagawa H, Takeda M, Takeuchi K, Yanagi Y (2002) SLAM (CD150)-independent measles virus entry as revealed by recombinant virus expressing green fluorescent protein. J Virol 76:6743–6749

Horvat B, Rivailler P, Varior-Krishnan G, Cardoso A, Gerlier D, Rabourdin-Combe C (1996) Transgenic mice expressing human measles virus (MV) receptor CD46 provide cells exhibit-ing different permissivities to MV infection. J Virol 70:6673–6681

Kemper C, Leung M, Stephensen CB, Pinkert CA, Liszewski MK, Cattaneo R, Atkinson JP (2001) Membrane cofactor protein (MCP; CD46) expression in transgenic mice. Clin Exp Immunol 124:180–189

Kerdiles YM, Sellin CI, Druelle J, Horvat B (2006) Immunosuppression caused by measles virus: role of viral proteins. Rev Med Virol 16:49–63

Kouomou DW, Wild TF (2002) Adaptation of wild-type measles virus to tissue culture. J Virol 76:1505–1509

Lawrence DM, Vaughn MM, Belman AR, Cole JS,Rall GF (1999) Immune response-mediated protection of adult but not neonatal mice from neuron-restricted measles virus infection and central nervous system disease. J Virol 73:1795–1801

b12325337-0006ztc.indd 125 10/3/2008 10:54:19 AM


Lawrence DM, Patterson CE, Gales TL, D’Orazio JL, Vaughn MM, Rall GF (2000) Measles virus spread between neurons requires cell contact but not CD46 expression syncytium formation or extracellular virus production. J Virol 74:1908–1918

Lorin C, Mollet L, Delebecque F, Combredet C, Hurtrel B, Charneau P, Brahic M, Tangy F (2004) A single injection of recombinant measles virus vaccines expressing human immunodeficiency virus (HIV) type 1 clade B envelope glycoproteins induces neutralizing antibodies and cellular immune responses to HIV. J Virol 78:146–157

Makhortova NR, Askovich P, Patterson CE, Gechman LA, Gerard NP, Rall GF (2007) Neurokinin-1 enables measles virus trans-synaptic spread in neurons. Virology 362:235–244

Manchester M, Eto DS, Oldstone MB (1999) Characterization of the inflammatory response dur-ing acute measles encephalitis in NSE-CD46 transgenic mice. J Neuroimmunol 96:207–217

Marie JC, Kehren J, Trescol-Biemont MC, Evlashev A, Valentin H, Walzer T, Tedone R, Loveland B, Nicolas JF, Rabourdin-Combe C, Horvat B (2001) Mechanism of measles virus-induced suppression of inflammatory immune responses. Immunity 14:69–79

Marie JC, Astier AL, Rivailler P, Rabourdin-Combe C, Wild TF, Horvat B (2002) Linking innate and acquired immunity: divergent role of CD46 cytoplasmic domains in T cell induced inflam-mation. Nat Immunol 3:659–666

Mrkic B, Pavlovic J, Rulicke T, Volpe P, Buchholz CJ, Hourcade D, Atkinson JP, Aguzzi A, Cattaneo R (1998) Measles virus spread and pathogenesis in genetically modified mice. J Virol 72:7420–7427

Mrkic B, Odermatt B, Klein MA, Billeter MA, Pavlovic J, Cattaneo R (2000) Lymphatic dissemi-nation and comparative pathology of recombinant measles viruses in genetically modified mice. J Virol 74:1364–1372

Naniche D, Varior-Krishnan G, Cervoni F, Wild TF, Rossi B, Rabourdin-Combe C, Gerlier D (1993) Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus. J Virol 67:6025–6032

Niewiesk S, Schneider-Schaulies J, Ohnimus H, Jassoy C, Schneider-Schaulies S, Diamond L, Logan JS, and ter Meulen V (1997) CD46 expression does not overcome the intracellular block of measles virus replication in transgenic rats. J Virol 71:7969–7973


Oldstone MB, Lewicki H, Thomas D, Tishon A, Dales S, Patterson J, Manchester M, Homann D, Naniche D, Holz A (1999) Measles virus infection in a transgenic model: virus-induced immu-nosuppression and central nervous system disease. Cell 98:629–640

Oldstone MB, Dales S, Tishon A, Lewicki H, Martin L (2005) A role for dual viral hits in causa-tion of subacute sclerosing panencephalitis. J Exp Med 202:1185–1190

Ono N, Tatsuo H, Tanaka K, Minagawa H, Yanagi Y (2001) V domain of human SLAM (CDw150) is essential for its function as a measles virus receptor. J Virol 75:1594–1600

Patterson JB, Thomas D, Lewicki H, Billeter MA, Oldstone MB (2000) V and C proteins of mea-sles virus function as virulence factors in vivo. Virology 267:80–89

Patterson JB, Cornu TI, Redwine J, Dales S, Lewicki H, Holz A, Thomas D, Billeter MA, Oldstone MB (2001) Evidence that the hypermutated M protein of a subacute sclerosing pan-encephalitis measles virus actively contributes to the chronic progressive CNS disease. Virology 291:215–225

Patterson CE, Lawrence DM, Echols LA, Rall GF (2002) Immune-mediated protection from measles virus-induced central nervous system disease is noncytolytic and gamma interferon dependent. J Virol 76:4497–4506

Patterson CE, Daley JK, Echols LA, Lane TE, Rall GF (2003) Measles virus infection induces chemokine synthesis by neurons. J Immunol 171:3102–3109

Peng KW, TenEyck CJ, Galanis E, Kalli KR, Hartmann LC, Russell SJ (2002) Intraperitoneal therapy of ovarian cancer using an engineered measles virus. Cancer Res 62:4656–4662

Rall GF, Manchester M, Daniels LR, Callahan EM, Belman AR, Oldstone MB (1997) A transgenic mouse model for measles virus infection of the brain. Proc Natl Acad Sci U S A 94:4659–4663

b12325337-0006ztc.indd 126 10/3/2008 10:54:19 AM


Rivailler P, Trescol-Biemont MC, Gimenez C, Rabourdin-Combe C, Horvat B (1998) Enhanced MHC class II-restricted presentation of measles virus (MV) hemagglutinin in transgenic mice expressing human MV receptor CD46. Eur J Immunol 28:1301–1314

Roscic-Mrkic B, Schwendener RA, Odermatt B, Zuniga A, Pavlovic J, Billeter MA, Cattaneo R (2001) Roles of macrophages in measles virus infection of genetically modified mice. J Virol 75:3343–3351

Santoro F, Kennedy PE, Locatelli G, Malnati MS, Berger EA, Lusso P (1999) CD46 is a cellular receptor for human herpesvirus 6. Cell 99:817–827

Sellin CI, Davoust N, Guillaume V, Baas D, Belin MF, Buckland R, Wild TF, Horvat B (2006) High pathogenicity of wild-type measles virus infection in CD150 (SLAM) transgenic mice. J Virol 80:6420–6429

Shingai M, Inoue N, Okuno T, Okabe M, Akazawa T, Miyamoto Y, Ayata M, Honda K, Kurita-Taniguchi M, Matsumoto M et al (2005) Wild-type measles virus infection in human CD46/CD150-transgenic mice: CD11c-positive dendritic cells establish systemic viral infection. J Immunol 175:3252–3261

Slifka MK, Homann D, Tishon A, Pagarigan R, Oldstone MB (2003) Measles virus infection results in suppression of both innate and adaptive immune responses to secondary bacterial infection. J Clin Invest 111:805–810

Takeuchi K, Miyajima N, Nagata N, Takeda M, Tashiro M (2003) Wild-type measles virus induces large syncytium formation in primary human small airway epithelial cells by a SLAM(CD150)-independent mechanism. Virus Res 94:11–16

Tatsuo H, Ono N, Tanaka K, Yanagi Y (2000) SLAM (CDw150) is a cellular receptor for measles virus. Nature 406:893–897

Thorley BR, Milland J, Christiansen D, Lanteri MB, McInnes B, Moeller I, Rivailler P, Horvat B, Rabourdin-Combe C, Gerlier D et al (1997) Transgenic expression of a CD46 (membrane cofactor protein) minigene: studies of xenotransplantation and measles virus infection. Eur J Immunol 27:726–734

Tishon A, Lewicki H, Andaya A, McGavern D, Martin L, Oldstone MB (2006) CD4 T cell control primary measles virus infection of the CNS: regulation is dependent on combined activity with either CD8 T cells or with B cells: CD4 CD8 or B cells alone are ineffective. Virology 347:234–245

Welstead GG, Iorio C, Draker R, Bayani J, Squire J, Vongpunsawad S, Cattaneo R, Richardson CD (2005) Measles virus replication in lymphatic cells and organs of CD150 (SLAM) trans-genic mice. Proc Natl Acad Sci U S A 102:16415–16420

WHO (2002) Scaling up the response to infectious diseases. www.who.int/infectious-disease-report/2002/introduction.html. Cited 26 May 2008

Yannoutsos N, Ijzermans JN, Harkes C, Bonthuis F, Zhou CY, White D, Marquet RL, Grosveld F (1996) A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection. Genes Cells 1:409–419

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Chapter 7 Molecular Epidemiology of Measles Virus

P. A. Rota( ) , D. A. Featherstone , and W. J. Bellini

Abstract Genetic characterization of wild-type measles viruses provides a means to study the transmission pathways of the virus and is an essential component of laboratory-based surveillance. Laboratory-based surveillance for measles and rubella, including genetic characterization of wild-type viruses, is performed throughout the world by the WHO Measles and Rubella Laboratory Network, which serves 166 countries in all WHO regions. In particular, the genetic data can help confirm the sources of virus or suggest a source for unknown-source cases as well as to establish links, or lack thereof, between various cases and outbreaks. Virologic surveillance has helped to document the interruption of transmission of endemic measles in some regions. Thus, molecular characterization of measles viruses has provided a valuable tool for measuring the effectiveness of measles

P.A. Rota Measles, Mumps, Rubella and Herpesvirus Laboratory Branch , Centers for Disease Control and Prevention , Atlanta , GA, USA, e-mail: [email protected]

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130Laboratory Surveillance for Measles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Background and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131Utility of Molecular Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131Standard Methods for Molecular Epidemiology of Measles . . . . . . . . . . . . . . . . . . . . . . 132Data Reporting and Surveillance Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133Virus Isolation and Clinical Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135New Molecular Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135Measles Vaccines and Antigenic Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136SSPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Global Distribution of Measles Genotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137Genotypes in Areas with Endemic Measles or Frequent Outbreaks . . . . . . . . . . . . . . . . 138Measles Elimination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141Measles Reintroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

Mapping Transmission Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144


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130 P.A. Rota et al.

control programs, and virologic surveillance needs to be expanded in all areas of the world and conducted during all phases of measles control.

Introduction

The widespread use of live attenuated vaccines for measles has dramatically reduced the worldwide incidence of measles. The disease has been eliminated in the Western hemisphere (Region of the Americas) since 2000 and the European, Eastern Mediterranean, and Western Pacific Regions of the World Health Organization (WHO) have set elimination goals for the near future (Anonymous 2005d). Despite these successes, measles remains endemic in many developing countries. Measles remains a major cause of childhood morbidity and mortality, accounting for an estimated 345,000 deaths in 2005, 87% of which were in the African and South East Asian Regions of WHO (Wolfson et al. 2007). Subsequently the African and Southeast Asian Regions have implemented a strategy of measles mortality reduction. Even counties that have measles vaccination programs experi-ence outbreaks because of the accumulation of susceptible individuals and the constant threat of viral importations from endemic areas. Maintaining measles elimination requires achieving and maintaining very high levels of population immunity and good laboratory-based surveillance to rapidly detect and control periodic outbreaks.

Laboratory Surveillance for Measles

When the incidence of measles is low, surveillance based on clinical presentation of cases has low sensitivity and specificity. Therefore, an essential component of any measles control program is laboratory-based surveillance to provide confirma-tion of cases and genetic characterization of circulating wild-type viruses. Routine laboratory confirmation of suspected cases is based on detection of measles-spe-cific IgM in a single blood sample taken as soon as possible after rash onset. In some cases, molecular techniques such as RT-PCR to detect viral RNA are used to complement serologic testing. Another important aspect of laboratory surveillance for measles, and the subject of this chapter, is the genetic characterization of circu-lating wild-type viruses to support molecular epidemiologic studies (World Health Organization 2005a, Anonymous 2005b; Rota and Bellini 2003).

Laboratory-based surveillance for measles and rubella is performed throughout the world by the WHO Measles and Rubella Laboratory Network (LabNet). This network provides for standardized testing and reporting with labs serving 166 coun-tries in all WHO regions. A system for monitoring indicators of laboratory perform-ance, including laboratory accreditation, and proficiency testing has been implemented in all regions (World Health Organization 2005a, Anonymous 2005b). The LabNet also supports genetic characterization of currently circulating strains of measles viruses and LabNet has been responsible for standardization of the nomenclature and

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7 Molecular Epidemiology of Measles Virus 131

laboratory procedures that are used to describe the genetic characteristics of wild-type measles and rubella viruses (World Health Organization 1998, 2001a, 2001b, 2003, 2005b, 2005c, 2006, 2007a). This standardization has allowed sharing of virologic surveillance data between laboratories and permitted efficient communication of this data throughout the measles control programs (World Health Organization 2007b).

Background and Methods

Measles virus is an RNA virus in the genus Morbillivirus within the family Paramyxoviridae . Although other members of the genus infect various animal species, measles only infects humans and non-human primates. The negative-sense, single-stranded RNA genome is contained within a helical nucleocapsid in the virion. The genome consists of 15,894 nucleotides, which code for the six structural proteins, the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemag-glutinin (H), and large protein (L), and two nonstructural proteins, C and V (Griffin 2001). Although measles is a monotypic virus, genetic and antigenic variation has been detected in wild-type viruses (Rota et al. 1992; Tamin et al. 1994; Taylor et al. 1991). The nucleotide sequences of the L, M, and F genes (Bankamp et al. 1999; Rota et al. 1994b) are much less variable than the sequences of the N, P, and H genes, which have 7%–10% variability (Bankamp et al. 2008; Rima et al. 1997; Rota et al. 1992). The N and H gene sequences are most commonly used for genetic characterization of wild-type viruses. In particular, one of the most variable parts of the measles genome is the 450-nucleotide region, which codes for the COOH-terminal 150 amino acids the N protein, where nucleotide variability can approach 12% between wild-type viruses (Xu et al. 1998).

Utility of Molecular Epidemiology

The combination of molecular epidemiologic techniques and standard case classi-fication and reporting provides a very sensitive means to describe the transmission pathways of measles. In particular, sequence data can help confirm the sources of virus or suggest a source for unknown-source cases as well as to establish links, or lack thereof, between various cases and outbreaks. Virologic surveillance is espe-cially beneficial when it is possible to observe the change in viral genotypes over time in a particular country or region because this information, when analyzed in conjunction with standard epidemiologic data, has helped to document the interrup-tion of transmission of endemic measles. Thus, molecular characterization of mea-sles viruses has provided a valuable tool for measuring the effectiveness of measles control programs (Mulders et al. 2001; Riddell et al. 2005; Rota et al. 1996, 2002; Rota and Bellini 2003).

Virologic surveillance can also help to classify suspected cases as vaccine reac-tions. A small proportion of measles vaccine recipients experience rash and fever 10–14 days following vaccination (Griffin 2001). During outbreaks, measles vaccine

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is administered to help control the outbreak, and in these situations, vaccine reac-tions may be mistakenly classified as measles cases. Since serologic methods cannot distinguish between a vaccine-induced antibody response and antibodies derived from natural disease, molecular characterization of viral isolates provides a method to confirm whether vaccine or wild-type measles virus caused the rash and fever. Molecular information is also useful for analyzing unusual or severe cases of mea-sles infection, suspected adverse events following vaccination, and severe sequelae of measles infection such as subacute sclerosing panencephalitis (SSPE) (Bellini et al. 2005). SSPE will be discussed in more detail in Sect. 2.7.

Standard Methods for Molecular Epidemiology of Measles

Before 1998, there was no uniform nomenclature or analysis protocol to describe the genetic characteristics of wild-type measles viruses. In 1998, the WHO made recom-mendations for a standard nomenclature for naming strains, describing genotypes, and conducting sequence analysis so that genetic data would be directly comparable between laboratories. These recommendations have been updated periodically since 1998 (World Health Organization 1998, 2001a, 2001b, 2003, 2005b, 2005c, 2006, 2007a). WHO recommends that the 450 nucleotides coding for the COOH-terminal 150 amino acids of N are the minimum amount of sequence data required for genotyp-ing a measles virus isolate or clinical specimen. Complete H gene sequences should be obtained from representative strains or if a new genotype is suspected. Phylogenetic analysis of the H gene sequences provides additional support for the genotype assign-ment while monitoring amino acid substitutions that could affect antigenicity.

For molecular epidemiologic purposes, the genotype designations are consid-ered the operational taxonomic unit, while related genotypes are grouped by clades. WHO currently recognizes eight clades designated A, B, C, D, E, F, G, and H. Within these clades, there are 23 recognized genotypes, designated A, B1, B2, B3, C1, C2, D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, E, F, G1, G2, G3, H1, and H2. Some clades contain only one genotype and, in such cases, the genotype designa-tion is the same as the clade name. Other clades contain multiple genotypes and are designated by using the clade letter (in uppercase) and genotype number. Several of the genotypes—B1, E, F, G1, D1—appear to be extinct or inactive since repre-sentatives of these genotypes have not been isolated for at least 15 years. However, the sequences of the inactive genotypes are maintained in the set of WHO reference sequences for completeness (World Health Organization 2005b). With the excep-tion of genotype F which is based only on sequences derived from a case of SSPE, all of the genotypes have an assigned reference strain (Table 7. 1 , Fig. 7. 1 ) chosen to represent the earliest isolation of virus from each genotype. Sequences from recent viral isolates are then compared to the set of WHO reference sequences, which are available from GenBank (World Health Organization 2005b) and the WHO Strain Banks, to determine the genotype. WHO has established guidelines based on both molecular biologic and epidemiologic criteria for the designation of new genotypes (World Health Organization 2001b, 2003, 2005b).

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Table 7.1 Reference strains to be used for genetic analysis of wild-type measles viruses: 2008

Genotype Statusa Reference strains (MVi)b

H gene accessionc

N gene accession

A Active Edmonston-wt.USA/54 U03669 U01987B1 Inactive Yaounde.CAE/12.83

‘‘Y-14”AF079552 U01998

B2 Active Libreville.GAB/84 “R-96”

AF079551 U01994

B3 Active New York.USA/94 L46752 L46753Ibadan.NIE/97/1 AJ239133 AJ232203

C1 Active Tokyo.JPN/84/K AY047365 AY043459C2 Active Maryland.USA/77 “JM” M81898 M89921

Erlangen.DEU/90 “WTF ”

Z80808 X84872

D1 Inactive Bristol.UNK/74 (MVP) Z80805 D01005D2 Active Johannesburg.SOA/88/1 AF085198 U64582D3 Active Illinois.USA/89/1

“Chicago-1”M81895 U01977

D4 Active Montreal.CAN/89 AF079554 U01976D5 Active Palau.BLA/93 L46757 L46758

Bangkok.THA/93/1 AF009575 AF079555D6 Active New Jersey.USA/94/1 L46749 L46750D7 Active Victoria.AUS/16.85 AF247202 AF243450

Illinois.USA/50.99 AY043461 AY037020D8 Active Manchester.UNK/30.94 U29285 AF280803D9 Active Victoria.AUS/12.99 AY127853 AF481485D10 Active Kampala.UGA/51.00/1 AY923213 AY923185E Inactive Goettingen.DEU/71

“Braxator ”Z80797 X84879

F Inactive MVs/Madrid.SPA/94 SSPE

Z80830 X84865

G1 Inactive Berkeley.USA/83 AF079553 U01974G2 Active Amsterdam.NET/49.97 AF171231 AF171232G3 Active Gresik.INO/17.02 AY184218 AY184217H1 Active Hunan.CHN/93/7 AF045201 AF045212H2 Active Beijing.CHN/94/1 AF045203 AF045217

a Active genotypes that have been isolated within the past 15 yearsb WHO name; other names that have been used in the literature appear in quotation marksc Sequences available at GenBank (http://www.ncbi.nlm.nih.gov) or from WHO strain banks Reproduced from World Health Organization 2005b

Data Reporting and Surveillance Guidelines

Through the efforts of LabNet, virologic surveillance has expanded on a global scale. Information on circulating genotypes has been reported from almost every country with endemic or widespread measles. Genbank contains over 1500 partial

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N gene sequences with over 80% of these sequences reported since 2000 (Rota and Tian, unpublished observations). WHO maintains a database of genotype informa-tion that is reported from national and regional laboratories within LabNet. Recently, it has become apparent that comparison of the sequence information is as important as having the genotype designation. Many genotypes contain multiple co-circulating lineages and assignment of a viral sequence to one of these lineages allows for more accurate mapping of transmission pathways. For this reason, rapid exchange of both genotype and sequence information on a global scale is critical. Databases that allow rapid exchange of sequence information are being developed by the Global Specialized Laboratories within LabNet (World Health Organization 2005a).

Surveillance guidelines recommend that countries involved in measles elimi-nation collect appropriate specimens for virus isolation from every chain of transmission, while countries involved in outbreak-control and mortality reduc-tion obtain representative specimens from measles outbreaks (World Health Organization 2007b). It is important to conduct virologic surveillance before acce-lerated control measures are initiated so that it will be possible to study the pattern of genotypes present both before and after vaccination campaigns. The best samples for virologic surveillance are throat or nasopharyngeal swabs or urine sediments since they can be used for virus isolation as well as direct RT-

C2

B3

B3B3

B3

D4D2

D5H1

H1

H2D5D4 D8

D4

D6D6D4

D5 D6

D4

B3

B3

B2

D10

D4

D4

D4D8

D4

B3

B3

B2

H1

D9

B2

D9, G2 G3

D3

D3

Fig. 7.1 Global distribution of measles genotypes: 1995–2008. Figure shows geographical distri-bution of measles genotypes for regions that have not yet eliminated measles transmission. Figure is adapted from the Weekly Epidemiologic record of WHO (2006). Figure is based on surveillance conducted between 1995 and 2008. The region of the Americas and Australia are not shown because these countries have eliminated measles and have detected multiple genotypes from imported cases. Because countries in the European Region have a complex pattern, only the genotypes associated with major outbreaks in 2005–2008 are indicated on the map

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PCR. Peripheral blood mononuclear cells are a good source of virus, but this sample requires specialized laboratory techniques and biosafety procedures that are not available throughout the laboratory network. RNA from measles virus can be detected in both oral fluid samples and dried blood spots on filter paper and these samples have been used to expand virologic surveillance when the collec-tion and transportation of the standard samples is not logistically feasible. It is noteworthy that the United Kingdom has used oral fluid for routine measles sur-veillance for the last decade and greatly expanded their molecular surveillance capabilities. In all cases, it is imperative to obtain the sample for virologic sur-veillance as soon as possible after onset of rash to maximize chances of isolating virus or detecting RNA (World Health Organization 2007b).

Virus Isolation and Clinical Samples

The Vero/hSLAM cell line is now recommended for routine isolation of measles in the WHO laboratory network. These cells are Vero cells that have been transfected with a plasmid encoding the gene for the human SLAM (signaling lymphocyte-activation molecule) protein (Ono et al. 2001). SLAM has been shown to be a receptor for both wild type and laboratory-adapted strains of measles. The sensitiv-ity of Vero/hSLAM cells for isolation of measles virus is equivalent to that of B95a cells (Kobune et al. 1990) and measles infection of Vero/hSLAM results in the characteristic CPE, syncytium formation. The advantage of the Vero/hSLAM cells compared to B95a cells is that they are not persistently infected with Epstein-Barr virus and therefore are not considered hazardous material. This provides a signifi-cant safety advantage for laboratory workers and greatly facilitates international shipments. Vero/hSLAM cells can also be used to isolate rubella viruses from clini-cal samples with a sensitivity that is similar to that of standard Vero cells.

Though virus isolation is encouraged, many laboratories use reverse tran-scriptase-polymerase chain reaction (RT-PCR) to amplify measles RNA directly from clinical specimens. The use of real-time RT-PCR assays has greatly improved sensitivity (Hubschen et al. 2008; Hummel et al. 2006).

New Molecular Techniques

Though viral genotypes are assigned based on analysis of sequence data, other techniques such as restriction fragment length polymorphism and heteroduplex mobility assays have been proposed (Kreis et al. 1997; Kremer et al. 2007; Mulders et al. 2003). However, it is doubtful that these methods would provide the level of sensitivity required to accurately map transmission pathways. Array-based tech-niques have been used for genotyping, but these techniques are more costly and technically challenging than standard sequencing assays (Neverov et al. 2006). A potential advantage to the array-based techniques would be the ability to rapidly obtain more sequence information from selected viral isolates or samples.

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Measles Vaccines and Antigenic Variation

Sequence analyses have shown that all of the measles vaccine strains are represent-atives of genotype A (Parks et al. 2001a, 2001b; Rota et al. 1994a). This includes both vaccines derived from the original Edmonston isolate of 1954 (e.g., Moraten, Schwarz, Edmonston-Zagreb, AIK-C) as well as vaccines derived from other wild-type viruses isolated during the 1950s and 1960s in China and Japan (e.g., Shanghai-191, Chanchun-47, CAM-70). While this suggests that genotype A viruses may have had a wide distribution in the pre-vaccine era, it is also possible that gen-otype A viruses were more frequently detected because they were easier to isolate in the cell culture systems available at the time. There are few samples or viruses from the pre-vaccine era that are available for genetic analysis. However, one study detected viruses with sequences in genotypes C2 and E in samples obtained during the pre-vaccine era in Denmark (Christensen et al. 2002).

Genotype A viruses have been isolated from a few sporadic measles cases in the last 10 years (Wairagkar et al. 2002), but there have been no reports that this geno-type has been associated with any large outbreaks. Though it is possible that wild-type genotype A viruses are still circulating, there is a strong likelihood that the more recently detected genotype A viruses are vaccine viruses or laboratory con-taminants. Efforts are underway to attempt to identify a set of genetic markers to distinguish wild-type, genotype A viruses from vaccine viruses.

The detection of genetic variation within wild-type measles viruses has led to the suggestion that these viruses have antigenic characteristics that allow them to circulate more efficiently in the presence of vaccine-induced immunity. Although antigenic dif-ferences between measles viruses from the various genotypes have been detected by using monoclonal antibodies and polyvalent antiserum (Giraudon et al. 1988; Santibanez et al. 2002, 2005; Tamin et al. 1994), contemporary wild-type viruses are neutralized by polyclonal antiserum to the vaccine virus (Santibanez et al. 2005; Xu et al. 1998). More importantly, measles vaccination programs, when properly admin-istered, have been exceptionally successful in all parts of the world irrespective of the endemic genotype of wild-type virus. Studies are in progress to explore the potential for biologic differences between measles viruses from different lineages.

The publication of the structure of the H protein, the major target of neutralizing antibodies, has allowed fine mapping of the antigenic sites on the molecule (Colf et al. 2007; Hashiguchi et al. 2007). The structural model shows that one of the regions that is accessible to antibody recognition includes the SLAM binding site and conservation of these key residues may account for the monotypic nature of the virus (Hashiguchi et al. 2007). Analysis of the amino acid sequences of the measles H gene show weak, if any, evidence for selective pressure (Woelk et al. 2001).

SSPE

SSPE, also called Dawson’s encephalitis, is a persistent measles infection of the central nervous system. SSPE is a progressive, invariably fatal, encephalopathy

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characterized by personality changes, mental deterioration, involuntary movements, muscular rigidity, and death. SSPE usually begins 4–10 years after the patient has recovered from naturally acquired measles. Successful isolation of measles virus from brain and lymphoid tissues of SSPE patients (Horta-Barbosa et al. 1969, 1971) clearly established measles virus as the etiologic agent of the disease. However, the introduction of the live-attenuated measles vaccine raised concerns that the vaccine virus might either cause, or in some manner influence the frequency of SSPE, and led to formation of SSPE registries in a number of countries. Epidemiologic studies demonstrated a dramatic decrease in uncomplicated measles as well as the frequency of SSPE (Halsey et al. 1980).

Molecular characterization of measles virus nucleic acid sequences derived from brain biopsy or autopsy has identified wild-type measles sequences with few excep-tions, and not those of the vaccine strains (see review by Campbell et al. 2007). Few genotype A virus sequences have been identified (Cattaneo et al. 1989), and it is interesting that those that have been identified were from the first few SSPE isolates ever obtained. Ironically, it was suggested that Halle, the measles isolate believed to provide direct evidence for defining measles involvement in SSPE, was likely a vaccine virus laboratory contaminant. Halle virus grew very well in cell culture, a characteristic unlike any other subsequent SSPE isolate (Rima et al. 1995a). Because genotype A wild-type viruses were widely circulating in the pre-vaccine era, it is difficult to say with certainty that Halle or any other genotype A virus was not the source of infection resulting in SSPE.

Measles virus genotypes found in association with SSPE clinical specimens reflect the sequences of those viruses that circulated in the geographic region where the patients acquired natural infection. For example, the measles resurgence occur-ring from 1989–1992 in the United States was dominated by measles virus belong-ing to genotype D3. Subsequent characterization of measles sequences associated with SSPE cases over the following 5–12 years identified only genotype D3 sequences. No vaccine sequences were identified from tissues of SSPE patients, regardless of the fact that literally millions of doses of measles containing vaccine were administered over the resurgence period (Bellini et al. 2005). This and other recent studies have clearly demonstrated that measles vaccine virus is not involved in the genesis of SSPE and that the use of measles vaccine not only is beneficial in preventing acute measles, but has all but eliminated SSPE from the United States (Bellini et al. 2005; Jin et al. 2002).

Global Distribution of Measles Genotypes

Virologic surveillance is now well established in all WHO regions. Though surveil-lance in some areas is still not adequate, a global picture has emerged (World Health Organization 2006). In general, three patterns of measles genotype distribution have been described. In countries that still have endemic transmission of measles, the majority of cases are caused by several endemic genotypes that are distributed geo-graphically (Fig. 7. 2 ). In these cases, multiple co-circulating lineages within the

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countries to provide baseline data (World Health Organization 2006). In countries with endemic measles, there is a geographic distribution of genotypes. Some countries have more than one endemic genotype and frequently multiple, co-circulating lineages of virus are present within a genotype, indicative of multiple chains of transmission.

Although the WHO African Region has made substantial progress with reducing measles mortality since 1999 (Wolfson et al. 2007), many African countries have endemic measles and several genotypes have been detected. Clade B viruses are endemic in the central and western parts of sub-Saharan Africa, and analysis of a large number of measles isolates from Nigeria, Ghana, the Gambia, Cameroon, and Sudan has supported the division of clade B into three genotypes: B1, B2, and B3 (World Health Organization 2006; El Mubarak et al. 2002; Hanses et al. 1999; Outlaw et al. 1997; Truong et al. 2001). Genotype B3 has been divided into two clusters. Genotype B3, cluster 1 viruses have been isolated from Cameroon, Ghana, and Nigeria, and as far east as Sudan, Kenya, and Tanzania, suggesting that clade B viruses are widely distributed throughout Africa (El Mubarak et al. 2002). The circulation of genotype B3 cluster 2 viruses appears to be more limited to western Africa (Kouomou et al. 2002). Genotype B2 was considered inactive since, until recently, no representative viruses had been isolated since 1984. However, genotype B2 viruses were detected in southeastern Africa in 2002–2003 primarily in association with cases and importation from Angola and in the Central African Republic (Gouandjika-Vasilache et al. 2006; Smit et al. 2005). This reemergence of what was thought to be an inactive genotype was likely the result of surveillance gaps in some countries in central Africa.

Genotypes D2 and D4 have been the most frequently detected genotypes in the southern part and eastern parts of the African continent (Kreis et al. 1997; Mbugua et al. 2003; Nigatu et al. 2001), though more recent outbreaks in Kenya, Somalia, and Tanzania have been caused by genotype B3 viruses (Rota et al. 2006). A new geno-type, D10, was initially detected in Uganda in 2000 and has been detected in mostly central African countries (World Health Organization 2006; Muwonge et al. 2005).

The northern African countries of Tunisia, Libya, and Algeria have detected genotype B3 presumably imported from sub-Saharan countries. Morocco has also reported continued circulation of genotype C2, once one of the prevalent genotypes in Western Europe (Alla et al. 2002, 2006; Waku-Kouomou et al. 2006). Genotype D4, the most prevalent genotype detected in the Middle East and the Arabian Peninsula (Djebbi et al. 2005), has been also reported in Egypt (2006).

Measles is endemic on the Indian subcontinent. Genotype D4 and D8 viruses have been isolated in India and Nepal, and genotype D4 was detected in Pakistan (Anonymous 2001b; Truong et al. 2001; Wairagkar et al. 2002). Genotype D4 and D8 viruses have also been detected in measles cases imported into the United States from both Pakistan and India (Rota et al. 1998, 2002). Recently, genotype D7 was detected in India (Vaidya et al. 2008). This genotype had been one of the most frequently detected genotypes in Europe in the early part of the decade. The recent detection in India suggests that India may have been the original source of genotype D7 in Europe, but expanded virologic surveillance in India is needed to establish the extent of circulation of genotype D7.

Extensive virologic surveillance in Japan demonstrated a succession of geno-types since surveillance activities began there in the early 1990s. Genotypes D3 and

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D5 had been co-circulating in Japan for most of the 1990s (Katayama et al. 1997; Kubo et al. 2003; Sakata et al. 1993; Takahashi et al. 2000; Yamaguchi 1997) and genotype D5 viruses had been associated with many measles cases imported from Japan (Rota et al. 1998, 2002). More recently, Japan has experienced large out-breaks associated with genotypes D9 and H1 (Kubo et al. 2002; Nakayama et al. 2003; Zhou et al. 2003). In 2007, genotype D5 was apparently reintroduced in Japan and was associated with large outbreaks (Morita et al. 2007).

Elsewhere in Asia, sequence analysis of wild-type measles viruses isolated in throughout the People’s Republic of China show widespread distribution of a single genotype of virus, H1, since 1993 (Xu et al. 1998; Zhang et al. 2007). Sequence analysis of over 300 samples obtained throughout the country has established that genotype H1 is clearly the endemic genotype in China. Viruses that were indistin-guishable from the Chinese genotype H1 viruses were isolated during the outbreak of measles in Korea during 2000–2001 (Na et al. 2001, 2003) and were associated with imported cases in a number of countries. However, endemic circulation of genotype H1 appears to be restricted to China. Wild-type measles viruses in Vietnam are also classified as clade H, but they are sufficiently different from the Chinese viruses to be designated as a separate genotype, H2 (Liffick et al. 2001). It is interesting to note that wild-type measles viruses isolated in Thailand and Cambodia are in geno-type D5 (Horm et al. 2003; Rota et al. 1998) and more closely related to viruses cir-culating in Japan than to viruses circulating in other parts of Asia.

Until 1997, the only measles viruses representing clade G had been isolated in 1983, and this clade was considered to be inactive. In 1997, a virus belonging to clade G was isolated from an Indonesian child who was being treated at a Dutch hospital. The H and N sequences of this virus were sufficiently different from the reference strain for it to be considered a new genotype (G2) within clade G (de Swart et al. 2000). Viruses belonging to genotype G2 have recently been isolated in Indonesia and Malaysia (Rota et al. 2000). In addition, viruses from genotypes G2 and G3 have been isolated in Indonesia and East Timor. RT-PCR and sequence analyses of clinical specimens obtained from measles cases imported into Australia confirmed that G3 was present in East Timor. Genotypes G2, G3, and D9 appear to be the endemic genotypes in Indonesia, East Timor, and possibly Malaysia (Chibo et al. 2002).

In some cases, the circulation of a genotype in a particular country has not been verified by virologic surveillance in the source country but was inferred based upon a consistent pattern of importations. For example, although genotype D3 viruses have never been isolated in the Philippines, there have been several instances of genotype D3 being detected in measles cases imported into the United States from the Philippines (Rota et al. 1996, 1998, 2002). In each of these cases, standard case investigation confirmed that the individuals were traveling in the Philippines during the incubation period.

Because of relatively low vaccination coverage rates in some countries and the constant importation of measles from endemic regions in Asia and Africa, European countries have frequent measles outbreaks. Europe has also experienced rapid change in circulating genotypes. In the early part of this decade, genotypes C2, D6, and D7 were most frequently detected (Hanses et al. 2000; Korukluoglu et al. 2005; Rima

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et al. 1995b; Sakata et al. 1993; Santibanez et al. 1999, 2002). Genotype D6 may be endemic in Turkey (Korukluoglu et al. 2005; Korukluoglu and Zarakolu 2006). In 2005–2006, there were major measles epidemics associated with genotypes B3, D6, and D4, and these genotypes were associated with measles cases exported from Europe to other parts of the world (Korukluoglu and Zarakolu 2006; Kremer et al. 2008). Genotype D6 was associated with a larger outbreak in the Ukraine, which fueled spread cases and small outbreaks throughout Europe, while genotype D4 was detected in a large outbreak in Romania. The decreased diversity of the genotype D6 viruses along with the rapid disappearance of the previously circulating C2 and D7 viruses suggest that vaccination programs had successfully interrupted several chains of transmission. However, measles virus was reintroduced by importation and spread via highly mobile, unvaccinated communities (Kremer et al. 2008).

Measles Elimination

Endemic transmission has been eliminated in many areas of the world, including the countries in the Western hemisphere. Both virologic and epidemiologic data col-lected in the United States between 1989 and 2000 indicated that interruption of transmission of the genotype D3 viruses that were associated with the measles resur-gence of the early 1990s was achieved in 1993 and subsequently maintained (Rota et al. 1996, 1998). Analysis of viruses isolated from measles cases and outbreaks in the United Stated between 1994 and 2007 failed to detect ongoing transmission of an endemic genotype. Rather, the diversity of genotypes detected in the last 15 years is indicative of multiple, imported sources of virus (Rota et al. 1998, 2002). Likewise, the diversity of genotypes detected in Australia and Canada, and the United Kingdom is similar to that observed in the United States, suggesting frequent importation of measles and lack of endemic circulation of virus (Chibo et al. 2000, 2003; Jin et al. 1997; Ramsay et al. 2003; Rota et al. 1998, 2002). However, gaps in the vaccination program may have allowed for re-establishment of endemic trans-mission of measles in the United Kingdom (Asaria and MacMahon 2006).

Though virologic surveillance has improved recently in South America, there is no record of the endemic genotypes that circulated before PAHO launched its very successful measles elimination efforts in the early 1990s. However, molecular epi-demiologic studies have demonstrated interruption of circulation of genotype D6 viruses that were responsible for the large measles outbreak in Sao Paulo in 1997 and subsequent outbreaks in Rio de Janeiro, Argentina, Chile, Bolivia, Haiti, and the Dominican Republic (Barrero et al. 2000; Baumeister et al. 2000; Canepa et al. 2000; Hersh et al. 2000; Oliveira et al. 2002; Siqueira et al. 2001). The record low number of cases and the identification of genotypes other than D6 in association with measles cases imported into South and Central America are consistent with regional elimination (Hersh et al. 2000).

During 2002, indigenous transmission in the Americas was limited to a large outbreak (>2000 cases) that started in Venezuela and spread to Colombia. Viruses isolated in Venezuela were found to be members of what was a previously

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unknown genotype, D9 (Anonymous 2002). At the time, genotype D9 was consid-ered to be a previously undetected endemic genotype in the Americas and its dis-covery cast doubts on the assertion that measles had been eliminated from the region. However, shortly after the outbreaks in Venezuela and Colombia, genotype D9 viruses were found to be circulating in Java, Indonesia and were associated with measles cases imported into Australia (Chibo et al. 2003). Since genotype D9 was found to be circulating in a country with endemic measles, the more likely scenario was that genotype D9 viruses were imported into Venezuela from an unidentified index case (Anonymous 2003).

Measles Reintroduction

Laboratory studies have estimated that the mutation rate of measles virus is similar to those of other RNA viruses (Schrag et al. 1999). However, the high level of genetic stability of measles field isolates was also noted by sequencing viruses from the same genotype that had been isolated several years apart (Rima et al. 1997). In addition, molecular epidemiologic studies of the measles resurgences in Brazil in 1997 (Oliveira et al. 2002) and the United States in 1989–1991 (Rota et al. 1996) suggest that there is very little sequence variation in the N and H genes within a single chain of measles transmission. Nucleotide sequences from the N genes of viruses isolated during the large outbreak in Sao Paulo, Brazil, in 1997 were nearly identical to the sequences obtained from viruses that had spread to other states in Brazil as well as other South American countries during 1997 and 1998 (Oliveira et al. 2002). The genotype D3 viruses that were isolated during the resurgence of measles in the United States during 1989–1991 showed very little sequence variability in both the H and N genes, suggesting that one strain of virus had seeded the entire country (Rota et al. 1996). Even in areas with recent endemic transmission of virus, there appears to be very little sequence variation present in viruses isolated from the same chain of trans-mission. For example, there was very little sequence heterogeneity in viruses obtained during outbreaks that occurred after a mass vaccination campaign in Burkina Faso, suggesting that a single introduction of virus was responsible for the outbreaks (Mulders et al. 2003). This is in contrast to the pattern observed in measles-endemic areas, which shows much more sequence variation within a genotype because the epidemiologic conditions favor maintenance of multiple chains of transmission.

Therefore, measles vaccination programs can reduce the number of co-circulating chains of transmission and eventually interrupt measles transmission. However, viruses are continually being introduced from external sources, and as the number of susceptible individuals increases, sustained transmission of the newly introduced viral genotype is possible. The result of introduction into regions where vaccination programs have failed to maintain a high level of population immunity are apparent rapid changes in the endemic genotype as observed in European countries and/or circulation of viruses with very limited genetic diversity (Kremer et al. 2008; Mulders et al. 2003; Rima et al. 1995b; Santibanez et al. 2002).

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Mapping Transmission Pathways

Molecular epidemiologic studies of measles virus have taught us several valuable lessons about transmission of the virus. One of these lessons is that if large measles out-breaks are occurring anywhere in the world, the viruses are soon detected throughout the world. The lineage of genotype B3 viruses that were associated with a large out-break in Kenya in 2005 were soon detected throughout Europe, the United States, Canada, and Mexico (Fig. 7. 3 ) (Rota et al. 2006). Likewise, there were a number of internationally spread cases and some small outbreaks that could be linked to the large outbreaks in Romania and Ukraine in 2006 (Kremer et al. 2008).

Another lesson is that measles transmission can occur anywhere and that molec-ular techniques are often the only method for identifying the source of an outbreak or case. Exposures can occur in airports or other areas frequented by international travelers such as amusement parks. In 2005, sequence information was used to link cases that occurred in the Netherlands to an exposure in an airport in the United States (Rota et al. 2006), while in 2007 sequence data were used to link cases that occurred in Texas and Michigan to an imported case at an international youth sport-ing event in Pennsylvania (Anonymous 2008a). In 2008, measles cases were imported into the United States primarily from large outbreaks associated with genotypes D5 and D4 in Europe (Anonymous 2008b).

Of course, there are limitations to the ability to map transmission pathways. Molecular studies can confirm independent sources of infection if different geno-types or clearly distinct lineages are detected. However, if viruses from the same lineage are detected in nonlinked cases in a particular country, the molecular data alone may not be able to differentiate between continuous circulation of virus and multiple introductions from the same source.

Challenges

Genetic characterization of wild-type measles viruses provides a means to study the transmission pathways of the virus and is an essential component of laboratory-based surveillance. Virologic surveillance needs to be expanded in all areas of the world and conducted during all phases of measles control.

The greatest challenge to expanding virologic surveillance for measles is to col-lect and transport specimens in a timely and efficient manner. The importance of obtaining samples for viral detection at first contact with the suspected case cannot be understated. In addition, local healthcare workers must have the proper supplies and training needed to obtain, process, and ship samples. This is particularly chal-lenging in developing countries with inadequate infrastructure. The targeted intro-duction of new sampling techniques such as collection of dried blood on filter paper or oral fluid will help to expand virologic surveillance into remote areas (World Health Organization 2008). The WHO Laboratory Network is expanding the capacity of the national and regional laboratories for virus isolation and viral detection. The Vero/hSLAM cell line has been distributed through the network.

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Another significant challenge is the development of protocols for rapid exchange of sequence information. Timely reporting and dissemination of the genotype infor-mation will enhance our ability to track transmission of measles and to evaluate the success of vaccination programs. The planned development of databases and web sites will permit near real-time exchange of genetic information and benefit both laboratorians and public health officials.

References

Anonymous (2002) Outbreak of measles – Venezuela and Colombia 2001–2002. MMWR Morb Mortal Wkly Rep 51:757–760

Anonymous (2003) Absence of transmission of the D9 measles virus – Region of the Americas, November 2002–March 2003. MMWR Morb Mortal Wkly Rep 52(11):228–229

Anonymous (2005a) Progress toward elimination of measles and prevention of congenital rubella infection – European region 1990–2004. MMWR Morb Mortal Wkly Rep 54(7):175–179

Fig. 7.3 a, b Transmission of measles genotype B3 in 2005 (adapted from Rota et al. 2006). In A , the dendrogram shows the relationships among the measles reference strains representing the 23 known measles genotypes. B shows the phylogenetic analysis of nucleoprotein genes (450 nucleotides) of measles viruses from cases in the United States, Mexico, the Netherlands, Canada, Germany, and Kenya during 2005–2006. The unrooted tree includes sequences from cases in Ivory Coast in 2004 and Benin and Nigeria in 2005 as well as selected B3 sequences available from GenBank for comparison. *Collected from the Dagoretti area of Nairobi; the other Nairobi sequences were from cases in the Eastleigh area. The insert to the right highlights the transmission pathways. Note that no epidemiologic link was established with western Africa ( dotted line ). A separate importation of genotype B3 with a link to Spain occurred in Venezuela

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Anonymous (2005b) Global Measles and Rubella Laboratory Network January 2004–June 2005. MMWR Morb Mortal Wkly Rep 54(43):1100–1104

Anonymous (2008a) Multistate measles outbreak associated with an international youth sporting event – Pennsylvania Michigan, and Texas August–September 2007. MMWR Morb Mortal Wkly Rep 57(7):169–173

Anonymous (2008b) Measles--United States, January -July 2008, MMWR Morb Mortal Wkly Rep 57(33):893-896

Alla A, Liffick SL, Newton BR, Elaouad R, Rota PA, Bellini WJ (2002) Genetic analysis of mea-sles viruses isolated in Morocco. J Med Virol 68:441–444

Alla A, Waku-Kouomou D, Benjouad A, Elaouad R, Wild TF (2006) Rapid diversification of measles virus genotypes circulating in Morocco during 2004–2005 epidemics. J Med Virol 78:1465–1472

Asaria P, MacMahon E (2006) Measles in the United Kingdom: can we eradicate it by 2010? BMJ 333:890–895

Bankamp B, Bellini WJ, Rota PA (1999) Comparison of L proteins of vaccine and wild-type measles viruses. J Gen Virol 80:1617–1625

Bankamp B, Lopareva EN, Kremer JR, Tian Y, Clemens MS, Pate R, LaMonte-Fowlkes AC, Kessler JR, Muller CP, Bellini WJ, Rota PA (2008) Genetic variability and mRNA editing frequencies of the phosphoprotein genes of wild-type measles viruses. Virus Res doi:10.1016/j.virusres.2008.04.008 (in press)

Barrero PR, de Wolff CD, Passeggi CA, Mistchenko AS (2000) Sequence analysis of measles virus hemagglutinin isolated in Argentina during the 1997–1998 outbreak. J Med Virol 60:91–96

Baumeister E, Siqueira MM, Savy V, Friedrich F (2000) Genetic characterization of wild-type mea-sles viruses isolated during the 1998 measles epidemic in Argentina. Acta Virol 44:169–174

Bellini WJ, Rota JS, Lowe LE, Katz RS, Dyken PR, Zaki SR, Shieh WJ, Rota PA (2005) Subacute sclerosing panencephalitis: more cases of this fatal disease are prevented by measles immuni-zation than was previously recognized. J Infect Dis 192:1686–1693

Campbell H, Andrews N, Brown KE, Miller E (2007) Review of the effect of measles vaccination on the epidemiology of SSPE. Int J Epidemiol 36:1334–48

Canepa E, Siqueira MM, Hortal M, Friedrich F (2000) Recent measles viral activity in Uruguai: serological and genetic approaches. Acta Virol 44:35–39

Cattaneo R, Schmid A, Spielhofer P, Kaelin K, Baczko K, ter Meulen V, Pardowitz J, Flanagan S, Rima BK, Udem SA, et al (1989) Mutated and hypermutated genes of persistent measles viruses which caused lethal human brain diseases. Virology 173:415–425

Chibo D, Birch CJ, Rota PA, Catton MG (2000) Molecular characterization of measles viruses isolated in Victoria Australia, between 1973 and 1998. J Gen Virol 81:2511–2518

Chibo D, Riddell M, Catton M, Birch C (2002) Novel measles virus genotype East Timor and Australia. Emerg Infect Dis 8:735–737

Chibo D, Riddell M, Catton M, Lyon M, Lum G, Birch C (2003) Studies of measles viruses cir-culating in Australia between 1999 and 2001 reveals a new genotype. Virus Res 91:213–221

Christensen LS, Scholler S, Schierup MH, Vestergaard BF, Mordhorst CH (2002) Sequence analy-sis of measles virus strains collected during the pre- and early-vaccination era in Denmark reveals a considerable diversity of ancient strains. APMIS 110:113–122

Colf LA, Juo ZS, Garcia KC (2007) Structure of the measles virus hemagglutinin. Nat Struct Mol Biol 14:1227–1228

de Swart RL, Wertheim-van Dillen PM, van Binnendijk RS, Muller CP, Frenkel J, Osterhaus AD (2000) Measles in a Dutch hospital introduced by an immuno-compromised infant from Indonesia infected with a new virus genotype. Lancet 355:201–202

Djebbi A, Bahri O, Mokhtariazad T, Alkhatib M, Ben Yahia A, Rezig D, Mohsni E, Triki H (2005) Identification of measles virus genotypes from recent outbreaks in countries from the Eastern Mediterranean Region. J Clin Virol 34:1–6

El Mubarak HS, van de Bildt MW, Mustafa OA, Vos HW, Mukhtar MM, Ibrahim SA, Andeweg AC, El Hassan AM, Osterhaus AD, de Swart RL (2002) Genetic characterization of wild-type measles viruses circulating in suburban Khartoum 1997–2000. J Gen Virol 83:1437–1443

b12325337-0007ztc.indd 145 10/3/2008 10:57:02 AM


Giraudon P, Jacquier MF, Wild TF (1988) Antigenic analysis of African measles virus field iso-lates: identification and localisation of one conserved and two variable epitope sites on the NP protein. Virus Res 10:137–152

Gouandjika-Vasilache I, Waku-Kouomou D, Menard D, Beyrand C, Guye F, Ngoay-Kossy JC, Selekon B, and Wild TF (2006) Cocirculation of measles virus genotype B2 and B3.1 in Central African Republic during the 2000 measles epidemic. J Med Virol 78:964–970

Griffin DE (2001) Measles virus. In: Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman Strauss SE (eds) Fields virology, 4th edn. Lippincott Williams, and Wilkins, Philadelphia, pp 1401–1441

Halsey NA, Modlin JF, Jabbour JT, Dubey L, Eddins DL, Ludwig DD (1980) Risk factors in suba-cute sclerosing panencephalitis: a case-control study. Am J Epidemiol 111:415–424

Hanses F, Truong AT, Ammerlaan W, Ikusika O, Adu F, Oyefolu AO, Omilabu SA, Muller CP (1999) Molecular epidemiology of Nigerian and Ghanaian measles virus isolates reveals a genotype circulating widely in western and central Africa. J Gen Virol 80:871–877

Hanses F, van Binnendijk R, Ammerlaan W, Truong AT, de Rond L, Schneider F, Muller CP (2000) Genetic variability of measles viruses circulating in the Benelux. Arch Virol 145:541–551

Hashiguchi T, Kajikawa M, Maita N, Takeda M, Kuroki K, Sasaki K, Kohda D, Yanagi Y, Maenaka K (2007) Crystal structure of measles virus hemagglutinin provides insight into effective vaccines. Proc Natl Acad Sci U S A 104:19535–19540

Hersh BS, Tambini G, Nogueira AC, Carrasco P, de Quadros CA (2000) Review of regional mea-sles surveillance data in the Americas 1996–99. Lancet 355:1943–1948

Horm SV, Dumas C, Svay S, Feldon K, Reynes JM (2003) Genetic characterization of wild-type measles viruses in Cambodia. Virus Res 97:31–37

Horta-Barbosa L, Fuccillo DA, Sever JL, Zeman W (1969) Subacute sclerosing panencephalitis: isolation of measles virus from a brain biopsy. Nature 221:974

Horta-Barbosa L, Hamilton R, Wittig B, Fuccillo DA, Sever JL, Vernon ML (1971) Subacute sclerosing panencephalitis: isolation of suppressed measles virus from lymph node biopsies. Science 173:840–841

Hubschen JM, Kremer JR, De Landtsheer S, Muller CP (2008) A multiplex TaqMan PCR assay for the detection of measles and rubella virus. J Virol Methods 149:246–250

Hummel KB, Lowe L, Bellini WJ, Rota PA (2006) Development of quantitative gene-specific real-time RT-PCR assays for the detection of measles virus in clinical specimens. J Virol Methods 132:166–173

Jin L, Brown DW, Ramsay ME, Rota PA, Bellini WJ (1997) The diversity of measles virus in the United Kingdom 1992–1995. J Gen Virol 78:1287–1294

Jin L, Beard S, Hunjan R, Brown DW, Miller E (2002) Characterization of measles virus strains causing SSPE: a study of 11 cases. J Neurovirol 8:335–344

Katayama Y, Shibahara K, Kohama T, Homma M, Hotta H (1997) Molecular epidemiology and changing distribution of genotypes of measles virus field strains in Japan. J Clin Microbiol 35:2651–2653


Korukluoglu G, Liffick S, Guris D, Kobune F, Rota PA, Bellini WJ, Ceylan A, Ertem M (2005) Genetic characterization of measles viruses isolated in Turkey during 2000 and 2001. Virol J 2:58

Korukluoglu G, Zarakolu P (2006) Antigenic analysis of wild-type measles viruses currently iso-lated in Turkey. Turk J Pediatr 48:105–108

Kouomou DW, Nerrienet E, Mfoupouendoun J, Tene G, Whittle H, Wild TF (2002) Measles virus strains circulating in Central and West Africa: geographical distribution of two B3 genotypes. J Med Virol 68:433–440

Kreis S, Vardas E, Whistler T (1997) Sequence analysis of the nucleocapsid gene of measles virus isolates from South Africa identifies a new genotype. J Gen Virol 78:1581–1587

Kremer JR, Nguyen GH, Shulga SV, Nguyen PH, Nguyen UT, Tikhonova NT, Muller CP (2007) Genotyping of recent measles virus strains from Russia and Vietnam by nucleotide-specific multiplex PCR. J Med Virol 79:987–994

b12325337-0007ztc.indd 146 10/3/2008 10:57:02 AM


Kremer JR, Brown KE, Jin L, Santibanez S, Shulga SV, Aboudy Y, Demchyshyna IV, Djemileva S, Echevarria JE, Featherstone DF, Hukic M, Johansen K, Litwinska B, Lopareva E, Lupulescu E, Mentis A, Mihneva Z, Mosquera MM, Muscat M, Naumova MA, Nedeljkovic J, Nekrasova LS, Magurano F, Fortuna C, de Andrade HR, Richard JL, Robo A, Rota PA, Samoilovich EO, Sarv I, Semeiko GV, Shugayev N, Utegenova ES, van Binnendijk R, Vinner L, Waku-Kouomou D, Wild TF, Brown DW, Mankertz A, Muller CP, Mulders MN (2008) High genetic diversity of measles virus. World Health Organization European Region 2005–2006. Emerg Infect Dis 14:107–114

Kubo H, Iritani N, Murakami T, Haruki K (2002) Isolation of a wild type measles virus classified as genotype H1 in Osaka City. Jpn J Infect Dis 55:177–179

Kubo H, Iritani N, Seto Y (2003) Co-circulation of two genotypes of measles virus and mutual change of the prevailing genotypes every few years in Osaka, Japan. J Med Virol 69:273–278

Liffick SL, Thi Thoung N, Xu W, Li Y, Phoung Lien H, Bellini WJ, Rota PA (2001) Genetic characterization of contemporary wild-type measles viruses from Vietnam and the People’s Republic of China: identification of two genotypes within clade H. Virus Res 77:81–87

Mbugua FM, Okoth FA, Gray M, Kamau T, Kalu A, Eggers R, Borus P, Kombich J, Langat A, Maritim P, Lesiamon J, Tipples GA (2003) Molecular epidemiology of measles virus in Kenya. J Med Virol 71:599–604

Morita Y, Suzuki T, Shiono M, Shiobara M, Saitoh M, Tsukagoshi H, Yoshizumi M, Ishioka T, Kato M, Kozawa K, Ttanaka-Taya K, Yasui Y, Noda M, Okabe N, Kimura H (2007) Sequence and phylogenetic analysis of the nucleoprotein (N) gene in measles viruses prevalent in Gunma Japan, in 2007. Jpn J Infect Dis 60:402–404

Mulders MN, Truong AT, Muller CP (2001) Monitoring of measles elimination using molecular epidemiology. Vaccine 19:2245–2249

Mulders MN, Nebie YK, Fack F, Kapitanyuk T, Sanou O, Valea DC, Muyembe-Tamfum JJ, Ammerlaan W, Muller CP (2003) Limited diversity of measles field isolates after a national immunization day in Burkina Faso: progress from endemic to epidemic transmission? J Infect Dis 187 Suppl 1:S277–82

Muwonge A, Nanyunja M, Rota PA, Bwogi J, Lowe L, Liffick SL, Bellini WJ, Sylvester S (2005) New measles genotype Uganda. Emerg Infect Dis 11:1522–1526

Na BK, Lee JS, Shin GC, Shin JM, Lee JY, Chung JK, Ha DR, Lee JK, Ma SH, Cho HW, Kang C, Kim WJ (2001) Sequence analysis of hemagglutinin and nucleoprotein genes of measles viruses isolated in Korea during the 2000 epidemic. Virus Res 81:143–149

Na BK, Shin JM, Lee JY, Shin GC, Kim YY, Lee JS, Lee JK, Cho HW, Lee HJ, Rota PA, Bellini WJ, Kim WJ, Kang C (2003) Genetic and antigenic characterization of measles viruses that circulated in Korea during the 2000–2001 epidemic. J Med Virol 70:649–654

Nakayama T, Zhou J, Fujino M (2003) Current status of measles in Japan. J Infect Chemother 9:1–7

Neverov AA, Riddell MA, Moss WJ, Volokhov DV, Rota PA, Lowe LE, Chibo D, Smit SB, Griffin DE, Chumakov KM, Chizhikov VE (2006) Genotyping of measles virus in clinical specimens on the basis of oligonucleotide microarray hybridization patterns. J Clin Microbiol 44:3752–3759

Nigatu W, Jin L, Cohen BJ, Nokes DJ, Etana M, Cutts FT, Brown DW (2001) Measles virus strains circulating in Ethiopia in 1998–1999: molecular characterisation using oral fluid sam-ples and identification of a new genotype. J Med Virol 65:373–380

Oliveira MI, Rota PA, Curti SP, Figueiredo CA, Afonso AM, Theobaldo M, Souza LT, Liffick SL, Bellini WJ, Moraes JC, Stevien KE, Durigon EL (2002) Genetic homogeneity of measles viruses associated with a measles outbreak Sao Paulo Brazil 1997. Emerg Infect Dis 8:808–813

Ono N, Tatsuo H, Hidaka Y, Aoki T, Minagawa H, Yanagi Y (2001) Measles viruses on throat swabs from measles patients use signaling lymphocytic activation molecule (CDw150) but not CD46 as a cellular receptor. J Virol 75:4399–4401

Outlaw MC, Jaye A, Whittle HC, Pringle CR (1997) Clustering of haemagglutinin gene sequences of measles viruses isolated in the Gambia. Virus Res 48:125–131

b12325337-0007ztc.indd 147 10/3/2008 10:57:02 AM


Parks CL, Lerch RA, Walpita P, Wang HP, Sidhu MS, Udem SA (2001a) Analysis of the noncod-ing regions of measles virus strains in the Edmonston vaccine lineage. J Virol 75:921–933

Parks CL, Lerch RA, Walpita P, Wang HP, Sidhu MS, Udem SA (2001b) Comparison of predicted amino acid sequences of measles virus strains in the Edmonston vaccine lineage. J Virol 75:910–920

Ramsay ME, Jin L, White J, Litton P, Cohen B, Brown D (2003) The elimination of indigenous measles transmission in England and Wales. J Infect Dis 187 Suppl 1:S198–S207

Riddell MA, Rota JS, Rota PA (2005) Review of the temporal and geographical distribution of measles virus genotypes in the prevaccine and postvaccine eras. Virol J 2:87

Rima BK, Earle JA, Baczko K, Rota PA, Bellini WJ (1995a) Measles virus strain variations. Curr Top Microbiol Immunol 191:65–83

Rima BK, Earle JA, Yeo RP, Herlihy L, Baczko K, ter Meulen V, Carabana J, Caballero M, Celma ML, Fernandez-Munoz R (1995b) Temporal and geographical distribution of measles virus genotypes. J Gen Virol 76:1173–1180

Rima BK, Earle JA, Baczko K, ter Meulen V, Liebert UG, Carstens C, Carabana J, Caballero M, Celma ML, Fernandez-Munoz R (1997) Sequence divergence of measles virus haemagglutinin during natural evolution and adaptation to cell culture. J Gen Virol 78:97–106

Rota PA, Bellini WJ (2003) Update on the global distribution of genotypes of wild type measles viruses. J Infect Dis 187 Suppl 1:S270–S276

Rota JS, Hummel KB, Rota PA, Bellini WJ (1992) Genetic variability of the glycoprotein genes of current wild-type measles isolates. Virology 188:135–142

Rota JS, Wang ZD, Rota PA, Bellini WJ (1994a) Comparison of sequences of the H, F, and N coding genes of measles virus vaccine strains. Virus Res 31:317–330

Rota PA, Bloom AE, Vanchiere JA, Bellini WJ (1994b) Evolution of the nucleoprotein and matrix genes of wild-type strains of measles virus isolated from recent epidemics. Virology 198:724–730

Rota JS, Heath JL, Rota PA, King GE, Celma ML, Carabana J, Fernandez-Munoz R, Brown D, Jin L, Bellini WJ (1996) Molecular epidemiology of measles virus: identification of pathways of transmission and implications for measles elimination. J Infect Dis 173:32–37

Rota JS, Rota PA, Redd SB, Redd SC, Pattamadilok S, Bellini WJ (1998) Genetic analysis of measles viruses isolated in the United States 1995–1996. J Infect Dis 177:204–208

Rota PA, Liffick S, Rosenthal S, Heriyanto B, Chua KB (2000) Measles genotype G2 in Indonesia and Malaysia. Lancet 355:1557–1558

Rota PA, Liffick SL, Rota JS, Katz RS, Redd S, Papania M, Bellini WJ (2002) Molecular epide-miology of measles viruses in the United States 1997–2001. Emerg Infect Dis 8:902–908

Rota J, Lowe L, Rota P, Bellini W, Redd S, Dayan G, van Binnendijk R, Hahne S, Tipples G, Macey J, Espinoza R, Posey D, Plummer A, Bateman J, Gudino J, Cruz-Ramirez E, Lopez-Martinez I, Anaya-Lopez L, Holy Akwar T, Giffin S, Carrion V, de Filippis AM, Vicari A, Tan C, Wolf B, Wytovich K, Borus P, Mbugua F, Chege P, Kombich J, Akoua-Koffi C, Smit S, Bukenya H, Bwogi J, Baliraine FN, Kremer J, Muller C, Santibanez S (2006) Identical genotype B3 sequences from measles patients in 4 countries 2005. Emerg Infect Dis 12:1779–1781

Sakata H, Kobune F, Sato TA, Tanabayashi K, Yamada A, Sugiura A (1993) Variation in field iso-lates of measles virus during an 8-year period in Japan. Microbiol Immunol 37:233–237

Santibanez S, Heider A, Gerike E, Agafonov A, Schreier E (1999) Genotyping of measles virus isolates from central Europe and Russia. J Med Virol 58:313–320

Santibanez S, Tischer A, Heider A, Siedler A, Hengel H (2002) Rapid replacement of endemic measles virus genotypes. J Gen Virol 83:2699–2708

Santibanez S, Niewiesk S, Heider A, Schneider-Schaulies J, Berbers GA, Zimmermann A, Halenius A, Wolbert A, Deitemeier I, Tischer A, Hengel H (2005) Probing neutralizing-anti-body responses against emerging measles viruses (MVs): immune selection of MV by H pro-tein-specific antibodies? J Gen Virol 86:365–374

Schrag SJ, Rota PA, Bellini WJ (1999) Spontaneous mutation rate of measles virus: direct estima-tion based on mutations conferring monoclonal antibody resistance. J Virol 73:51–54

b12325337-0007ztc.indd 148 10/3/2008 10:57:03 AM


Siqueira MM, Castro-Silva R, Cruz C, Oliveira IC, Cunha GM, Mello M, Rota PA, Bellini WJ, Friedrich F (2001) Genomic characterization of wild-type measles viruses that circulated in different states in Brazil during the 1997 measles epidemic. J Med Virol 63:299–304

Smit SB, Hardie D, Tiemessen CT (2005) Measles virus genotype B2 is not inactive: evidence of continued circulation in Africa. J Med Virol 77:550–557

Takahashi M, Nakayama T, Kashiwagi Y, Takami T, Sonoda S, Yamanaka T, Ochiai H, Ihara T, Tajima T (2000) Single genotype of measles virus is dominant whereas several genotypes of mumps virus are co-circulating. J Med Virol 62:278–285

Tamin A, Rota PA, Wang ZD, Heath JL, Anderson LJ, Bellini WJ (1994) Antigenic analysis of current wild type and vaccine strains of measles virus. J Infect Dis 170:795–801

Taylor MJ, Godfrey E, Baczko K, ter Meulen V, Wild TF, Rima BK (1991) Identification of sev-eral different lineages of measles virus. J Gen Virol 72:83–88

Truong AT, Mulders MN, Gautam DC, Ammerlaan W, de Swart RL, King CC, Osterhaus AD, Muller CP (2001) Genetic analysis of Asian measles virus strains – new endemic genotype in Nepal. Virus Res 76:71–78

Vaidya SR, Wairagkar NS, Raja D, Khedekar DD, Gunasekaran P, Shankar S, Mahadevan A, Ramamurty N (2008) First detection of measles genotype D7 from India. Virus Genes 36:31–34

Wairagkar N, Rota PA, Liffick S, Shaikh N, Padbidri VS, Bellini WJ (2002) Characterization of measles sequences from Pune India. J Med Virol 68:611–614

Waku-Kouomou D, Alla A, Blanquier B, Jeantet D, Caidi H, Rguig A, Freymuth F, Wild FT (2006) Genotyping measles virus by real-time amplification refractory mutation system PCR represents a rapid approach for measles outbreak investigations. J Clin Microbiol 44:487–494

Woelk CH, Jin L, Holmes EC, Brown DW (2001) Immune and artificial selection in the haemag-glutinin (H) glycoprotein of measles virus. J Gen Virol 82:2463–2474

Wolfson LJ, Strebel PM, Gacic-Dobo M, Hoekstra EJ, McFarland JW, Hersh BS (2007) Has the 2005 measles mortality reduction goal been achieved? A natural history modelling study. Lancet 369:191–200

World Health Organization (1998) Standardization of the nomenclature for describing the genetic characteristics of wild-type measles viruses. Wkly Epidemiol Rec 73:265–272

World Health Organization (2001a) Nomenclature for describing the genetic characteristics of wild-type measles viruses (update) Part I. Wkly Epidemiol Rec 76(32):242–247

World Health Organization (2001b) Nomenclature for describing the genetic characteristics of wild-type measles viruses (update) Wkly Epidemiol Rec 76(33):249–251

World Health Organization (2003) Update of the nomenclature for describing the genetic charac-teristics of wild-type measles viruses: new genotypes and reference strains. Wkly Epidemiol Rec 78(27):229–232

World Health Organization (2005a) Global Measles and Rubella Laboratory Network – update. Wkly Epidemiol Rec 80(44):384–388

World Health Organization (2005b) New genotype of measles virus and update on global distribu-tion of measles genotypes. Wkly Epidemiol Rec 80(40):347–351

World Health Organization (2005c) Standardization of the nomenclature for genetic characteris-tics of wild-type rubella viruses. Wkly Epidemiol Rec 80(14):126–132

World Health Organization (2006) Global distribution of measles and rubella genotypes – update. Wkly Epidemiol Rec 81(51/52):474–479

World Health Organization (2007a) Update of standard nomenclature for wild-type rubella viruses 2007. Wkly Epidemiol Rec 82(24):216–222

World Health Organization (2007b) Manual for the diagnosis of measles and rubella infection, 2nd edn. World Health Organization, Geneva

World Health Organization (2008) Measles and rubella laboratory network: 2007 meeting on the use of alternative sampling techniques for surveillance. Wkly Epdemiol Rec 83:225–232

Xu W, Tamin A, Rota JS, Zhang L, Bellini WJ, Rota PA (1998) New genetic group of measles virus isolated in the People’s Republic of China. Virus Res 54:147–156

b12325337-0007ztc.indd 149 10/3/2008 10:57:03 AM


Yamaguchi S (1997) Identification of three lineages of wild measles virus by nucleotide sequence analysis of N, P, M, F, and L genes in Japan. J Med Virol 52:113–120

Zhang Y, Zhu Z, Rota PA, Jiang X, Hu J, Wang J, Tang W, Zhang Z, Li C, Wang C, Wang T, Zheng L, Tian H, Ling H, Zhao C, Ma Y, Lin C, He J, Tian J, Ma Y, Li P, Guan R, He W, Zhou J, Liu G, Zhang H, Yan X, Yang X, Zhang J, Lu Y, Zhou S, Ba Z, Liu W, Yang X, Ma Y, Liang Y, Li Y, Ji Y, Featherstone D, Bellini WJ, Xu S, Liang G, Xu W (2007) Molecular epidemiology of measles viruses in China 1995–2003. Virol J 4:14

Zhou J, Fujino M, Inou Y, Kumada A, Aoki Y, Iwata S, Nakayama T (2003) H1 genotype of mea-sles virus was detected in outbreaks in Japan after 2000. J Med Virol 70:642–648

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Chapter 8 Human Immunology of Measles Virus Infection

D. Naniche

Abstract Measles is a highly contagious disease, which was responsible for high infant mortality before the advent of an effective vaccine in 1963. In immuno-competent individuals, measles virus (MV) infection triggers an effective immune response that starts with innate responses and then leads to successful adaptive immunity, including cell-mediated immunity and humoral immunity. The virus is cleared and lifelong protection is acquired. However, changing epidemiology of measles due to vaccination as well as severe immunodeficiency has created new pockets of individuals vulnerable to measles. This chapter reviews the knowledge on effective measles-specific immune responses induced by natural infection and vaccination and explores problems arising in specific cases of immunodeficiency, infant immunity, and ineffective vaccination against measles.

D. Naniche Barcelona Center for International Health Research (CRESIB), Hospital Clinic , Institut d’Investigacions Biomedicas August Pi i Sunyer (IDIBAPS) , C/Rossello 132, 4 08036 , Barcelona , Spain, e-mail: [email protected].

D.E. Grippin and M.B.A. Oldstone (eds.) Measles – Pathogenesis and Control. 151© Springer-Verlag Berlin Heidelberg 2009

Contents

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152Epidemiology of Measles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152Natural History of Clinical Measles Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153Immune Response to Natural Measles Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

Types of Immunity Involved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154Humoral Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155Cellular Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157Immunogenetic Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158Longevity and Measles Vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

Measles Immunity in Infants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160Measles Immunity in Immunocompromised Hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

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152 D. Naniche

Abbreviations

MV Measles virus IL Interleukin IFN Interferon TNF Tumor necrosis factor TLR Toll-like receptors Th1 T-helper type 1 Th2 T-helper type 2 CTL Cytotoxic T cell PBMC Peripheral blood mononuclear cells HIV Human immunodeficiency virus HAART Highly active antiretroviral therapy

Introduction

Before the development of an effective vaccine in 1963, measles was responsible for a high level of mortality throughout the world. Essentially a rite of passage dur-ing childhood, those who survived measles infection acquired a lifelong immunity to measles in the absence of re-exposure to the virus. The landmark study of mea-sles in the Faroe Island population during the 1846 epidemic established the basic principles of measles epidemiology, transmissibility, incubation period, and life-long immunity. Measles entered an isolated island population that had not had an measles epidemic in 65 years. The attack rate was nearly 100% for susceptible individuals, but all persons over 65 years of age were protected by immunity from the previous outbreak (Panum 1938). Since this crucial observation regarding mea-sles immunology, great advances have been made in the understanding of immune responses to natural measles infection and to the licensed measles vaccine; how-ever, many fundamental questions still remain unanswered.

Epidemiology of Measles

The advent of an effective attenuated measles vaccine in the 1960s and worldwide generalization of vaccination through the expanded program of immunization (EPI) in the following decades led to a dramatic decrease in measles-related mortality. In the early 1960s, over 135 million cases of measles and over 6 million measles-related deaths were estimated to have occurred yearly (Clements and Hussey 2004). Through intensive international efforts scaled up in the late 1990s to diversify immunization strategies with a goal of reducing the mortality due to measles, the annual number of measles deaths was reduced by 60% between 1999 and 2005 from an estimated 873,000 (634,000–1,140,000) to 345,000 (247,000–458,000) (Centers

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8 Human Immunology of Measles Virus Infection 153

for Disease Control 2006; Wolfson et al. 2007). Despite high vaccine coverage lev-els, a small pool of susceptible individuals is sufficient for measles to occur in the form of sporadic outbreaks spaced further and further apart in time as vaccination coverage is increased. Thus occasional clusters of cases in industrialized countries and outbreaks in endemic countries occur. This is mainly due to the clusters of susceptible individuals remaining as a result of heterogeneous worldwide vaccine coverage, a 2%–5% vaccine failure, and increased international travel.

Measles case fatality ratios (CFRs) vary depending on the age of infection, the nutritional status, and access to healthcare. Measles CFR was historically as high as 30% and currently may be as low as 1 in 1000 in industrialized countries, 5%–10% in endemic areas in sub-Saharan Africa, and may reach 25% in refugee camps or other situations of overcrowding (Aaby 1988; Aaby and Clements 1989; Cutts et al. 1991; Moss 2007).

Prior to mass vaccination, major epidemics occurred approximately every 2–3 years, and the highest occurrence of the disease was in children, with the highest risk of complications and death in children younger than 5 years of age and in adults over 20 years of age (Perry and Halsey 2004). In the United States, it was estimated that more than 90% of individuals had contracted measles by the age of 15 (Langmuir 1962). Vaccination dramatically changed the epidemiology of measles by shifting the peak age of infection to older children. Vaccination also shifted the burden of disease out of the age group with the highest case-fatality (infancy) and thus contributed to reducing measles-associated mortality (Moss 2007). However, this changing epidemiology created new pockets of vulnerability to measles in partially protected or unprotected infants below immunization age, and in the young adult populations with waning vaccine-induced immunity (Anders et al. 1996).

Natural History of Clinical Measles Infection

Measles virus is a highly contagious disease transmitted from person to person by respiratory droplets or airborne spray to mucous membranes in the upper respiratory tract. Measles usually presents clinically with prodromal symptoms of fever, cough, and conjunctivitis occurring 10–12 days after exposure and lasting 2–4 days prior to the appearance of a characteristic rash and high fever (Griffin 2007). The virus ini-tially spreads to the regional lymphoid tissue, where the virus undergoes extensive replication and establishes a primary viremia allowing spread of the virus to other lymphoid tissue including spleen and liver. A second phase of viremia occurs approximately 5–7 days after exposure accompanied by characteristic lymphopenia and dissemination of virus to multiple organs and epithelium. The rash marks the onset of the immune response to measles, coinciding with the appearance of mea-sles-specific humoral and cellular immunity. The uncomplicated clinical course of measles accompanied by an effective immune response leads to decreased symptoms and clearance of detectable virus by 7–10 days after initiation of the rash. However,

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the transient immunosuppression caused by measles can lead to severe complica-tions including otitis media, laryngotracheobronchitis, pneumonia, diarrhea, febrile seizures, and encephalitis (reviewed in Perry and Halsey 2004). In a vaccinated population, children under 5 years of age, adults over 20 years, as well as malnour-ished and immunocompromised individuals are at greater risk of serious complica-tions (reviewed in Perry and Halsey 2004). The high complication rate and mortality historically associated with measles is in large part due to opportunistic infections that arise during measles-induced immunosuppression. Measles can also cause a rare fatal degenerative persistent central nervous system disease called subacute sclerosing panencephalitis (SSPE). SSPE occurs 5–15 years after measles infection in 1 per 100,000 cases of measles and results in motor disabilities, coma, and death (Jabour et al. 1969; Termeulen et al. 1983).

Immune Response to Natural Measles Infection

Types of Immunity Involved

In immunocompetent individuals, measles (MV) infection elicits a strong measles-specific immune response that clears infection. Concomitantly, MV is responsible for a generalized immunosuppression that dampens immune responses to other pathogens. Much research has been dedicated to the immunological paradox by which MV-specific immune activation co-exists with nonspecific immune suppression. The mechanisms of MV-induced immunosuppression are discussed in chapter 12 of this publication and the present chapter further discusses MV-specific immune responses.

The first immune response to be activated by many viral infections, including MV, is the innate immune response. Specific receptors including the Toll-like receptors (TLRs) of the innate immune system detect the presence of viruses and trigger production of proinflammatory cytokines, interferons, and TNF. However, many viruses have developed strategies to subvert these innate responses and pur-sue their replication.

Measles infection triggers the activation of TLRs, type I interferons, cytokines, and RNA-binding proteins (Berghall et al. 2006; Bieback et al. 2002). Wild-type measles has been shown to activate TLR2, which leads to the activation of TLR responsive genes in macrophages (IL-1, IL-6, IL-12p40), thus contributing to immune activation and to the start of the adaptive immune response (Bieback et al. 2002). However, wild-type measles has been shown to induce very low levels of type I IFN and possesses mechanisms to suppress type I IFN production (Naniche et al. 2000; Shingai et al. 2007) and signaling (Fontana et al. 2008; Palosaari et al. 2003; Shaffer et al. 2003; Takeuchi et al. 2003). In parallel to activation of TLRs, MV can suppress cytokine production due to activation of TLRs such as TLR4-activated IL-12 production (Hahm et al. 2007). IL-12 is thought to be a cytokine that participates in linking the innate and adaptive immune responses and its

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production can be inhibited by MV both in vitro and in vivo (Atabani et al. 2001; Karp et al. 1996; Polack et al. 2002). This reflects the generalized immunosuppres-sion induced by MV, which impairs responses to other pathogens (discussed in chapter 12).

The immune correlates of protection against measles have been defined but the relative contributions of nonspecific innate immunity and specific cellular and humoral immunity are not completely understood. The humoral arm of the immune response has been the prime focus for determining vaccine efficacy and assessing protection against measles. However, several experiments of nature and studies in monkey models have demonstrated that cell immunity is crucial for recovery from measles infection (Markowitz et al. 1988; Mitus et al. 1962; Permar et al. 2003). Children with agammaglobulinemia have a normal course of measles infection and clear the virus with no increased complications, whereas children with cellular immune deficiencies, such as leukemia, are unable to clear measles virus infection and develop progressive illness and death from measles (Burnet 1968; Good and Zak 1956; Markowitz et al. 1988). Furthermore, in CD8-lymphocyte-depleted rhe-sus macaques, infection with measles virus leads to more extensive viremia, disease, and death (Permar et al. 2003, 2004).

Humoral Immunity

Measles-specific antibody is first detectable at the onset of the rash and titers rise rapidly thereafter (Bech 1959). Detailed kinetic studies have shown that 77% of individuals develop measles-specific IgM within 72 h after rash onset. By 11 days, 100% have detectable IgM and over 90% maintain IgM for 28 days (Helfand et al. 1997, 1999). IgG responses are generated shortly thereafter, peak at 3–4 weeks, and are lifelong (Stokes 1961).

Antibody is induced to most MV proteins but appears first and most abundantly to the nucleocapsid (N) protein (Norrby and Gollmar 1972) and then to the hemag-glutinin (H) and fusion (F) proteins and very little to the matrix (M) protein (Norrby et al. 1981; Stephenson and ter Meulen 1979). Measles is a monotypic virus and thus is considered to have one serotype and be subdivided into various genotypes. Thus a polyclonal measles-specific serum can neutralize all strains of measles (Bellini et al. 1994; Bellini and Rota 1998). The majority of neutralizing antibodies are specific for the H protein, although there are F-specific antibodies that neutral-ize MV (Black 1989; Malvoisin and Wild 1990).

Measles-specific antibody can be either neutralizing or non-neutralizing. The gold standard for measuring protective neutralizing antibodies to measles is by the plaque reduction neutralizing (PRN) assay, although other ELISA and hemaggluti-nation-inhibition assays are also used (Griffin 2007; Ratnam et al. 1995). Based on studies in outbreaks in partially vaccinated populations, a specific neutralizing antibody titer has been assigned by WHO at 200 mIU/ml as a necessary level con-ferring protection to measles (Chen et al. 1990; Samb et al. 1995; World Health

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Organization 1993). Quantitative aspects of humoral immunity are well character-ized and there is growing information that the quality of the antibody response, including factors such as IgG subclass, complement-fixing capacity, and antibody avidity, is also important for protection. Isotype selection arises from recombina-tion of IgG heavy chain exons resulting in antibodies containing Fc regions with different effector functions, including capacity for complement activation and opsonization (Paul 2003). IgG1 and IgG3 have been found to be the predominating antibodies elicited during acute measles, similar to other respiratory viral infec-tions. During the convalescent phase of measles and long-term humoral memory, IgG1 and then IgG4 appear to remain relatively high, whereas IgG3 decreases (El Mubarak et al. 2004). MV neutralization activity is primarily mediated by H- specific IgG1 and IgG3 isotypes with little contribution from IgG2 and IgG4 (Audet et al. 2006). Complement-activating capacity is primarily mediated by IgG4 (Paul 1989).

Another parameter of the qualitative antibody response is an antibody’s avidity for antigen, which stems from somatic hypermutation of the variable region of the IgG (responsible for epitope recognition), leading to the process of affinity matura-tion, whereby antibody-producing cells secrete antibodies with progressively higher affinities for their antigen (Paul 2003). There is increasing evidence that high antibody avidity is correlated with protection or better prognosis for several viral infections and vaccine responses (Bouche et al. 2002; Griffin 2007). Measles-spe-cific IgG undergo affinity maturation after infection, which appears to increase throughout the first 3 months after infection (El Mubarak et al. 2004). It has been shown that improper measles vaccine design can lead to short-lived inadequate antibody responses. This was the case of the formalin-inactivated measles vaccine in use in the United States between 1963 and 1968 and withdrawn because of poor protection and atypical measles syndrome upon exposure to natural measles infec-tion (Fulginiti et al. 1967). It was demonstrated that this formalin-inactivated vac-cine induced production of non-neutralizing antibodies with low-affinity and high complement-fixing capacity. This process resulted in immune complex-mediated disease leading to atypical measles pathology upon natural measles infection. This example thus highlights the importance of the quality of the antibody response in conferring protection to re-infection (Polack et al. 2003).

MV-specific neutralizing antibodies correlate with protection at the time of virus exposure and play a key role in preventing measles infection following natural infection, vaccination, or passive transfer of antibodies (Albrecht et al. 1977; Black 1989; Chen et al. 1990; Halsey et al. 1985; Panum 1938). However, the role of antibodies in clearance of MV once the infection is initiated remains unclear. Studies in non-human primates have suggested that humoral immunity plays a minimal role in clearance of replicating MV (Permar et al. 2003, 2004). However, others suggest that antibodies may contribute and/or accelerate the clearance of measles infection mainly driven by the cellular immune responses (Forthal et al. 1994; Fujinami and Oldstone 1979, 1980).

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Cellular Immunity

Early work has shown evidence of detectable T cell activity during the measles rash when plasma levels of IFN-gamma and soluble IL-2 receptor increase (Griffin et al. 1990). The cellular immune response has been shown to bloom at the onset of the rash as plasma levels of soluble CD8 and beta-2 microglobulin increase (Griffin et al. 1989, 1990, 1992) and measles-specific cytotoxic T cells are detected (Jaye et al. 1998; Nanan et al. 1995; Nanan et al. 2000; Vanbinnendijk et al. 1990). Acute measles has been described to generate a T-helper type 1 (Th1) response profile, including IFN-gamma and IL-2, whereas the convalescent phase of MV infection appears to be skewed towards T-cell helper type 2 (Th2) responses, including the type 2 cytokines IL-4 and IL-5 (Griffin and Ward 1993; Griffin et al. 1989, 1990). In patients with measles infection, from both industrialized and developing coun-tries, type 2 cytokine skewing has been shown by abundant production of IL-4, presence of IL-10, and little IFN-γ after in vitro stimulation with T cell mitogens (Griffin and Ward 1993; Moss et al. 2002; Ward et al. 1991). Furthermore, MV has been shown to suppress mitogen-induced IL-12 production after in vitro infection and during measles in humans and in rhesus macaques (Atabani et al. 2001; Karp et al. 1996; Polack et al. 2002).

Great advances in cellular immunology have shown that T cell immune responses are complex and involve various phases, including the effector phase, effector memory phase, and central memory phase. Effector cell types are also more hetero-geneous than initially thought and encompass capacities for cytotoxicity, cytokine-production, suppression, proliferation, and differential homing (Reiner et al. 2007). Studies have confirmed that after in vitro restimulation with measles antigens, IFN-γ-producing T cells can be detected during acute measles and measles vaccination, confirming the existence of T cells with a Th1 profile during the acute response (Gans et al. 1999; Ovsyannikova et al. 2003). At later phases, months to years after natural infection or vaccination, restimulation of peripheral blood mononuclear cells (PBMCs) with MV antigens detects measles-specific T cells that can produce IFN-γ or IL-4 (Dhiman et al. 2005a; Howe et al. 2005; Nanan et al. 2000; Ovsyannikova et al. 2003). One study detected a predominance of IFN-γ-producing cells with a small percentage of individuals who exclusively produced IL-4 after MV stimulation (Dhiman et al. 2005b). Early studies specifically assessed Th1/Th2 polarization during and after measles infection or vaccination with relation to immunosuppression of responses to other non-MV antigens and nonspecific stimu-lation rather than as a study of the polarization of MV-specific responses. Measles-induced Th2 polarization may thus be a manifestation of measles-induced immunosuppression occurring later in the course of infection after an effective MV-specific response has been initiated. Thus Th2 skewing may not necessarily affect an uncomplicated immune response to natural measles infection or vaccination. The kinetics of Th1/Th2 polarization during and after measles infection or vaccination

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may depend on the stage of the T cell response (acute, memory), on methods of antigen restimulation used for detection in vitro, and/or on the type of memory cell detected (effector or central memory). At a given time postinfection or vaccination, antigen-specific effector and memory T lymphocytes are present at many differen-tiation stages ranging from the uncommitted Th0 cells to pre-Th2/pre-Th1 and committed cells (Lanzavecchia and Sallusto 2005).

Both CD8 and CD4 cells specific for various measles proteins and specific epitopes have been described in murine and non-human primate models (reviewed in van Els and Nanan 2002). All main structural MV proteins are recognized by T cells in the context of either MHC class I or class II molecules. A number of these epitopes have been confirmed in humans during natural infection or vaccination, including epitopes in the H, M, and C proteins (Jaye et al. 2003; Ota et al. 2007; van Els and Nanan 2002). Since HLA-A2 is an abundant class I histocompatibility antigen, many epitopes have been characterized for this human class I molecule (Jaye et al. 2003; Ota et al. 2007). There is no gold standard for measuring cellular immunity to measles and the diverse techniques used include ex vivo cytotoxicity assays that detect ongoing immune responses, as well as in vitro restimulation with MV antigens followed by cytotoxicity assays with ELISPOT or flow cytometry to detect memory cytokine-producing CD8 and/or CD4 T cells. The variation of techniques has introduced a degree of difficulty in comparing studies and in drawing conclusions regarding the correlates of protective immunity to measles infection both during acute infection and for long-term memory. Furthermore, it is not clear whether natural measles-induced T cell responses differ from vaccine-induced T cell responses in terms of the quantity, quality, and longevity of the T cell response.

Immunogenetic Factors

Genetic factors play a role in the immune response and disease progression in vari-ous viral infections such as hepatitis B, West Nile, EBV, herpes, and HIV, as well as in vaccine responses (Helminen et al. 2001; Hurme et al. 2003; Kaslow et al. 2005; Thio et al. 2003; Yakub et al. 2005). Genetic polymorphisms that could potentially have an impact on immune response to measles infection and/or to vac-cination are present in many steps of the immune response, including T cell recog-nition, cell signaling, cytokine production, antibody isotype switching, and many others. Several studies have assessed polymorphisms in HLA genes, cytokine, cytokine receptor genes, and MV receptor genes (SLAM and CD46) and their potential association with parameters of cellular and humoral measles immune response after measles vaccination.

Associations have been identified between HLA class I and class II alleles and levels of measles-specific IFN-γ and IL-4 production (Ovsyannikova et al. 2005a; Ovsyannikova et al. 2005b). Other associations have been identified between genetic polymorphisms in IL-2, IL-10, and IL-12b receptor genes and levels of

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measles-specific antibody production and lymphoproliferation (Dhiman et al. 2007a; Ovsyannikova et al. 2006). Furthermore, polymorphisms in measles SLAM and CD46 have been found to be associated with lower measles-specific antibody responses when measured at a median of 4.8 years after a second dose of measles vaccine (Dhiman et al. 2007b).

These studies have been conducted in children years after having received one dose or two doses of measles vaccine and living in areas with very little to no natu-ral boosting from outbreaks of measles. It is not known whether these polymor-phisms only play a role in immune memory or are also applicable to the initial immune response upon vaccination. It is likely, although unknown, that these poly-morphisms and others can be extrapolated to the immune response after natural measles infection. Genetic polymorphisms could thus impact the efficiency, breadth, and the quality of the measles-specific immune response, which could have consequences on viral replication, transmission, and disease severity.

Recently, gene expression array studies during acute measles virus infection have been conducted and have identified genes potentially involved in immunological changes both in PBMCs and in dendritic cells (Zilliox et al. 2006, 2007). Many genes were observed to be up- or downregulated during measles infection and the 1st month of follow-up. Genes included inflammatory cytokines and their recep-tors, chemokines and their receptors, cell surface adhesion, signaling, and transcrip-tion regulation. These studies open interesting areas of research into the immunogenetics involved in the ability to control acute measles virus infection, including in immunosuppressed patients.

Longevity and Measles Vaccination

As shown by the Faroe Island example, measles immunity after natural infection is lifelong in the absence of natural boosting. However, it appears that measles- vaccine induced protective immunity after vaccination with a single dose may not be lifelong and it is yet unknown whether two doses of the vaccine can elicit life-long immunity. Several studies have evaluated the duration of measles-specific T cell immunity after vaccination. The cytotoxic T lymphocytes (CTLs) elicited immediately after vaccination are indistinguishable from those induced after natu-ral measles in terms of lysis, restimulation capacity, and recognition of measles proteins (Jaye et al. 1998). Short-term prospective studies have analyzed T cell responses up to 8 months after vaccination and showed that PBMCs from infants vaccinated at 6, 9, and 12 months of age were able to proliferate and secrete cytokines (IL-2 and IFN-γ) in response to in vitro measles stimulation assessed at 3 months postvaccination (Gans et al. 1999). T cell proliferative capacity has also been detected in subjects up to 15 years after vaccination (Bautista-Lopez et al. 2000; Toyoda et al. 1999). A study conducted in the United States suggested that CTL activity might be weaker in vaccinated individuals 16 years postvaccination as compared to those having had natural measles over 23 years earlier (Wu et al.

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1993). Another study showed that both measles- specific CD4 and CD8 T cells could be detected up to 34 years after vaccination and up to 47 years after natural measles infection. It was also suggested that vaccine-induced measles-specific CD4 cells may wane over time (Naniche et al. 2004).

There are a greater number of studies assessing the waning of measles-specific antibody after vaccination than the waning of cell immunity including studies assessing measles-specific antibody titer years after a single dose. Studies have documented both stable and waning titers of vaccine-induced measles-specific antibody and models have been established to estimate the decay rate of MV- vaccine-induced antibody titer (Bautista-Lopez et al. 2001; Dai et al. 1991; Lee et al. 2001; Lee and Nokes 2001; Mossong et al. 2000; Weibel et al. 1980; Whittle et al. 1999). Two studies estimated the half-life of anti-MV antibody in two different geographic regions: 12 years in Canada and 5.5 years in Taiwan (Lee and Nokes 2001; Mossong et al. 2000). It is likely that the longevity of vaccine-induced humoral immunity may differ according to genetic, demo-graphic, and environmental factors as well as to the immunocompetence status of individuals.

Measles-Specific Immunity in Infants

Infant immunity to measles is a special situation whereby immunity depends on the presence and transfer of maternal antibody to the infant. Levels of transpla-centally acquired measles-specific antibody depend on the amount of maternal antibody transferred and the rate of antibody decay in the infant. This results in a variable window of vulnerability between the waning of the protection con-ferred by maternal antibodies and generation of infant antibodies after exposure to measles or measles vaccination. The recommendation for vaccination at 12–15 months of age was made at a time when mothers had measles-specific anti-body induced by natural infection, which they transferred to their infants transplacentally. Vaccination at 12–15 months ensured that no passively trans-ferred maternal antibody remained in the infant that could hamper vaccine effi-cacy. In contrast, after four decades of extensive measles vaccination, currently most infants are born to mothers who have vaccine-induced immunity, thus transferring less maternal antibody to their infants. Studies in highly vaccinated populations have shown higher susceptibility of infants to measles and a win-dow of vulnerability with little to no protective measles antibodies as early as 4–6 months (Dabis et al. 1989; de Francisco et al. 1998; Markowitz et al. 1996; Nicoara et al. 1999; Papania et al. 1999; Tapia et al. 2005). Furthermore, in developing countries, other factors such as poor nutrition, high parity, low birth weight, HIV infection, and anemia can further contribute to lower measles antibody titers in infants (de Moraes-Pinto et al. 1998; Farquhar et al. 2005; Okoko et al. 2001a, b; Owens et al. 2006; Scott et al. 2005, 2007; Wesumperuma et al. 1999).

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In many developing countries with high exposure to measles, vaccination is rec-ommended at 9 months of age (World Health Organization 2006), which may not be completely effective. There has been intense evaluation of the possibility of vaccinat-ing at the youngest possible age, including trials at 6 months. However, at 6 months, there appears to be an intrinsic deficiency in generating a proper humoral response to measles (Gans et al. 2001, 2003, 2004). Compared to infants at 9 and 12 months, 6-month-old infants with no residual maternal antibody develop lower protective antibody responses after vaccination and lower IFN-γ production after MV stimula-tion (Gans et al. 1998). Further work has shown that it is not necessarily the quantita-tive production of IgG that is affected by age but rather affinity maturation and avidity of measles-vaccine-specific antibody that is lower in 6-month-old infants independent of the presence of maternal antibody (Nair et al. 2007). This is likely to be due to an impairment of the somatic hypermutation mechanism in human B cells early in life, which develops during an infant’s 1st year (Ridings et al. 1998).

Overall, until vaccination at 9 or 12 months, infants initially depend on maternal antibody for protection from measles prior to the development of a long-lasting response to measles infection or vaccination. Many research efforts are aimed at the development of vaccines that could give an effective response when given at an earlier age than the current measles vaccine.

Measles Immunity in Immunocompromised Hosts

In individuals with impaired cellular immunity (HIV infection, congenital immuno-deficiency, leukemia, etc.), measles infection can lead to giant cell pneumonia, measles inclusion body encephalitis, and death (Markowitz et al. 1988; Mustafa et al. 1993; Rand et al. 1976; Siegel et al. 1977). Many regions of the developing world where measles remains endemic are also regions burdened with a high preva-lence of human immunodeficiency virus (HIV) infection. The impact of the HIV epidemic on the immune response to natural measles infection and to vaccination is the subject of intense study, as it is unknown whether the HIV epidemic will impede measles elimination efforts.

Natural measles infection in HIV-infected children leads to a prolonged phase of illness and MV shedding in PBMCs, nasopharyngeal swabs, and in urine as com-pared to HIV-negative children (Permar et al. 2001). This suggests that the impaired CD4 and CD8 T cell response present in HIV-infected children results in slower clearance of MV, which could thus increase in the measles transmissibility window. Extremely immunosuppressed children may not be able to clear the virus and can die from measles-induced giant-cell pneumonia (Krasinski and Borkowsky 1989; Markowitz et al. 1988; Nadel et al. 1991; Palumbo et al. 1992). Furthermore, clini-cal manifestations of measles in HIV-infected individuals have been described to be altered (Moss et al. 1999). Since the cellular immune response to measles is respon-sible for the hallmark rash, HIV-infected individuals with more marked immuno-suppression may become ill with measles without developing the rash (Centers for

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Disease Control 1988; Markowitz et al. 1988; Moss et al. 1999). The rate of com-plications and measles-related mortality is also significantly higher in HIV-positive children as compared to HIV-negative children. Initial studies assessed measles mortality with relation to infant HIV-antibody positivity. Further studies assessing HIV infection by virological methods demonstrated that HIV-negative children born to HIV-positive mothers also have higher mortality after measles infection as compared to children born to HIV-negative women (Moss et al. 2008).

Measles vaccination of HIV-infected infants is hampered by impaired immune responses and it has been observed that response rates to measles vaccination range from 25% to 72% as measured by protective measles-specific antibodies (Arpadi et al. 1996; Brena et al. 1993; Krasinski and Borkowsky 1989; Palumbo et al. 1992; Rudy et al. 1994), whereas 95% of healthy children mount protective immunity to the measles vaccine (Krugman 1977). Measles-specific antibody levels elicited by vaccination also decay more rapidly in HIV-infected children (al-Attar et al. 1995) than in HIV-negative infants. A severe dysfunction of long-term humoral memory caused by a progressive memory B cell loss during chronic HIV infection may be involved in measles-specific antibody decay (Titanji et al. 2006). However, several studies have shown effective responses and antibody maintenance after measles revaccination in children undergoing successful highly active antiretroviral therapy (HAART) (Aurpibul et al. 2007; Berkelhamer et al. 2001).

Prior to measles immunization, infants are protected from measles by mater-nally transferred antibody. Levels of MV-protective antibodies are lower in infants born to HIV-positive women as compared to infants born to HIV-negative women, thus increasing the window of vulnerability to natural measles infection prior to vaccination age. Lower levels of MV-specific antibodies in infants born of HIV-positive mothers are likely to be due to both lower maternal antibody levels and/or impaired placental transfer (de Moraes-Pinto et al. 1996, 1998; Scott et al. 2005, 2007).

Overall the HIV epidemic poses obstacles to measles elimination by (1) increas-ing transmissibility of natural measles infection, (2) modifying clinical manifesta-tions, thus disrupting case-finding efforts, (3) increasing vulnerability of infants born to HIV-positive mothers, and (4) reducing efficacy of the measles vaccine in HIV-positive individuals.

However, models have suggested that the impact of the HIV pandemic on popula-tion immunity to measles should not introduce an insurmountable barrier to measles control (Helfand et al. 2005; Scott et al. 2008). This is partially due to the high mor-tality rate of these children, which counteracts the increased vaccine failure, shorter duration of maternal antibody protection, and longer duration of MV infectiousness among HIV-infected children. However, survival is largely determined by HAART and one published model suggests that HAART treatment could lead to an increase in the population susceptible to measles if vaccine-induced protection in HIV-1-infected children wanes to an equal extent in HIV-infected children irrespective of HAART (Scott et al. 2008). A combination of HAART and revaccination against measles may improve the situation. More studies on measles vaccine- induced immunity in HIV-infected infants under HAART are necessary, as are studies assessing revaccination after HAART response.

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Conclusions

The immune response to natural measles infection is a complex cascade of innate, cellular, and humoral immune responses leading to lifelong immunity (Fig. 8. 1 ). The humoral arm of the immune response has been the prime focus for determining vaccine efficacy and assessing protection against measles. However, it has clearly been demonstrated that cellular immunity is essential for effective recovery from measles infection. The quality as well as the intensity of the response to MV is cru-cial such that the T cell effector functions elicited, antibody IgG isotypes, and avid-ity play a role in determining whether the MV-specific response will be protective. Individuals with cellular immunodeficiencies have impaired immune responses to measles infection and are at increased risk of severe complications and death due to measles infection. Immunodeficiencies such as HIV infection also impair responses to measles vaccination.

As a result of four decades of extensive measles vaccination, currently most infants are born to mothers who have vaccine-induced immunity rather than immu-nity induced by natural measles infection. It has been shown that these mothers transfer less maternal antibody to their infants, thus leaving young infants suscepti-ble to measles in areas where measles outbreaks still occur. Two doses of measles

TLR activationIFN type I / IL-12 production

CD4-Th1(early): IFN-g, IL-2 CD8-CTL CD4-Th2(late): IL-4,IL-5,IL-10

NeutralizingIgG1 IgG3

Serologic memoryIgG1 IgG4>200mIU/ml

Short-termEM

Long-termCM

Maintenance by TCD4

YYYY

Innate immunity

CMI

DC

mφ

Measles infection

recoveryprotection from re-infection

Affinity maturation

Humoral

Fig. 8.1 Schema of the components of an effective anti-measles immune response. Immunodefi-ciency, improper vaccine design, vaccination, and severe measles-induced immunosuppression can affect various aspects of the immune cascade. CMI cell-mediated immunity, EM effector memory, CM central memory

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vaccine is currently thought to induce long-lasting protection against measles infection and studies are currently underway to develop a vaccine that would be immunogenic for infants younger than 9 months of age.

References

Anonymous (2006) Progress in reducing global measles deaths: 1999–2004. Wkly Epidemiol Rec 81:90–94

Aaby P (1988) Malnutrition and overcrowding/intensive exposure in severe measles infection: review of community studies. Rev Infect Dis 10:478–491

Aaby P, Clements CJ (1989) Measles immunization research: a review. Bull World Health Organ 67:443–8

al-Attar I, Reisman J, Muehlmann M, McIntosh K (1995) Decline of measles antibody titers after immunization in human immunodeficiency virus-infected children. Pediatr Infect Dis J 14:149–151

Albrecht P, Ennis FA, Saltzman EJ, Krugman S (1977) Persistence of maternal antibody in infants beyond 12 months: mechanism of measles vaccine failure. J Pediatr 91:715–718

Anders JF, Jacobson RM, Poland GA, Jacobsen SJ, Wollan PC (1996) Secondary failure rates of measles vaccines: a metaanalysis of published studies. Pediatr Infect Dis J 15:62–66

Arpadi SM, Markowitz LE, Baughman AL, Shah K, Adam H, Wiznia A, Lambert G, Dobroszycki J, Heath JL, Bellini WJ (1996) Measles antibody in vaccinated human immunodeficiency virus type 1-infected children. Pediatrics 97:653–657

Atabani SF, Byrnes AA, Jaye A, Kidd IM, Magnusen AF, Whittle H, Karp CL (2001) Natural measles causes prolonged suppression of interleukin-12 production. J Infect Dis 184:1–9

Audet S, Virata-Theimer ML, Beeler JA, Scott DE, Frazier DJ, Mikolajczyk MG, Eller N, Chen FM, Yu MY (2006) Measles-virus-neutralizing antibodies in intravenous immunoglobulins. J Infect Dis 194:781–789

Aurpibul L, Puthanakit T, Sirisanthana T, Sirisanthana V (2007) Response to measles, mumps, and rubella revaccination in HIV-infected children with immune recovery after highly active antiretroviral therapy. Clin Infect Dis 45:637–642

Bautista-Lopez N, Ward BJ, Mills E, McCormick D, Martel N, Ratnam S (2000) Development and durability of measles antigen-specific lymphoproliferative response after MMR vaccina-tion. Vaccine 18 1393–1401

Bautista-Lopez NL, Vaisberg A, Kanashiro R, Hernandez H, Ward BJ (2001) Immune response to measles vaccine in Peruvian children. Bull World Health Organ 79:1038–1046

Bech V (1959) Studies on the development of complement fixing antibodies in measles patients. Observations during a measles epidemic in Greenland. J Immunol 83:267–275

Bellini WJ, Rota PA (1998) Genetic diversity of wild-type measles viruses: implications for global measles elimination programs. Emerg Infect Dis 4:29–35

Bellini WJ, Rota JS, Rota PA (1994) Virology of measles virus. J Infect Dis 170 [Suppl 1]:S15–S23

Berghall H, Siren J, Sarkar D, Julkunen I, Fisher PB, Vainionpaa R, Matikainen S (2006) The interferon-inducible RNA helicase, mda-5, is involved in measles virus-induced expression of antiviral cytokines. Microbes Infect 8:2138–2144

Berkelhamer S, Borock E, Elsen C, Englund J, Johnson D (2001) Effect of highly active antiret-roviral therapy on the serological response to additional measles vaccinations in human immu-nodeficiency virus-infected children. Clin Infect Dis 32:1090–1094

Bieback K, Lien E, Klagge IM, Avota E, Schneider-Schaulies J, Duprex WP, Wagner H, Kirschning CJ, Ter Meulen V, Schneider-Schaulies S (2002) Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signaling. J Virol 76:8729–8736

Black FL (1989) Measles active and passive immunity in a worldwide perspective. Prog Med Virol 36:1–33

b12325337-0008ztc.indd 164 10/3/2008 10:58:22 AM


Bouche FB, Ertl OT, Muller CP (2002) Neutralizing B cell response in measles. Viral Immunol 15:451–471

Brena AE, Cooper ER, Cabral HJ, Pelton SI (1993) Antibody response to measles and rubella vaccine by children with HIV infection. J Acquir Immune Defic Syndr 6:1125–1129

Burnet FM (1968) Measles as an index of immunological function. Lancet 2:610–613 Centers for Disease Control (1988) Measles in HIV-infected children, United States. MMWR

Morb Mortal Wkly Rep 37:183–186 Chen RT, Markowitz LE, Albrecht P, Stewart JA, Mofenson LM, Preblud SR, Orenstein WA

(1990) Measles antibody: reevaluation of protective titers. J Infect Dis 162:1036–1042 Clements CJ, Hussey GD (2004) Measles. In: Murray CJL, Lopez AD, Mathers CD (eds) The burden

of disease and injury series. Volume IV. Global epidemiology of infectious diseases. http://whqlibdoc.who.int/publications/2004/9241592303_chap4.pdf . Cited 10 June 2008. World Health Organization, Geneva

Cutts FT, Henderson RH, Clements CJ, Chen RT, Patriarca PA (1991) Principles of measles con-trol. Bull World Health Organ 69:1–7

Dabis F, Waldman RJ, Mann GF, Commenges D, Madzou G, Jones TS (1989) Loss of maternal measles antibody during infancy in an African city. Int J Epidemiol 18:264–268

Dai B, Chen ZH, Liu QC, Wu T, Guo CY, Wang XZ, Fang HH, Xiang YZ (1991) Duration of immunity following immunization with live measles vaccine: 15 years of observation in Zhejiang Province, China. Bull World Health Organ 69:415–423

de Francisco A, Hall AJ, Unicomb L, Chakraborty J, Yunus M, Sack RB (1998) Maternal measles antibody decay in rural Bangladeshi infants--implications for vaccination schedules. Vaccine 16:564–568

de Moraes-Pinto MI, Almeida AC, Kenj G, Filgueiras TE, Tobias W, Santos AM, Carneiro-Sampaio MM, Farhat CK, Milligan PJ, Johnson PM, Hart CA (1996) Placental transfer and maternally acquired neonatal IgG immunity in human immunodeficiency virus infection. J Infect Dis 173:1077–1084

de Moraes-Pinto MI, Verhoeff F, Chimsuku L, Milligan PJ, Wesumperuma L, Broadhead RL, Brabin BJ, Johnson PM, Hart CA (1998) Placental antibody transfer: influence of maternal HIV infection and placental malaria. Arch Dis Child Fetal Neonatal Ed 79:F202–F205

Dhiman N, Ovsyannikova IG, Jacobson RM, Vierkant RA, Pankratz VS, Jacobsen SJ, Poland GA (2005a) Correlates of lymphoproliferative responses to measles, mumps, and rubella (MMR) virus vaccines following MMR-II vaccination in healthy children. Clin Immunol 115:154–161

Dhiman N, Ovsyannikova IG, Ryan JE, Jacobson RM, Vierkant RA, Pankratz VS, Jacobsen SJ, Poland GA (2005b) Correlations among measles virus-specific antibody, lymphoproliferation and Th1/Th2 cytokine responses following measles-mumps-rubella-II (MMR-II) vaccination. Clin Exp Immunol 142:498–504

Dhiman N, Ovsyannikova IG, Cunningham JM, Vierkant RA, Kennedy RB, Pankratz VS, Poland GA, Jacobson RM (2007a) Associations between measles vaccine immunity and single-nucle-otide polymorphisms in cytokine and cytokine receptor genes. J Infect Dis 195:21–29

Dhiman N, Poland GA, Cunningham JM, Jacobson RM, Ovsyannikova IG, Vierkant RA, Wu Y, Pankratz VS (2007b) Variations in measles vaccine-specific humoral immunity by polymor-phisms in SLAM and CD46 measles virus receptors. J Allergy Clin Immunol 120:666–672

El Mubarak HS, Ibrahim SA, Vos HW, Mukhtar MM, Mustafa OA, Wild TF, Osterhaus AD, de Swart RL (2004) Measles virus protein-specific IgM, IgA, and IgG subclass responses during the acute and convalescent phase of infection. J Med Virol 72:290–298

Farquhar C, Nduati R, Haigwood N, Sutton W, Mbori-Ngacha D, Richardson B, John-Stewart G (2005) High maternal HIV-1 viral load during pregnancy is associated with reduced placental transfer of measles IgG antibody. J Acquir Immune Defic Syndr 40:494–497

Fontana JM, Bankamp B, Bellini WJ, Rota PA (2008) Virology 28:28 Forthal DN, Landucci G, Habis A, Zartarian M, Katz J, Tilles JG (1994) Regulation of interferon

signaling by the C and V proteins from attenuated and wild-type strains of measles virus. J Infect Dis 169:1377–1380

Fujinami RS, Oldstone MB (1979) Antiviral antibody reacting on the plasma membrane alters measles virus expression inside the cell. Nature 279:529–530

b12325337-0008ztc.indd 165 10/3/2008 10:58:22 AM

166 D. Naniche

Fujinami RS, Oldstone MB (1980) Alterations in expression of measles virus polypeptides by antibody: molecular events in antibody-induced antigenic modulation. J Immunol 125:78–85

Fulginiti VA, Eller JJ, Downie AW, Kempe CH (1967) Altered reactivity to measles virus. Atypical measles in children previously immunized with inactivated measles virus vaccines.JAMA 202:1075–1080

Gans HA, Arvin AM, Galinus J, Logan L, DeHovitz R, Maldonado Y (1998) Deficiency of the humoral immune response to measles vaccine in infants immunized at age 6 months. JAMA 280:527–532

Gans HA, Maldonado Y, Yasukawa LL, Beeler J, Audet S, Rinki MM, DeHovitz R, Arvin AM (1999) IL-12, IFN-gamma, and T cell proliferation to measles in immunized infants. J Immunol 162:5569–5575

Gans H, Yasukawa L, Rinki M, DeHovitz R, Forghani B, Beeler J, Audet S, Maldonado Y, Arvin AM (2001) Immune responses to measles and mumps vaccination of infants at 6, 9, and 12 months. J Infect Dis 184:817–826

Gans H, DeHovitz R, Forghani B, Beeler J, Maldonado Y, Arvin AM (2003) Measles and mumps vaccination as a model to investigate the developing immune system: passive and active immu-nity during the first year of life. Vaccine 21:3398–3405

Gans HA, Yasukawa LL, Alderson A, Rinki M, DeHovitz R, Beeler J, Audet S, Maldonado Y, Arvin AM (2004) Humoral and cell-mediated immune responses to an early 2-dose measles vaccination regimen in the United States. J Infect Dis 190:83–90

Good RA, Zak SJ (1956) Disturbances in gamma globulin synthesis as experiments of nature. Pediatrics 18:109–149

Griffin DE (2007) Measles virus. In: Knipe DM (ed) Fields virology. Lippincott Williams and Wilkins, Philadelphia, pp 1551

Griffin DE, Ward BJ (1993) Differential CD4 T cell activation in measles. J Infect Dis 168:275–281

Griffin DE, Ward BJ, Jauregui E, Johnson RT, Vaisberg A (1989) Immune activation in measles. N Eng J Med 320:1667–1672

Griffin DE, Ward BJ, Jauregui E, Johnson T, Vaisberg A (1990) Immune activation during mea-sles: interferon-gamma and neopterin in plasma and cerebrospinal fluid in complicated and uncomplicated disease. J Infect Dis 161:449–453

Griffin DE, Ward BJ, Juaregui E, Johnson RT, Vaisberg A (1992) Immune activation during mea-sles: beta 2-microglobulin in plasma and cerebrospinal fluid in complicated and uncomplicated disease. J Infect Dis 166:1170–1173


Halsey NA, Boulos R, Mode F, Andre J, Bowman L, Yaeger RG, Toureau S, Rohde J, Boulos C (1985) Response to measles vaccine in Haitian infants 6 to 12 months old. Influence of mater-nal antibodies, malnutrition, and concurrent illnesses. N Engl J Med 313:544–549

Helfand RF, Heath JL, Anderson LJ, Maes EF, Guris D, Bellini WJ (1997) Diagnosis of measles with an IgM capture EIA: the optimal timing of specimen collection after rash onset. J Infect Dis 175:195–199

Helfand RF, Kebede S, Gary HE Jr, Beyene H, Bellini WJ (1999) Timing of development of mea-sles-specific immunoglobulin M and G after primary measles vaccination. Clin Diagn Lab Immunol 6:178–180

Helfand RF, Moss WJ, Harpaz R, Scott S, Cutts F (2005) Evaluating the impact of the HIV pan-demic on measles control and elimination. Bull World Health Organ 83:329–337

Helminen ME, Kilpinen S, Virta M, Hurme M (2001) Susceptibility to primary Epstein-Barr virus infection is associated with interleukin-10 gene promoter polymorphism. J Infect Dis 184:777–780

Howe RC, Dhiman N, Ovsyannikova IG, Poland GA (2005) Induction of CD4 T cell proliferation and in vitro Th1-like cytokine responses to measles virus. Clin Exp Immunol 140:333–342

b12325337-0008ztc.indd 166 10/3/2008 10:58:22 AM


Hurme M, Haanpaa M, Nurmikko T, Wang XY, Virta M, Pessi T, Kilpinen S, Hulkkonen J, Helminen M (2003) IL-10 gene polymorphism and herpesvirus infections. J Med Virol 70 [Suppl 1]:S48–S50

Jabbour JT, Carcia HJ, Lemmi H, Ragland J, Duenas DA, Sever JL (1969) Subacute sclerosing panencephalitis. A multidisciplinary study of eight cases. JAMA 207:2248–2254

Jaye A, Magnusen AF, Sadiq AD, Corrah T, Whittle HC (1998) Ex vivo analysis of cytotoxic T lymphocytes to measles antigens during infection and after vaccination in Gambian children. J Clin Invest 102:1969–1977

Jaye A, Herberts CA, Jallow S, Atabani S, Klein MR, Hoogerhout P, Kidd M, van Els CA, Whittle HC (2003) Vigorous but short-term gamma interferon T-cell responses against a dominant HLA-A*02-restricted measles virus epitope in patients with measles. J Virol 77:5014–5016


Kaslow RA, Dorak T, Tang JJ (2005) Influence of host genetic variation on susceptibility to HIV type 1 infection. J Infect Dis 191 [Suppl 1]:S68–S77

Krasinski K, Borkowsky W (1989) Measles and measles immunity in children infected with human immunodeficiency virus. JAMA 261:2512–2516

Krugman S (1977) Present status of measles and rubella immunization in the United States: a medical progress report. J Pediatr 90:1–12

Langmuir AD (1962) Medical importance of measles. Am J Dis Child 103:224–226 Lanzavecchia A, Sallusto F (2005) Understanding the generation and function of memory T cell

subsets. Curr Opin Immunol 17:326–332 Lee MS, Nokes DJ (2001) Predicting and comparing long-term measles antibody profiles of dif-

ferent immunization policies. Bull World Health Organ 79:615–624 Lee MS, Chien LJ, Yueh YY, Lu CF (2001) Measles seroepidemiology and decay rate of vaccine-

induced measles IgG titers in Taiwan, 1995–1997. Vaccine 19:4644–4651 Malvoisin E, Wild F (1990) Contribution of measles virus fusion protein in protective immunity:

anti-F monoclonal antibodies neutralize virus infectivity and protect mice against challenge. J Virol 64:5160–5162

Markowitz LE, Chandler FW, Roldan EO, Saldana M, Roach KC, Hutchins SS, Preblud SR, Mitchell CD, Scott GB (1988) Fatal measles pneumonia without rash in a child with AIDS. J Infect Dis 158:480–483

Markowitz LE, Albrecht P, Rhodes P, Demonteverde R, Swint E, Maes EF, Powell C, Patriarca PA (1996) Changing levels of measles antibody titers in women and children in the United States: impact on response to vaccination. Kaiser Permanente Measles Vaccine Trial Team. Pediatrics 97:53–58

Mitus A, Holloway A, Evans AE, Enders JF (1962) Attenuated measles vaccine in children with acute leukemia. Am J Dis Child 103:413–418

Moss WJ (2007) Measles still has a devastating impact in unvaccinated populations. PLoS Med 4:e24

Moss WJ, Cutts F, Griffin DE (1999) Implications of the human immunodeficiency virus epidemic for control and eradication of measles. Clin Infect Dis 29:106–112


Moss WJ, Fisher C, Scott S, Monze M, Ryon JJ, Quinn TC, Griffin DE, Cutts FT (2008) HIV type 1 infection is a risk factor for mortality in hospitalized Zambian children with measles. Clin Infect Dis 46:523–527

Mossong J, O’Callaghan CJ, Ratnam S (2000) Modelling antibody response to measles vaccine and subsequent waning of immunity in a low exposure population. Vaccine 19:523–529

Mustafa MM, Weitman SD, Winick NJ, Bellini WJ, Timmons CF, Siegel JD (1993) Subacute measles encephalitis in the young immunocompromised host: report of two cases diagnosed by polymerase chain reaction and treated with ribavirin and review of the literature. Clin Infect Dis 16:654–660

b12325337-0008ztc.indd 167 10/3/2008 10:58:22 AM

168 D. Naniche

Nadel S, McGann K, Hodinka RL, Rutstein R, Chatten J (1991) Measles giant cell pneumonia in a child with human immunodeficiency virus infection. Pediatr Infect Dis J 10:542–544

Nair N, Gans H, Lew-Yasukawa L, Long-Wagar AC, Arvin A, Griffin DE (2007) Age-dependent differences in IgG isotype and avidity induced by measles vaccine received during the first year of life. J Infect Dis 196:1339–1345

Nanan R, Carstens C, Kreth HW (1995) Demonstration of virus-specific CD8+ memory T cells in measles-seropositive individuals by in vitro peptide stimulation. Clin Exp Immunol 102:40–45

Nanan R, Rauch A, Kampgen E, Niewiesk S, Kreth HW (2000) A novel sensitive approach for frequency analysis of measles virus-specific memory T-lymphocytes in healthy adults with a childhood history of natural measles. J Gen Virol 81:1313–1319

Naniche D, Yeh A, Eto D, Manchester M, Friedman RM, Oldstone MB (2000) Evasion of host defenses by measles virus: wild-type measles virus infection interferes with induction of Alpha/Beta interferon production. J Virol 74:7478–7484

Naniche D, Garenne M, Rae C, Manchester M, Buchta R, Brodine SK, Oldstone MB (2004) Decrease in measles virus-specific CD4 T cell memory in vaccinated subjects. J Infect Dis 190:1387–1395

Nicoara C, Zach K, Trachsel D, Germann D, Matter L (1999) Decay of passively acquired mater-nal antibodies against measles, mumps, and rubella viruses. Clin Diagn Lab Immunol 6:868–871

Norrby E, Gollmar Y (1972) Appearance and persistence of antibodies against different virus components after regular measles infections. Infect Immun 6:240–247

Norrby E, Orvell C, Vandvik B, Cherry JD (1981) Antibodies against measles virus polypeptides in different disease conditions. Infect Immun 34:718–724

Okoko BJ, Wesuperuma LH, Ota MO, Banya WA, Pinder M, Gomez FS, Osinusi K, Hart AC (2001a) Influence of placental malaria infection and maternal hypergammaglobulinaemia on materno-foetal transfer of measles and tetanus antibodies in a rural west African population. J Health Popul Nutr 19:59–65

Okoko JB, Wesumperuma HL, Hart CA (2001b) The influence of prematurity and low birthweight on transplacental antibody transfer in a rural West African population. Trop Med Int Health 6:529–534

Ota MO, Ndhlovu Z, Oh S, Piyasirisilp S, Berzofsky JA, Moss WJ, Griffin DE (2007) Hemagglutinin protein is a primary target of the measles virus-specific HLA-A2-restricted CD8+ T cell response during measles and after vaccination. J Infect Dis 195:1799–1807

Ovsyannikova IG, Dhiman N, Jacobson RM, Vierkant RA, Poland GA (2003) Frequency of mea-sles virus-specific CD4+ and CD8+ T cells in subjects seronegative or highly seropositive for measles vaccine. Clin Diagn Lab Immunol 10:411–416

Ovsyannikova IG, Jacobson RM, Ryan JE, Vierkant RA, Pankratz VS, Jacobsen SJ, Poland GA (2005a) HLA class II alleles and measles virus-specific cytokine immune response following two doses of measles vaccine. Immunogenetics 56:798–807

Ovsyannikova IG, Jacobson RM, Vierkant RA, Jacobsen SJ, Pankratz VS, Poland GA (2005b) Human leukocyte antigen class II alleles and rubella-specific humoral and cell-mediated immunity following measles-mumps-rubella-II vaccination. J Infect Dis 191:515–519

Ovsyannikova IG, Ryan JE, Jacobson RM, Vierkant RA, Pankratz VS, Poland GA (2006) Human leukocyte antigen and interleukin 2, 10 and 12p40 cytokine responses to measles: is there evi-dence of the HLA effect? Cytokine 36:173–179

Owens S, Harper G, Amuasi J, Offei-Larbi G, Ordi J, Brabin BJ (2006) Placental malaria and immunity to infant measles. Arch Dis Child 91:507–508


b12325337-0008ztc.indd 168 10/3/2008 10:58:22 AM


Palumbo P, Hoyt L, Demasio K, Oleske J, Connor E (1992) Population-based study of measles and measles immunization in human immunodeficiency virus-infected children. Pediatr Infect Dis J 11:1008–1014

Panum P (1938) Med Classics 3:829–886 Papania M, Baughman AL, Lee S, Cheek JE, Atkinson W, Redd SC, Spitalny K, Finelli L,

Markowitz L (1999) Increased susceptibility to measles in infants in the United States. Pediatrics 104:e59

Paul WE (1989) In: Paul WE (ed) Peripheral T lymphocytes. Fundamental immunology. Raven Press, New York, pp 398–399

Paul WE (2003) Fundamental immunology. Lippincott Raven, Philadelphia Permar SR, Moss WJ, Ryon JJ, Monze M, Cutts F, Quinn TC, Griffin DE (2001) Prolonged mea-

sles virus shedding in human immunodeficiency virus-infected children, detected by reverse transcriptase-polymerase chain reaction. J Infect Dis 183:532–538



Perry RT, Halsey NA (2004) The clinical significance of measles: a review. J Infect Dis 189 [Suppl 1]:S4–S16

Polack FP, Hoffman SJ, Moss WJ, Griffin DE (2002) Altered synthesis of interleukin-12 and type 1 and type 2 cytokinesin rhesus macaques during measles and atypical measles. J Infect Dis 185:13–19

Polack FP, Hoffman SJ, Crujeiras G, Griffin DE (2003) A role for nonprotective complement-fix-ing antibodies with low avidity for measles virus in atypical measles. Nat Med 9:1209–1213

Rand KH, Emmons RW, Merigan TC (1976) Measles in adults. An unforeseen consequence of immunization? JAMA 236:1028–1031

Ratnam S, Gadag V, West R, Burris J, Oates E, Stead F, Bouilianne N (1995) Comparison of com-mercial enzyme immunoassay kits with plaque reduction neutralization test for detection of measles virus antibody. J Clin Microbiol 33:811–815

Reiner SL, Sallusto F, Lanzavecchia A (2007) Division of labor with a workforce of one: chal-lenges in specifying effector and memory T cell fate. Science 317:622–625

Ridings J, Dinan L, Williams R, Roberton D, Zola H (1998) Somatic mutation of immunoglobulin V(H)6 genes in human infants. Clin Exp Immunol 114:33–39

Rudy BJ, Rutstein RM, Pinto-Martin J (1994) Responses to measles immunization in children infected with human immunodeficiency virus. J Pediatr 125:72–74

Samb B, Aaby P, Whittle HC, Seck AM, Rahman S, Bennett J, Markowitz L, Simondon F (1995) Serologic status and measles attack rates among vaccinated and unvaccinated children in rural Senegal. Pediatr Infect Dis 14:203–209

Scott S, Cumberland P, Shulman CE, Cousens S, Cohen BJ, Brown DW, Bulmer JN, Dorman EK, Kawuondo K, Marsh K, Cutts F (2005) Neonatal measles immunity in rural Kenya: the influ-ence of HIV and placental malaria infections on placental transfer of antibodies and levels of antibody in maternal and cord serum samples. J Infect Dis 191:1854–1860

Scott S, Moss WJ, Cousens S, Beeler JA, Audet SA, Mugala N, Quinn TC, Griffin DE, Cutts FT (2007) The influence of HIV-1 exposure and infection on levels of passively acquired antibod-ies to measles virus in Zambian infants. Clin Infect Dis 45:1417–1424

Scott S, Mossong J, Moss WJ, Cutts FT, Cousens S (2008) Predicted impact of the HIV-1 epi-demic on measles in developing countries: results from a dynamic age-structured model. Int J Epidemiol 30:356–367

b12325337-0008ztc.indd 169 10/3/2008 10:58:22 AM

170 D. Naniche

Shaffer JA, Bellini WJ, Rota PA (2003) The C protein of measles virus inhibits the type I inter-feron response. Virology 315:389–397

Shingai M, Ebihara T, Begum NA, Kato A, Honma T, Matsumoto K, Saito H, Ogura H, Matsumoto M, Seya T (2007) Differential type I IFN-inducing abilities of wild-type versus vaccine strains of measles virus. J Immunol 179:6123–6133

Siegel MM, Walter TK, Ablin AR (1977) Measles pneumonia in childhood leukemia. Pediatrics 60:38–40

Stephenson JR, ter Meulen V (1979) Antigenic relationships between measles and canine distem-per viruses: comparison of immune response in animals and humans to individual virus-spe-cific polypeptides. Proc Natl Acad Sci U S A 76:6601–6605

Stokes J, Reilly CM, Bunyak EB, Hilleman MR (1961) Immunologic studies of measles. Am J Hyg 74:293–303

Takeuchi K, Kadota SI, Takeda M, Miyajima N, Nagata K (2003) Measles virus V protein blocks interferon (IFN)-alpha/beta but not IFN-gamma signaling by inhibiting STAT1 and STAT2 phosphorylation. FEBS Lett 545:177–182

Tapia MD, Sow SO, Medina-Moreno S, Lim Y, Pasetti MF, Kotloff K, Levine MM (2005) A serosurvey to identify the window of vulnerability to wild-type measles among infants in rural Mali. Am J Trop Med Hyg 73:26–31

Termeulen V, Stephenson JR, Kreth HW (1983) Subacute sclerosing panencephalitis. In: Fraenkel-Conrat H, Wagner RR (eds) Comprehensive virology. Plenum, New York, pp 105–159

Thio CL, Thomas DL, Karacki P, Gao X, Marti D, Kaslow RA, Goedert JJ, Hilgartner M, Strathdee SA, Duggal P, O’Brien SJ, Astemborski J, Carrington M (2003) Comprehensive analysis of class I and class II HLA antigens and chronic hepatitis B virus infection. J Virol 77:12083–12087

Titanji K, De Milito A, Cagigi A, Thorstensson R, Grutzmeier S, Atlas A, Hejdeman B, Kroon FP, Lopalco L, Nilsson A, Chiodi F (2006) Loss of memory B cells impairs maintenance of long-term serologic memory during HIV-1 infection. Blood 108:1580–1587

Toyoda M, Ihara T, Nakano T, Ito M, Kamiya H (1999) Expression of interleukin-2 receptor alpha and CD45RO antigen on T lymphocytes cultured with rubella virus antigen, compared with humoral immunity in rubella vaccinees. Vaccine 17:2051–2058

van Els CA, Nanan R (2002) T cell responses in acute measles. Viral Immunol 15:435–450 Van Binnendijk RS, Poelen MCM, Kuijpers KC, Osterhaus ADME, Uytdehaag FGCM (1990)

The predominance of CD8+ T cells after infection with measles virus suggests a role for CD8+ class I MHC-restricted cytotoxic T lymphocytes (CTL) in recovery from measles. Clonal analyses of human CD8+ class I MHC-restricted CTL. J Immunol 144:2394–2399

Ward BJ, Johnson RT, Vaisberg A, Jauregui E, Griffin DE (1991) Cytokine production in vitro and the lymphoproliferative defect of natural measles virus infection. Clin Immunol Immunopathol 61:236–248

Weibel RE, Buynak EB, McLean AA, Roehm RR, Hilleman MR (1980) Persistence of antibody in human subjects for 7 to 10 years following administration of combined live attenuated mea-sles, mumps, and rubella virus vaccines. Proc Soc Exp Biol Med 165:260–263

Wesumperuma HL, Perera AJ, Pharoah PO, Hart CA (1999) The influence of prematurity and low birthweight on transplacental antibody transfer in Sri Lanka. Ann Trop Med Parasitol 93:169–177

Whittle HC, Aaby P, Samb B, Cisse B, Kanteh F, Soumare M, Jensen H, Bennett J, Simondon F (1999) Poor serologic responses five to seven years after immunization with high and standard titer measles vaccines. Pediatr Infect Dis J 18:53–57


World Health Organization (1993) Immunological Basis for Immunization Module 7: Measles. WHO/EPI/GEN/98.17, World Health Organization, Geneva

World Health Organization (2006) WHO AFRO Measles SIA Field Guide. World Health Organization, Geneva

b12325337-0008ztc.indd 170 10/3/2008 10:58:22 AM


Wu VH, McFarland H, Mayo K, Hanger L, Griffin DE, Dhib-Jalbut S (1993) Measles virus-specific cellular immunity in patients with vaccine failure. J Clin Microbiol 31:118–122

Yakub I, Lillibridge KM, Moran A, Gonzalez OY, Belmont J, Gibbs RA, Tweardy DJ (2005) Single nucleotide polymorphisms in genes for 2'–5'-oligoadenylate synthetase and RNase L inpatients hospitalized with West Nile virus infection. J Infect Dis 192:1741–1748

Zilliox MJ, Parmigiani G, Griffin DE (2006) Gene expression patterns in dendritic cells infected with measles virus compared with other pathogens. Proc Natl Acad Sci U S A 103:3363–3368

Zilliox MJ, Moss WJ, Griffin DE (2007) Gene expression changes in peripheral blood mononuclear cells during measles virus infection. Clin Vaccine Immunol 14:918–923

b12325337-0008ztc.indd 171 10/3/2008 10:58:22 AM

Chapter 9 Measles Control and the Prospect of Eradication

W. J. Moss

Abstract Remarkable progress has been made in reducing measles incidence and mortality as a consequence of implementing the measles mortality reduction strat-egy of the World Health Organization (WHO) and United Nations Children’s Fund (UNICEF). The revised global measles mortality reduction goal set forth in the WHO-UNICEF Global Immunization Vision and Strategy for 2006–2015 is to reduce measles deaths by 90% by 2010 compared to the estimated 757,000 deaths in 2000. The possibility of measles eradication has been discussed for almost 40 years, and measles meets many of the criteria for eradication. Global measles eradication will face a number of challenges to achieving and sustaining high levels

W. J. Moss Department of Epidemiology and the W. Harry Feinstone Department of Molecular Microbiology and Immunology , Johns Hopkins University Bloomberg School of Public Health , Baltimore MD, USA , e-mail: [email protected]

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174Goals and Strategies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

Mortality Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175Regional Elimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

Progress in Measles Mortality Reduction and Elimination . . . . . . . . . . . . . . . . . . . . . . . . . . . 178Global Goals and Progress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178Regional Progress in Mortality Reduction and Elimination . . . . . . . . . . . . . . . . . . . . . . . . 179

Feasibility of Measles Eradication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180Biological Feasibility of Measles Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181Technical Feasibility of Measles Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181Logistical Feasibility of Measles Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

Challenges to Global Measles Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182Challenges to Achieving High Levels of Measles Vaccine Coverage . . . . . . . . . . . . . . . . . 182Challenges to Achieving High Levels of Population Immunity . . . . . . . . . . . . . . . . . . . . . 184Challenges to Sustained Measles Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

Prospects for Measles Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186


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of vaccine coverage and population immunity, including population growth and demographic changes, conflict and political instability, and public perceptions of vaccine safety. To achieve the measles mortality reduction goal, continued progress needs to be made in delivering measles vaccines to the world’s children.

Introduction

Measles virus (MV) has caused millions of deaths since its emergence as a zoonosis thousands of years ago. Prior to the introduction of measles vaccine, more than 130 million cases and 7–8 million deaths due to measles were estimated to have occurred annually, and almost everyone was infected during childhood. Measles mortality declined in the first half of the twentieth century in developed countries as a consequence of improvements in living conditions, better nutritional status, and the availability of antibiotics for secondary bacterial infections. The introduction of measles vaccines beginning in the 1960s led to substantial reductions in measles incidence, morbidity, and mortality in both developed and developing countries. Measles vaccines were not available for many of the world’s children, however, until the World Health Organization (WHO) launched the Expanded Programme on Immunization (EPI) in 1974, which provided vaccines against six target diseases including measles. Global vaccine coverage against EPI targeted diseases increased from less than 5% at the start of the program to almost 80% by 1990.

More recently, remarkable progress has been made in reducing measles inci-dence and mortality as a consequence of implementing the measles mortality reduction strategy of the WHO and United Nations Children’s Fund (UNICEF). This strategy focuses on 47 priority countries and includes: (1) achieving and maintaining more than 90% coverage with the first dose of measles vaccine in every district by the age of 12 months; (2) ensuring that all children receive a sec-ond opportunity for measles vaccination; (3) surveillance for measles cases and serological confirmation; and (4) provision of appropriate case management (WHO/UNICEF 2001). Support for these efforts comes from the Measles Initiative, a partnership started in 2001 and led by the American Red Cross, the United Nations Foundation, UNICEF, the United States Centers for Disease Control and Prevention, and the WHO.

Goals and Strategies

The key to measles control is achieving and sustaining high levels of measles vac-cine coverage (Fig. 9.1 ). Different goals for measles control have been established, necessitating different vaccination strategies. Three broad goals can be defined: mortality reduction, regional elimination, and global eradication.

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9 Measles Control and the Prospect of Eradication 175

Mortality Reduction

Mortality reduction, the least demanding of the three goals, calls for a reduction in measles mortality from a predetermined level through reductions in incidence and case fatality. Although reducing case fatality through appropriate case management is an important component, measles mortality reduction is achieved largely through a reduction in incidence. To reduce incidence, measles vaccine is administered as a single dose through routine immunization services (Fig. 9. 2 ), with the optimal age

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Fig. 9.1 Global annual reported measles incidence and measles vaccine coverage, 1986–2006. From World Health Organization 2007a

Fig. 9.2 Immunization coverage with measles containing vaccines in infants, 2006. From World Health Organization 2008

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176 W.J. Moss

of immunization determined by the average age of infection and the rate of decline of maternal antibodies. In areas where measles is endemic, and the average age of infection is low, measles vaccine is routinely administered at 9 months of age in accordance with the EPI schedule (WHO 2004). In countries where MV transmis-sion has been significantly reduced, the age at first vaccination is often increased to 12–15 months, resulting in a higher proportion of children who develop protective immunity. If vaccination coverage is sufficiently high, substantial reductions in incidence and mortality occur, the period between epidemics lengthens, and the age distribution shifts toward older children and young adults, further contributing to a reduction in measles case fatality.

Mortality reduction can also be achieved by proper case management, including the administration of vitamin A to persons with measles (D’Souza and D’Souza 2002) and prompt antibiotic treatment of secondary bacterial pneumonia (Duke and Mgone 2003). Provision of vitamin A through polio and measles vaccination cam-paigns further contributes to the reduction in measles mortality (WHO 2005).

Regional Elimination

Measles elimination is the interruption of MV transmission within a defined geo-graphic area, such as country, continent, or WHO region. Small outbreaks of pri-mary and secondary cases may occur following importation from outside the region, but sustained transmission does not occur. Because of the high infectivity of MV and the fact that not all persons develop protective immunity following vac-cination, a single dose of measles vaccine does not achieve a sufficient level of population immunity to eliminate measles. A second opportunity for measles immunization is necessary to provide protective immunity to children who fail to respond to the first dose, as well as to immunize those children who were not previ-ously vaccinated. Two strategies to administer the second dose of measles vaccine have been used. In countries with sufficient health infrastructure, and where chil-dren routinely receive well-child care beyond the 1 st year of life, the second dose of measles vaccine is administered through routine immunization services, typi-cally prior to the start of school (4–6 years of age). High coverage levels can be ensured by school entry requirements (CDC 2007). A second approach, first devel-oped by the Pan American Health Organization (PAHO) for South and Central America (PAHO 1999) and modeled after polio eradication strategies, involves mass immunization campaigns (called supplementary immunization activities or SIA) to deliver the second dose of measles vaccine. This strategy was successful in eliminating measles in South and Central America (de Quadros et al. 1996, 2004) and has resulted in a marked reduction in measles incidence and mortality in much of sub-Saharan Africa (Otten et al. 2005; WHO 2006b).

The PAHO strategy consists of four subprograms: catch-up, keep-up, follow-up, and mop-up. The catch-up activity consists of a one-time, mass immunization cam-paign that targets all children within a broad age range regardless of whether they

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have previously had wild-type MV infection or measles vaccination. The goal is to rapidly achieve a high level of population immunity and interrupt MV transmission. These campaigns are conducted over a short period of time, usually several weeks, and during a low transmission season. Under the PAHO strategy, children 9 months to 14 years of age were targeted for vaccination, a substantial proportion of the total population in many countries. The appropriate target age range depends upon the age distribution of measles cases. In regions where measles is endemic, the major-ity of older children are likely to be immune. Nevertheless, seroprevalence studies usually are not conducted prior to catch-up campaigns, and this broad age range first adopted by PAHO has been widely used in sub-Saharan Africa and Asia. These campaigns require significant financial investments and the commitment of large numbers of personnel; extensive logistical planning to transport and store vaccines, maintain cold chains, and dispose of syringes and needles; and community mobili-zation to ensure participation. If successful, SIA are cost effective (Dayan et al. 2004; Uzicanin et al. 2004) and can abruptly interrupt MV transmission, with dra-matic declines in incidence and mortality.

Keep-up refers to the need to maintain greater than 90% coverage with the first dose of measles vaccine through routine immunization services. Follow-up refers to periodic mass campaigns to prevent the accumulation of susceptible children. Follow-up campaigns typically target children 9 months to 4 years of age, a narrower age group than targeted in catch-up campaigns. Follow-up campaigns should be con-ducted when the estimated number of susceptible children reaches the size of one birth cohort, generally every 3–5 years after the catch-up campaign. These campaigns need to be conducted indefinitely because of the potential risk of MV importation, or until the health infrastructure is sufficiently developed so that the second opportunity can be provided through routine immunization services. Mop-up campaigns target difficult-to-reach children in areas of measles outbreaks or low vaccine coverage. Difficult to reach children include those living on the street or in areas of conflict.

Measles elimination also requires active surveillance for measles cases to assist outbreak response (Grais et al. 2008) and to monitor progress in mortality reduction and elimination. Blood samples should be collected from all suspected measles cases for confirmation by detection of IgM antibodies to MV. Measles outbreaks require serological investigation of the first five to ten cases (WHO/UNICEF 2001), with other cases linked epidemiologically. Less invasive oral fluid or dried blood spot samples also may be used, and urine or nasopharyngeal specimens should be obtained for virus isolation and genetic characterization (Bellini and Helfand 2003). To support these activities, the WHO established the Global Measles Laboratory network in 2000 and the Measles and Rubella Laboratory Network (LabNet) in 2003, comprising over 700 laboratories in more than 160 countries (CDC 2005).

Eradication

The Dahlem Conference on Disease Eradication (1997) defined eradication as the permanent reduction to zero of the global incidence of infection caused by a specific

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178 W.J. Moss

pathogen as a result of deliberate efforts, with the consequence that interventions would no longer be necessary (Dowdle and Hopkins 1998). The pathogen need not be extinct. Smallpox virus, for example, exists in government laboratories in the United States and Russia, although the disease is eradicated and vaccination activi-ties have ceased (Stone 2002). Eradication is the most ambitious goal, requiring sustained high levels of financial investment, political commitment, and public cooperation. Presumably, measles eradication would be achieved using strategies similar to those described for elimination but applied and coordinated globally.

Progress in Measles Mortality Reduction and Elimination

Global Goals and Progress

In 2003, the World Health Assembly endorsed a resolution to reduce the number of deaths attributed to measles by 50% by the end of 2005 compared with 1999 esti-mates. This target was met. Global measles mortality in 2005 was estimated to be 345,000 deaths (uncertainty bounds 247,000 and 458,000 deaths), a 60% decrease from 1999 (Wolfson et al. 2007). Further reductions in global measles mortality were achieved in 2006, with an estimated 242,000 deaths (uncertainty bounds 173,000 and 325,000 deaths) (Fig. 9.3 ) (WHO 2007b). These estimates are not based on active surveillance for measles deaths but instead are derived from models of measles inci-dence and mortality, using estimated measles vaccine coverage and case fatality rates.

The revised global measles mortality reduction goal set forth in the WHO-UNICEF Global Immunization Vision and Strategy (GIVS) for 2006–2015 is to reduce measles deaths by 90% by 2010 compared to the estimated 757,000 deaths in 2000 (WHO/UNCF 2005). The financial resources required to achieve the

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Fig. 9.3 Estimated global measles deaths, 2000–2006. Note the estimated number of global deaths is similar to the reported incidence shown in Fig. 1 because of the different methods used to derive the estimates. From World Health Organization 2007a

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measles control goals for 2001–2005 was estimated to be US $984 million, with approximately two-thirds for the operational costs of conducting SIA and one-third for the purchase of vaccines and injection equipment (WHO/UNICEF 2001). The cost of scaling up childhood immunization services to reach the WHO-UNICEF GIVS goal of reducing mortality due to all vaccine-preventable diseases by two-thirds by 2015 was recently estimated to be US $35 billion (range US $ 13–40 bil-lion) for the 72 poorest countries of the world, including US $8.7 billion for the purchase of vaccines (Wolfson et al. 2008).

Regional Progress in Mortality Reduction and Elimination

Four WHO regions set measles elimination goals: Americas (2000), Europe (2010), Eastern Mediterranean (2010), and Western Pacific (2012). Measles elimination was achieved in the United States in 2000 and in the Americas by November 2002. The two regions where most measles deaths occur (Table 9. 1 ), Africa and South-East Asia, have measles mortality reduction goals, although many countries in these regions have implemented the WHO-UNICEF strategy of providing a second opportunity for measles vaccination through SIA, resulting in dramatic declines in

Table 9.1 Estimated number of deaths from measles and coverage of first-dose measles vaccine through routine immunization services, by WHO region, 2000 and 2006

WHO region 2000 2006 % Decrease in measles deaths 2000–2006

% Coverage with 1st dose of measles vaccine

Measles deaths (uncertainty bounds)

% Coverage with 1st dose of measles vaccine

Measles deaths (uncertainty bounds)

Africa 56 396,000 (290–514,000)

73 36,000 (26–49,000)

91

Americas 92 <1000 93 <1000 –

Eastern Mediterranean

73 96,000 (71–124,000)

83 23,000 (16–34,000)

76

Europe 91 <1000 94 <1000 –

South-East Asia 60 240,000 (173–316,000)

65 178,000 (128–234,000)

26

Western Pacific 86 25,000 (17–35,000)

93 5,000 (3–7,000)

81

Total 72 757,000 (551–990,000)

80 242,000 (173–325,000)

68

Adapted from World Health Organization 2007b

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180 W.J. Moss

incidence and mortality. The largest decrease in measles mortality was in Africa, where measles mortality decreased 91% from 2000 to 2006, accounting for 70% of the global reduction in measles mortality (WHO 2007b). Progress in measles mor-tality reduction in the South-East Asia region was not as marked because several populous countries have not conducted SIA and routine immunization coverage remains suboptimal (WHO 2007b). Low vaccination coverage and susceptibility among adults hinder measles elimination in parts of Europe and Australia (Andrews et al. 2008). Conflicts and political instability in several countries of the Eastern Mediterranean region, including Afghanistan, Iraq, Lebanon, Somalia, and Sudan, have impeded measles control, but a catch-up SIA was conducted in parts of Pakistan in 2007 (CDC 2008c).

Although measles was declared eliminated from the United States in 2000, recent outbreaks highlight the challenges in sustaining elimination. First, despite very high levels of measles vaccine coverage and population immunity overall (McQuillan et al. 2007), clustering of susceptible persons can lead to outbreaks. In 2005, a 17-year-old girl with measles returned to Indiana from Romania and attended a church gathering of over 500 people (Parker et al. 2006). Despite 98% measles vaccination coverage in the state of Indiana (among sixth-graders), 34 cases of measles resulted, of whom 94% were unvaccinated. Second, the widespread cir-culation of MV and ease of global travel allows for MV importation. Perhaps sur-prisingly, these importations frequently arise from countries with ample resources for measles control. A multistate outbreak of measles in 2007 occurred at an inter-national sporting event after importation from Japan (CDC 2008a), the country responsible for the most imported cases of measles into the US over the past several years (Takahashi and Saito 2008). Thousands of measles cases occur annually in Japan as a consequence of low vaccination coverage resulting from the relaxing of vaccination requirements after an outbreak of aseptic meningitis from the Urabe mumps vaccine strain (Gomi and Takahashi 2004). Third, as measles control efforts are increasingly successful in reducing disease incidence, public perceptions of the risk of measles diminish and are replaced by concerns of possible adverse events associated with measles vaccine. An outbreak of measles in San Diego in 2008, imported from Switzerland, resulted in 11 additional cases in unvaccinated children and two generations of secondary cases (CDC 2008b). Among the nine cases older than 1 year of age, eight were unvaccinated because of personal exemption beliefs.

Feasibility of Measles Eradication

The possibility of measles eradication has been discussed for almost 40 years (Sencer et al. 1967), beginning in the late 1960s when smallpox eradication was nearing completion and the long-term protective immunity induced by measles vaccine became evident. Three criteria are deemed important for disease eradica-tion: (1) humans must be critical to transmission; (2) sensitive and specific diag-nostic tools must exist; and (3) an effective intervention must be available

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(Dowdle and Hopkins 1998). Interruption of transmission in large geographical areas for prolonged periods further supports the feasibility of eradication. Measles is thought by many experts to meet these criteria (de Quadros 2004; Orenstein et al. 2000).

Biological Feasibility of Measles Eradication

MV meets many of the biological criteria for disease eradication. MV has no non-human reservoir, infection can be readily diagnosed after onset of a rash, and MV has not mutated to significantly alter immunogenic epitopes (Moss and Griffin 2006). Although MV displays sufficient genetic variation to conduct molecular epidemiologic analyses (WHO 2006a), the epitopes against which protective anti-bodies develop have remained stable, likely because of functional constraints on the amino acid sequence and tertiary structure of the MV surface proteins (Frank and Bush 2007).

Where MV differs from smallpox and polio viruses is that it is more highly infectious, necessitating higher levels of population immunity to interrupt transmis-sion. Outbreaks can occur in populations in which less than 10% of individuals are susceptible. The contagiousness of MV is best expressed by the basic reproductive number R

o , which represents the mean number of secondary cases that would arise

if an infectious agent were introduced into a completely susceptible population. R o

is a function not only of the infectious agent, but also of the host population. The estimated R

o for MV is 12–18, in contrast to 5–7 for smallpox and polio viruses and

2–3 for SARS-coronavirus. The high infectivity of MV implies that a high level of population immunity (approximately 92%–94%) is required to interrupt MV trans-mission (Gay 2004).

Technical Feasibility of Measles Eradication

Measles vaccines are safe and effective and have interrupted MV transmission in large geographic areas, providing the critical tool for measles eradication. Despite progress in measles mortality reduction and elimination, there are several limitations of the licensed vaccines that may make measles eradication more challenging. First, attenu-ated measles vaccines are inactivated by heat and a cold chain must be maintained to support measles immunization activities. Second, in contrast to oral polio vaccines, measles vaccines must be injected subcutaneously or intramuscularly, necessitating trained healthcare workers, needles, syringes, and proper disposal of hazardous waste. Third, both maternally acquired antibodies and immunological immaturity reduce the protective efficacy of measles vaccination in early infancy, hindering effective immu-nization of young infants (Gans et al. 1998). Fourth, the attenuated measles vaccine has the potential to cause serious adverse events, such as lung or brain infection, in

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182 W.J. Moss

severely immunocompromised persons (Angel et al. 1998; Monafo et al. 1994). Lastly, a second opportunity for measles vaccination must be provided to achieve suf-ficient levels of population immunity to interrupt MV transmission.

The duration of immunity following measles vaccination is more variable and shorter than following wild-type MV infection, but persists for decades even in countries where measles is no longer endemic and immunological boosting from wild-type MV infection does not occur (Amanna et al. 2007; Dine et al. 2004). Although antibody levels induced by vaccination may decline over time and become undetectable, immunological memory persists and most vaccinated per-sons produce a MV-specific immune response without clinical symptoms following exposure to wild-type MV.

Logistical Feasibility of Measles Eradication

The elimination of measles in large areas, such as the Americas, suggests that mea-sles eradication is feasible with current vaccination strategies (CDC 1997; de Quadros 2004; Meissner et al. 2004). Perhaps the major logistical challenge to measles eradication will be sustaining the financial resources, political will, and public confidence to implement widespread and coordinated measles vaccination and surveillance activities.

Challenges to Global Measles Eradication

Global measles eradication will face a number of challenges to achieving and sus-taining high levels of vaccine coverage and population immunity. Serious discus-sion of measles eradication is not likely to take place before polio eradication is achieved. Garnering the necessary political and public support for measles eradica-tion will be extremely difficult should polio eradication efforts fail.

Challenges to Achieving High Levels of Measles Vaccine Coverage

Sustainability of Current Measles Control and Elimination Strategies

To eradicate measles, high levels of population immunity need to be sustained through coverage with two doses of measles vaccine. Because the first dose of measles vaccine is administered through routine immunization services, strength-ening the primary healthcare system will further this goal. However, the long-term sustainability of mass vaccination campaigns is unclear as these activities make

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additional demands on the resources and staff of the primary healthcare system and require continued public support in the face of decreasing disease burden and per-ception of public health importance. The polio eradication campaign in Nigeria, for example, was perceived negatively to trigger a massive outbreak response for just three confirmed cases of polio in Adamawa State in 2005, whereas hundreds of deaths due to measles did not result in a comparable response (Schimmer and Ihekweazu 2006). Countries dependent upon SIA to deliver the second dose of measles vaccine will need to strengthen their primary health care systems to pro-vide this dose beyond the 1st year of life. Ensuring the necessary supply of measles vaccine as eradication efforts progress will be critical (Costa et al. 2003), requiring close collaboration with vaccine manufacturers and the understanding that vaccina-tion may decrease or cease should eradication be achieved.

Public Perceptions of Vaccine Safety and Public Health

Loss of public confidence in vaccines can significantly impair control efforts, as demonstrated by the poliovirus outbreaks in northern Nigeria, which subsequently spread across several continents (Katz 2006). Numerous measles outbreaks have occurred in communities opposed to vaccination on religious or philosophical grounds (CDC 2000) or because of unfounded fears of serious adverse events (Feikin et al. 2000). Garnering the political will and public support for measles eradication is likely to be difficult in countries where the burden of disease due to measles is not recognized and unfounded fears of serious vaccine adverse events are common.

Much public attention has focused on a purported association between measles-mumps-rubella (MMR) vaccine and autism following publication of a report in 1998 hypothesizing that MMR vaccine may cause a syndrome of autism and intes-tinal inflammation (Wakefield et al. 1998). The events that followed, and the public concern over the safety of MMR vaccine, led to diminished vaccine coverage in the United Kingdom and provide important lessons in the misinterpretation of epide-miologic evidence and the communication of scientific results to the public (Offit and Coffin 2003). As a consequence, measles outbreaks became more frequent and larger in size (Jansen et al. 2003). Several epidemiological studies and comprehen-sive reviews of the evidence rejected a causal relationship between MMR vaccina-tion and autism (DeStefano and Thompson 2004; Madsen et al. 2002).

Conflict and Political Instability

Maintaining high levels of measles vaccine coverage in areas of conflict and political instability is challenging (Senessie et al. 2007), and devastating measles outbreaks occur frequently in refugee populations and internally displaced populations (Connolly et al. 2004). Measles case fatality rates in such settings have been as high as 20%–30% (Salama et al. 2001). Progress in global control has made outbreaks of

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measles less likely in some regions, although outbreaks continue to occur in refugee and internally displaced populations with low levels of immunity (Kamugisha et al. 2003). Polio vaccination campaigns have been successfully conducted during sched-uled cease-fires in regions of conflict (“days of tranquility”) (Tangermann et al. 2000) and similar results should be achievable for measles vaccination campaigns, although more highly skilled healthcare workers are needed to administer parenteral measles vaccine than oral poliovirus vaccine.

Population Growth and Demographic Changes

The world’s population is predicted to increase 2.5 billion from 2007 to 2050, from the current 6.7 billion to 9.2 billion (UN 2007). This increase is equivalent to the total number of people in the world in 1950. Population growth will be greatest in the less developed regions of the world, increasing from 5.4 billion in 2007 to 7.9 billion in 2050 (UN 2007), where the proportion of people younger than 15 years of age is greatest. Specifically, the population of the 50 least developed countries will likely more than double, increasing from 0.8 billion in 2007 to 1.7 billion in 2050 (UN 2007). This increase in global population, particularly in less developed coun-tries, will require additional resources, personnel, and vaccine supplies just to main-tain current levels of vaccine coverage.

Most of the predicted population growth will take place in urban areas of less developed countries (Cohen 2003). Currently, more than half of the world’s popula-tion lives in urban areas, and one in three urban residents live in slums, with a much higher proportion in sub-Saharan Africa and Asia (Dye 2008; UNPF 2007). Measles elimination may be particularly difficult in impoverished areas of large cities in Africa and Asia where several factors converge to facilitate MV transmission, including the high population density and difficulties in achieving high vaccination coverage. A critical question regarding measles eradication is whether the epidemi-ological conditions are sufficiently different in the large, densely populated cities of Africa and Asia than in the Americas to hinder measles eradication efforts.

Challenges to Achieving High Levels of Population Immunity

Impact of the HIV-1 Pandemic

In regions of high HIV prevalence and crowding, such as urban centers in sub-Saharan Africa, HIV-infected children may play a role in the sustaining MV trans-mission (Moss et al. 1999). Children with defective cell-mediated immunity can develop measles without the characteristic rash (Moss et al. 1999), hampering diagnosis, and HIV-infected children have prolonged shedding of MV RNA (Permer et al. 2001), potentially increasing the period of infectivity. Children born to HIV-infected mothers have lower levels of passively acquired maternal antibodies,

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increasing susceptibility to measles at an earlier age than children born to uninfected mothers (Moss et al. 2002; Scott et al. 2007), and protective antibody levels follow-ing vaccination wane within 2–3 years in many HIV-infected children not receiving antiretroviral therapy (Moss et al. 2007), creating a potential pool of susceptible children (Tejiokem et al. 2007). Thus, population immunity could be reduced in regions of high HIV-1 prevalence despite high levels of measles vaccine coverage.

Counteracting the increased susceptibility of HIV-infected children is their high mortality rate, particularly in sub-Saharan Africa, such that these children do not live long enough to build up a sizeable pool of susceptible children (Helfand et al. 2005). Successful control of measles in the countries of southern Africa suggests that the HIV-1 epidemic is not a major barrier to measles control (Biellik et al. 2002; Otten et al. 2005). This may change with increased access to antiretroviral therapy, which may prolong survival without enhancing protective immunity in the absence of revaccination. Using a dynamic, age-structured mathematical model, the prevalence of measles increased after introduction of antiretroviral therapy into a hypothetical population of children with a high prevalence of HIV-1 infection (Scott et al. 2008).

Challenges to Sustained Measles Eradication

Potential Use of Measles Virus as an Agent of Bioterrorism

The high infectivity of MV is a characteristic suitable to a bioterrorist agent, but high levels of measles vaccination coverage throughout the world would protect many persons from the deliberate release of MV. Genetic engineering of a MV strain that was not neutralized by antibodies induced by the current measles vac-cines would likely have reduced infectivity, as suggested by the fact that wild-type MV has not mutated to alter neutralizing epitopes. Whether the threat from bioter-rorism precludes stopping measles vaccination after eradication is unclear but, at the least, a single-dose rather than a two-dose measles vaccination strategy could be adopted (Meissner et al. 2004).

Prospects for Measles Eradication

The measles eradication end-game is likely to be different than that for smallpox or polio viruses (Gounder 1998; Morgan 2004). In contrast to polioviruses, pro-longed shedding of potentially virulent vaccine viruses and environmental viral reservoirs will not be challenges to measles eradication. Although MV can be carried by persons during the incubation period, transmission from mobile, asymptomatic carriers is not as common as with poliovirus. However, higher lev-els of population immunity are necessary to interrupt MV transmission, more

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highly skilled healthcare workers are required to administer measles vaccines, and containment through case detection and ring vaccination will be more difficult for MV than smallpox virus because of infectivity before rash onset. New tools, such as aerosol administration of measles vaccines (Low et al. 2008), will facilitate mass vaccination campaigns, allowing less highly trained workers to administer vaccine and diminishing the medical waste disposal problems. Critics of eradica-tion programs claim they can divert resources from primary healthcare and are imposed on countries or communities from outside. Enormous resources and efforts may be required to eradicate the few remaining measles cases, and the economic and social costs of eradication need to be carefully considered (Cutts and Steinglass 1998). Despite enormous progress, measles remains a leading vac-cine-preventable cause of childhood mortality worldwide, and continues to cause outbreaks in communities with low vaccination coverage rates in industrialized nations. To achieve the measles mortality reduction goal, continued progress needs to be made in delivering measles vaccines to the world’s children.

References

Amanna IJ, Carlson NE, Slifka MK (2007) Duration of humoral immunity to common viral and vaccine antigens. N Engl J Med 357:1903–1915

Andrews N, Tischer A, Siedler A, Pebody RG, Barbara C, Cotter S, Duks A, Gacheva N, Bohumir K, Johansen K, Mossong J, Ory F, Prosenc K, Slacikova M, Theeten H, Zarvou M, Pistol A, Bartha K, Cohen D, Backhouse J, Griskevicius A (2008) Towards elimination: measles sus-ceptibility in Australia and 17 European countries. Bull World Health Organ 86:197–204

Angel JB, Walpita P, Lerch RA, Sidhu MS, Masuredar M, DeLellis RA, Noble JT, Snydman DR, Udem SA (1998) Vaccine-associated measles pneumonitis in an adult with AIDS. Ann Intern Med 129:104–106

Bellini WJ, Helfand RF (2003) The challenges and strategies for laboratory diagnosis of measles in an international setting. J Infect Dis 187 [Suppl 1]:S283–S290

Biellik R, Madema S, Taole A, Kutsulukuta A, Allies E, Eggers R, Ngcobo N, Nxumalo M, Shearley A, Mabuzane E, Kufa E, Okwo-Bele JM (2002) First 5 years of measles elimination in southern Africa: 1996–2000. Lancet 359:1564–1568

Centers for Disease Control (1997) Measles eradication: recommendations from a meeting cosponsored by the World Health Organization, the Pan American Health Organization, and the CDC. MMWR 46:1–20

Centers for Disease Control (2000) Measles outbreak—Netherlands, April 1999 – January 2000. MMWR Morb Mortal Wkly Rep 49:299–303

Centers for Disease Control and Prevention (2005) Global Measles and Rubella Laboratory Network, January 2004–June 2005. MMWR Morb Mortal Wkly Rep 54:1100–1104

Centers for Disease Control and Prevention (2007) Vaccination coverage among children in kin-dergarten – United States, 2006–07 school year. MMWR Morb Mortal Wkly Rep 56:819–821

Centers for Disease Control and Prevention (2008a) Multistate measles outbreak associated with an international youth sporting event – Pennsylvania, Michigan, and Texas, August–September 2007. MMWR Morb Mortal Wkly Rep 57:169–173

Centers for Disease Control and Prevention (2008b) Outbreak of measles – San Diego, California, January–February 2008. MMWR Morb Mortal Wkly Rep 57:203–206

b12325337-0009ztc.indd 186 10/3/2008 10:59:57 AM


Centers for Disease Control and Prevention (2008c) Progress toward measles mortality reduction and elimination – Eastern Mediterranean Region, 1997–2007. MMWR Morb Mortal Wkly Rep 57:262–267

Cohen JE (2003) Human population: the next half century. Science 302:1172–1175 Connolly MA, Gayer M, Ryan MJ, Salama P, Spiegel P, Heymann DL (2004) Communicable dis-

eases in complex emergencies: impact and challenges. Lancet 364:1974–1983 Costa A, Henao-Restrepo AM, Hall SM, Jarrett S, Hoekstra EJ (2003) Determining measles-con-

taining vaccine demand and supply: an imperative to support measles mortality reduction efforts. J Infect Dis 187 [Suppl 1]:S22–S28

Cutts FT, Steinglass R (1998) Should measles be eradicated? BMJ 316:765–767 Dowdle WR, Hopkins DR (1998) The eradication of infectious diseases: report of the Dahlem

Workshop on the eradication of infectious diseases, Berlin, March 16–22, 1997. Wiley, New York

D’Souza RM, D’Souza R (2002) Vitamin A for the treatment of children with measles – a system-atic review. J Trop Pediatr 48:323–327

Dayan GH, Cairns L, Sangrujee N, Mtonga A, Nguyen V, Strebel P (2004) Cost-effectiveness of three different vaccination strategies against measles in Zambian children. Vaccine 22:475–484

de Quadros CA (2004) Can measles be eradicated globally? Bull World Health Organ 82:134–138 de Quadros CA, Olive JM, Hersh BS, Strassburg MA, Henderson DA, Brandling-Bennett D, Alleyne

GA (1996) Measles elimination in the Americas. Evolving strategies. JAMA 275:224–229 de Quadros CA, Izurieta H, Venczel L, Carrasco P (2004) Measles eradication in the Americas:

progress to date. J Infect Dis 189 [Suppl 1]:S227–S235 DeStefano F, Thompson WW (2004) MMR vaccine and autism: an update of the scientific evi-

dence. Expert Rev Vaccines 3:19–22 Dine MS, Hutchins SS, Thomas A, Williams I, Bellini WJ, Redd SC (2004) Persistence of vac-

cine-induced antibody to measles 26–33 years after vaccination. J Infect Dis 189 [Suppl 1]:S123–S130

Duke T, Mgone CS (2003) Measles: not just another viral exanthem. Lancet 361:763–773 Dye C (2008) Health and urban living. Science 319:766–769 Feikin DR, Lezotte DC, Hamman RF, Salmon DA, Chen RT, Hoffman RE (2000) Individual and

community risks of measles and pertussis associated with personal exemptions to immuniza-tion. JAMA 284:3145–3150

Frank SA, Bush RM (2007) Barriers to antigenic escape by pathogens: trade-off between repro-ductive rate and antigenic mutability. BMC Evol Biol 7:229


Gay NJ (2004) The theory of measles elimination: implications for the design of elimination strategies. J Infect Dis 189 [Suppl 1]:S27–S35

Gomi H, Takahashi H (2004) Why is measles still endemic in Japan? Lancet 364:328–329 Gounder C (1998) The progress of the Polio Eradication Initiative: what prospects for eradicating

measles? Health Policy Plan 13:212–233 Grais RF, Conlan AJ, Ferrari MJ, Djibo A, Le Menach A, Bjornstad ON, Grenfell BT (2008) Time

is of the essence: exploring a measles outbreak response vaccination in Niamey, Niger. J R Soc Interface 5:67–74

Helfand RF, Moss WJ, Harpaz R, Scott S, Cutts F (2005) Evaluating the impact of the HIV pan-demic on measles control and elimination. Bull World Health Organ 83:329–337

Jansen VA, Stollenwerk N, Jensen HJ, Ramsay ME, Edmunds WJ, Rhodes CJ (2003) Measles outbreaks in a population with declining vaccine uptake. Science 301:804

Kamugisha C, Cairns KL, Akim C (2003) An outbreak of measles in Tanzanian refugee camps. J Infect Dis 187 [Suppl 1]:S58–S62

Katz SL (2006) Polio: new challenges in 2006. J Clin Virol 36:163–165 Low N, Kraemer S, Schneider M, Restrepo AM (2008) Immunogenicity and safety of aerosolized

measles vaccine: systematic review and meta-analysis. Vaccine 26:383–398

b12325337-0009ztc.indd 187 10/3/2008 10:59:58 AM

188 W.J. Moss

Madsen KM, Hviid A, Vestergaard M, Schendel D, Wohlfahrt J, Thorsen P, Olsen J, Melbye M (2002) A population-based study of measles, mumps, and rubella vaccination and autism. N Engl J Med 347:1477–1482

McQuillan GM, Kruszon-Moran D, Hyde TB, Forghani B, Bellini W, Dayan GH (2007) Seroprevalence of measles antibody in the US population, 1999–2004. J Infect Dis 196:1459–1464

Meissner HC, Strebel PM, Orenstein WA (2004) Measles vaccines and the potential for worldwide eradication of measles. Pediatrics 114:1065–1069

Monafo WJ, Haslam DB, Roberts RL, Zaki SR, Bellini WJ, Coffin CM (1994) Disseminated measles infection after vaccination in a child with a congenital immunodeficiency. J Pediatr 124:273–276

Morgan OW (2004) Following in the footsteps of smallpox: can we achieve the global eradication of measles? BMC Int Health Hum Rights 4:1

Moss WJ, Griffin DE (2006) Global measles elimination. Nat Rev Microbiol 4:900–908 Moss WJ, Cutts F, Griffin DE (1999) Implications of the human immunodeficiency virus epidemic

for control and eradication of measles. Clin Infect Dis 29:106–112 Moss WJ, Monze M, Ryon JJ, Quinn TC, Griffin DE, Cutts F (2002) Prospective study of measles

in hospitalized human immunodeficiency virus (HIV)-infected and HIV-uninfected children in Zambia. Clin Infect Dis 35:189–196

Moss WJ, Scott S, Mugala N, Ndhlovu Z, Beeler JA, Audet SA, Ngala M, Mwangala S, Nkonga-Mwangilwa, Ryon JJ, Monze M, Kasolo F, Quinn TC, Cousens S, Griffin DE, Cutts FT (2007) Immunogenicity of standard-titer measles vaccine in HIV-1-infected and uninfected Zambian children: an observational study. J Infect Dis 196:347–355

Offit PA, Coffin SE (2003) Communicating science to the public: MMR vaccine and autism. Vaccine 22:1–6

Orenstein WA, Strebel PM, Papania M, Sutter RW, Bellini WJ, Cochi SL (2000) Measles eradica-tion: is it in our future? Am J Public Health 90:1521–1525

Otten M, Kezaala R, Fall A, Masresha B, Martin R, Cairns L, Eggers R, Biellik R, Grabowsky M, Strebel P, Okwo-Bele JM, Nshimirimana D (2005) Public-health impact of accelerated mea-sles control in the WHO African Region 2000–03. Lancet 366:832–839

Pan American Health Organization (1999) Measles eradication. Field guide. Washington, DC, Pan American Health Organization

Parker AA, Staggs W, Dayan GH, Ortega-Sanchez IR, Rota PA, Lowe L, Boardman P, Teclaw R, Graves C, LeBaron CW (2006) Implications of a 2005 measles outbreak in Indiana for sus-tained elimination of measles in the United States. N Engl J Med 355:447–455

Permar SR, Moss WJ, Ryon JJ, Monze M, Cutts F, Quinn TC, Griffin DE (2001) Prolonged mea-sles virus shedding in human immunodeficiency virus-infected children, detected by reverse transcriptase-polymerase chain reaction. J Infect Dis 183:532–538

Salama P, Assefa F, Talley L, Spiegel P, van Der V, Gotway CA (2001) Malnutrition, measles, mortality, and the humanitarian response during a famine in Ethiopia. JAMA 286:563–571

Schimmer B, Ihekweazu C (2006) Polio eradication and measles immunisation in Nigeria. Lancet Infect Dis 6:63–65

Scott S, Moss WJ, Cousens S, Beeler JA, Audet SA, Mugala N, Quinn TC, Griffin DE, Cutts FT (2007) The influence of HIV-1 exposure and infection on levels of passively acquired antibod-ies to measles virus in Zambian infants. Clin Infect Dis 45:1417–1424

Scott S, Mossong J, Moss WJ, Cutts FT, Cousens S (2008) Predicted impact of the HIV-1 epi-demic on measles in developing countries: results from a dynamic age-structured model. Int J Epidemiol 37:356–367

Sencer DJ, Dull HB, Langmuir AD (1967) Epidemiologic basis for eradication of measles in 1967. Public Health Rep 82:253–256

Senessie C, Gage GN, von Elm E (2007) Delays in childhood immunization in a conflict area: a study from Sierra Leone during civil war. Confl Health 1:14

b12325337-0009ztc.indd 188 10/3/2008 10:59:58 AM


Stone R (2002) Smallpox. WHO puts off destruction of U.S., Russian caches. Science 295:598–599

Takahashi H, Saito H (2008) Measles exportation from Japan to the United States, 1994 to 2006. J Travel Med 15:82–86

Tangermann RH, Hull HF, Jafari H, Nkowane B, Everts H, Aylward RB (2000) Eradication of poliomyelitis in countries affected by conflict. Bull World Health Organ 78:330–338

Tejiokem MC, Gouandjika I, Beniguel L, Zanga MC, Tene G, Gody JC, Njamkepo E, Kfutwah A, Penda I, Bilong C, Rousset D, Pouillot R, Tangy F, Baril L (2007) HIV-infected children living in central Africa have low persistence of antibodies to vaccines used in the Expanded Program on Immunization. PLoS ONE:e1260

United Nations, Department of Economic and Social Affairs Population Division (2007) World population prospects: the 2006 revision, highlights. United Nations, New York

United Nations Population Fund (2007) State of the world population 2007: unleashing the poten-tial of urban growth. United Nations Population Fund

Uzicanin A, Zhou F, Eggers R, Webb E, Strebel P (2004) Economic analysis of the 1996–1997 mass measles immunization campaigns in South Africa. Vaccine 22:3419–3426

Wakefield AJ, Murch SH, Anthony A, Linnell J, Casson DM, Malik M, Berelowitz M, Dhillon AP, Thomson MA, Harvey P, Valentine A, Davies SE, Walker-Smith JA (1998) Ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children. Lancet 351:637–641


Wolfson LJ, Gasse F, Lee-Martin SP, Lydon P, Magan A, Tibouti A, Johns B, Hutubessy R, Salama P, Okwo-Bele JM (2008) Estimating the costs of achieving the WHO-UNICEF Global Immunization Vision and Strategy, 2006–2015. Bull World Health Organ 86:27–39

World Health Organization (2004) Measles vaccines. Wkly Epidemiol Rec 79:130–142 World Health Organization (2005) Progress in reducing measles mortality – worldwide 1999–

2003. Weekly Epidemiol Rec 80:78–81 World Health Organization (2006a) Global distribution of measles and rubella genotypes – update.

Wkly Epidemiol Rec 81:474–479 World Health Organization (2006b) Impact of measles control activities in the WHO African

Region, 1999–2005. Wkly Epidemiol Rec 81:365–371 World Health Organization (2007a) Expanded Programme on Immunization of the Department of

Immunization, Vaccines and Biologicals. WHO vaccine-preventable diseases: monitoring sys-tem. 2007 global summary. WHO/IVB/2007. World Health Organization, Geneva

World Health Organization (2007b) Progress in global measles control and mortality reduction, 2000–2006. Wkly Epidemiol Rec. 82:418–424

World Health Organization (2008) Immunization surveillance, assessment and monitoring. http://www.who.int/immunization_monitoring/diseases/measles/en/. Cited 28 May 2008. World Health Organization, Geneva

World Health Organization/United Nations Children’s Fund (2001) Measles mortality reduction and regional elimination strategic plan 2001–2005. World Health Organization, Geneva

World Health Organization/United Nations Children’s Fund (2005) Global Immunization Vision and Strategy 2006–2015. World Health Organization, Geneva

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Chapter 10 Measles: Old Vaccines, New Vaccines

D. E. Griffin ( ) and C.-H. Pan

Abstract Isolation of measles virus in tissue culture by Enders and colleagues in the 1960s led to the development of the first measles vaccines. An inactivated vac-cine provided only short-term protection and induced poor T cell responses and antibody that did not undergo affinity maturation. The response to this vaccine primed for atypical measles, a more severe form of measles, and was withdrawn. A live attenuated virus vaccine has been highly successful in protection from measles and in elimination of endemic measles virus transmission with the use of two doses. This vaccine is administered by injection between 9 and 15 months of age. Measles control would be facilitated if infants could be immunized at a younger age, if the vaccine were thermostable, and if delivery did not require a needle and syringe. To these ends, new vaccines are under development using macaques as an animal model and various combinations of the H, F, and N viral proteins. Promising studies have been reported using DNA vaccines, subunit vaccines, and virus-vectored vaccines.

D. E. Griffin Department of Molecular Microbiology and Immunology , Johns Hopkins Bloomberg School of Public Health , 615 N. Wolfe St. Rm E5132 Baltimore , MD 21205 , USA , e-mail: [email protected]

Contents

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192Current Understanding of Protective Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193Cellular Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Inactivated Vaccines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194Atypical Measles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

Live Attenuated Virus Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195Determinants of Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

Development of New Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199Rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199Types of Vaccines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

Summary and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203


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192 D.E. Griffin, C.-H. Pan

Abbreviations

FIMV Formalin-inactivated measles vaccine HI Hemagglutination inhibition HIV Human immunodeficiency virus LAV Live attenuated virus vaccine MV Measles virus

Introduction

Measles is a childhood rash disease associated with substantial morbidity and mor-tality and was an early target for development of a vaccine. The first attempts at vaccination by the Scottish physician Francis Home in 1749 were based on the principles of variolation. He inoculated individuals with blood taken from measles patients during the early stages of the rash with the reasoning that introduction of disease through the skin would lessen the effects on the lung (Home 1759). However, morbillization was in general unsuccessful and tended to result in trans-mission of measles (Hektoen 1905). Subsequent approaches between 1920 and 1940 designed to inactivate or attenuate the virus by culture in chick embryos also met with limited success (Maris et al. 1949).

In 1954, the isolation of measles virus (MV) in tissue culture from the blood of a child with measles by Enders and Peebles opened the way for vaccine develop-ment (Enders and Peeble 1954). The Edmonston strain of MV was successfully propagated in human and monkey cells and led to the simultaneous development of both inactivated and live attenuated virus vaccines. Inactivated virus vaccines were developed using formalin or tween-ether for viral inactivation, but proved unsuc-cessful (Rauh and Schmidt 1965; Warren and Gallian 1962). A live attenuated virus vaccine (LAV), based on adaptation of MV to growth in chick cells (Enders et al. 1962), provided long-term protection from measles, and derivatives of this vaccine are used worldwide today (Peradze and Smorodintsev 1983).

Prior to the widespread use of measles vaccine, measles was estimated to result in 5–8 million deaths each year. The decline in mortality from measles in developed countries can be attributed in part to improved nutrition and medical care, but mostly to effective delivery of LAV. However, in resource-poor countries, measles remains the second most common cause of vaccine-preventable deaths in children (CDC 2006). This is due in part to difficulty with delivery of two doses of vaccine to a high proportion of the population and to the inability to effectively immunize infants under the age of 9 months, leaving a window of vulnerability to infection (Moss and Griffin 2006).

The adverse outcomes associated with certain vaccines have hampered new approaches to measles immunization, but have also increased interest in an under-standing of the determinants of vaccine-induced protective immunity (Lambert et al. 2005). On the one hand, the early formalin-inactivated measles vaccine was not only poorly protective, but it also primed for a more severe disease known as atypical

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10 Measles: Old Vaccines, New Vaccines 193

measles (Nader et al. 1968; Rauh and Schmidt 1965). Further, LAV was associated with increased mortality in girls when given in a high dose to young infants in coun-tries with high rates of childhood mortality (Garenne et al. 1991; Holt et al. 1993). The biologic bases for these problems with prior MV vaccines are incompletely understood. Definition of protective immunity to measles could enlighten not only the development of new measles vaccines, but also the development of other viral vaccines.

Current Understanding of Protective Immunity

Antibody

Antibody can protect from MV infection and may contribute to recovery from infection (Albrecht et al. 1997; Endo et al. 2001). Antibody to MV is sufficient for protection because infants are protected by maternal antibody (Albrecht et al. 1997) and passive transfer of immune serum can modify or interfere with measles vacci-nation and can partially protect children from measles after exposure (Reilly et al. 1961). In infants, the level of maternal antibody correlates with failure of response to vaccination (Albrecht et al. 1997). In outbreaks, antibody levels correlate with protection from disease with a plaque reduction neutralizing titer (PRNT) of 120 mIU/ml generally considered the level needed to prevent disease (Chen et al. 1990). Furthermore, in vaccine studies the best correlate of protection from infec-tion is the level of neutralizing antibody (Polack et al. 2000).

Most neutralizing antibodies are directed to the H glycoprotein. Major confor-mational epitopes have been localized to regions between amino acids 368 and 396 and in the SLAM-binding region (Erlt et al. 2003; Liebert et al. 1994). Most of these epitopes are predicted to be a part of exposed surfaces on top of the molecule (Erlt et al. 2003; Langedijk et al. 1997). Although the H gene sequence is variable (Rota et al. 1992), MV is relatively stable in the regions required for receptor bind-ing (Hashiguchi et al. 2007) and the current vaccines developed in the 1960s con-tinue to provide protection from all wild-type strains of MV. Antibody to the highly conserved F protein also contributes to virus neutralization, probably by preventing fusion of the virus membrane with the cell membrane at the time of virus entry (Malvoisin and Wild 1990; Polack et al. 2000). Antibodies to other viral proteins, particularly the nucleocapsid (N) protein, are induced after infection with wild-type or vaccine strains of MV, but their role in protection is unclear (Graves et al. 1984).

Cellular Immunity

The cellular immune response is also important for development of protective immunity. CD4 + T cells are essential for development of an antibody response that is mature with respect to avidity, isotype, and durability. Formation of germinal

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centers, a major site for class switch recombination and somatic hypermutation of immunoglobulin genes and for development of long-lived plasma cells is T cell-dependent (Fazilleau et al. 2007). CD8 + T cells are important for the early control of virus replication (Permar et al. 2003) and are also likely to contribute to protec-tive immunity. T cell epitopes are present in many MV proteins and are particularly abundant in the H protein (Oh et al. 2006; Ota et al. 2007).

Inactivated Vaccines

Development

Inactivated MV vaccines were developed using formalin or tween-ether and the Edmonston B strain of MV (Warren and Gallian 1962). The alum-precipitated for-malin-inactivated measles vaccine (FIMV) developed in the United States was given in a three-dose regimen and licensed in 1963 (Carter et al. 1962; Warren and Gallian 1962). Recipients of the inactivated vaccine developed moderate levels of neutralizing and hemagglutination inhibiting (HI) antibodies, but low levels of complement fixing (CF) antibodies (Carter et al. 1962; Feldman et al. 1962; Norrby et al. 1975). The vaccine was protective when exposure to measles occurred within several months after immunization (Feldman et al. 1962; Fulginiti and Kempe 1963; Measles Vaccines Committee 1968). However, antibody titers declined rap-idly, and recipients again became susceptible to measles (Fulginiti and Kempe 1963; Rauh and Schmidt 1965). When infected, these previously FIMV-vaccinated individuals had a tendency to develop a more severe disease, atypical measles (Nader et al. 1968; Rauh and Schmidt 1965).

Atypical Measles

Atypical measles was characterized by a higher and more prolonged fever, unusual skin lesions and severe pneumonitis compared to measles in unvaccinated persons (Fulginiti et al. 1967; Nader et al. 1968). The rash was often accompanied by evi-dence of hemorrhage or vesiculation and began on the extremities rather than the trunk. The pneumonitis included distinct nodular parenchymal lesions and hilar adenopathy (Gremillion and Crawford 1981; Young et al. 1970). Abdominal pain, hepatic dysfunction, headache, eosinophilia, pleural effusions, and edema were also described. Atypical measles was reported up to16 years after receipt of the inacti-vated vaccine. Administration of LAV after two to three doses of FIMV did not eliminate subsequent susceptibility to atypical measles and was sometimes associ-ated with severe reactions at the site of LAV inoculation (Buser 1967; Chatterji and Mankad 1977; Fulginiti et al. 1968).

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Hypotheses about the pathogenesis of atypical measles included an abnormally intense cellular immune response (Fulginiti et al. 1968), an inability of the inacti-vated vaccine to induce local respiratory tract immunity (Bellanti et al. 1969) and a lack of production of antibody to F, which allowed virus to spread from cell to cell despite the development of antibody to H (Merz et al. 1980; Norrby et al. 1975). Studies in rhesus macaques have shown that FIMV induces no cytotoxic T cell response and that antibody does not undergo affinity maturation and production is short-lived (Polack et al. 1999, 2003a). The low-avidity antibody induced by FIMV can neutralize in vitro infection with viruses that use CD46 as a receptor, as routinely measured by PRNT assays in Vero cells, but cannot neutralize infection with wild-type viruses that primarily use SLAM as a receptor (Polack et al. 2003a). Subsequent infection with MV induces an anamnestic antibody response, but the antibody is also of low avidity and cannot neutralize wild-type virus. This leads to formation of complexes of non-neutralizing antibody and MV, resulting in immune complex deposition, vasculitis, and pneumonitis (Plowright and Ferris 1962; Polack et al. 2003a). The nature of the defect in immune priming exhibited by FIMV has not yet been identified.

These problems of short-lived immunity and predisposition to enhanced disease, in the face of simultaneous development of the more successful LAV, led to the withdrawal of this vaccine in 1967.

Live Attenuated Virus Vaccines

Development

The process of adaptation of MV grown in primary human and monkey kidney and amnion cells to cells of nonsusceptible hosts, such as the chick embryo, led success-fully to the development of LAV strains (Enders et al. 1960; Katz et al. 1958, 1959; Milovanovic et al. 1957; Schwarz and Zirbel 1959) (see the chapter by S.L. Katz, this volume). The first attenuated live measles vaccine was developed by passage of the Edmonston strain of MV in chick embryo fibroblasts to produce the Edmonston B virus (Enders et al. 1962). Inoculation of this virus into primates produced no clinical symptoms, no detectable viremia, and no spread to the respiratory tract (Auwaerter et al. 1999), but did induce an immune response that protected the mon-keys from subsequent challenge with wild-type virus (Enders et al. 1960).

The Edmonston B strain of LAV protected children from measles (Krugman 1971) and was licensed in March 1963, but induced fever and rash in a large pro-portion of children (Katz et al. 1960). Reactions were reduced when MV antibody was given at the same time as the vaccine (Ad Hoc Advisory committee on Measles Control 1963; Milovanovic 1965; Reilly et al. 1961; Stokes et al. 1961). More extensive passage of the Edmonston B virus in chick embryo fibroblasts at reduced temperature produced the more attenuated vaccines, Schwarz and Moraten, in

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extensive use today (Rota et al. 1994; Schwarz 1962). Other Edmonston-derived vaccine strains (e.g., Zagreb, AIK-C) and attenuated strains developed independ-ently (e.g., CAM, Leningard-16, Shanghai-191) are also successful LAVs (Hirayama 1983; Jianzhi and Zhihui 1983; Peradze and Smorodintsev 1983; Sakata et al. 1978). Few differences have been described among MV vaccine strains (all geno-type A) regardless of the geographic origin of the parent virus (Rota et al. 1994). However, there may be some biologic differences. For instance, Edmonston-Zagreb is produced in human diploid cells, rather than chick embryo fibroblasts, and may be more immunogenic in young infants and when delivered by the aerosol route than other strains (Cutts et al. 1997).

Lyophilized LAV is relatively stable, but the reconstituted vaccine rapidly loses infectivity. LAV is inactivated by light and heat and after reconstitution loses about half of its potency at 20°C and almost all potency at 37°C within 1 h (Melnick 1996). Therefore, a cold chain must be maintained for the vaccine prior to and after reconstitution. Attenuated strains replicate less efficiently in vivo than wild-type MV (Auwaerter et al. 1999; Van Binnendijk et al. 1994), perhaps due to altered cellular tropism (Condack et al. 2007). However, LAV induces both neutralizing antibody and cellular immune responses qualitatively similar to that induced by natural disease, although titers are lower (Krugman 1971; Ovsyannikova et al. 2003). Antibodies first appear 12–15 days after vaccination and peak at 1–3 months. In many countries, LAV is combined with other live attenuated virus vaccines such as those for mumps, rubella (MMR) and varicella (MMRV). These measles-containing vaccines have proven safe and effective and have saved the lives of many millions of children (Bellini et al. 1994; Wolfson et al. 2007).

Determinants of Response

Age

The recommended age of vaccination varies from 6 to 15 months. The probability of seroconversion and the amounts of antibody induced are determined by the levels of persisting MV-specific maternal antibody and the age of the infant at the time of vaccination (Cutts et al. 1995; Gans et al. 1998, 2004; Redd et al. 2004; Reilly et al. 1961). Levels of passively acquired antibody are dependent on the mother’s level of antibody, on the transfer of antibody across the placenta and on the rate of decay in the infant (Caceres et al. 2000). The cellular immune response is induced more read-ily than antibody in young infants with maternal antibody (Gans et al. 2004). As measles is controlled in a region, an increasing proportion of mothers will have measles immunity induced by vaccination rather than natural infection. This will result in lower levels of passively acquired antibody in infants and the possibility of lowering the age of vaccination (Lennon and Black 1986; Maldonado et al. 1995; Markowitz et al. 1996; Pabst et al. 1992). Currently, the proportions of children that

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develop protective levels of antibody based on age of vaccination are approximately 85% at 9 months and 95% at 12 months (Cutts et al. 1995). The recommended age for measles vaccination varies regionally and is a balance between the optimum age for seroconversion and the probability of acquiring measles before that age (Cutts et al. 1995). In areas where measles remains prevalent, measles vaccination is rou-tinely performed at 9 months, whereas in areas with little measles, vaccination is often at 12–15 months. During epidemics and for human immunodeficiency virus (HIV)-infected infants in developing countries, vaccination at 6 months with a second dose at 9 or 12 months is recommended (Hutchins et al. 2001; WHO/UNICEF 1989).

Route

LAV is administered subcutaneously or intramuscularly. However, there is substan-tial interest in alternate routes of delivery that would not require needles and syringes. Neither oral nor intranasal administration is effective (Black and Sheridan 1960; Stittelaar et al. 2002a), but respiratory delivery may be more promising. There are several ongoing efforts to develop and evaluate aerosol delivery of aque-ous and dry powder forms of LAV (Cutts et al. 1997; de Swart et al. 2006, 2007). Aerosol administration of the aqueous vaccine is highly effective in boosting pre-existing antibody and may hold promise for use in older children (Cutts et al. 1997; Sabin 1991; Sabin et al. 1983). Respiratory routes of vaccination have also been advocated as a means to lower the age of immunization (Sabin et al. 1983, 1985). However, the primary immune response to aerosolized measles vaccine, particu-larly in young infants, is lower than it is to subcutaneous administration (Low et al. 2008; Wong et al. 2004, 2006). The reasons for this are not known, but may be related to dose, efficiency of delivery, or ability of the vaccine virus to establish infection in the respiratory tract. Responses of macaques to respiratory delivery of a dry powder vaccine are lower than to aqueous vaccine for reasons that are not yet clear (de Swart et al. 2007).

Host Determinants

Genetic background affects the likelihood of seroconversion and antibody titers (Dhiman et al. 2007; Ovsyannikova et al. 2004, 2006). Common childhood illnesses at the time of vaccination may also have an effect (Migasena et al. 1997). Any potential decrease in seroconversion must be balanced against the loss of the oppor-tunity for vaccination and the consequent risk of the child acquiring measles. Similar compromises must be considered with respect to immunizing individuals infected with HIV-1 (Cutts et al. 1999). LAV is contraindicated in individuals with severe deficiencies of cellular immunity because of the possibility of progressive pulmonary or CNS infection (Bitnun et al. 1999; Mawhinney et al. 1971; Monafo et al. 1994). However, LAV has generally been well tolerated in HIV-infected

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children and adults, although the antibody responses are of lower avidity and less durable than those of HIV-uninfected children (Brena et al. 1993; McLaughlin et al. 1988; Moss et al. 2007; Rudy et al. 1994). Because of the potential severity of wild-type MV infection in HIV-infected individuals (Budka et al. 1996; CDC 1988; Moss et al. 2008), LAV is recommended for administration without respect to HIV status in most countries. However, LAV is not recommended for those with known low CD4 + T cell counts (Committee on Infectious Diseases and Committee on Pediatric AIDS 1999) because of the possibility of progressive infection (Angel et al. 1998; Goon et al. 2001).

Dose

The dose of MV routinely used for immunization is between 10 3 and 10 4 plaque-forming units. Overall, the efficacy of a single dose of measles vaccine in infancy is estimated at 80%–95% (Gans et al. 2004). When 100-fold higher doses were used, seroconversion in younger infants improved and in 1990 the WHO recom-mended the high-titer vaccine for use in countries with significant measles transmis-sion (WHO 1990). However, subsequent follow-up of children receiving high-titer vaccines in countries with high childhood mortality showed an increased mortality in girls over the subsequent 2–3 years and the recommendation was withdrawn (Cutts and Markowitz 1994; Holt et al. 1993; Knudsen et al. 1996). Mortality was due to a relative increase in deaths due to other infections (Aaby et al. 1996). The pathogenesis of delayed increased mortality after the high-titer vaccine is not under-stood, but occurred primarily in those who developed a rash after vaccination and may be related to long-term suppression of immune responses to other pathogens similar to that induced by measles (Seng et al. 1999) or to a change in the sequence of vaccine delivery (Aaby et al. 2003a, 2003b, 2006).

The duration of vaccine-induced immunity is variable. In general, levels of anti-body are lower after vaccination than after recovery from natural disease and MV-specific antibody and CD4 + T cells decay with time (Christenson and Bottiger 1994; Eghafona et al. 1991; Krugman 1971; Naniche et al. 2004). Secondary vac-cine failure rates of 5% have been estimated at 10–15 years after vaccination, but are probably lower when vaccine is given after the age of 1 year (Anders et al. 1996; Mathias et al. 1989; Ozanne and d’Halewyn 1992). Decreasing antibody tit-ers do not necessarily imply a complete loss of protective immunity, as a secondary immune response usually develops after re-exposure to MV, with a rapid rise in antibody and absence of clinical disease (Ozanne and d’Halewyn 1992).

Because of the high infectivity of MV and the fact that not everyone develops protective immunity following vaccination, a single dose of measles vaccine does not achieve a sufficient level of population immunity to eliminate endemic MV transmission (see the chapter by P.A. Rota et al., this volume). Therefore, to achieve 95% immunity, a second dose of vaccine is necessary to immunize persons who missed or did not respond to the first dose (CDC 2000; Cutts et al. 1999; Gay 2004; Orenstein et al. 2006). The two-dose strategy has been credited with elimination of

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indigenous measles in many countries in which it has been employed (Cutts et al. 1999).

Two broad strategies to administer the second dose have been used. In coun-tries with sufficient infrastructure, the second dose can be delivered as a part of routine vaccination, typically prior to the start of school with school entry require-ments enforcing the policy (Orenstein 2006). A second approach uses mass sup-plementary immunization campaigns to deliver the second dose of vaccine in a wide geographic area (de Quadros et al. 2004). The goal is to rapidly achieve a high level of population immunity and interrupt MV transmission. However, even highly immunized populations in countries that have eliminated endemic trans-mission are vulnerable to localized outbreaks associated with importation from areas where measles remains endemic (Rota et al. 2002).

Development of New Vaccines

Rationale

A new vaccine would be advantageous if it would allow vaccination of infants before 6 months of age. This would both close the window of susceptibility between decay of maternal antibody and vaccination and facilitate vaccine delivery by allowing measles vaccine to be given along with other WHO Expanded Program for Immu-nization (EPI) vaccines (El Kasmi and Muller 2001). Additional motivations for development of a new vaccine would be to eliminate the need for a cold chain, to avoid the use of needles and syringes for delivery (Mitragotri 2005), and to provide a vaccine that would be safe for immunocompromised individuals. Such a vaccine would need to induce protective immunity and overcome the obstacles of immuno-logic immaturity and maternal antibody present in young infants.

Several animal models, including transgenic mice and cotton rats, have been used for testing potential new measles vaccines, but only non-human primates, par-ticularly rhesus macaques, develop a disease similar to that of humans and offer the opportunity for assessing both protection from wild-type MV challenge and prim-ing for enhanced disease (Auwaerter et al. 1999; Combredet et al. 2003; El Mubarak et al. 2007; Polack et al. 1999; Van Binnenkijk et al. 1995). A number of experi-mental vaccines have been developed and there is an increasing understanding of measles protective immunity. Vaccinations with individual MV proteins expressed in viral or bacterial vectors or as DNA, peptides, or proteins have been explored in mice and cotton rats (El Kasmi and Muller 2001; Wyde et al. 2000). A more limited portfolio of experimental vaccines have been studied in macaques (Polack et al. 2000; Premenko-Lanier et al. 2006;Stittelaar et al. 2000b, 2002c; Van Binnenkijk et al. 1997).

The choices of MV antigens to be used in a new vaccine have focused on pro-teins required for induction of neutralizing antibody and CD4 + and CD8 + T cell responses. Neutralizing antibody is directed primarily against H and to a lesser

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degree against F. However, in several studies it has been observed that the level of neutralizing antibody is higher when H is used alone for vaccination rather than in combination with F (Pan et al. 2005; Pasetti et al. 2007; Polack et al. 2000). The reason for this is not clear, but may be related to the fact that H primes for a type 2 cytokine response while F primes for a type 1 response and that these responses are cross-modulated when H and F are given together (Polack et al. 2003b). H contains many human epitopes presented by HLA-class I (Oh et al. 2006; Ota et al. 2007) and induces both CD4 + and CD8 + T cell responses in macaques (Pan et al. 2005, 2008; Polack et al. 2000); N has occasionally been included to enhance the oppor-tunity for induction of T cell responses.

Types of Vaccines

DNA

Delivery of viral genes into host cells for processing and antigen presentation with-out the need for virus infection make DNA vaccines an attractive possibility for development. In addition, DNA vaccines are thermostable, inexpensive to manufac-ture, do not induce antivector immunity, and induce strong T cell responses. Immu-nization with DNA expressing N did not protect against intracerebral challenge with rodent-adapted MV (Fooks et al. 1996), but DNA expressing H or F delivered by gene gun, intramuscularly or by various mucosal routes induced good humoral and cellular responses in mice and cotton rats (Cardoso et al. 1996; Etchart et al. 1997; Pasetti et al. 2003; Yang et al. 1996).

Several DNA vaccines have been tested in juvenile cynomolgus or rhesus macaques. Transdermal delivery of two doses of plasmids encoding MV proteins elicited low serum antibody responses, but priming for more rapid antibody and cellular immune responses and a tendency for lower viremia after challenge (Stittelaar et al. 2002b). Intradermal or gene gun delivery of two doses of nonadju-vanted DNA encoding H, F, or H and F elicited cytotoxic T cell responses and sus-tained antibody responses of variable titer (Polack et al. 2000). Protection from challenge 2 years after the initial immunization correlated with the levels of neu-tralizing antibody. All monkeys having antibody levels less than 120 mIU/ml (one immunized with F, one with H, and one with H + F) developed a rash and viremia. However, the rashes were mild and there was no suggestion of atypical measles. Monkeys with intermediate levels of antibody developed viremia without a rash (partial protection) and monkeys with high levels of antibody did not develop a viremia or rash (full protection). These studies indicated that DNA vaccines could protect from measles and that DNA immunization did not predispose to atypical measles. Studies of the same or similar vaccines in infant macaques have shown induction of lower levels of antibody, particularly in the face of maternal antibody, and limited protection from challenge (Premenko-Lanier et al. 2006).

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More recent studies have focused on improved plasmid design and the use of adjuvants to enhance immunogenicity of DNA vaccines and more reliably exceed the threshold of antibody needed for complete protection. New plasmids have used different promoter and expression strategies. Adjuvants have included cytokines (Premenko-Lanier et al. 2004) and formulation of the DNA with complex carbohy-drates or lipids designed to improve DNA entry into cells, to provide a depot for prolonged release or to stimulate B cell production of antibody (Kaslow 2004).

Improved plasmid design has included codon optimization and use of alphavirus DNA vectors (e.g., SINCP) for a layered DNA/RNA expression of antigen (Dubensky et al. 1996). In this approach, the DNA used for immunization consists of the genes coding for the alphavirus nonstructural proteins, the subgenomic pro-moter, and the MV protein. When the DNA is transcribed to mRNA, the nonstruc-tural proteins are translated, replicate the RNA, and produce abundant subgenomic mRNAs that code for the antigen. Amplification of the mRNAs results in large amounts of synthesized protein (Boorsma et al. 2003; Dubensky et al. 1996; Herweijer et al. 1995). In addition, these vaccines activate innate immune pathways to increase immunogenicity of the antigens encoded (Leitner et al. 2003). Sindbis virus-based replicon plasmid (SINCP)-based vaccines expressing H and F have been used successfully to prime responses of juvenile and infant macaques to LAV (Pasetti et al. 2007).

A number of DNA adjuvants have also been studied. Adsorption of DNA onto biodegradable cationic polylactide co-glycolide (PLG) microparticles (Denis-Mize et al. 2000; O’Hagan et al. 2001) delivers the DNA vaccine to and activates antigen-presenting cells, increases DNA persistence and recruits mononuclear cells to the site (Denis-Mize et al. 2003). An improvement over naked DNA for induction of both antibody and T cell responses has been demonstrated in rodents and primates for other candidate vaccines (Mollenkipf et al. 2004; O’Hagan et al. 2001, 2004; Otten et al. 2005). PLG formulation improved the antibody responses of mice, but not the responses of macaques, to MV DNA vaccination (Pan et al. 2008). PLG/SINCP-H given intramuscularly elicited short-lived neutralizing antibody and memory T cells in juvenile rhesus macaques that provided partial protection from challenge. The PLG/SINCP-H vaccine was less effective when given intradermally with lower antibody and T cell responses than after intramuscular inoculation. Monkeys vaccinated intradermally were not protected from rash or viremia after challenge. Monkeys vaccinated with low-dose PLG/SINCP-H intradermally had higher viremias, more severe rashes and higher eosinophil counts after challenge than monkeys that received the control vaccine, suggesting exacerbated disease (Pan et al. 2008).

Another class of adjuvants that has been explored is cationic lipids. Cationic lipids are safe and well tolerated in humans and other animals (Nabel et al. 1993; Parker et al. 1995). Vaxfectin consists of an equimolar mixture of the cationic lipid GAP-DMORIE [(+)- N -(3-aminopropyl)- N,N -dimethyl-2,3-bis(cis-9-tetradeceny-loxy)-1-propanaminium bromide)] and a neutral co-lipid DPyPE (1,2-diphytanoyl-sn-glydero-3-phosphoethanolamine) (Hartikka et al. 2001). Immunization with

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Vaxfectin-formulated, codon-optimized DNAs expressing the MV H and F proteins elicited strong antibody and T cell responses in mice and monkeys and provided complete protection against rash and viremia after challenge with wild-type MV in both juvenile and infant rhesus macaques. Two doses of vaccine delivered either intradermally or intramuscularly to juvenile or infant rhesus macaques induced MV-specific antibody responses that were durable, neutralizing, and of high avidity, as well as MV-specific interferon (IFN)-γ-producing memory T cells.

Recombinant and Subunit Proteins and Peptides

Recombinant proteins, peptides, and proteins purified from MV have been used for vaccine development. In mice, peptides from H and F have been used alone and in combination to induce neutralizing antibody (El Kasmi and Muller 2001) and H protein expressed in tobacco plants and delivered orally could boost the response to a DNA vaccine (Webster et al. 2002). Immune-stimulating complexes (iscoms) incorporating the F and H proteins into a matrix with quilaja saponins, phospholip-ids, and cholesterol stimulated HI and hemolysis-inhibiting antibodies in mice (Stittelaar et al. 2000a). Immunized mice could be protected from intracerebral challenge with a neurotropic strain of MV (Condack et al. 2007; Varsanyi et al. 1987). In cynomolgus macaques, iscoms induced durable MV-specific antibody in the presence and absence of passively transferred antibody and provided partial protection from challenge (Stittelaar et al. 2002c; Van Binnendijk et al. 1997). MV proteins from infected cells mixed with a proteosome adjuvant consisting of Neisseria meningitidis outer membrane proteins and Shigella flexneri 2a lipopoly-saccharide stimulated neutralizing antibody in mice after intranasal administration (Chabot et al. 2005) and could boost the responses of rhesus macaques to a DNA vaccine (Pasetti et al. 2007).

Vectored by Other Viruses or Bacteria

Several viruses have been used to express MV proteins and tested as experimental vaccines. The first studies were done with vaccinia virus expressing H, F, and/or N and induced immune responses in mice, rats, and macaques (El Kasmi and Muller 2001). This vaccine was not able to stimulate an antibody response in the presence of passively acquired antibody, but did provide partial protection from challenge, presumably mediated by the observed MV-specific T cell responses (Van Binnendijk et al. 1997). Subsequently, studies of the replication-defective modified vaccinia virus Ankara (MVA) expressing H and F showed that two doses of 10 8 pfu delivered intramuscularly and intranasally 1 month apart elicited neutralizing antibody and T cell responses in juvenile cynomolgus macaques in the presence and absence of passively transferred antibody (Sittelaar et al. 2000b). These monkeys were all pro-tected from challenge 1 year after vaccination. This vaccine is safe in immunosup-pressed macaques (Stittelaar et al. 2001).

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Alphavirus replicon particle vaccines expressing MV H induced high-titered, dose-dependent, MV-neutralizing antibody after a single vaccination in mice. Vaccination of juvenile rhesus macaques with a single dose, and infant macaques with two doses, of 10 8 particles induced sustained levels of high-titered MV-neutralizing antibody and IFN-γ-producing memory T cells. Most monkeys were protected from disease, but not from viremia when challenged 18 months later (Pan et al. 2005).

Recombinant Bacille-Calmette-Guérin, the mycobacteria used for neonatal immunization against tuberculosis, has been engineered to express the MV N protein for immunization of infant rhesus macaques (Zhu et al. 1997). Cellular immune responses were elicited after challenge and there was reduced lung inflammation, but monkeys were not protected from systemic infection.

Summary and Future Directions

The current live attenuated measles virus vaccine is safe and has been effective in eliminating endemic MV transmission in regions that have successfully delivered two doses of vaccine to a high proportion of the population. Challenges to measles control remain in many regions due to difficulty with delivery of two doses as a part of routine childhood vaccination programs. Development of a new vaccine that did not require a cold chain or delivery with needle and syringe and that could be safely and effectively used in infants under the age of 6 months could facilitate measles control in many parts of the world. Investigation of the efficacy of experimental measles vaccines has advanced our knowledge of the determinants of protective immunity and may lead to new vaccines to aid in the control of measles.

Acknowledgements Work from the authors’ laboratory was supported by research grants from the National Institutes of Health (AI 23047) and the Bill and Melinda Gates Foundation.

References

Aaby P, Samb B, Simondon F, Knudsen K, Seck AM, Bennett J, Markowitz L, Whittle H (1996) A comparison of vaccine efficacy and mortality during routine use of high-titre Edmonston-Zagreb and Schwarz standard measles vaccines in rural Senegal. Trans R Soc Trop Med Hyg 90:326–330

Aaby P, Jensen H, Samb B, Cisse B, Sodemann M, Jakobsen M, Poulsen A, Rodrigues A, Lisse IM, Simondon F, Whittle H (2003a) Differences in female-male mortality after high-titre measles vaccine and association with subsequent vaccination with diphtheria-tetanus-pertussis and inactivated poliovirus: reanalysis of West African studies. Lancet 361:2183–2188

Aaby P, Jensen H, Simondon F, Whittle H (2003b) High-titer measles vaccination before 9 months of age and increased female mortality: do we have an explanation? Semin Pediatr Infect Dis 14:220–232

Aaby P, Ibrahim SA, Libman MD, Jensen H (2006) The sequence of vaccinations and increased female mortality after high-titre measles vaccine: trials from rural Sudan and Kinshasa. Vaccine 24:2764–2771

b12325337-0010ztc.indd 203 10/3/2008 11:01:41 AM


Ad Hoc Advisory Committee on Measles Control (1963) Statement of the status of measles vac-cines. JAMA 183:1112–1113

Albrecht P, Ennis FA, Saltzman EJ, Krugman S (1977) Persistence of maternal antibody in infants beyond 12 months: mechanism of measles vaccine failure. J Pediatr 91:715–718

Anders JF, Jacobson RM, Poland GA, Jacobsen SJ, Wollan PC (1996) Secondary failure rates of measles vaccines: a metaanalysis of published studies. Pediatr Infect Dis J 15:62–66

Angel JB, Walpita P, Lerch RA, Sidhu MS, Masurekar M, DeLellis RA, Noble JT, Snydman DR, Udem SA (1998) Vaccine-associated measles pneumonitis in an adult with AIDS. Ann Intern Med 129:104–106

Auwaerter PG, Rota PA, Elkins WR, Adams RJ, DeLozier T, Shi Y, Bellini WJ, Murphy BR, Griffin DE (1999) Measles virus infection in rhesus macaques: altered immune responses and comparison of the virulence of six different virus strains. J Infect Dis 180:950–958

Bellanti JA, Sanga RL, Klutinis B, Brandt B, Artenstein MS (1969) Antibody responses in serum and nasal secretions of children immunized with inactivated and attenuated measles-virus vac-cines. N Engl J Med 280:628–633

Bellini WJ, Rota JS, Rota PA (1994) Virology of measles virus. J Infect Dis 170:S15–S23 Bitnun A, Shannon P, Durward A, Rota PA, Bellini WJ, Graham C, Wang E, Ford-Jones EL, Cox

P, Becker L, Fearon M, Petric M, Tellier R (1999) Measles inclusion-body encephalitis caused by the vaccine strain of measles virus. Clin Infect Dis 29:855–861

Black FL, Sheridan SR (1960) Studies on an attenuated measles-virus vaccine. N Engl J Med 263:165–169

Boorsma M, Saudan P, Pfruender H, Bailey JE, Schlesinger S, Renner WA, Bachmann MF (2003) Alphavirus cDNA-based expression vectors: effects of RNA transcription and nuclear export. Biotechnol Bioeng 81:553–562

Brena AE, Cooper ER, Cabral HJ, Pelton SI (1993) Antibody response to measles and rubella vaccine by children with HIV infection. J Acquir Immune Defic Syndr 6:1125–1129

Budka H, Urbanits S, Liberski PP, Eichinger S, Popow-Kraupp T (1996) Subacute measles virus encephalitis: a new and fatal opportunistic infection in a patient with AIDS. Neurology 46:586–587

Buser F (1967) Side reaction to measles vaccination suggesting the Arthus phenomenon. N Engl J Med 277:250–251

Caceres VM, Strebel PM, Sutter RW (2000) Factors determining prevalence of maternal antibody to measles virus throughout infancy: a review. Clin Infect Dis 31:110–119

Cardoso AI, Blixenkrone-Moller M, Fayolle J, Liu M, Buckland R, Wild TF (1996) Immunization with plasmid DNA encoding for the measles virus hemagglutinin and nucleoprotein leads to humoral and cell-mediated immunity. Virology 225:293–299

Carter CH, Conway TJ, Cornfeld D, Iezzoni DG, Kempe CH, Moscovici C, Rauh LW, Vignec AJ, Warren J (1962) Serologic response of children to inactivated measles vaccine. JAMA 179:848–853

Centers for Disease Control (1988) Measles in HIV-infected children, United States. MMWR 37:183–186

Centers for Disease Control (2000) Recommendations from meeting on strategies for improving global measles control, May 11–12, 2000. MMWR 49 (12):1116–1118

Centers for Disease Control (2006) Vaccine preventable deaths and the Global Immunization Vision and Strategy, 2006–2015. MMWR Morb Mortal Wkly Rep 55:511–515

Chabot S, Brewer A, Lowell G, Plante M, Cyr S, Burt DS, Ward BJ (2005) A novel intranasal Protollin-based measles vaccine induces mucosal and systemic neutralizing antibody responses and cell-mediated immunity in mice. Vaccine 23:1374–1383

Chatterji M, Mankad V (1977) Failure of attenuated viral vaccine in prevention of atypical mea-sles. JAMA 238:2635

Chen RT, Markowitz LE, Albrecht P, Stewart JA, Mofenson LM, Preblud SR, Orenstein WA (1990) Measles antibody: reevaluation of protective titers. J Infect Dis 162:1036–1042

b12325337-0010ztc.indd 204 10/3/2008 11:01:41 AM


Christenson B, Bottiger M (1994) Measles antibody: comparison of long-term vaccination titres, early vaccination titres and naturally acquired immunity to and booster effects on the measles virus. Vaccine 12:129–133


Committee on Infectious Diseases and Committee on Pediatric AIDS (1999) Measles immuniza-tion in HIV-infected children. Pediatrics 103:1057–1060

Condack C, Grivel JC, Devaux P, Margolis L, Cattaneo R (2007) Measles virus vaccine attenua-tion: suboptimal infection of lymphatic tissue and tropism alteration. J Infect Dis 196:541–549

Cutts FT, Markowitz LE (1994) Successes and failures in measles control. J Infect Dis 170:S32–S41

Cutts FT, Grabowsky M, Markowitz LE (1995) The effect of dose and strain of live attenuated measles vaccines on serological responses in young infants. Biologicals 23:95–106

Cutts FT, Clements CJ, Bennett JV (1997) Alternative routes of measles immunization: a review. Biologicals 25:323–338

Cutts FT, Henao-Restrepo A, Olive JM (1999) Measles elimination: progress and challenges. Vaccine 17 [Suppl 3]:S47–S52

de Quadros CA, Izurieta H, Venczel L, Carrasco P (2004) Measles eradication in the Americas: progress to date. J Infect Dis 189 [Suppl 1]:S227–S235

de Swart RL, Kuiken T, Fernandez-de Castro J, Papania MJ, Bennett JV, Valdespino JL, Minor P, Witham CL, Yuksel S, Vos H, van Amerongen G, Osterhaus AD (2006) Aerosol measles vac-cination in macaques: preclinical studies of immune responses and safety. Vaccine 24:6424–6436

de Swart RL, LiCalsi C, Quirk AV, van Amerongen G, Nodelman V, Alcock R, Yuksel S, Ward GH, Hardy JG, Vos H, Witham CL, Grainger CI, Kuiken T, Greenspan BJ, Gard TG, Osterhaus AD (2007) Measles vaccination of macaques by dry powder inhalation. Vaccine 25:1183–1190

de Vries P, Van Binnendijk RS, Van der Marel P, Van Wezel AL, Voorma HO, Sundquist B, UytdeHaag FG, Osterhaus AD (1988) Measles virus fusion protein presented in an immune-stimulating complex (iscom) induces haemolysis-inhibiting and fusion-inhibiting antibodies, virus-specific T cells and protection in mice. J Gen Virol 69:549–559

Denis-Mize KS, Dupuis M, MacKichan ML, Singh M, Doe B, O’Hagan D, Ulmer JB, Donnelly JJ, McDonald DM, Ott G (2000) Plasmid DNA adsorbed onto cationic microparticles mediates target gene expression and antigen presentation by dendritic cells. Gene Ther 7:2105–2112

Denis-Mize KS, Dupuis M, Singh M, Woo C, Ugozzoli M, O’Hagan DT, Donnelly JJ, III, Ott G, McDonald DM (2003) Mechanisms of increased immunogenicity for DNA-based vaccines adsorbed onto cationic microparticles. Cell Immunol 225:12–20

Dhiman N, Ovsyannikova IG, Cunningham JM, Vierkant RA, Kennedy RB, Pankratz VS, Poland GA, Jacobson RM (2007) Associations between measles vaccine immunity and single-nucle-otide polymorphisms in cytokine and cytokine receptor genes. J Infect Dis 195:21–29

Dubensky T, Driver D, Polo J, Belli B, Latham E, Ibanez C, Chada S, Brumm D, Banks T, Mento S, Jolly D, Chang S (1996) Sindbis virus DNA-based expression vectors: utility for in vitro and in vivo gene transfer. J Virol 70:508–519

Eghafona NO, Ahmad AA, Ezeokoli CD, Emejuaiwe SO (1991) Haemagglutination inhibition antibody levels one year after natural measles infection and vaccination. Microbios 68:33–36

El Kasmi KC, Muller CP (2001) New strategies for closing the gap of measles susceptibility in infants: towards vaccines compatible with current vaccination schedules. Vaccine 19:2238–2244

El Mubarak HS, Yuksel S, van Amerongen G, Mulder PG, Mukhtar MM, Osterhaus AD, de Swart RL (2007) Infection of cynomolgus macaques ( Macaca fascicularis ) and rhesus macaques ( Macaca mulatta ) with different wild-type measles viruses. J Gen Virol 88:2028–2034

b12325337-0010ztc.indd 205 10/3/2008 11:01:41 AM


Enders JF, Peebles TC (1954) Propagation in tissue cultures of cytopathic agents from patients with measles. Proc Soc Exp Biol Med 86:277–286

Enders JF, Katz SL, Holloway A (1962) Development of attenuated measles-virus vaccines. Am J Dis Child 103:335–340

Enders JF, Katz SL, Milovanovic MV, Holloway A (1960) Studies of an attenuated measles virus vaccine: I. Development and preparation of the vaccine: technics for assay of effects of vacci-nation. N Engl J Med 263:153–159

Endo A, Izumi H, Miyashita M, Taniguchi K, Okubo O, Harada K (2001) Current efficacy of pos-texposure prophylaxis against measles with immunoglobulin. J Pediatr 138:926–928

Ertl OT, Wenz DC, Bouche FB, Berbers GA, Muller CP (2003) Immunodominant domains of the measles virus hemagglutinin protein eliciting a neutralizing human B cell response. Arch Virol 148:2195–2206

Etchart N, Buckland R, Liu MA, Wild TF, Kaiserlian D (1997) Class I-restricted CTL induction by mucosal immunization with naked DNA encoding measles virus haemagglutinin. J Gen Virol 78:1577–1580

Fazilleau N, McHeyzer-Williams LJ, McHeyzer-Williams MG (2007) Local development of effector and memory T helper cells. Curr Opin Immunol 19:259–267

Feldman HA, Novack A, Warren J, Haute T (1962) Inactivated measles virus vaccine. II. Prevention of natural and experimental measles with the vaccine. JAMA 179:391–397

Fooks AR, Jeevarajah D, Warnes A, Wilkinson GW, Clegg JC (1996) Immunization of mice with plasmid DNA expressing the measles virus nucleoprotein gene. Viral Immunol 9:65–71

Fulginiti V, Kempe CH (1963) Measles exposure among vaccine recipients. Am J Dis Child 160:62–73

Fulginiti VA, Eller JJ, Downie AW, Kempe CH (1967) Altered reactivity to measles virus: atypical measles in children previously immunized with inactivated measles virus vaccines. JAMA 202:1075–1080

Fulginiti VA, Arthur JH, Pearlman DS, Kempe CH (1968) Altered reactivity to measles virus: local reactions following attenuated measles virus immunization in children who previously received a combination of inactivated and attenuated vaccines. Am J Dis Child 115:67–72


Gans HA, Yasukawa LL, Alderson A, Rinki M, DeHovitz R, Beeler J, Audet S, Maldonado Y, Arvin AM (2004) Humoral and cell-mediated immune responses to an early 2-dose measles vaccination regimen in the United States. J Infect Dis 190:83–90

Garenne M, Leroy O, Beau J-P, Sene I (1991) Child mortality after high-titre measles vaccines: prospective study in Senegal. Lancet 338:903–907

Gay NJ (2004) The theory of measles elimination: implications for the design of elimination strategies. J Infect Dis 189 [Suppl 1]:S27–S35

Goon P, Cohen B, Jin L, Watkins R, Tudor-Williams G (2001) MMR vaccine in HIV-infected children – potential hazards? Vaccine 19:3816–3819

Graves MC, Griffin DE, Johnson RT, Hirsch RL, Lindo de Soriano I, Roedenbeck S, Vaisberg A (1984) Development of antibody to measles virus polypeptides during complicated and uncomplicated measles virus infections. J Virol 49:409–412

Gremillion DH, Crawford GE (1981) Measles pneumonia in young adults: an analysis of 106 cases. Am J Med 71:539–542

Hartikka J, Bozoukova V, Ferrari M, Sukhu L, Enas J, Sawdey M, Wloch MK, Tonsky K, Norman J, Manthorpe M, Wheeler CJ (2001) Vaxfectin enhances the humoral immune response to plasmid DNA-encoded antigens. Vaccine 19:1911–1923

Hashiguchi T, Kajikawa M, Maita N, Takeda M, Kuroki K, Sasaki K, Kohda D, Yanagi Y, Maenaka K (2007) Crystal structure of measles virus hemagglutinin provides insight into effective vaccines. Proc Natl Acad Sci U S A 104:19535–19540

Hektoen L (1905) Experimental measles. J Infect Dis 2:238–255

b12325337-0010ztc.indd 206 10/3/2008 11:01:42 AM


Herweijer H, Latendresse JS, Williams P, Zhang G, Danko I, Schlesinger S, Wolff JA (1995) A plasmid-based self-amplifying Sindbis virus vector. Hum Gene Ther 6:1161–1167

Hirayama M (1983) Measles vaccines used in Japan. Rev Infect Dis 5:495–503 Holt EA, Moulton LH, Siberry GK, Halsey NA (1993) Differential mortality by measles vaccine

titer and sex. J Infect Dis 168:1087–1096 Home F (1759) Medical facts and experiments. Miller, London Hutchins SS, Dezayas A, Le Blond K, Heath J, Bellini W, Audet S, Beeler J, Wattigney W,

Markowitz L (2001) Evaluation of an early two-dose measles vaccination schedule. Am J Epidemiol 154:1064–1071

Jianzhi X, Zhihui C (1983) Measles vaccine in the People’s Republic of China. Rev Infect Dis 5:506–510

Kaslow DC (2004) A potential disruptive technology in vaccine development: gene-based vac-cines and their application to infectious diseases. Trans R Soc Trop Med Hyg 98:593–601

Katz SL, Milovanovic MV, Enders JF (1958) Propagation of measles virus in cultures of chick embryo cells. Proc Soc Exp Biol Med 97:23–29

Katz SL, Enders JF, Kibrick S (1959) Immunization of children with a live attenuated measles virus. Am J Dis Child 98:605–607

Katz SL, Enders JF, Holloway A (1960) Studies on an attenuated measles-virus vaccine. II. Clinical, virologic and immunologic effects of vaccine in institutionalized children. N Engl J Med 263:159–160

Knudsen KM, Aaby P, Whittle H, Rowe M, Samb B, Simondon F, Sterne J, Fine P (1996) Child mortality following standard, medium or high titre measles immunization in West Africa. Int J Epidemiol 25:665–673

Krugman S (1971) Present status of measles and rubella immunization in the United States: a medical progress report. J Pediatr 78:1–16

Lambert PH, Liu M, Siegrist CA (2005) Can successful vaccines teach us how to induce efficient protective immune responses? Nat Med 11:S54–S62

Langedijk JPM, Daus FJ, van Oirschot JT (1997) Sequence and structure alignment of Paramyxoviridae attachment proteins and discovery of enzymatic activity for a morbillivirus hemagglutinin. J Virol 71:6155–6167

Leitner WW, Hwang LN, deVeer MJ, Zhou A, Silverman RH, Williams BR, Dubensky TW, Ying H, Restifo NP (2003) Alphavirus-based DNA vaccine breaks immunological tolerance by activating innate antiviral pathways. Nat Med 9:33–39

Lennon JL, Black FL (1986) Maternally derived measles immunity in era of vaccine-protected mothers. J Pediatr 108:671–676

Liebert UG, Flanagan SG, Loffler S, Baczko K, ter Meulen V, Rima BK (1994) Antigenic deter-minants of measles virus haemagglutinin associated with neurovirulence. J Virol 68:1486–1493

Low N, Kraemer S, Schneider M, Restrepo AM (2008) Immunogenicity and safety of aerosolized measles vaccine: systematic review and meta-analysis. Vaccine 26:383–398

Maldonado YA, Lawerence EC, DeHovitz R, Hartzell H, Albrecht P (1995) Early loss of passive measles antibody in infants of mothers with vaccine-induced immunity. Pediatrics 96:447–450

Malvoisin E, Wild F (1990) Contribution of measles virus fusion protein in protective immunity: anti-F monoclonal antibodies neutralize virus infectivity and protect mice against challenge. J Virol 64:5160–5162

Maris EP, Gellis SS, Shaffer F, Dunham WB, Stokes J Jr, Rake G (1949) Vaccination of children with various chorioallantoic passages of measles virus. Pediatrics 4:1–8

Markowitz LE, Albrecht P, Rhodes P, Demonteverde R, Swint E, Maes EF, Powell C, Patriarca PA (1996) Changing levels of measles antibody titers in women and children in the United States: impact on response to vaccination. Pediatrics 97:53–58

Mathias RG, Meekison WG, Arcand TA (1989) The role of secondary vaccine failures in measles outbreaks. Am J Public Health 79:475–478

b12325337-0010ztc.indd 207 10/3/2008 11:01:42 AM


Mawhinney H, Allen IV, Beare JM, Bridges JH, Connolly MH, Nevin NC, Neill DW, Hobbs JR (1971) Dysgammaglobulinaemia complicated by disseminated measles. BMJ 2:380–381

McLaughlin M, Thomas P, Onorato I, Rubinstein A, Oleske J, Nicholas S, Krasinski K, Guigli P, Orenstein W (1988) Live virus vaccines in human immunodeficiency virus-infected children: a retrospective survey. Pediatrics 82:229–233

Measles Vaccines Committee (1968) Vaccination against measles: clinical trial of live measles vaccine given alone and live vaccine preceded by killed vaccine. BMJ 2:449–452

Melnick JL (1996) Thermostability of poliovirus and measles vaccines. Dev Biol Stand 87:155–160

Merz DC, Scheid A, Choppin PW (1980) Importance of antibodies to the fusion glycoprotein of paramyxoviruses in the prevention of spread of infection. J Exp Med 151:275–288

Migasena S, Simasathien S, Samakoses R, Pitisuttitham P, Heath J, Bellini W, Bennet J (1997) Adverse impact of infections on antibody responses to measles vaccination. Vaccine 15:72–77

Milovanovic MV (1965) Live measles vaccine trials in Yugoslavia. Archges Virusforsch 16:231–242

Milovanovic MV, Enders JF, Mitus A (1957) Cultivation of measles virus in human amnion cells and in developing chick embryo. Proc Soc Exp Biol Med 95:120–127

Mitragotri S (2005) Immunization without needles. Nat Rev Immunol 5:905–916 Mollenkopf HJ, Dietrich G, Fensterle J, Grode L, Diehl KD, Knapp B, Singh M, O’Hagan DT,

Ulmer JB, Kaufmann SH (2004) Enhanced protective efficacy of a tuberculosis DNA vaccine by adsorption onto cationic PLG microparticles. Vaccine 22:2690–2695

Monafo WJ, Haslam DB, Roberts RL, Zaki SR, Bellini WJ, Coffin CM (1994) Disseminated measles infection after vaccination in a child with a congenital immunodeficiency. J Pediatr 124:273–276

Moss WJ, Griffin DE (2006) Global measles elimination. Nat Rev Microbiol 4:900–908 Moss WJ, Scott S, Mugala N, Ndhlovu Z, Beeler JA, Audet SA, Ngala M, Mwangala S, Nkonga-

Mwangilwa C, Ryon JJ, Monze M, Kasolo F, Quinn TC, Cousens S, Griffin DE, Cutts FT (2007) Immunogenicity of standard-titer measles vaccine in HIV-1-infected and uninfected Zambian children: an observational study. J Infect Dis 196:347–355

Moss WJ, Fisher C, Scott S, Monze M, Ryon JJ, Quinn TC, Griffin DE, Cutts FT (2008) HIV type 1 infection is a risk factor for mortality in hospitalized Zambian children with measles. Clin Infect Dis 46:523–527

Nabel GJ, Nabel EG, Yang ZY, Fox BA, Plautz GE, Gao X, Huang L, Shu S, Gordon D, Chang AE (1993) Direct gene transfer with DNA-liposome complexes in melanoma: expression, bio-logic activity, and lack of toxicity in humans. Proc Natl Acad Sci U S A 90:11307–11311

Nader PR, Horwitz MS, Rousseau J (1968) Atypical exanthem following exposure to natural mea-sles: eleven cases in children previously inoculated with killed vaccine. J Pediatr 72:22–28

Naniche D, Garenne M, Rae C, Manchester M, Buchta R, Brodine SK, Oldstone MB (2004) Decrease in measles virus-specific CD4 T cell memory in vaccinated subjects. J Infect Dis 190:1387–1395

Norrby E, Enders-Ruckle G, ter Meulen V (1975) Differences in the appearance of antibodies to structural components of measles virus after immunization with inactivated and live virus. J Infect Dis 132:262–269

O’Hagan D, Singh M, Ugozzoli M, Wild C, Barnett S, Chen M, Schaefer M, Doe B, Otten GR, Ulmer JB (2001) Induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus DNA vaccines. J Virol 75:9037–9043

O’Hagan DT, Singh M, Dong C, Ugozzoli M, Berger K, Glazer E, Selby M, Wininger M, Ng P, Crawford K, Paliard X, Coates S, Houghton M (2004) Cationic microparticles are a potent delivery system for a HCV DNA vaccine. Vaccine 23:672–680

Oh S, Stegman B, Pendleton CD, Ota MO, Pan CH, Griffin DE, Burke DS, Berzofsky JA (2006) Protective immunity provided by HLA-A2 epitopes for fusion and hemagglutinin proteins of measles virus. Virology 352:390–399

b12325337-0010ztc.indd 208 10/3/2008 11:01:42 AM


Orenstein WA (2006) The role of measles elimination in development of a national immunization program. Pediatr Infect Dis J 25:1093–1101

Orenstein WA, Hinman AR, Strebel PJ (2006) Measles: the need for 2 opportunities for preven-tion. Clin Infect Dis 42:320–321

Ota MO, Ndhlovu Z, Oh S, Piyasirisilp S, Berzofsky JA, Moss WJ, Griffin DE (2007) Hemagglutinin protein is a primary target of the measles virus-specific HLA-A2-restricted CD8 + T cell response during measles and after vaccination. J Infect Dis 195:1799–1807

Otten GR, Schaefer M, Doe B, Liu H, Srivastava I, Megede J, Kazzaz J, Lian Y, Singh M, Ugozzoli M, Montefiori D, Lewis M, Driver DA, Dubensky T, Polo JM, Donnelly J, O’Hagan DT, Barnett S, Ulmer JB (2005) Enhanced potency of plasmid DNA microparticle human immunodeficiency virus vaccines in rhesus macaques by using a priming-boosting regimen with recombinant proteins. J Virol 79:8189–8200

Ovsyannikova IG, Dhiman N, Jacobson RM, Vierkant RA, Poland GA (2003) Frequency of mea-sles virus-specific CD4 + and CD8 + T cells in subjects seronegative or highly seropositive for measles vaccine. Clin Diagn Lab Immunol 10:411–416

Ovsyannikova IG, Jacobson RM, Poland GA (2004) Variation in vaccine response in normal pop-ulations. Pharmacogenomics. 5:417–427

Ovsyannikova IG, Pankratz VS, Vierkant RA, Jacobson RM, Poland GA (2006) Human leukocyte antigen haplotypes in the genetic control of immune response to measles-mumps-rubella vac-cine. J Infect Dis 193:655–663

Ozanne G, d’Halewyn M-A (1992) Secondary immune response in a vaccinated population during a large measles epidemic. J Clin Microbiol 30:1778–1782

Pabst HF, Spady DW, Maruskyk RG, Carson MM, Chui LW-L, Joffres MR, Grimsrud KM (1992) Reduced measles immunity in infants in a well-vaccinated population. Pediatr Infect Dis J 11:525–529

Pan CH, Valsamakis A, Colella T, Nair N, Adams RJ, Polack FP, Greer CE, Perri S, Polo JM, Griffin DE (2005) Modulation of disease, T cell responses, and measles virus clearance in monkeys vaccinated with H-encoding alphavirus replicon particles. Proc Natl Acad Sci U S A 102:11581–11588

Pan CH, Nair N, Adams RJ, Zink MC, Lee EY, Polack FP, Singh M, O’Hagan DT, Griffin DE (2008) Dose-dependent protection against or exacerbation of disease by a polylactide glycol-ide microparticle-adsorbed, alphavirus-based measles virus DNA vaccine in rhesus macaques. Clin Vaccine Immunol 15:697–706

Parker SE, Vahlsing HL, Serfilippi LM, Franklin CL, Doh SG, Gromkowski SH, Lew D, Manthorpe M, Norman J (1995) Cancer gene therapy using plasmid DNA: safety evaluation in rodents and non-human primates. Hum. Gene Ther 6:575–590

Pasetti MF, Barry EM, Losonsky G, Singh M, Medina-Moreno SM, Polo JM, Ulmer J, Robinson H, Sztein MB, Levine MM (2003) Attenuated Salmonella enterica serovar Typhi and Shigella flexneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemag-glutinin, inducing specific immune responses and protection in cotton rats. J Virol 77:5209–5217


Peradze TV, Smorodintsev AA (1983) Epidemiology and specific prophylaxis of measles. Rev Infect Dis 5:487–490


Plowright W, Ferris RD (1962) Studies with rinderpest virus in tissue culture: the use of attenuated cultured virus as a vaccine for cattle. Res Vet Sci 3:172–182

b12325337-0010ztc.indd 209 10/3/2008 11:01:42 AM


Polack FP, Auwaerter PG, Lee S-H, Nousari HC, Valsamakis A, Leiferman KM, Diwan A, Adams RJ, Griffin DE (1999) Production of atypical measles in rhesus macaques: evidence for disease mediated by immune complex formation and eosinophils in the presence of fusion-inhibiting antibody. Nat Med 5:629–634

Polack F, Lee S, Permar S, Manyara E, Nousari H, Jeng Y, Mustafa F, Valsamakis A, Adams R, Robinson H, Griffin D (2000) Successful DNA immunization against measles: neutralizing antibody against either the hemagglutinin or fusion glycoprotein protects rhesus macaques without evidence of atypical measles. Nat Med 6:776–781

Polack FP, Hoffman SJ, Crujeiras G, Griffin DE (2003a) A role for nonprotective complement-fixing antibodies with low avidity for measles virus in atypical measles. Nat Med 9:1209–1213

Polack FP, Hoffman SJ, Moss WJ, Griffin DE (2003b) Differential effects of priming with DNA vaccines encoding the hemagglutinin and/or fusion proteins on cytokine responses after mea-sles virus challenge. J Infect Dis 187:1794–1800

Premenko-Lanier M, Rota P, Rhodes G, Bellini W, McChesney M (2004) Prior DNA vaccination does not interfere with the live-attenuated measles vaccine. Vaccine 22:762–765

Premenko-Lanier M, Hodge G, Rota P, Tamin A, Bellini W, McChesney M (2006) Maternal anti-body inhibits both cellular and humoral immunity in response to measles vaccination at birth. Virology 350:429–432

Rauh LW, Schmidt R (1965) Measles immunization with killed virus vaccine. Am J Dis Child 109:232–237

Redd SC, King GE, Heath JL, Forghani B, Bellini WJ, Markowitz LE (2004) Comparison of vac-cination with measles-mumps-rubella vaccine at 9, 12, and 15 months of age. J Infect Dis 189 [Suppl 1]:S116–S122

Reilly CM, Stokes J Jr, Buynak EB, Goldner H, Hilleman MR (1961) Living attenuated measles-virus vaccine in early infancy. Studies of the role of passive antibody in immunization. N Engl J Med 265:165–169

Rota JS, Hummel KB, Rota PA, Bellini WJ (1992) Genetic variability of the glycoprotein genes of current wild-type measles isolates. Virology 188:135–142

Rota JS, Wang ZD, Rota PA, Bellini WJ (1994) Comparison of sequences of the H, F, and N cod-ing genes of measles virus vaccine strains. Virus Res 31:317–330

Rota PA, Liffick SL, Rota JS, Katz RS, Redd S, Papania M, Bellini WJ (2002) Molecular epide-miology of measles viruses in the United States, 1997–2001. Emerg Infect Dis 8:902–908

Rudy BJ, Rutstein RM, Pinto-Martin J (1994) Responses to measles immunization in children infected with human immunodeficiency virus. J Pediatr 125:72–74

Sabin AB (1991) Measles, killer of millions in developing countries: strategy for rapid elimination and continuing control. Eur J Epidemiol 7:1–22

Sabin AB, Arechiga AF, de Castro JF, Sever JL, Madden DL, Shekarchi I, Albrecht P (1983) Successful immunization of children with and without maternal antibody by aerosolized mea-sles vaccine. JAMA 249:2651–2662

Sabin AB, Albrecht P, Takeda AK, Ribeiro EM, Veronesi R (1985) High effectiveness of aero-solized chick embryo fibroblast measles vaccine in seven-month-old and older infants. J Infect Dis 152:1231–1237

Sakata H, Hirayama M, Kimura M (1978) A ten-year follow-up study on measles vaccination in Japan: evaluation of the efficacy analyzed on a computer system. Japan. J Med Sci Biol 31:339–356

Schwarz AJF (1962) Preliminary tests of a highly attenuated measles vaccine. Am J Dis Child 103:216–219

Schwarz AJF, Zirbel LW (1959) Propagation of measles virus in non-primate tissue culture. I. Propagation in bovine kidney tissue culture. Proc Soc Exp Biol Med 102:711–714

Seng R, Samb B, Simondon F, Cisse B, Soumare M, Jensen H, Bennett J, Whittle H, Aaby P (1999) Increased long term mortality associated with rash after early measles vaccination in rural Senegal. Pediatr Infect Dis J 18:48–52

b12325337-0010ztc.indd 210 10/3/2008 11:01:42 AM


Stittelaar KJ, Boes J, Kersten GF, Spiekstra A, Mulder PG, de Vries P, Roholl PJ, Dalsgaard K, van den DG, van Alphen L, Osterhaus AD (2000a) In vivo antibody response and in vitro CTL activation induced by selected measles vaccine candidates, prepared with purified Quil A components. Vaccine 18:2482–2493

Stittelaar KJ, Wyatt LS, de Swart RL, Vos HW, Groen J, van Amerongen G, Van Binnendijk RS, Rozenblatt S, Moss B, Osterhaus AD (2000b) Protective immunity in macaques vaccinated with a modified vaccinia virus Ankara-based measles virus vaccine in the presence of pas-sively acquired antibodies. J Virol 74:4236–4243

Stittelaar KJ, Kuiken T, de Swart RL, van Amerongen G, Vos HW, Niesters HG, van Schalkwijk P, van der KT, Wyatt LS, Moss B, Osterhaus AD (2001) Safety of modified vaccinia virus Ankara (MVA) in immune-suppressed macaques. Vaccine 19:3700–3709

Stittelaar KJ, de Swart RL, Vos HW, van Amerongen G, Agafonov AP, Nechaeva EA, Osterhaus AD (2002a) Enteric administration of a live attenuated measles vaccine does not induce pro-tective immunity in a macaque model. Vaccine 20:2906–2912

Stittelaar KJ, de Swart RL, Vos HW, van Amerongen G, Sixt N, Wild TF, Osterhaus AD (2002b) Priming of measles virus-specific humoral- and cellular-immune responses in macaques by DNA vaccination. Vaccine 20:2022–2026

Stittelaar KJ, Vos HW, van Amerongen G, Kersten GF, Osterhaus AD, de Swart RL (2002c) Longevity of neutralizing antibody levels in macaques vaccinated with Quil A-adjuvanted measles vaccine candidates. Vaccine 21:155–157

Stokes J Jr, Hilleman MR, Weibel RE, Buynak EB, Halenda R, Goldner H (1961) Efficacy of live, attenuated measles-virus vaccine given with human immune globulin. A preliminary report. N Engl J Med 265:507–513

Van Binnendijk RS, van der Heijden RWJ, van Amerongen G, Uytdehaag FGCM, Osterhaus ADME (1994) Viral replication and development of specific immunity in macaques after infection with different measles virus strains. J Infect Dis 170:443–448

Van Binnendijk RS, van der Heijden RWJ, Osterhaus ADME (1995) Monkeys in measles research. Curr Top Microbiol Immunol 191:135–148

Van Binnendijk RS, Poelen MCM, van Amerongen G, de Vries P, Osterhaus ADME (1997) Protective immunity in macaques vaccinated with live attenuated, recombinant, and subunit measles vaccines in the presence of passively acquired antibodies. J Infect Dis 175:524–532

Varsanyi TM, Morein B, Love A, Norrby E (1987) Protection against lethal measles virus infec-tion in mice by immune-stimulating complexes containing the hemagglutinin or fusion pro-tein. J Virol 61:3896–3901

Warren J, Gallian MJ (1962) Concentrated inactivated measles-virus vaccine: preparation and antigenic potency. Am J Dis Child 103:248–253

Webster DE, Cooney ML, Huang Z, Drew DR, Ramshaw IA, Dry IB, Strugnell RA, Martin JL, Wesselingh SL (2002) Successful boosting of a DNA measles immunization with an oral plant-derived measles virus vaccine. J Virol 76:7910–7912

World Health Organization (1990) Expanded programme on immunization. Global Advisory Report, Part 1. Wkly Epidemiol Rep 65:5–12

WHO/UNICEF (1989) Joint WHO/UNICEF statement on early immunization for HIV-infected children. Global programme on AIDS and expanded programme on immunization. Wkly Epidemiol Rec 7:48


Wong-Chew RM, Islas-Romero R, Garcia-Garcia ML, Beeler JA, Audet S, Santos-Preciado JI, Gans H, Lew-Yasukawa L, Maldonado YA, Arvin AM, Valdespino-Gomez JL (2004) Induction of cellular and humoral immunity after aerosol or subcutaneous administration of Edmonston-Zagreb measles vaccine as a primary dose to 12-month-old children. J Infect Dis 189:254–257

Wong-Chew RM, Islas-Romero R, Garcia-Garcia ML, Beeler JA, Audet S, Santos-Preciado JI, Gans H, Lew-Yasukawa L, Maldonado YA, Arvin AM, Valdespino-Gomez JL (2006)

b12325337-0010ztc.indd 211 10/3/2008 11:01:42 AM


Immunogenicity of aerosol measles vaccine given as the primary measles immunization to nine-month-old Mexican children. Vaccine 24:683–690

Wyde PR, Stittelaar KJ, Osterhaus AD, Guzman E, Gilbert BE (2000) Use of cotton rats for pre-clinical evaluation of measles vaccines. Vaccine 19:42–53

Yang K, Mustafa F, Valsamakis A, Santoro JC, Griffin DE, Robinson HL (1996) Early studies on DNA-based immunizations for measles virus. Vaccine 15:888–892

Young LW, Smith DI, Glasgow LA (1970) Pneumonia of atypical measles. Residual nodular lesions. Am J Roentgenol 110:439–448

Zhu Y, Fennelly G, Miller C, Tarara R, Saxe I, Bloom B, McChesney M (1997) Recombinant Bacille Calmette-Guérin expressing the measles virus nucleoprotein protects infant Rhesus macaques from measles virus pneumonia. J Infect Dis 176:1445–1453

b12325337-0010ztc.indd 212 10/3/2008 11:01:42 AM

D.E. Grippin and M.B.A. Oldstone (eds.) Measles – Pathogenesis and Control. 213© Springer-Verlag Berlin Heidelberg 2009

Chapter 11 Measles Virus for Cancer Therapy

S. J. Russell ( ) and K. W. Peng

Abstract Measles virus offers an ideal platform from which to build a new genera-tion of safe, effective oncolytic viruses. Occasional so-called spontaneous tumor regressions have occurred during natural measles infections, but common tumors do not express SLAM, the wild-type MV receptor, and are therefore not suscepti-ble to the virus. Serendipitously, attenuated vaccine strains of measles virus have adapted to use CD46, a regulator of complement activation that is expressed in higher abundance on human tumor cells than on their nontransformed counterparts.

S.J. Russell Mayo Clinic , Department of Molecular Medicine , 200 1st Street SW, Rochester , MN 55905 , USA, e-mail: [email protected]

Contents

Oncolytic Viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214Why Attenuated Measles Viruses Are Attractive Oncolytic Agents . . . . . . . . . . . . . . . . . . . . 215

Tumor Targeting Through CD46 Density Discrimination. . . . . . . . . . . . . . . . . . . . . . . . . . 215Safety to Population. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217Oncolytic Activity of Wild-Type and Attenuated (Vaccine Strain) Measles Viruses . . . . . 218

Engineering Attenuated Measles Viruses to Enhance Their Utility as Oncolytic Agents. . . . 220Noninvasive Monitoring of Measles Virus Spread . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220Arming the Virus to Combat Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223Engineering Measles Virus Tropism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

Clinical Translation of Recombinant Oncolytic Measles Viruses . . . . . . . . . . . . . . . . . . . . . . 227Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227Choice of Animal Models for Toxicology Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227Pharmacology and Toxicology Testing of MV-CEA and MV-NIS . . . . . . . . . . . . . . . . . . . 229Current Status of Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

Enhancing the Efficacy of Oncolytic Measles Viruses: Immune Evasion and Immune Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

Use of Cell Carriers to Deliver Oncolytic Measles Viruses to Sites of Tumor Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231Combination Therapy with Oncolytic Measles Viruses and Immunosuppressive Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Current Status and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

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214 S.J. Russell, K.W. Peng

For this reason, attenuated measles viruses are potent and selective oncolytic agents showing impressive antitumor activity in mouse xenograft models. The viruses can be engineered to enhance their tumor specificity, increase their antitumor potency, and facilitate noninvasive in vivo monitoring of their spread. A major impediment to the successful deployment of oncolytic measles viruses as anticancer agents is the high prevalence of preexisting anti-measles immunity, which impedes blood-stream delivery and curtails intratumoral virus spread. It is hoped that these prob-lems can be addressed by delivering the virus inside measles-infected cell carriers and/or by concomitant administration of immunosuppressive drugs. From a safety perspective, population immunity provides an excellent defense against measles spread from patient to carers and, in 50 years of human experience, reversion of attenuated measles to a wild-type pathogenic phenotype has not been observed. Clinical trials testing oncolytic measles viruses as an experimental cancer therapy are currently underway.

Oncolytic Viruses

Viruses that replicate selectively in neoplastic tissues (oncolytic viruses) hold con-siderable promise as novel therapeutic agents for the treatment of human malignan-cies and many such agents are currently under investigation, both in preclinical studies and in human clinical trials (Aghi and Martuza 2005; Hermiston 2006; Liu et al. 2007; Tai and Kasahara 2008). The existence of viruses was not recognized until the turn of the nineteenth century, but ever since that time, they have contin-ued to attract considerable interest as possible agents of tumor destruction (Kelly and Russell 2007; Sinkovics and Horvath 2000). Clinical observations suggested that, given the right set of conditions, cancers would sometimes regress during naturally acquired virus infections (Kelly and Russell 2007; Bluming and Ziegler 1971; Zygiert 1971; Hoster et al. 1949). Clinical trials were therefore conducted in which a variety of different human and animal viruses were administered to cancer patients (Kelly and Russell 2007; Sjoùozi et al. 1988; Asada 1974; Okuno et al. 1978; Newman and Southam 1954; Southam and Moore 1952). Most often, these viruses were arrested by the host immune system and did not significantly impact tumor growth (Huebner et al. 1956). However, in a few immunosuppressed pat-ients, the infection took and tumors regressed, although all too often, this was associated with unacceptable morbidities due to infection of normal tissues. Attempts to address the specificity problem continued throughout the 1950s and 1960s, but the results, although encouraging, were not compelling, and with the advent of anticancer chemotherapy, the concept of using replication competent viruses as anticancer agents was largely eclipsed (Kelly and Russell 2007). However, by the 1980s it was clear that even the combination of surgery, radio-therapy, and anticancer chemotherapy was failing to substantially impact cancer mortality and with the advent of modern virology accompanied by powerful reverse genetic systems, there came a resurgence of interest in oncolytic viruses (Russell and Peng 2007; Campbell and Gromeier 2005; Lichty et al. 2004). During

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11 Measles Virus for Cancer Therapy 215

the past two decades, oncolytic virotherapy has reestablished itself as a respectable field of research and there are new and numerous ongoing early-phase clinical tri-als testing a wide variety of oncolytic viruses representing many virus families (Liu et al. 2007; Lorence et al. 2007; Thorne et al. 2007; Forsyth et al. 2008; Yu and Fang 2007; Shen and Neumunaitis 2006; Kinoh and Inoue 2008; Nakamura and Russell 2004).

Why Attenuated Measles Viruses Are Attractive Oncolytic Agents

Safety concerns arising from the use of oncolytic viruses for human cancer therapy can be divided into two areas: risk to the patient and risk to the population (Russell 1994). To minimize risk to the patient, an ideal oncolytic virus should be selective for the tumor, nonpathogenic for normal host tissues, nonpersistent, and genetically stable. To minimize risk to the population, in addition to the above characteristics, the virus should be nontransmissible and preferably derived from a virus to which the population is generally immune (Russell 1994).

Attenuated measles viruses fulfill the above requirements. During the past 50 years, live attenuated measles viruses have been administered as vaccines to more than a billion people and the safety record has been outstanding (Griffin 2001; Nakamura and Russell 2004). Very occasionally, in people with severely compro-mised immune functions, the viral vaccine has propagated and caused disease in the recipient. However, even in this extreme circumstance, as in the case of an HIV-infected patient with virtually no CD4 lymphocytes who succumbed to measles pneumonia 9 months after vaccination (Anonymous 1996), there was no evidence that the offending virus had reverted to a pathogenic phenotype capable of spreading and causing disease in normal people.

Tumor Targeting Through CD46 Density Discrimination

Wild-type pathogenic and attenuated measles viruses have different receptor trop-isms (Yanagi et al. 2006). Most importantly, attenuated vaccines strains such as MV-Edm are capable of using CD46 as a cell entry receptor (Naniche et al. 1993; Dorig et al. 1993). Wild-type measles viruses do not, in general, use CD46 as a cell entry receptor, but acquire the CD46 tropism during tissue culture adaption via a mutation in the H-attachment protein coding sequence that changes the amino acid at position 481 in the H-protein, from asparagine to tyrosine (Hsu et al. 1998; Xie et al. 1999; Rota et al. 1994). Attenuated measles virus strains carrying this muta-tion are typically selected when wild-type measles stocks are applied to CD46- positive SLAM-negative cell monolayers (for example, Vero cells) (Nielsen et al. 2001). CD46, also known as membrane cofactor protein, is ubiquitously expressed by all human cells except erythrocytes (Riley-Vargas et al. 2004). CD46 plays an

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important role in protecting autologous cells from complement attack by serving as a cofactor for Factor I-mediated inactivation of C3b and C4b, thus blocking the complement cascade at the C3 activation stage (Liszewski and Atkinson 1992). Fortuitously for those wishing to use attenuated measles as a oncolytic agent, CD46 is frequently overexpressed on human cancer cells compared with their normal nontransformed counterparts, possibly as a mechanism to protect the cancer cells from complement mediated lysis (Fishelson et al. 2003; Durrant and Spendlove 2001; Hara et al. 1995; Seya et al. 1990). Overexpression of CD46 has been docu-mented in gastrointestinal, hepatocellular, colorectal, endometrial, cervical, ovarian, breast, renal, and lung carcinomas, also in leukemias and multiple myeloma, and has been found to limit the therapeutic potential of monoclonal antibody therapy (Seya et al. 1990; Ong et al. 2006; Bjorge et al. 1997; Varsano et al. 1998; Blok et al. 2000; Simpson et al. 1997; Murray et al. 2000; Kinugasa et al. 1999; Juhl et al. 1997; Gorter et al. 1996; Thorsteinson et al. 1998; Yamakawa et al. 1994).

CD46 mediates not only the attachment and entry of attenuated measles viruses, but also drives the process of virus-induced cell-to-cell fusion between a virus-infected cell and its neighboring cells. Using engineered Chinese hamster ovary (CHO) cells expressing a range of CD46 densities, it was shown that intercellular fusion between infected and uninfected cells was minimal at low CD46 receptor density, but increased dramatically above a threshold CD46 expression level (Anderson et al. 2004). Virus entry, by contrast, increased progressively with increasing CD46 receptor density, showing no dramatic all-or-nothing threshold effects. Thus, at low CD46 receptor densities typical of normal cells, attenuated measles virus is able to infect but intercellular fusion is negligible. At higher CD46 receptor densities typical of tumor cells, infection leads to extensive intercellular fusion (the classical cytopathic effect of measles virus), culminating in a dramati-cally increased level of cell killing (Anderson et al. 2004). In one recent study, the levels of CD46 expression on myeloma cells was found to be much higher (49,130 per cell) than levels on corresponding normal bone marrow cells (7340 per cell) from 38 myeloma patients (Ong et al. 2006). Potent cytopathic effects of extensive intercellular fusion were observed in the myeloma cells after measles infection, but not in the normal bone marrow cells (Fig. 11. 1 ). Also, the extent of measles virus-induced cell fusion varied from patient to patient, but correlated with CD46 expres-sion levels on the myeloma cells, colony-forming assays demonstrated that measles was not cytotoxic to the normal bone marrow progenitor cells (Ong et al. 2006). Discrimination between cells with high and low CD46 receptor densities provides a compelling basis for the oncolytic specificity of attenuated measles viruses and establishes them as a highly promising CD46 targeted cancer therapeutic agent (Ong et al. 2006; Anderson et al. 2004).

There may be additional factors contributing to the tumor specificity of attenu-ated measles virus besides CD46 receptor density. One possible contributing factor could be a higher intrinsic membrane fusogenicity in tumor cells, making them more likely to undergo intercellular fusion when infected by measles virus, regard-less of the surface density of CD46 (Ong et al. 2006). Another factor might be that there are deficiencies in the innate antiviral responses of tumor cells that render

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them more highly susceptible to virus infection (Balachandran and Barber 2007). For example, the interferon-α/β and RNA-dependent protein kinase response path-ways are often impaired in tumor cells but not in normal cells, and this is the mechanism underlying the tumor selectivity of a number of RNA viruses currently being tested for cancer therapy, such as vesicular stomatitis virus and reovirus (Campbell and Gromeier 2005; Basler and Garcia-Sastre 2002; Strong et al. 1998; Stojdl et al. 2003). These same innate viral control mechanisms may also serve, albeit to a lesser extent, to control the propagation of attenuated measles viruses.

Safety to Population

The main public safety concern associated with the therapeutic use of an oncolytic virus is accidental emergence of a new viral pathogen capable of epidemic spread in the human population (Russell 1994). Serious epidemics arise when pathogenic viruses gain access to susceptible populations under conditions that favor transmis-sion between individual members of the population. In selecting a virus from which to develop an oncolytic antitumor agent for human use, consideration should be

MM cell

A

B

PC NPC10

100

1000

C

CD

46 d

en

sit

y

Groups

Fig. 11.1 A–C Attenuated measles virus preferentially targets CD46, which is overexpressed on cancer cells, including multiple myeloma cells. A Cytospin of a bone marrow aspirate showing myeloma cells. B Bone marrow aspirates obtained from myeloma patients were separated into plasma cells (myeloma) and nonplasma cells (all normal hemapoietic cells in the marrow). Cells were stained with an anti-CD46 antibody and the numbers of CD46 receptors/cell were deter-mined using BD-QuantiBrite Beads. (Ong et al. 2006). Primary myeloma cells express sevenfold higher CD46 receptors than normal nonplasma cells in the bone marrow. C Primary myeloma cells expressing high levels of CD46 receptors ( left ) are more susceptible to the cytopathic effects of measles induced syncytial formation compared to normal bone marrow cells isolated from the same bone marrow aspirates. (Ong et al. 2006)

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given to its mutability, potential pathogenicity, potential transmissibility, and preva-lence in the population. As mentioned above, even though the measles vaccine has been given to more than one billion people over the past 50 years, there has never yet been a documented reversion to wild-type measles (Griffin 2001). Moreover, in contrast to viruses such as influenza which are inherently unstable, requiring new vaccines every year, measles virus has remained very stable and has been effectively controlled by essentially the same vaccine for decades (Griffin et al. 2008). Even if one considers the worst case scenario wherein an attenuated measles virus used for cancer therapy might revert back to the wild-type pathogenic strain, the risk of virus transmission from patients to carers and then into the population is limited by the high prevalence of anti-measles immunity. At the current time, because of the suc-cessful childhood measles vaccination programs, which give lifelong protection, more than 80% of the people in the world are currently measles immune (McQuillan et al. 2007). For this reason, attenuated measles viruses raise significantly fewer safety concerns than oncolytic viruses derived from other virus families.

Oncolytic Activity of Wild-Type and Attenuated (Vaccine Strain) Measles Viruses

Occasional so-called spontaneous tumor regressions of Hodgkin’s disease and Burkitt’s lymphoma have been documented after measles infections (Bluming and Ziegler 1971; Zygiert 1971; Taqui et al. 1981; Mota 1973; Ziegler 1976). Perhaps the most compelling was the case history of an 8-year-old African boy who pre-sented to a clinic in Uganda with a 4-month history of painless right orbital swelling. A biopsy specimen of the right retroorbital tumor was histologically diagnostic of Burkitt’s lymphoma but at the time of planned initiation of therapy, he was noted to have a generalized measles rash. On the same day, the right orbital tumor was noted to be regressing and because of the presumed measles infection, he was given no chemotherapy for the Burkitt’s lymphoma. During the course of the next 2 weeks, his rash disappeared and he seroconverted to measles. At the same time, the tumor regressed completely and remained in complete remission for at least 4 months after the measles infection in the absence of antineoplastic therapy. The mechanism underlying the rapid tumor regression that was observed in this remarkable case history was never elucidated, but Burkitt’s lymphomas are known to express high levels of SLAM and are therefore susceptible to infection by wild-type measles viruses (Nagy et al. 2002; Nakamura et al. 2005). The timing of the regression, coinciding with the period during which measles virus burden and measles-induced immunosuppression are at their peak, supports the contention that the tumor cells were directly destroyed by the virus.

It could be argued on the basis of case histories such as the one described above that there would be some value to treating cancer patients with a wild-type patho-genic strain of measles. However, there are strong safety and efficacy arguments against this. From a safety perspective, measles is a serious and unpleasant illness,

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which is highly transmissible to nonimmune subjects and is sometimes fatal (Griffin et al. 2008). Regarding efficacy, as discussed previously, most human malignancies lack receptors for wild-type measles viruses (Cocks et al. 1995) and therefore are not even theoretically susceptible to its possible oncolytic actions. Attenuated vac-cine strains of measles virus, by contrast, have far greater appeal as possible oncolytic agents for intentional administration to human cancer patients. Most of the measles vaccine strains in current use belong to the Edmonston lineage, which com-prises a number of closely interrelated laboratory-adapted substrains derived from a 1954 clinical isolate (from the throat of a child named David Edmonston) that has been passaged extensively in tissue culture, resulting in a loss of pathogenicity (Anonymous 1996; Griffin et al. 2008; Enders and Peebles 1954). The MV-Edm vaccine was initially licensed in 1963 but was found to be reactogenic, causing fever and rash in measles-naïve children (Katz 1965, 1996). The virus was passaged fur-ther in other cells, including chick embryo fibroblasts, giving rise to more highly attenuated vaccine strains, including MV-Moraten, which was licensed as Attenuvax in the United States in 1968 and the Edmonston-Zagreb strain (MV-EZ), a strain which has been used extensively in Europe (Griffin 2001). In contrast to wild-type measles viruses, all the laboratory-adapted Edmonston strains of measles virus have acquired the ability to use CD46 as a receptor to mediate virus entry and intercellular fusion (Naniche et al. 1993; Dorig et al. 1993; Nielsen et al. 2001).

Preclinical studies to test the oncolytic potential of attenuated Edmonston line-age viruses were first conducted at the turn of the twentieth century using a geneti-cally modified derivative of the Edmonston-B strain (MV-Edm tag), which was rescued from a molecular clone of the viral genome and was subsequently amplified on Vero cells (Radecke et al. 1995). The Edm-tag strain and its genetically engi-neered derivatives (see below in this paragraph) were found to be selectively destructive to human tumor cells in culture (Ong et al. 2006) and showed promising antitumor activity in several mouse xenograft models of different human malignan-cies, including lymphoma, multiple myeloma, ovarian, colorectal, breast, and liver cancer and glioma (Grote et al. 2001; Peng et al. 2001, 2002a; Phuong et al. 2003; Blechacz et al. 2006; McDonald et al. 2006; Hoffmann et al. 2006). However, clini-cal testing of these engineered measles strains presented considerable logistic chal-lenges, including the need to develop new manufacturing processes, validate the clinical products, and perform extensive toxicology testing to confirm their safety. For these reasons, there was a strong impetus to take the simple path and explore the possibility of using agents that had already been approved for human use, such as the Moraten and Edmonston-Zagreb measles vaccines, as oncolytic agents for cancer therapy. For this reason, the oncolytic potential of the commercially available Moraten measles vaccine was tested in a murine intraperitoneal model of human ovarian cancer and was compared with the efficacy of a recombinant MV-Edm tag-derived viral strain (Myers et al. 2005). In vitro, the Moraten strain was able to infect human ovarian cancer cells but caused intercellular fusion with greatly delayed kinetics compared to the MV-Edm tag strain. However, in vivo studies in ovarian cancer models showed that both viruses had equivalent antitumor potency causing significant prolongations in survival after intraperitoneal administration of

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a total dose of 10 6 infectious units (Myers et al. 2005). Unfortunately, since the Moraten vaccine is packaged and sold commercially in doses of only 10 3 TCID

50 , it

was considered impractical to proceed to a clinical trial in which, it was anticipated, MV-Moraten doses in excess of 10 8 TCID

50 (i.e., 100,000 times the vaccination

dose) would be required for oncolytic efficacy. While there have been no human studies evaluating the oncolytic potential of

the MV-Moraten vaccine strain, a phase I clinical study using the Edmonston-Zagreb vaccine strain was conducted in Zurich in patients with cutaneus T cell lymphoma (Heinzerling et al. 2005). A total of five patients were enrolled in this study and the Edmonston-Zagreb vaccine strain of measles virus was administered directly into accessible cutaneous tumors for up to a total of four doses. Each virus injection was preceded by two subcutaneous injections of interferon-alpha (9 mil-lion units subcutaneously), at 72 h and 24 h previously. This was a dose escalation study with a minimum intratumoral dose of 100 TCID

50 and a maximum dose of

1000 TCID 50

of the MV-Edmonston-Zagreb virus. The treatment was very well tolerated and five of six MV-injected lesions showed clear regression. In two of the patients, distant noninjected lesions improved, but they remained unchanged in the other three of five. In all five patients, there was a slight increase in the anti-mea-sles antibody titer after therapy. These very promising data were published in October 2005 and follow-on studies are eagerly awaited (Heinzerling et al. 2005).

Engineering Attenuated Measles Viruses to Enhance Their Utility as Oncolytic Agents

A reverse genetic system for the generation of recombinant measles viruses derived from MV-Edm tag was first reported in 1996 (Radecke et al. 1995). This opened the door for the generation of recombinant measles viruses encoding additional transcription units as well as the engineering of viral structural and nonstructural protein coding sequences to modulate virus biology (Fig. 11. 2 ). Genome engineer-ing has therefore emerged as an important approach whereby new versions of the MV-Edm tag virus can be generated as a way to enhance its performance in cancer therapy. Efforts to date have focused on the generation of recombinant viruses whose in vivo spread can be noninvasively monitored as well as the generation of viruses that can more effectively combat host innate immune responses or that can more accurately recognize and destroy neoplastic tissues. Each of these approaches is discussed separately in Sect. 3.1–3.3.

Noninvasive Monitoring of Measles Virus Spread

Pharmacokinetics describes the fate of a drug in the body, including its absorption, distribution, biotransformation, and excretion. Unfortunately, pharmacokinetic issues

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have not been adequately addressed in previous human virotherapy studies and this is proving to be a significant impediment to the intelligent clinical development of these agents. Ideally, it should be possible to noninvasively monitor the in vivo spread and elimination of an oncolytic virus in a treated cancer patient and to determine the profile of viral gene expression over time. In the absence of data on virus kinetics, it is not possible to know whether a failed response to therapy is due to lack of infec-tion, inadequate virus spread, weakened cytopathic effect, or premature virus elimi-nation. Noninvasive monitoring should therefore be an integral component of human studies of oncolytic measles viruses and has the potential to facilitate the tailoring of virotherapy protocols for individual patients.

Two approaches have been taken to facilitate in vivo monitoring of the spread of oncolytic measles viruses (Fig. 11.2). In the first approach, additional transcription

LN P M F

NIS

B. Imaging

D. Targeting

C. Arming

CEA

Pwt

A. Monitoring ablate CD46 & SLAM binding

display celltargeting ligands

x HF

x x x x

CD46, SLAM blind

H F

CD46, SLAM able

ligand

H

Fig. 11.2 Genetic engineering of attenuated measles virus for cancer therapy. A The virus can be engineered to express soluble marker proteins (e.g., MV-CEA), which are secreted into the circu-lation, thus enabling noninvasive monitoring of the profiles of viral gene expression over time by sampling body fluids (Peng et al. 2002). B The virus can be engineered to express the sodium iodide symporter (MV-NIS), which concentrates radioiodine in the infected cell, thus enabling noninvasive monitoring of the sites of MV infection by gamma camera, SPECT-CT, or PET-CT imaging (Dingli et al. 2004). Virotherapy can also be enhanced by a timely dose of beta-emitting I-131 to result in synergistic killing of MV-infected tumors. C Arming of the virus with genes (e.g., P gene from wild-type measles) that enable the virus to combat the innate antiviral immunity (Haralambieva et al. 2007). D Targeting virus entry, the H glycoprotein of measles virus can accommodate addition of large polypeptides (e.g., single-chain antibodies) as C-terminal exten-sions on the H protein. Mutations in the H protein that ablate fusion via CD46 and SLAM have been identified and incorporated in the retargeted viruses. The displayed ligand redirects binding of the virus to the new receptor to mediate virus entry and syncytial formation via the targeted receptor (Nakamura et al. 2005)

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units coding for soluble marker peptides were inserted into the viral genome (Peng et al. 2002b). It was reasoned that ideal marker peptides should be nonimmuno-genic with no biological function and a constant circulation half-life. They should also be efficiently secreted from virus-infected cells into the blood stream. The sol-uble extracellular domain of human carcinoembryonic antigen (CEA) and the β subunit of human chorionic gonadotrophin (βhCG) were therefore chosen for this approach. Oncolytic measles viruses expressing each of these transgenes from an additional transcription unit inserted upstream of the N gene were constructed (Peng et al. 2002b). The marker peptides did not compromise virus replication and the kinetics of virus propagation in CD46 transgenic mice could be easily followed by measuring concentrations of the virally encoded marker peptides in serum. When mice bearing human tumor xenografts were challenged with the trackable viruses, different kinetic profiles of marker gene expression could be correlated with distinct therapeutic outcomes (Peng et al. 2002b, 2006). In subsequent studies, the MV-CEA virus was shown to retain its potent oncolytic activity in preclinical models of ovarian cancer (Peng et al. 2002a; Hasegawa et al. 2006b) and subse-quently in brain cancer (Phuong et al. 2003), which provided the impetus for its advancement to phase I clinical testing in patients with each of these malignancies (see later).

While virally encoded soluble marker peptides do provide for noninvasive moni-toring of the total burden of virus-infected cells and tissues in the body, they do not provide any anatomical information about the location of virus-infected cells (Fig. 11.2). To this end, a second recombinant measles virus was generated coding for the human thyroidal sodium iodide symporter (NIS) (Dingli et al. 2004). NIS is a membrane ion channel expressed on thyroid follicular cells, which efficiently transports iodine (required for thyroxine production) into cells against its concentra-tion gradient (Dadachova and Carrasco 2004). Thyroidal NIS expression has been exploited for more than 50 years in clinical practice for thyroid imaging (with I-123) or ablation (with I-131) and for systemic therapy of well-differentiated thyroid malignancies (Riesco-Eizaguirre and Santisteban 2006; Mazzaferri and Kloos 2001). MV-NIS-infected cells are able to concentrate radioactive iodine from the bloodstream, enabling the status of an infection to be monitored by serial noninva-sive single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging using I-123 or I-124 as tracers, respectively (Dingli et al. 2005a; Carlson et al. 2006). The approach has been used for noninvasive moni-toring of intratumoral virus propagation in preclinical models of multiple myeloma, pancreatic, hepatocellular, and ovarian carcinoma, and has further been used to enhance the therapeutic potency of measles virotherapy by judiciously timed admin-istration of the β-emitting radioiodine isotope I-131 (Blechacz et al. 2006; Hasegawa et al. 2006b; Dingli et al. 2003, 2004; Carlson et al. 2006). Interestingly the recom-binant measles virus in which NIS was inserted between the H and L cistrons is able to propagate as efficiently as the MV-Edm tag strain from which it was derived and is potently oncolytic even in the absence of I-131 (Dingli et al. 2004). Based on these favorable characteristics, the MV-NIS virus has been advanced to phase I clinical testing in patients with multiple myeloma (see later).

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The interaction of a replicating measles virus with tumor cells and with the host immune system represents a complex dynamical system that can be analyzed math-ematically as a problem in population dynamics (Dingli et al. 2006; Wein et al. 2003). The outcome of this type of therapy depends in a complex way on the intri-cate interactions between the various populations involved and modeling of the kinetics of virotherapy may aid in improved understanding of treatment outcomes and in the design of improved therapeutic protocols. Mathematical models of can-cer virotherapy with recombinant measles viruses and of radiovirotherapy using the combination of MV-NIS and radioactive I-131 have been developed and provide a reasonably good fit to experimental data (Dingli et al. 2006; Bajzer et al. 2008).

Arming the Virus to Combat Innate Immunity

Pathogenic measles viruses are capable of combating the cellular innate immune response. They do this by means of the P/VC proteins encoded in the phospho pro-tein (P) transcription unit (Gotoh et al. 2001). The P and V proteins are particularly implicated in measles immune evasion and have been shown to inhibit interferon-induced STAT nuclear translocation and to suppress STAT1 and STAT2 phosphor-ylation (Palosaari et al. 2003; Shaffer et al. 2003; Takeuchi et al. 2003; Devaux et al. 2007). Attenuated measles viruses are typically mutated in their P and V proteins and are therefore unable to efficiently suppress innate immune responses (Ohno et al. 2004). As a consequence, wild-type measles isolates induce signifi-cantly lower release of interferon upon infecting peripheral blood lymphocytes when compared to attenuated strains (Ohno et al. 2004; Waehler et al. 2007).

As a general rule, tumor cells are thought to have defects in their interferon response pathways such that they are unable to mount effective innate antiviral responses (Stojdl et al. 2000). However, this is not an absolute deficiency. Oncolytic measles viruses derived from the MV-Edm tag infectious clone were shown to induce significantly higher levels of interferon in both normal and tumor cells when compared to a wild-type measles virus (Haralambieva et al. 2007). Moreover, pre-treatment of tumor cells with interferon prior to measles infection did significantly compromise viral gene expression. Based on these observations, a chimeric mea-sles virus based on the Edm-tag platform, but armed with a wild-type P gene, was generated and evaluated both in vitro and in vivo for antineoplastic activity (Haralambieva et al. 2007). As expected, the chimeric virus exhibited a reduced capacity to induce interferon in infected cells, and when intravenously administered to SCID mice bearing human myeloma xenografts, showed greater oncolytic potency. This study served to emphasize that oncolytic measles viruses can be sub-ject to control by the innate immune defenses of human tumor cells and may there-fore be more effective when engineered to rearm them with wild-type proteins that silence the innate immune response. This engineering strategy does raise the legiti-mate concern that rearming an attenuated measles virus with a wild-type P gene to permit more effective immune evasion may generate a more pathogenic agent and

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thereby compromise patient safety. However, measles virus virulence and patho-genicity are known to be complex and do not depend solely on P/V/C proteins (Bankamp et al. 2008). Clinical testing of oncolytic measles viruses that have been engineered to more effectively combat the innate intracellular immune response is not currently being pursued. However, depending upon the clinical outcomes (i.e., efficacy vs toxicity) of ongoing clinical trials that are using highly attenuated mea-sles viruses, there may be a good rationale for testing such fortified viruses in the future.

Engineering Measles Virus Tropism

At this time, measles is still the only virus that can be efficiently retargeted through a broad range of cellular receptors without significant reductions in entry efficiency (Nakamura and Russell 2004; Waehler et al. 2007). However, while the retargeting of measles virus entry is without question an extraordinary technical triumph, its utility has yet to be proven. The original rationale for attempting to redirect the trop-isms of oncolytic measles viruses was the incorrect assumption that, because it is expressed ubiquitously, CD46 would not be tumor-selective. However, as outlined previously, the oncolytic strains of measles virus in current use do efficiently dis-criminate between the high density of CD46 expressed on tumor cells and the lower densities of this receptor present on nontransformed cells (Ong et al. 2006; Anderson et al. 2004; Peng et al. 2002a). As such, these viruses already discriminate and selectively destroy cancer cells by targeting CD46. For this reason, unless significant toxicities arising from collateral damage to normal tissues are encountered in ongo-ing clinical studies using the nontargeted viruses MV-CEA and MV-NIS in patients with ovarian cancer, glioma, and multiple myeloma, it will be difficult to justify the use of fully retargeted viruses. However, it is possible that certain tumor types will prove to have lower levels of CD46 receptor expression, in which case there may be a stronger case for using viruses with alternative, engineered receptor tropisms. In addition, for intravenously administered viruses, the ability to interact with the lumenal surface of vascular endothelial cells lining tumor blood vessels might lead to significant enhancements in virus uptake at sites of tumor growth.

Initial efforts to engineer foreign polypeptides into the measles virus coat were focused on the fusion (F) protein. But even small modifications to the extreme N-terminus of this protein completely destroyed its ability to provide fusion support (F. Morling and S.J. Russell, unpublished observations). In contrast to the F protein, the Hemagglutinin (H), a type II membrane glycoprotein, was able to tolerate the inser-tion of large polypeptide sequences at or close to its extreme C-terminus without compromising its ability to be incorporated into virus particles and to provide fusion support functions. Building upon this observation, recombinant viral genomes cod-ing for chimeric H-glycoproteins with a variety of different C-terminal extensions were constructed and rescued as recombinant infectious viral particles. In this way, it was possible to display a wide variety of different ligands on the viral surface,

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including growth factors (human EGF and IGF 1), single-chain antibodies (against CEA CD38, CD20, EGF receptor, and others), single-chain T cell receptors, and snake venom peptides such as Echistatin (Schneider et al. 2000; Hammond et al. 2001; Peng et al. 2003a, 2004; Bucheit et al. 2003; Hallak et al. 2005). In each case, the recombinant viruses were shown to display multiple copies of the respective polypeptides on the surface and were endowed not only with new binding specifici-ties, but also with expanded tropisms. Thus each recombinant virus was able to bind and enter cells via its targeted receptor and thereafter to drive the process of inter-cellular fusion between the infected cell and neighboring receptor positive cells.

Having determined that the tropisms of recombinant measles viruses could be expanded by surface display of cell binding polypeptides, the next step was to engineer the underlying H protein to ablate the natural virus tropisms for CD46 and SLAM (Vongpunsawad et al. 2004; Nakamura et al. 2004). Several different muta-tions known to interfere with binding to either CD46 (Xie et al. 1999; Lecouturier et al. 1996) or SLAM (Vongpunsawad et al. 2004) were therefore engineered into an H-expression construct coding for a chimeric H protein displaying a single-chain antibody against CD38. Numerous different permutations of CD46- and SLAM-ablating mutations were tested in a screening system, wherein the H-expression constructs were co-transfected with an F-expression plasmid into cells expressing receptors for CD46, SLAM, or CD38, respectively (T. Nakamura and Russell, unpublished observations). In this way, a construct with mutations at residues 481 (Y to A) and 533 (R to A) was found to efficiently mediate antibody-targeted cell fusion, even when the displayed domain was replaced with single-chain antibodies recognizing alternative cellular targets (EGFR and EGFRvIII) (Nakamura et al. 2004). Subsequently, these fully retargeted H-coding sequences were engineered into recombinant measles virus genomes and the corresponding viruses were recov-ered (Hadac et al. 2004). In each case, recombinant viruses displaying single-chain antibodies on a doubly ablated H protein displayed the expected (fully retargeted) host range properties and could be used to mediate targeted destruction of tumors expressing the appropriate cognate receptor in living mice.

One technical issue associated with the rescue and propagation of fully retar-geted viruses was the requirement that the targeted receptor should be expressed on the cells being used as a substrate for virus growth. This was addressed in two ways, either by generating Vero cells expressing the targeted receptors (Hadac et al. 2004) or, alternatively, by means of a pseudo-receptor system using Vero-α His cells expressing a membrane-anchored single-chain antibody that recognizes a hexahis-tidine peptide (H6) (Kaufmann et al. 2002). The H6 peptide was then incorporated at the extreme C-terminus of the chimeric H proteins of fully retargeted viruses, such that they could be efficiently rescued and propagated on the Vero-α His cells (Nakamura et al. 2005). This system has subsequently been used to generate fully retargeted viruses displaying single-chain antibodies with diverse receptor specifici-ties. At the current time, the list includes viruses retargeted to CD38, EGFR, EGFRvIII, α-folate receptor, HER2/neu, CD20, CD19, CD52, prostate specific membrane antigen (PSMA), and the myeloma antigens HM1.24 and Wue-1 (S.J. Russell, unpublished observations) (Nakamura et al. 2005; Hasegawa et al. 2006a,

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2007; Ungerechts et al. 2007b; Paraskevakou et al. 2007; Allen et al. 2006). Interestingly, where tested, the new tropisms conferred on measles viruses by dis-played scFvs are stably maintained during multiple serial virus passages without reversion to native receptor usage. While one conclusion from the aforementioned studies is that measles virus has a remarkably flexible and adaptable entry mecha-nism that can utilize multiple alternative cellular receptors, it has also become apparent that there can be significant differences in the efficiency of virus entry and intercellular fusion depending on the precise specificity and affinity of the displayed ligand. To further explore the relationship between the affinity of a displayed ligand for its targeted receptor and the behavior of the retargeted viruses, a panel of six recombinant HER2/neu retargeted measles viruses was generated displaying a panel of single-chain antibodies with identical HER2/neu receptor specificities but with dissociation constants ranging from 1.6 × 10 −6 M to 1.5 × 10 −11 M (Hasegawa et al. 2007). Comparisons of the infectivities and cytopathic effects of these viruses on a panel of cell lines expressing different surface densities of the HER2/neu receptor gave quite unexpected results, showing that there was a functional threshold affinity level above which infection and intercellular fusion proceeded with equal efficiency, even when affinity increased over 1000-fold above the threshold level. Below the threshold, infection was minimal. This affinity threshold was shown to correlate inversely with receptor density such that higher affinities were required to fuse cells with lower receptor densities. Thus, depending on their receptor affinities, retar-geted measles viruses are able to discriminate efficiently between cells expressing different densities of a targeted receptor (Hasegawa et al. 2007).

Several in vivo therapy studies have been published in which fully retargeted viruses have been administered to mice bearing human tumor xenografts by intra-tumoral, intraperitoneal, or intravenous routes, resulting in retardation of tumor growth and prolongation of survival. Examples include the use of measles viruses targeted to EGF receptor in ovarian cancer and glioma models, viruses targeted to the α-folate receptor in an ovarian cancer model, EGFRvIII-targeted viruses for glioma, and CD38- or CD20-targeted viruses for the treatment of two different mouse lymphoma models (Nakamura et al. 2005; Hasegawa et al. 2006a; Paraskevakou et al. 2007; Allen et al. 2006; Ungerechts et al. 2007a). In general, the retargeted viruses showed at least equivalent antitumor activity compared to the parental MV-Edm tag strain, but with reduced capacity to cause damage to the brains of CD46 transgenic mice.

As discussed previously, it is still an open question whether fully retargeted measles viruses can offer therapeutic advantages over the CD46 specific viruses that are currently undergoing clinical testing. However, it is hoped that measles viruses that can interact more efficiently with markers expressed on the lumenal surfaces of the endothelial cells lining tumor blood vessels will more efficiently localize to sites of tumor growth when infused into the bloodstream. Exploratory studies using measles viruses displaying αvβ

3 integrin-binding peptides (cyclic

RGD and Echistatin) as extensions of their H proteins have shown that they can, at least, interact with the lumenal surface of developing neovessels in the chick chori-oallantoic membrane and that they can be transcystosed across the vascular

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endothelium of these vessels into the interstitial space (K.W. Peng and S.J. Russell, unpublished observations).

Clinical Translation of Recombinant Oncolytic Measles Viruses

Of the recombinant measles viruses discussed in the previous section, two (MV-CEA and MV-NIS) are currently being administered to cancer patients in phase I clinical trials. MV-CEA is being infused into the peritoneal cavities of patients with advanced treatment-refractory ovarian cancer, and in a second trial, it is being administered into the tumor bed after surgical excision of high-grade tumors of the brain. MV-NIS is being administered intravenously to patients with advanced treat-ment-refractory multiple myeloma. Approval for these clinical protocols was, in each case, preceded by a detailed FDA review of the clinical protocol design, the manufacturing process, the purity, identity, and sterility of the manufactured prod-uct, the results of preclinical efficacy studies, and the results of comprehensive toxicology and biodistribution studies conducted in appropriate animal models.

Manufacture

Based on the doses of MV-CEA and MV-NIS that were required to mediate tumor regression in mouse xenograft models of human cancer, it was projected that doses of virus in the region of 10 9 TCID

50 would be required for clinical efficacy. A single

dose of the Moraten measles vaccine contains somewhere between 10 3 and 10 4 TCID

50 and existing manufacturing processes used by major measles vaccine sup-

pliers were unsuitable for the generation of high titer viral stocks required for onco-lytic applications (Meyers et al. 2005). A new process was therefore developed for the scaled manufacture and partial purification of oncolytic measles viruses using the immortal Vero cell line as substrate. Manufacture of clinical grade lots of both MV-CEA and MV-NIS was then completed in a pilot manufacturing facility at Mayo Clinic Rochester, adhering to the principles of Good Manufacturing Practices (GMP).

Choice of Animal Models for Toxicology Studies

Because they are derived from attenuated laboratory adapted strains of measles virus, the host range properties of MV-CEA and MV-NIS are quite distinct from those of wild-type pathogenic measles virus strains. As mentioned previously, these viruses efficiently exploit CD46 as a receptor for binding, cell entry, and cell fusion and this receptor usage is a critical factor influencing the choice of an appropriate animal model for pharmacology and toxicology studies. As for other vaccine strains of

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measles viruses, because they are highly attenuated (i.e., nonpathogenic), there is no model in which to study their pathogenesis. The three animal models that have proven to be of considerable value for FDA-mandated preclinical studies of biodistribution and toxicity are rhesus monkeys (Old World primates), squirrel monkeys (New World primates), and interferon α/β receptor knockout CD46 transgenic mice.

CD46 transgenic mouse models were originally created in the hope that they might facilitate the study of measles virus pathogenesis (Mrkic et al. 1998; Oldstone et al. 1999; Manchester and Rall 2001). However, dissemination of viruses belong-ing to the Edmonston lineage has only ever been observed in CD46 mice lacking the interferon α/β receptor (IFNAR ko xCD46-Ge) (Mrkic et al. 2000). These mice express human CD46 in a tissue distribution that mimics the pattern of CD46 expression in humans, including low to absent expression on erythrocytes (Mrkic et al. 1998; Kemper et al. 2001). After intranasal challenge with MV-tag, the ani-mals showed local virus replication in the respiratory epithelium followed by dis-semination via the lymph node system, similar to the pattern of wild-type measles virus dissemination in the human host (Mrkic et al. 1998, 2000). Infection of cells of the monocyte macrophage lineage is prominent in this model (Mrkic et al. 2000; Kemper et al. 2001; Peng et al. 2003b).

An alternative and better-established model for the study of measles virus pathogenesis is the rhesus monkey, which, like other Old World non-human pri-mates, develops a measles-like illness when challenged with wild-type measles virus (Kobune et al. 1996; van Binnendijk et al. 1994). However, rhesus monkeys do not provide, by any means, a perfect model in which to test the virulence of tis-sue culture adapted measles viruses that use CD46 as a receptor (Kobune et al. 1996; Auwaerter et al. 1999; Takeda et al. 1998; Kobune et al. 1990). Previous studies have shown repeatedly that rhesus monkey virulence studies of attenuated measles viruses are not predictive of their reactogenicity (i.e., the ability to cause measles-like illness) in humans (Aldous et al. 1961; Collard et al. 1961; Katz et al. 1960; Schwarz et al. 1960). Numerous attenuated measles viruses have been tested in primates during the development of new measles virus vaccines (Schwarz et al. 1960; Enders et al. 1960; Goffe and Laurence 1961) and even though they have scored completely negative in this macaque-virulence test, they have proven to be reactogenic in humans, causing a morbilliform rash, often accompanied by fever and malaise in a high percentage of recipients (Aldous et al. 1961; Collard et al. 1961; Katz et al. 1960; Schwarz et al. 1960). The key deficiency of the macaque-virulence model is that the tissue distribution of CD46 in macaque differs signifi-cantly from humans. Most importantly, CD46 is abundant on macaque red blood cells, but is absent from human red blood cells (Hsu et al. 1997). CD46 binding strains of measles virus, therefore, are agglutinated on macaque, but not human red blood cells, an interaction that most likely impedes virus dissemination in the pri-mates. The macaque model was therefore considered to be suboptimal for toxicity studies in which recombinant measles viruses are administered into the blood-stream, but was considered relevant when studying the toxicity of intracerebral MV-CEA (see Sect. 4.3).

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Unlike Old World primates, New World primates such as the squirrel monkey ( Saimiri sciureus ) express a truncated CD46 molecule to which MV-Edm does not attach (Hsu et al. 1997). The red cells of New World monkeys are, therefore, not agglutinated by attenuated measles viruses, but these animals do get a measles-like illness when challenged with wild-type measles virus, making them an informative model in which to test the toxicities of oncolytic measles viruses arising from their interactions with the wild-type measles receptor SLAM. Squirrel monkeys chal-lenged with wild-type measles virus develop a classical measles-like illness char-acterized by fever, coryza rash, immunosuppression, and subsequent recovery (Kobune et al. 1996).

Pharmacology and Toxicology Testing of MV-CEA and MV-NIS

To support the first clinical protocol, a phase I trial of intraperitoneal administra-tion of MV-CEA in patients with recurrent ovarian cancer, the formal biodistribu-tion and toxicology studies were conducted in IFNAR ko xCD46-Ge mice (Peng et al. 2003b). The key findings were that MV-CEA administered into the peritoneal cavity efficiently infected peritoneal macrophages and these trafficked to abdomi-nal draining lymph nodes, as well as to the marginal zones of the spleen. CEA expression peaked between days 2 and 5 and returned to baseline by day 10, due to virus elimination. There was no evidence of virus shedding in the urine or respira-tory secretions, but there was some evidence for long-term persistence of viral genomes in the spleens of infected animals. Measles viruses encoding different marker genes were also employed for biodistribution studies and green fluorescent protein proved to be highly informative. Mesothelium and ovarian surface epithe-lium were remarkably resistant to infection but peritoneal macrophages were sus-ceptible. Large numbers of infected macrophages could be detected in the greater omentum concentrated in milky spots. Infected macrophages were also identified outside the peritoneal cavity at diaphragmatic stomata along lymphatic vessels and in the parathymic lymph nodes. Eventually, the cells escaped into the blood stream and could be identified in the marginal zones of the white pulp of the spleen.

In toxicology studies, MV-CEA was administered into the peritoneal cavity (maximum dose 10 7 TCID

50 ) and the animals were closely monitored for activity

level, general appearance, body weight, key hematological and biochemical param-eters, and serum CEA levels (K.W. Peng et al., unpublished observations). Necropsies were conducted on days 14, 28, and 91 after virus administration and all organs were sent for histopathological analysis. The study was essentially negative, with no significant toxicities encountered at any dose level. Dose-response studies in a SKOV3.ip1 intraperitoneal ovarian cancer xenograft model revealed that the virus was equally effective over a wide range of dose levels, from 6 × 10 7 down to 6 × 10 3 TCID

50 . Analysis of CEA profiles in the treated mice was highly informative, illus-

trating the variability of virus kinetics at different dose levels. The highest doses of

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virus were associated with higher initial levels of tumor cell killing, but the final outcome of MV-CEA therapy at all dose levels was a partial equilibrium between virus and tumor, resulting in significant slowing of tumor growth and enhanced sur-vival of the mice (Peng et al. 2006).

In support of the phase I brain cancer study of intratumoral and/or resection cavity administration of MV-CEA in patients with recurrent glioblastoma multi-forme, toxicology studies were conducted both in IFNAR ko xCD46-Ge mice and in rhesus monkeys (Allen et al. 2008). In both cases, the virus was delivered by intracerebral inoculation and animals were carefully observed for signs of neu-rological impairment or other organ damage. MV-Edm is known to be neu-ropathogenic in measles-naïve IFNAR ko xCD46-Ge mice and the animals were there fore preimmunized by intraperitoneal administration of MV-GFP for 1 month prior to the initiation of toxicology. The animals were closely monitored for 90 days after intracerebral inoculation with MV-CEA, but there was no evi-dence for neurotoxicity, neither clinically nor upon histological examination of brain sections at various time points after virus administration. For toxicity test-ing in monkeys, five adult measles-immune rhesus macaques received intracere-bral injections of MV-CEA (10 5 or 10 6 TCID

50 ) or vehicle control into the frontal

lobe on days 1 and 5. The animals were monitored closely thereafter. Monitoring studies included clinical observation, analysis of blood samples, throat swabs, and cerebral-spinal fluid and MRI ima ging of the brain. There was no evidence in this study for MV-CEA-mediated neurotoxicity.

In support of the phase I clinical trial testing intravenous administration of MV-NIS, with or without cyclophosphamide, in patients with multiple myeloma, pre-clinical pharmacology and toxicology studies were conducted in SCID mice bearing subcutaneous myeloma xenografts, nontumor-bearing IFNAR ko xCD46-Ge mice, and in measles-naïve squirrel monkeys (Myers et al. 2007). Dose-response studies conducted in the KAS-6/1 myeloma xenograft model demonstrated that a single intravenous dose of 4 × 10 6 TCID

50 /kg of MV-NIS was the minimum dose that reli-

ably led to tumor regression and prolongation of survival. Toxicity studies in IFNAR ko xCD46-Ge mice were negative up to a single intravenous dose of 4 × 10 8 TCID

50 /kg. A single dose of cyclophosphamide given prior to virus administration

did significantly impede the antiviral immune response in this model, leading to delayed virus elimination. Virus-associated toxicities were not observed in measles-naïve squirrel monkeys, even at very high intravenous doses of 10 8 TCID

50 /kg of

MV-NIS given alone or in combination with cyclophosphamide. Viral mRNA was detected in cheek swabs harvested from the squirrel monkeys on days 1, 2, 8, 15, and 22 after MV-NIS administration and copy numbers were higher at all time points in the cyclophosphamide-treated animals, with peak levels seen on day 8. Based on these studies, a safe starting dose of MV-NIS for the clinical protocol was set at 1.5 by 10 4 TCID

50 /kg (10 6 TCID

50 per patient) increasing to a maximum of

1.5 × 10 7 TCID 50

/kg (10 9 TCID 50

per patient) with a single-dose of 10 mg/kg cyclo-phosphamide administered 24 h earlier by intravenous infusion.

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Current Status of Clinical Trials

Each of the three phase I clinical trials discussed above is progressing satisfactorily. The first patient to receive a recombinant measles virus had advanced ovarian can-cer and received a series of six intraperitoneal infusions of MV-CEA starting in July 2004. To date, 20 patients have been treated in the ovarian cancer study at seven dose levels (10 3 –10 9 TCID

50 ). The study is progressing satisfactorily and the results

will be published in the near future. Two patients with glioblastoma multiforme have so far been enrolled in the intracerebral MV-CEA study and six patients with multiple myeloma have been enrolled for intravenous administration of MV-NIS at two dose levels (10 6 TCID

50 and 10 7 TCID

50 ). Each of these clinical studies is pro-

gressing satisfactorily, but detailed reports of the trial outcomes will not be pub-lished until the trials have been completed.

Enhancing the Efficacy of Oncolytic Measles Viruses: Immune Evasion and Immune Suppression

For successful virotherapy of disseminated malignancies, oncolytic measles viruses will have to be delivered via the bloodstream. Optimal treatment outcomes will require efficient delivery via the bloodstream to sites of tumor growth, and this should be followed by efficient intratumoral spread, leading to tumor destruction. Each of these processes can be severely constrained by anti-measles immunity; delivery by humoral immunity, and spread by cell-mediated immunity (Fig. 11. 3 ). Considerable attention has therefore been focused on the use of cells as delivery vehicles to circumvent the humoral anti-measles immune response, and on the use of immunosuppressive drugs to combat cell-mediated anti-measles immunity. These two approaches are discussed below.

Use of Cell Carriers to Deliver Oncolytic Measles Viruses to Sites of Tumor Growth

Irrespective of the presence of antiviral antibodies, many intravenously adminis-tered viruses are sequestered in the microcirculations of lung, liver, and spleen, where they are phagocytosed by macrophages (Wang and Yuan 2006; Fisher 2006). It is the small percentage of intravenously administered viruses escaping this fate that infect the tumor tissue and mediate the regressions that are seen in preclinical models. However, these viruses are highly vulnerable to the neutralizing activities of antiviral antibodies, which have a dramatic titer-dependent effect on the speed of virus inactivation (Scallan et al. 2006; Klasse and Sattentau 2002). Also, antibody

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titers increase progressively with each successive exposure to the virus (Reid et al. 2002; Hangartner et al. 2006). Because of prior measles infection or measles vacci-nation, approximately 90% of Americans have protective titers of anti-measles antibodies, which may limit the therapeutic efficacy of systemically administered measles viruses (McQuillan et al. 2007; Audet et al. 2006). In contrast to the gen-eral population, patients with multiple myeloma have profound suppression of their humoral immune responses and low antibody titers to measles virus, making them ideal candidates for systemic oncolytic measles virus therapy (Dingli et al. 2005b; Jacobson and Zolla-Pazner 1986). However, most patients suffering from other cancers do have healthy protective titers of anti-measles antibodies. The protective properties of anti-measles antibodies were well demonstrated in the prevaccination era when serum from convalescing measles patients was used as postexposure prophylaxis for children at risk (Zingher and Mortimer 2005; Zordman et al. 1944). Interestingly, serotherapy was only effective if administered within 6 days of mea-sles virus exposure, indicating that it cannot prevent the cell-associated viremia and dissemination of measles virus via the bloodstream that occurs toward the end of

Fig. 11.3 Considerations for future improvements of measles virotherapy. Ideally, intravascularly administered viruses will reach the tumor sites to result in infection of tumor cells, viral spread, and elimination of the tumors. However, anti-measles antibodies can potentially inhibit delivery of the viruses and viral spread in the infected tumor can be inhibited by cell-mediated immunity. To combat these barriers to successful therapy, virus-infected cell carriers can be exploited to act as Trojan horses to deliver virus to the tumor sites and cell-mediated immunity can be controlled by judicious use of immunosuppressive agents such as cyclophosphamide

MV

Noanti-MV

Immunity

Delivery

Spread

Anti-MVCTLs

Delivery

Spread

Anti-MVAntibodies

Delivery

Spread

Use cellsasdelivery vehicle

GiveImmuno-suppressivedrugs

Solution

X

XX

Tumor Eliminated!

Tumor Remains

Tumor Remains

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the incubation period (days 10–14 after initial exposure, coincident with the pro-dromal symptoms and rash) (Griffin 2001). Importantly, cell-free viremia has not been recorded in natural measles infections (Griffin 2001; Osunkoya et al. 1990). The virus enters via the respiratory tract, migrates locally to lymphoid tissues and then enters the bloodstream, only inside cells, typically monocytes or lymphocytes, which transport it to distant sites where it causes the typical rash and other disease features (Griffin 2001).

On the basis of the above insights into how measles virus can travel via the bloodstream, even the presence of antiviral antibodies, there is considerable interest in the use of infected cell carriers to deliver oncolytic measles viruses to sites of tumor growth (Munguia et al. 2008). Not only does this approach have the potential to avoid neutralization by antiviral antibodies, but might also prevent mislocaliza-tion of the virus in liver and spleen, as well as aiding extravasation from tumor blood vessels. In vitro studies demonstrated that in contrast to infection by naked virions, heterofusion between infected cell carriers and tumor cells was more resist-ant to antibody neutralization (Iankov et al. 2007; Ong et al. 2007). Moreover, sys-temic and intraperitoneal injection of measles-infected cells successfully transferred infection in vivo to sites of tumor growth (using xenograft models of lymphoma, hepatocellular carcinoma, and multiple myeloma) even in the presence of neutral-izing antibodies (Iankov et al. 2007; Ong et al. 2007). Monocytes, endothelial pro-genitor cells, and T lymphocytes were also shown to have potential as measles virus carriers, but considerable optimization of the approach is required since, to date, the enhancements in efficacy that have been achieved using this approach have been very small.

Combination Therapy with Oncolytic Measles Viruses and Immunosuppressive Drugs

There are many drugs with immunosuppressive properties currently being used in the clinic, either for the treatment of autoimmune disease or to suppress the rejec-tion of transplanted organs. Their profiles of activity vary, some being more effec-tive against a particular subset of lymphocytes, with others having a broader spectrum of activity. While there is a strong rationale for combining immunosup-pressive therapy with oncolytic measles virotherapy, progress in this research endeavor has been hampered by the lack of suitable animal models in which a measles-susceptible tumor is allowed to grow in a measles-susceptible animal with an intact immune system. However, despite the lack of suitable experimental mod-els, attempts have nevertheless been made to determine whether cyclophosphamide might be capable of suppressing anti-measles immune responses and thereby enhancing its therapeutic potency.

Cyclophosphamide is known to be highly toxic to proliferating lymphocytes and can modulate both primary and anamnestic immune responses to a variety of antigenic challenges, including virus infection (Steinberg 2001). Precise

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effects vary with the dose of antigen, the dose of cyclophosphamide, and the timing of cyclophosphamide administration relative to the antigenic challenge (Hill 1975). Suppression of B cell and T cell responses is most pronounced when cyclophosphamide is administered at the same time or within 3 days following antigen challenge, but is also seen when the drug is administered 1 or 2 days before exposure to antigen, particularly when higher doses of cyclophosphamide are used (Aisenberg and Davis 1968). When a single dose of cyclophosphamide was administered to IFNAR ko xCD46-Ge mice a few hours prior to intraperito-neal or intravenous injection of MV-CEA or MV-NIS, respectively, elimination of the viruses was substantially delayed and the primary anti-measles antibody response was suppressed. A similar delay in virus elimination was observed when squirrel monkeys were pretreated with cyclophosphamide prior to infusion of MV-NIS, and these data provided part of the justification for the ongoing MV-NIS clinical trial in which, in later cohorts, the patients will be pretreated with cyclophosphamide before they receive the virus. Additional preclinical studies were conducted in which cyclophosphamide was compared with other immuno-suppressive agents and found to be substantially superior to dexamethasone, an equipotent but less toxic than whole-body irradiation (R.M. Myers et al., unpub-lished observations).

Toward the development of an informative immunoincompetent murine model to experimentally test the role of immunosuppressive agents in measles virus therapy, a CEA-retargeted measles virus was used to treat a CEA-positive murine colon adenocarcinoma implanted in syngeneic C57BL/6 mice (Ungerechts et al. 2007a). This targeted virus was also armed with the prodrug convertase purine nucleotide phosphorylase (PNP). Systemic delivery of MV-PNP-anti CEA had no substantial oncolytic activity, but in combination with the prodrug, 6-methyl purine-2'-deoxyroboside, it was therapeutic, revealing synergistic affects between virus and prodrug. Immunosuppression with cyclophosphamide was shown to retard the appearance of measles-neutralizing antibodies and also to enhance onc-olytic efficacy.

Current Status and Future Prospects

Measles virotherapy has recently emerged as a safe and highly promising experi-mental approach to the treatment of human cancer. Clinical testing is still at an early stage and it remains possible that efficacy will be limited by preexisting anti-measles immunity. However, ongoing research emphasizing the use of engineered viruses, infected cell carriers, and supplemental therapy within immunosuppressive drugs is already offering viable strategies to enhance the potency of these agents, even in the face of preexisting anti-measles immunity.

Acknowledgements Dr. Stephen Russell and Dr. Kah-Whye Peng are supported by funds from the NIH/NCI (CA100634 and CA129966 to Dr. Stephen Russell and CA118488 to Dr. Kah-Whye Peng). A grant from the Rapid Access to Intervention Development (RAID) Program of

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the NCI supported the manufacture of MV-NIS and toxicology studies in transgenic mice and squirrel monkeys. Efforts of numerous individuals including clinical collaborators (Drs. Evanthia Galanis, Angela Dispenzieri, Lynn C. Hartmann, David Dingli), members of the virus manufac-turing facility (Drs. Mark Federspiel and Linda Gregory, Guy Griesmann, Kirsten Langfield, Julie Sauer, Sharon Stephan, Henry Walker, Troy Wegman, Cindy Whitcomb) and members of the pharmacology/toxicology group (Rae Myers, Suzanne Greiner, Mary Harvey, Pam Ryno, Nathan Jenks, Emily Mader) are gratefully acknowledged.

References

Aghi M, Martuza RL (2005) Oncolytic viral therapies—the clinical experience. Oncogene 24:7802–7816

Aisenberg AC, Davis C (1968) The thymus and recovery from cyclophosphamide-induced toler-ance to sheep erythrocytes. J Exp Med 128:35–46

Aldous IR, Kirman BH, Butler N, et al (1961) Vaccination against measles. III. Clinical trial in British children. BMJ 2(5262):1250–1253

Allen C, Vongpungsawad S, Nakamura T, et al (2006) Retargeted oncolytic measles strains enter-ing via the EGFRvIII receptor maintain significant antitumor activity against gliomas with increased tumor specificity. Cancer Res 66:11840–11850

Allen C, Paraskevakou G, Liu C, et al (2008) Oncolytic measles virus strains in the treatment of gliomas. Expert Opin Biol Ther 8:213–220

Anderson BD, Nakamura T, Russell SJ, et al (2004) High CD46 receptor density determines pref-erential killing of tumor cells by oncolytic measles virus. Cancer Res 64:4919–4926

Anonymous (1996) Measles pneumonitis following measles-mumps-rubella vaccination of a patient with HIV infection. MMWR Morb Mortal Wkly Rep 45:603–606

Asada T (1974) Treatment of human cancer with mumps virus. Cancer 34:1907–1928 Audet S, Virata-Theimer ML, Beeler MJ, et al (2006) Measles-virus-neutralizing antibodies in

intravenous immunoglobulins. J Infect Dis 194:781–789 Auwaerter PG, Rota PA, Elkins WR, et al (1999) Measles virus infection in rhesus macaques:

altered immune responses and comparison of the virulence of six different virus strains. J Infect Dis 180:950–958

Bajzer Z, Carr T, Josic K, et al (2008) Modeling of cancer virotherapy with recombinant measles viruses. J Theor Biol 252:109–122

Balachandran S, Barber GN (2007) PKR in innate immunity, cancer, and viral oncolysis. Methods Mol Biol 383:277–301

Bankamp B, Hodge G, McChesney MB, et al (2008) Genetic changes that affect the virulence of measles virus in a rhesus macaque model. Virology 373:39–50

Basler CF, Garcia-Sastre A (2002) Viruses and the type I interferon antiviral system: induction and evasion. Int Rev Immunol 21:305–337

Bjorge L, Hakulinen J, Walström T, et al (1997) Complement-regulatory proteins in ovarian malignancies. Int J Cancer 70:14–25

Blechacz B, Splinter PL, Greiner S, et al (2006) Engineered measles virus as a novel oncolytic viral therapy system for hepatocellular carcinoma. Hepatology 44:1465–1477

Blok VT, Daha MR, Tijsma O, et al (2000) A possible role of CD46 for the protection in vivo of human renal tumor cells from complement-mediated damage. Lab Invest 80:335–344

Bluming A, Ziegler J (1971) Regression of Burkitt’s lymphoma in association with measles infec-tion. Lancet 2:105–106

Bucheit AD, Kumar S, Grote DM, et al (2003) An oncolytic measles virus engineered to enter cells through the CD20 antigen. Mol Ther 7:62–72

Campbell SA, Gromeier M (2005a) Oncolytic viruses for cancer therapy. I. Cell-external factors: virus entry and receptor interaction. Onkologie 28:144–149

b12325337-0011ztc.indd 235 10/3/2008 11:03:35 AM


Campbell SA, Gromeier M (2005b) Oncolytic viruses for cancer therapy. II. Cell-internal factors for conditional growth in neoplastic cells. Onkologie 28:209–215

Carlson SK, Classic KL, Hadac EM, et al (2006) In vivo quantitation of intratumoral radioisotope uptake using micro-single photon emission computed tomography/computed tomography. Mol Imaging Biol 8:324–332

Cocks BG, Chang CC, Carballido JM, et al (1995) A novel receptor involved in T-cell activation. Nature 376(6537):260–263

Collard P, Hendrickse RG, Montefiore D, et al (1961) Vaccination against measles. II. Clinical trial in Nigerian children. BMJ 2(5262):1246–1250

Dadachova E, Carrasco N (2004) The Na/I symporter (NIS): imaging and therapeutic applications. Semin Nucl Med 34:23–31

Devaux P, von Messling V, Songsungthong W, et al (2007) Tyrosine 110 in the measles virus phosphoprotein is required to block STAT1 phosphorylation. Virology 360:72–83

Dingli D, Diaz RM, Bergert ER, et al (2003) Genetically targeted radiotherapy for multiple mye-loma. Blood. 102:489–496

Dingli D, Peng KW, Harvey ME, et al (2004) Image-guided radiovirotherapy for multiple mye-loma using a recombinant measles virus expressing the thyroidal sodium iodide symporter. Blood 103:1641–1646

Dingli D, Kemp BJ, O’Connor MK, et al (2005a) Combined I-124 positron emission tomography/computed tomography imaging of NIS gene expression in animal models of stably transfected and intravenously transfected tumor. Mol Imaging Biol 8:16–23

Dingli D, Peng KW, Harvey ME, et al (2005b) Interaction of measles virus vectors with Auger electron emitting radioisotopes. Biochem Biophys Res Commun 337:22–29

Dingli D, Cascino MD, Josic K, et al (2006) Mathematical modeling of cancer radiovirotherapy. Math Biosci 199:55–78

Dorig RE, Marcil A, Chropa A, et al (1993) The human CD46 molecule is a receptor for measles virus (Edmonston strain). Cell 75:295–305

Durrant LG, Spendlove I (2001) Immunization against tumor cell surface complement-regulatory proteins. Curr Opin Investig Drugs 2:959–966

Enders JF, Peebles TC (1954) Propagation in tissue cultures of cytopathogenic agents from patients with measles. Proc Soc Exp Biol Med 86:277–286

Enders JF, Katz SL, Milovanovic MV, et al (1960) Studies on an attenuated measles-virus vaccine. I. Development and preparations of the vaccine: technics for assay of effects of vaccination. N Engl J Med 263:153–159

Fishelson Z, Donin N, Zell S, et al (2003) Obstacles to cancer immunotherapy: expression of membrane complement regulatory proteins (mCRPs) in tumors. Mol Immunol 40:109–123

Fisher K (2006) Striking out at disseminated metastases: the systemic delivery of oncolytic viruses. Curr Opin Mol Ther 8:301–313

Forsyth P, Roldan G, George D, et al (2008) A phase I trial of intratumoral administration of reo-virus in patients with histologically confirmed recurrent malignant gliomas. Mol Ther 16:627–632

Goffe AP, Laurence GD (1961) Vaccination against measles. I. Preparation and testing of vaccines consisting of living attenuated virus. BMJ 2(5262):1244–1246

Gorter A, Blok VT, Haasnoot WH, et al (1996) Expression of CD46, CD55, and CD59 on renal tumor cell lines and their role in preventing complement-mediated tumor cell lysis. Lab Invest 74:1039–1049

Gotoh B, Komatsu T, Takeuchi K, et al (2001) Paramyxovirus accessory proteins as interferon antagonists. Microbiol Immunol 45:787–800

Griffin D (2001) Measles virus. In: Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE (eds) Fields virology, 5th edn, Lippincott Williams & Wilkins, Philadelphia, pp 1551–1585

Griffin DE, Pan CH, Moss WJ (2008) Measles vaccines. Front Biosci 13:1352–1370 Grote D, Russell SJ, Cornu TI, et al (2001) Live attenuated measles virus induces regression of

human lymphoma xenografts in immunodeficient mice. Blood 97:3746–3754

b12325337-0011ztc.indd 236 10/3/2008 11:03:35 AM


Hadac EM, Peng KW, Nakamura T, et al (2004) Reengineering paramyxovirus tropism. Virology 329:217–225

Hallak LK, Merchen JR, Storgard CM, et al (2005) Targeted measles virus vector displaying Echistatin infects endothelial cells via alpha(v)beta3 and leads to tumor regression. Cancer Res 65:5292–5300

Hammond AL, Plemper RK, Zhang J, et al (2001) Single-chain antibody displayed on a recom-binant measles virus confers entry through the tumor-associated carcinoembryonic antigen. J Virol 75:2087–2096

Hangartner L, Zinkernagel RM, Hengartner H (2006) Antiviral antibody responses: the two extremes of a wide spectrum. Nat Rev Immunol 6:231–243

Hara T, Suzuki Y, Semba T, et al (1995) High expression of membrane cofactor protein of comple-ment (CD46) in human leukaemia cell lines: implication of an alternatively spliced form con-taining the STA domain in CD46 up-regulation. Scand J Immunol 42:581–590

Haralambieva I, Iankov I, Hasegawa K, et al (2007) Engineering oncolytic measles virus to cir-cumvent the intracellular innate immune response. Mol Ther 15:588–597

Hasegawa K, Nakamura T, Harvey M, et al (2006a) The use of a tropism-modified measles virus in folate receptor-targeted virotherapy of ovarian cancer. Clin Cancer Res 12:6170–6178

Hasegawa K, Pham L, O’Connor MK, et al (2006b) Dual therapy of ovarian cancer using measles viruses expressing carcinoembryonic antigen and sodium iodide symporter. Clin Cancer Res 12:1868–1875

Hasegawa K, Hu C, Nakamura T, et al (2007) Affinity thresholds for membrane fusion triggering by viral glycoproteins. J Virol 81:13149–13157

Heinzerling L, Künzi V, Oberholzer PA, et al (2005) Oncolytic measles virus in cutaneous T-cell lymphomas mounts antitumor immune responses in vivo and targets interferon-resistant tumor cells. Blood. 106:2287–2294

Hermiston T (2006) A demand for next-generation oncolytic adenoviruses. Curr Opin Mol Ther 8:322–330

Hill DL (1975) Pharmacology. In: Thomas CC (ed) A review of cyclophosphamide. Thomas Publishers, Springfield, IL pp 60–85

Hoffmann D, Bangan JM, Bayer W, et al (2006) Synergy between expression of fusogenic mem-brane proteins, chemotherapy and facultative virotherapy in colorectal cancer. Gene Ther 13:1534–1544

Hoster HA, Zanes RP Jr, Von Haam E (1949) Studies in Hodgkin’s syndrome; the association of viral hepatitis and Hodgkin’s disease: a preliminary report. Cancer Res 9:473–480

Hsu EC, Dörig RE, Sarangi F, et al (1997) Artificial mutations and natural variations in the CD46 molecules from human and monkey cells define regions important for measles virus binding. J Virol 71:6144–6154

Hsu EC, Sarangi F, Iorio C, et al (1998) A single amino acid change in the hemagglutinin protein of measles virus determines its ability to bind CD46 and reveals another receptor on marmoset B cells. J Virol 72:2905–2916

Huebner RJ, Rowe WP, Schatten WE, et al (1956) Studies on the use of viruses in the treatment of carcinoma of the cervix. Cancer 9:1211–1218

Iankov ID, Blechacz B, Liu C, et al (2007) Infected cell carriers: a new strategy for systemic delivery of oncolytic measles viruses in cancer virotherapy. Mol Ther 15:114–122

Jacobson DR, Zolla-Pazner S (1986) Immunosuppression and infection in multiple myeloma. Semin Oncol 13:282–290

Juhl H, Helmig F, Baltzer K, et al (1997) Frequent expression of complement resistance factors CD46, CD55, and CD59 on gastrointestinal cancer cells limits the therapeutic potential of monoclonal antibody 17–1A. J Surg Oncol 64:222–230

Katz SL (1965) Immunization with live attenuated measles virus vaccines: five years’ experience. Arch Gesamte Virusforsch 16:222–230

Katz S (1996) The history of measles virus and the development and utilization of measles virus vaccines. In: Plotkins S, Fantini B (eds) Vaccinia, vaccination and vaccinology: Jenner Pasteur and their successors. Elsevier, Paris, pp 265–270

b12325337-0011ztc.indd 237 10/3/2008 11:03:35 AM


Katz SL, Kempe CH, Black FL, et al (1960) Studies on an attenuated measles-virus vaccine. VIII. General summary and evaluation of the results of vaccine. N Engl J Med 263:180–184

Kaufmann M, Lindner P, Honegger A, et al (2002) Crystal structure of the anti-His tag antibody 3D5 single-chain fragment complexed to its antigen. J Mol Biol 318:135–147

Kelly E, Russell SJ (2007) History of oncolytic viruses: genesis to genetic engineering. Mol Ther 15:651–659

Kemper C, Leung M, Stephensen CB, et al (2001) Membrane cofactor protein (MCP; CD46) expression in transgenic mice. Clin Exp Immunol 124:180–189

Kinoh H, Inoue M (2008) New cancer therapy using genetically-engineered oncolytic Sendai virus vector. Front Biosci 13:2327–2334

Kinugasa N, Higashi T, Nouso K, et al (1999) Expression of membrane cofactor protein (MCP, CD46) in human liver diseases. Br J Cancer 80:1820–1825

Klasse PJ, Sattentau QJ (2002) Occupancy and mechanism in antibody-mediated neutralization of animal viruses. J Gen Virol 83:2091–2108


Kobune F, Takahashi H, Terao K, et al (1996) Nonhuman primate models of measles. Lab Anim Sci 46:315–320

Lecouturier V, Fayolle J, Caballero M, et al (1996) Identification of two amino acids in the hemag-glutinin glycoprotein of measles virus (MV) that govern hemadsorption HeLa cell fusion, and CD46 downregulation: phenotypic markers that differentiate vaccine and wild-type MV strains. J Virol 70:4200–4204

Lichty BD, Power AT, Stojdl DF, et al (2004) Vesicular stomatitis virus: re-inventing the bullet. Trends Mol Med 10:210–216

Liszewski MK, Atkinson JP (1992) Membrane cofactor protein. Curr Top Microbiol Immunol 178:45–60

Liu TC, Galanis E, Kirn D (2007) Clinical trial results with oncolytic virotherapy: a century of promise, a decade of progress. Nat Clin Pract Oncol 4:101–117

Lorence RM, Roberts MS, O’Neil JD, et al (2007) Phase 1 clinical experience using intravenous administration of PV701, an oncolytic Newcastle disease virus. Curr Cancer Drug Targets 7:157–167

Manchester M, Rall GF (2001) Model systems: transgenic mouse models for measles pathogene-sis. Trends Microbiol 9:19–23

Mazzaferri EL, Kloos RT (2001) Clinical review 128: current approaches to primary therapy for papillary and follicular thyroid cancer. J Clin Endocrinol Metab 86:1447–1463

McDonald CJ, Erlichman C, Ingle JM, et al (2006) A measles virus vaccine strain derivative as a novel oncolytic agent against breast cancer. Breast Cancer Res Treat 99:177–184

McQuillan GM, Kruszon-Moran D, Hyde TB, et al (2007) Seroprevalence of measles antibody in the US population, 1999–2004. J Infect Dis 196:1459–1464

Mota HC (1973) Infantile Hodgkin’s disease: remission after measles. BMJ 2(5863):421 Mrkic B, Pavolvic J, Rülicke T, et al (1998) Measles virus spread and pathogenesis in genetically

modified mice. J Virol 72:7420–7427 Mrkic B, Odermatt B, Klein MA, et al (2000) Lymphatic dissemination and comparative pathol-

ogy of recombinant measles viruses in genetically modified mice. J Virol 74:1364–1672 Munguia A, Ota T, Meist T, Russell SJ (2008) Cell carriers to deliver oncolytic viruses to sites of

myeloma tumor growth. Gene Ther 15:797–806 Murray KP, Mathure S, Kaul R, et al (2000) Expression of complement regulatory proteins-CD

35, CD 46, CD 55, and CD 59-in benign and malignant endometrial tissue. Gynecol Oncol 76:176–182

Myers R, Greiner S, Harvey M, et al (2005) Oncolytic activities of approved mumps and measles vaccines for therapy of ovarian cancer. Cancer Gene Ther 12:593–599

Myers RM, Greiner S, Harvey M, et al (2007) Preclinical pharmacology and toxicology of intra-venous MV-NIS, an oncolytic measles virus administered with or without cyclophosphamide. Clin Pharmacol Ther 82:700–710

b12325337-0011ztc.indd 238 10/3/2008 11:03:35 AM


Nagy N, Maeda A, Bandobashi K, et al (2002) SH2D1A expression in Burkitt lymphoma cells is restricted to EBV positive group I lines and is downregulated in parallel with immunoblastic transformation. Int J Cancer 100:433–440

Nakamura T, Russell SJ (2004) Oncolytic measles viruses for cancer therapy. Expert Opin Biol Ther 4:1685–1692

Nakamura T, Peng KW, Vongpunsawad S, et al (2004) Antibody-targeted cell fusion. Nat Biotechnol 22:331–336

Nakamura T, Peng KW, Harvey M, et al (2005) Rescue and propagation of fully retargeted onco-lytic measles viruses. Nat Biotechnol 23:209–214

Naniche D, Varior-Krishnan G, Cervoni F, et al (1993) Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus. J Virol 67:6025–6032

Naniche D, Yeh A, Eto D, et al (2000) Evasion of host defenses by measles virus: wild-type mea-sles virus infection interferes with induction of alpha/beta interferon production. J Virol 74:7478–7484

Newman W, Southam CM (1954) Virus treatment in advanced cancer; a pathological study of fifty-seven cases. Cancer 7:106–118

Nielsen L, Blixenkrone-Møller M, Thylstrup M, et al (2001) Adaptation of wild-type measles virus to CD46 receptor usage. Arch Virol 146:197–208

Ohno S, Ono N, Takeda M, et al (2004) Dissection of measles virus V protein in relation to its ability to block alpha/beta interferon signal transduction. J Gen Virol 85:2991–2999

Okuno Y, Asada T, Yamanishi K, et al (1978) Studies on the use of mumps virus for treatment of human cancer. Biken J 21:37–49

Oldstone MB, Lewicki H, Thomas D, et al (1999) Measles virus infection in a transgenic model: virus-induced immunosuppression and central nervous system disease. Cell 98:629–640

Ong HT, Timm MM, Greip PR, et al (2006) Oncolytic measles virus targets high CD46 expression on multiple myeloma cells. Exp Hematol 34:713–720

Ong HT, Hasagawa K, Dietz KB, et al (2007) Evaluation of T cells as carriers for systemic mea-sles virotherapy in the presence of antiviral antibodies. Gene Ther 14:324–333

Ordman CW, Jennings CG, Janeway CA (1944) Chemical clinical, and immunological studies on the products of human plasma fractionation. XII. The use of concentrated normal human serum gamma globulin (human immune serum globulin) in the prevention and attenuation of measles. J Clin Invest 23:541–549

Osunkoya BO, Ukaejiofo EO, Ajayi O, et al (1990) Evidence that circulating lymphocytes act as vehicles or viraemia in measles. West Afr J Med 9:35–39

Palosaari H, Parisien JP, Rodriguez JJ, et al (2003) STAT protein interference and suppression of cytokine signal transduction by measles virus V protein. J Virol 77:7635–7644

Paraskevakou G, Allen C, Nakamura T, et al (2007) Epidermal growth factor receptor (EGFR)-retargeted measles virus strains effectively target EGFR- or EGFRvIII-expressing gliomas. Mol Ther 15:677–686

Peng KW, Ahmann GJ, Pham L, et al (2001) Systemic therapy of myeloma xenografts by an attenuated measles virus. Blood 98:2002–2007

Peng KW, Facteau S, Wegman T, et al (2002a) Intraperitoneal therapy of ovarian cancer using an engineered measles virus. Cancer Res 62:4656–4662

Peng KW, TenEyck CJ, Gallanis E, et al (2002b) Non-invasive in vivo monitoring of trackable viruses expressing soluble marker peptides. Nat Med 8:527–531

Peng KW, Donovan KA, Schneider U, et al (2003a) Oncolytic measles viruses displaying a single-chain antibody against CD38, a myeloma cell marker. Blood 101:2557–2562

Peng KW, Frenzke M, Meyers R, et al (2003b) Biodistribution of oncolytic measles virus after intra-peritoneal administration into Ifnar-CD46Ge transgenic mice. Hum Gene Ther 14:1565–1577

Peng K-W, Holler PD, Orr BA, et al (2004) Targeting membrane fusion to specific peptide/MHC complexes through a high-affinity T-cell receptor. Gene Ther 11:1234–1239

Peng KW, Hada EM, Anderson BD, et al (2006) Pharmacokinetics of oncolytic measles virother-apy: eventual equilibrium between virus and tumor in an ovarian cancer xenograft model. Cancer Gene Ther 13:732–738

b12325337-0011ztc.indd 239 10/3/2008 11:03:35 AM


Peng KW, Pham L, Ye H, et al (2008) Organ distribution of gene expression after intravenous infusion of targeted and untargeted lentiviral vectors. Gene Ther 8:1456–1463

Phuong LK, Allen C, Peng KW, et al (2003) Use of a vaccine strain of measles virus genetically engineered to produce carcinoembryonic antigen as a novel therapeutic agent against glioblas-toma multiforme. Cancer Res 63:2462–2469

Radecke F, Spielhofer P, Schneider H, et al (1995) Rescue of measles viruses from cloned DNA. Embo J 14:5773–5784

Reid T, Galanis E, Abbruzzese J, et al (2002) Hepatic arterial infusion of a replication-selective oncolytic adenovirus (dl1520): phase II viral, immunologic, and clinical endpoints. Cancer Res 62:6070–6079

Riesco-Eizaguirre G, Santisteban P (2006) A perspective view of sodium iodide symporter research and its clinical implications. Eur J Endocrinol 155:495–512

Riley-Vargas RC, Gill DB, Kemper C, et al (2004) CD46: expanding beyond complement regula-tion. Trends Immunol 25:496–503

Rota JS, Wang ZD, Rota PA, et al (1994) Comparison of sequences of the H, F, and N coding genes of measles virus vaccine strains. Virus Res 31:317–330

Russell S (1994) Replicating vectors for cancer therapy: a question of strategy. Semin Cancer Biol 5:437–443

Russell SJ, Peng KW (2007) Viruses as anticancer drugs. Trends Pharmacol Sci 28:326–333 Scallan CD, Jiang H, Liu T, et al (2006) Human immunoglobulin inhibits liver transduction by

AAV vectors at low AAV2 neutralizing titers in SCID mice. Blood 107:1810–1817 Schneider U, Bulloughh F, Vongpunsawad S, et al (2000) Recombinant measles viruses efficiently

entering cells through targeted receptors. J Virol 74: 9928–9936 Schwarz AJ, Boyer PA, Zirbel LW, et al (1960) Experimental vaccination against measles. I. Tests

of live measles and distemper vaccine in monkeys and two human volunteers under laboratory conditions. JAMA 173:861–867

Seya T, Hara T, Matsumoto M, et al (1990) Quantitative analysis of membrane cofactor protein (MCP) of complement. High expression of MCP on human leukemia cell lines, which is down-regulated during cell differentiation. J Immunol 145:238–245

Shaffer JA, Bellini WJ, Rota PA (2003) The C protein of measles virus inhibits the type I inter-feron response. Virology 315:389–397

Shen Y, Nemunaitis J (2006)Herpes simplex virus 1 (HSV-1) for cancer treatment. Cancer Gene Ther 13:975–992

Shimizu Y, Hasumi K, Okudaira Y, et al (1988) Immunotherapy of advanced gynecologic cancer patients utilizing mumps virus. Cancer Detect Prev 12:487–495

Simpson KL, Jones A, Norman S, et al (1997) Expression of the complement regulatory proteins decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59 in the normal human uterine cervix and in premalignant and malignant cervical disease. Am J Pathol 151:1455–1467

Sinkovics JG, Horvath JC (2000) Newcastle disease virus (NDV): brief history of its oncolytic strains. J Clin Virol 16:1–15

Southam CM, Moore AE (1952) Clinical studies of viruses as antineoplastic agents with particular reference to Egypt 101 virus. Cancer 5: 1025–1034

Steinberg A (2001) Cyclophosphamide. In: Austen K et al. (eds) Therapeutic immunology. Blackwell, Malden, pp 31–50

Stojdl DF, Lichty B, Knowles S, et al (2000) Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus. Nat Med 6(7):821–85

Stojdl DF,, et al (2003) VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents. Cancer Cell 4:263–275

Strong JE, Lichty B, ten Oever BR, et al (1998) The molecular basis of viral oncolysis: usurpation of the Ras signaling pathway by reovirus. EMBO J 17:3351–3362

Tai CK, Kasahara N (2008) Replication-competent retrovirus vectors for cancer gene therapy. Front Biosci 13:3083–3095

b12325337-0011ztc.indd 240 10/3/2008 11:03:35 AM


Takeda M, Kato A, Kobune F, et al (1998) Measles virus attenuation associated with transcrip-tional impediment and a few amino acid changes in the polymerase and accessory proteins. J Virol 72:8690–8696

Takeuchi K, Kadota SI, Takeda M, et al (2003) Measles virus V protein blocks interferon (IFN)-alpha/beta but not IFN-gamma signaling by inhibiting STAT1 and STAT2 phosphorylation. FEBS Lett 545:177–182

Taqi AM, Abdurrahman AB, Yakubu AM, et al (1981) Regression of Hodgkin’s disease after measles. Lancet 1:1112

Thorne SH, Hwang TH, O’Gorman WE, et al (2007) Rational strain selection and engineering creates a broad-spectrum, systemically effective oncolytic poxvirus JX-963. J Clin Invest 117:3350–3358

Thorsteinsson L, O’Dowd GM, Harrington PM, et al (1998) The complement regulatory proteins CD46 and CD59, but not CD55, are highly expressed by glandular epithelium of human breast and colorectal tumour tissues. APMIS 106:869–878

Ungerechts G, Springfield C, Frenzke ME, et al (2007a) An immunocompetent murine model for oncolysis with an armed and targeted measles virus. Mol Ther 15:1991–1997

Ungerechts G, Springfield C, Frenzke ME, et al (2007b) Lymphoma chemovirotherapy: CD20-targeted and convertase-armed measles virus can synergize with fludarabine. Cancer Res 67:10939–10947

van Binnendijk RS, van der Heijden RW, van Amerongen G, et al (1994) Viral replication and development of specific immunity in macaques after infection with different measles virus strains. J Infect Dis 170:443–448

Varsano S, Rashkovsky L, Shapiro H, et al (1998) Human lung cancer cell lines express cell membrane complement inhibitory proteins and are extremely resistant to complement-medi-ated lysis; a comparison with normal human respiratory epithelium in vitro, and an insight into mechanism(s) of resistance. Clin Exp Immunol 113:173–182

Vongpunsawad S, Oezgun M, Braun W, et al (2004) Selectively receptor-blind measles viruses: identification of residues necessary for SLAM- or CD46-induced fusion and their localization on a new hemagglutinin structural model. J Virol 78:302–313

Waehler R, Russell SJ, Curiel DT (2007) Engineering targeted viral vectors for gene therapy. Nat Rev Genet 8:573–587

Wang Y, Yuan F (2006) Delivery of viral vectors to tumor cells: extracellular transport, systemic distribution, and strategies for improvement. Ann Biomed Eng 34:114–127

Wein LM, Wu JT, Kirn DH (2003) Validation and analysis of a mathematical model of a replica-tion-competent oncolytic virus for cancer treatment: implications for virus design and delivery. Cancer Res 63:1317–1324

Xie M, Tanaka K, Ono N, et al (1999) Amino acid substitutions at position 481 differently affect the ability of the measles virus hemagglutinin to induce cell fusion in monkey and marmoset cells co-expressing the fusion protein. Arch Virol 144:1689–1699

Yamakawa M, Yamada K, Tsuge T, et al (1994) Protection of thyroid cancer cells by complement-regulatory factors. Cancer 73:2808–2817

Yanagi Y, Takeda M, Ohno S et al (2006) Measles virus receptors and tropism. Jpn J Infect Dis 59:1–5

Yu W, Fang H (2007) Clinical trials with oncolytic adenovirus in China. Curr Cancer Drug Targets 7:141–148

Ziegler JL (1976) Spontaneous remission in Burkitt’s lymphoma. Natl Cancer Inst Monogr 44:61–65

Zingher A, Mortimer P (2005) Convalescent whole blood, plasma and serum in the prophylaxis of measles: JAMA, 12 April, 1926; 1180–1181. Rev Med Virol 15:407–418; discussion 418–421

Zygiert Z (1971) Hodgkin’s disease: remissions after measles. Lancet 1(7699):593

b12325337-0011ztc.indd 241 10/3/2008 11:03:35 AM

Chapter 12 Measles Virus-Induced Immunosuppression

S. Schneider-Schaulies( ) and J. Schneider-Schaulies

Abstract Immunosuppression is the major cause of infant death associated with acute measles and therefore of substantial clinical importance. Major hallmarks of this generalized modulation of immune functions are (1) lymphopenia, (2) a prolonged cytokine imbalance consistent with suppression of cellular immunity to secondary infections, and (3) silencing of peripheral blood lymphocytes, which cannot expand in response to ex vivo stimulation. Lymphopenia results from depletion, which can occur basically at any stage of lymphocyte development, and evidently, expression of the major MV receptor CD150 plays an important role in targeting these cells. Virus transfer to T cells is thought to be mediated by dendritic cells (DCs), which are considered central to the induction of T cell silencing and functional skewing. As a consequence of MV interaction, viability and functional differentiation of DCs and thereby their expression pattern of co-stimulatory mol-ecules and soluble mediators are modulated. Moreover, MV proteins expressed by these cells actively silence T cells by interfering with signaling pathways essential for T cell activation.

S. Schneider-Schaulies Institute for Virology and Immunobiology , University of Würzburg , Versbacher Str. 7, 97078 Würzburg , Germany , e-mail: [email protected]

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244General Features Associated with MV-Induced Immunosuppression. . . . . . . . . . . . . . . . . . . 244Lymphopenia Mechanisms of Lymphocyte Depletion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245MV Regulation of APC Viability and Function-Modulated Instruction and/or Active Lymphocyte Silencing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

Dendritic Cells: Maturation Program and General Functions . . . . . . . . . . . . . . . . . . . . . . . 247MV-Induced Effects on Dendritic Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

Viral Proteins Effective at T Cell Silencing and Modulation. . . . . . . . . . . . . . . . . . . . . . . . . . 255The N Protein. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256The Glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

D.E. Griffin and M.B.A. Oldstone (eds.) Measles – History and Basic Biology. 243© Springer-Verlag Berlin Heidelberg 2009

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244 S. Schneider-Schaulies, J. Schneider-Schaulies

Introduction

Measles virus (MV) is the causative agent of acute measles, a well-defined clinical entity usually contracted by children. The virus is highly contagious, yet causes acute infection only once in the lifetime of an individual, indicating that anti-viral immune responses are efficiently generated and persistent. In spite of the availabil-ity of this efficient vaccine, more than 30 million cases of acute measles are annu-ally reported worldwide with approximately 345,000 cases of infant deaths.

Although MV can and does cause severe and sometimes lethal complications, including pneumonia and encephalitis also in industrialized countries, the majority of fatal cases develop in Third World countries on the basis of the transient, pro-nounced general immunosuppression induced by the virus during and for several weeks after acute measles (Katz 1995). There, exposure occurs early in infancy when fading maternal antibodies prevent successful vaccination but are no longer protective against wild-type MV infection (Black et al. 1986; Lennon and Black 1986; Ryon et al. 2002) . Further aggravated by malnutrition, secondary infections then often follow a severe or fatal course (Dollimore et al. 1997). Furthermore, MV-induced immunosuppression also favors reactivation and exacerbation of per-sistent infections in children and young adults (Tamashiro et al. 1987). More recently, immunosuppression caused by viral infection in infants where the immune system is still immature has been proposed to play an important role in the estab-lishment of persistent CNS MV infections (Oldstone et al. 2005).

General Features Associated with MV-Induced Immunosuppression

In the immunocompetent host, MV induces an efficient virus-specific immunity that controls spread and leads to clearance of MV and to a most likely lifelong immunity to clinical reinfection. An initial Th1 cell activation as marked by sol-uble CD4, CD8, IL-2R, and β2, microglobulin switches to a prolonged Th2-dominated response (reviewed in Griffin 1995 and the chapters by P.A. Rota et al. and W.J. Moss, this volume). The contribution of components of the innate immunity in MV control in humans is less well understood.

The term “anergy” was first coined by von Pirquet in 1908 to describe the loss of DTH reactions to tuberculin in MV-infected individuals. MV-induced immunosup-pression is commonly characterized by (1) a marked lymphopenia, which is rapidly resolved (Ryon et al. 2002), (2) a cytokine imbalance skewed toward a prolonged Th2 response, which suppresses cellular immunity to secondary infections (Griffin and Ward 1993), and (3), the inability of peripheral blood lymphocytes (PBL) to expand in response to polyclonal or antigen-specific stimulation ex vivo (Borrow and Oldstone 1995; Schneider-Schaulies et al. 2001). The extent to which these parameters are mechanistically decisive for or represent surrogates of MV-induced immunosuppression has not been resolved because skewing to an early Th2 response

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12 Measles Virus-Induced Immunosuppression 245

and slightly impaired proliferative responses of PBLs ex vivo have also been related to MV vaccination which, when applied at standard doses, does not cause immuno-suppression (Ward and Griffin 1993; Hussey et al. 1996). Thus, there is still a need to understand basic mechanisms underlying MV-induced immunosuppression such as targeting of specific host cells and their relative importance for immunomodula-tion in suitable experimental systems. These involve small models mainly involving genetically modified mice, ferrets, and cotton rats (detailed in the chapters by S. Pillet et al., S. Niewiesk, C.I. Sellin and B. Horvat, and B. Hahm, this volume) and experimental infection in non-human primates, which has continued to provide exciting insights into cell targeting in primary infection (reviewed in the chapter by R.L. de Swart, this volume). It will be extremely important to evaluate to what extent the plethora of findings obtained in vitro, mainly referred to within this chapter, are reflected or at least compatible with an in vivo infection.

Lymphopenia Mechanisms of Lymphocyte Depletion

Measles is associated with lymphopenia affecting B cells, monocytes, neutrophils, as well as CD4 and CD8 T cells. No genetic or gender-specific factors are known, yet the extent of lymphopenia correlates with age and severity of the disease (Okada et al. 2000, 2001). In contrast to B cell frequencies, which can still be below control levels for up to 6 weeks, T cell numbers return to normal after 10 days and the CD4/CD8 ratio remains constant over time (Arneborn et al. 1983; Okada et al. 2001; Ryon et al. 2002). Figures published on the frequency of infected peripheral blood mononuclear cells (PBMCs) vary considerably depending on the stage of disease analyzed and the methods used, however, generally do not exceed 2% at the onset of the acute phase (Schneider-Schaulies et al. 1991; Forthal et al. 1992; Esolen et al. 1993). This correlates with findings recently obtained in experimen-tally infected rhesus macaques (de Swart et al. 2007; see the chapter by R.L. de Swart, this volume). Not surprisingly, expression patterns of the major MV uptake receptor, CD150, directly correlate with infected cell types. Thus, the peripheral CD150 + B cell compartment was identified as preferentially targeted, and this may account for B cell lymphopenia as observed in humans (Okada et al. 2000). Peripheral blood monocytes do not express CD150 and were found not to be infected in this model, while earlier findings in humans suggested these were a major infected peripheral cell population (Esolen et al. 1993). In addition, infection of T lymphocytes both in PBMCs and secondary lymphatic tissues was documented in macaques (de Swart et al. 2007), and this mirrors findings obtained after in vitro infection of human tonsil slices (Condack et al. 2007). There, infection of both B and T lymphocytes directly segregated with expression of CD150 and targeted the CD45R0 T cell compartment, which was found to be preferentially depleted.

Mechanisms other than loss of mature lymphocytes due to infection have also been proposed to contribute to lymphopenia; these were mainly studied for T cells (schematically depicted in Fig. 12. 1 ). Thymocyte generation, viability, or differen-tiation into T cells can be viewed as effective targets for depletion. Consequences

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of MV interaction with CD34 + human hematopoietic stem cells (HSCs) were addressed in vitro (Manchester et al. 2002). While in mice members of the SLAM family distinguish defined HSC differentiation stages (Kiel et al. 2005), it is unknown whether the SLAM code also applies to corresponding cells in humans. MV infected with a very small subpopulation of human HSCs, however, did not affect their ability to differentiate into colony-forming cells in vitro. Interestingly, the ability of MV-infected stroma cells to support HSC development was impaired (Manchester et al. 2002). Since this study was conducted using a wild-type MV strain able to also interact with CD46, it is unclear if and how CD150-dependent wild-type viruses could access these cells.

Studies on the interaction of MV with thymocytes, which express CD150 in humans (Aversa et al. 1997; Sidorenko and Clark 2003), revealed that thymo-cytes (and CD4 and CD8 T cells) of mice, in which transgenic expression of CD150 was driven by an Lck-promoter, expressed MV proteins after in vivo or in vitro infection (Hahm et al. 2003). Though not analyzed in this study, apopto-sis of these cells likely occurs, since this was massively seen in MV-infected human thymus/liver xenografts in SCID mice where, however, thymocytes them-selves remained uninfected (Valsamakis et al. 1999). Thymocyte viability and/or differentiation into functional T cells can also be affected by as yet undefined viral components released from infected thymic epithelial cells (Valentin et al. 1999). Since CD150 ligation by antibodies can also transmit inhibitory signals (Sidorenko and Clark 2003), engagement by the MV H protein could also affect thymocyte viability. Indeed, prolonged passage of avirulent MV strains in human thymic tissue enhanced their ability to cause thymocyte apoptosis, and this

thymic epithelial cells

thymocytes

secondary lymphoid tissue

hsc

stroma cellsperipheral

lymphocytes

peripheral tissue

activation

priming forapoptosis

loss by fusion

MV transmission

1

2

3

4

Fig. 12.1 Potential mechanisms for MV-induced lymphopenia. Few hematopoietic stem cells (HSCs) express CD150 ( light green membrane) and remain largely unaffected by infection. Infected stroma cells can, however, release unknown components ( red ), which target HSC dif-ferentiation. Thymocytes express high levels of CD150 and may be depleted by infection and/or apoptosis ( dark grey ) induced by components released from infected thymic epithelial cells ( red ). Upon entry into secondary lymphatic tissues, T cells may be ( 1 ) activated normally, yet subse-quently home aberrantly to peripheral tissues, ( 2 ) primed for apoptosis ( dark grey ), ( 3 ) lost by fusion with ( dotted membrane infected dendritic cells), or ( 4 ) infection ( red cells) after MV transmission from these cells

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correlated with an amino acid exchange within the H protein ectodomain (Valsamakis et al. 1999).

Strikingly, thymic output determined by T cell receptor excision circle frequencies was found to be even increased in measles patients, indicating that depletion of pre-cursors possibly is not a major cause of peripheral T cell lymphopenia (Permar et al. 2003). Rather, cell loss due to infection may substantially contribute to this phenom-enon. They most likely acquire MV in secondary lymphatic tissues, eventually by dendritic cells (DCs) carrying infectious virus. Indeed, infected T lymphocytes were isolated from lymphoid tissues of experimentally infected rhesus macaques (de Swart et al. 2007). As for MV acquisition from DCs, surface expression of CD150 would also be a prerequisite for loss of T cells by fusion with infected DCs, as occurs in mixed DC/T cell cultures to some extent (Fugier-Vivier et al. 1997; Grosjean et al. 1997; Servet-Delprat et al. 2000; Vidalain et al. 2000, 2001a). Giant cell formation and necrosis were reported in lymphoid tissues of MV-infected rhesus macaques, albeit to a moderate extent (McChesney et al. 1989; Kobune et al. 1996). Giant cell formation in hyperplastic lymphoid tissue was documented in a measles patient at autopsy; however, MV could not be detected in this area (Nozawa et al. 1994). Thus, T cell loss in secondary lymphoid tissues may occur, as may induction of apoptosis as seen in cocultures of infected DCs and T cells (Fugier-Vivier et al. 1997). In addi-tion, as yet unidentified signals may prime T cells for apoptosis since peripheral T cells of measles patients revealed an enhanced sensitivity to CD3-ligation-induced cell death (Addae et al. 1995, 1998; Okada et al. 2000).

Lastly, a preferential decrease in the frequency of LFA-1 high T cells has been measured both in infected children and vaccinees, suggesting that these cells may aberrantly home to peripheral tissues, though activation-induced death is certainly also a possible explanation for the loss of these cells (Nanan et al. 1999).

MV Regulation of APC Viability and Function-Modulated Instruction and/or Active Lymphocyte Silencing

Dendritic Cells: Maturation Program and General Functions

MV-induced immunosuppression is thought to be centrally determined by its inter-action with professional antigen-presenting cells (APCs), especially DCs. DCs of myeloid origin (further referred to DCs as opposed to plasmacytoid DCs [pDCs]), which can be isolated from peripheral blood or generated from precursors, are con-ductors of the adaptive immunity orchestrating lymphocytes to specifically recog-nize and handle antigens in the most effective manner. In an immature state, DCs reside in peripheral tissues and initiate a maturation program in response to danger signals such as viral components or inflammatory cytokines. This includes dra-matic alteration of their receptor repertoire, acquisition of a migratory phenotype and secretion of soluble mediators to attract to T cells to which they present proc-essed antigens (Steinman et al. 1997; Banchereau and Steinman 1998; Steinman

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2003). Immature DCs express a variety of surface and intracellular pathogen recog-nition receptors (PRRs), some of which (such as C-type lectin receptors) are spe-cialized to internalize pathogens for subsequent processing and presentation, while other PRRs, such as TLRs, trigger maturation signals. Morphological changes associated with DC maturation include downregulation of actin-based ruffles and cell polarization, which allows distinction of a leading edge and an uropod (Fig. 12. 2 A). Surface receptors and molecules regulating cytoskeletal reorganiza-tion also polarize. Those essentially promoting directed migration (such as chem-okine receptors and small GTPases Rac and Cdc42) localize to the leading edge, while the uropod concentrates adhesion receptors such as CD43 and RhoA, respec-tively (Sanchez-Madrid and del Pozo 1999; Arrieumerlou and Meyer 2005). Directed movement from peripheral tissues along chemokine gradients allows DC

IL-1,-6,-10,-12,TNF-α

IFN-α/β/ωTNF-α, IL-6

pDC

TLRs 1-6

DC

CD209

TLRs 7/8, 9

NF-κB, IRF-3IRF3/IRF-7

CCR1,2,4,5,6,7,8

CXCR3,4

CCR7,

CXCR3,4

MV

MOCK

LPS NF-κB,

a b

Fig. 12.2 A, B DC/pDC functional differences and DC morphological maturation. A DCs gener-ated from monocytes by culture in GM-CSF/IL-4 were treated with uninfected cell extract ( MOCK ), lipopolysaccharide ( LPS ) or infectious wild-type MV for 24 h, seeded onto fibronectin, and analyzed by scanning electron microscopy. Immature DCs (MOCK, upper panel ) reveal large ruffled membrane protrusions while mature DCs (LPS, bottom panel ) appear polarized with lamellar protrusions on their leading edge. Although overall slightly smaller, MV-infected DCs morphologically resemble LPS DCs in showing clear front-rear polarization and lamellar protru-sions (MV, middle panel ). B Myelopid DCs ( DC ) and plasmacytoid DCs ( pDC ) differ in their expression profiles of pattern recognition receptors ( upper triangles ), chemokine receptors ( mid-dle triangles ) (both of which are regulated with DC maturation, e.g., CCR5 on immature, and CCR7 on mature DCs) and transcription factors, especially IRF-7 ( bottom triangles ). Supporting is important role in homing to secondary lymphatics, CCR7 is upregulated both on DC and pDCs. DCs and pDCs also differ substantially with regard to cytokine production

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homing to secondary lymphoid tissues where characteristic large actin based pro-trusions (veils) provide contact planes for scanning T cells (Shutt et al. 2000; Burns et al. 2004) (Fig. 12.2A). Antigen-processing and -presentation pathways are acti-vated, and finally, mature DCs display and present loaded MHC class II molecules at their surface (Banchereau and Steinman 1998). Activation of MHC class I restricted CD8 + T cells by DCs relies on viral replication, or cross-priming after uptake of cell-bound or cell free antigens (for review see Wilson and Villadangos 2005). Mature DCs do not leave but rather die in lymphoid tissues thereafter and are not found in the efferent lymph. Plasmacytoid DCs (pDCs) are mainly found in blood from where they directly access secondary lymphatic tissues (Banchereau and Steinman 1998). They also mature after pathogen encounter, yet their ability to present antigen is very limited. Their distinct repertoire of PRRs, chemokine recep-tors, and transcription factors rather coins them as effector cells of the innate immune system particularly in viral infections, because they readily produce high amounts of type I IFN, whereas (myeloid) DCs act rather as major producers of inflammatory cytokines such as IL-12, TNF-α, IL-6, and IL-1α/β as important for effector cell activation (Asselin-Paturel and Trinchieri 2005) (Fig. 12.2B).

MV-Induced Effects on Dendritic Cells

DC Infection In Vivo

Direct evidence for MV infection of DCs during acute measles in humans has not yet been obtained. These cells may represent prime targets during MV transmission since they localize within and can even project through the respiratory epithelium, and in contrast to epithelial cells, can express CD150 at least in vitro (Minagawa et al. 2001; Ohgimoto et al. 2001; de Witte et al. 2006; Rethi et al. 2006). If targeted for infection, these cells would and have been suggested to be good candidates for MV transport to local lymphatic tissues.

Indirect evidence for wild-type MV targeting secondary lymphoid tissues and there, determined by its H protein, DCs was obtained in experimentally infected cotton rats ( Sigmodon hispidus ) (Niewiesk et al. 1997; Pfeuffer et al. 2003; the chapter by S. Niewiesk, this volume). In CD46 transgenic mice, splenic DCs were found strongly activated and infected by attenuated MV (strain Edmonston), yet this required prior deletion of monocyte/macrophages (Mrkic et al. 1998, 2000). MV antigens were also detected in very limited numbers of splenic CD11c + DCs after intravenous MV wild-type infection of mice transgenic for CD150 driven by the CD11c promotor (Hahm et al. 2004; the chapter by B. Hahm, this volume). In this system, CD11 + DCs were, however, the only cells accessible to MV entry, and thus potential infection of other cell types also expressing CD150 could not be evaluated. Though ablation of type I IFN receptor or associated signaling pathways has led to a significant improvement in detection of MV RNAs of proteins in sec-ondary lymphoid tissues in mice, infection of CD11c + cells in vivo was, if at all, barely detectable (Shingai et al. 2005; Welstead et al. 2005; Ohno et al. 2007).

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However, transgenic expression of CD150 on murine DCs directly isolated or gen-erated from bone marrow precursors conferred susceptibility to MV infection in vitro, and this was strongly enhanced if type I IFN signaling was additionally ablated, indicating that this system is one of the major restriction factors for MV in murine cells. More recently, in macaques experimentally infected with a wild-type MV encoding eGFP, strong evidence for infection of CD11c + MHC class II-positive cells with DC morphology in secondary lymphoid tissues, the dermis, or emigrat-ing from skin explants from these animals was obtained (de Swart et al. 2007; the chapter by R.L. de Swart, this volume). Assuming that MV tropism in this model reflects this in humans, it appears thus very likely that DCs (and/or Langerhans cells) might indeed be targeted by MV infection in vivo. Early studies in non-human primates found MV associated with follicular dendritic cells (FDCs) which are of nonhematopoietic origin and may, though this has not been established, serve as long-term repositories for MV (McChesney et al. 1997).

MV Interaction with Receptors on DCs

Langerhans cells and DCs isolated from peripheral blood or generated in vitro from monocytes or CD34 + precursor cells are susceptible to MV infection (Fugier-Vivier et al. 1997; Grosjean et al. 1997; Schnorr et al. 1997b; Steineur et al. 1998; Klagge et al. 2000; Servet-Delprat et al. 2000; Dubois et al. 2001; Ohgimoto et al. 2001). Infection with CD46-adapted strains is not surprising since this molecule is expressed on DCs of any maturation stage. MV wild-type strains and recombinant MVs expressing wild-type-derived H proteins rely on CD150 for entry, and thus, CD150-specific antibodies inhibit MV uptake (de Witte et al. 2006) (Fig. 12. 3 ). As detailed in Sect. 4.2.1), this molecule is either expressed at low levels on cultured immature DCs or induced upon activation by LPS exposure or proinflammatory cytokines (Kruse et al. 2001; Minagawa et al. 2001). CD150 can also be induced by MV wild-type strains themselves as shown in monocytes (and probably in DCs) since these strains act as Toll-like receptor 2 (TLR2) agonists (Minagawa et al. 2001; Bieback et al. 2002). Interestingly, their TLR2 agonistic activity seems to inversely correlate with their ability to interact with CD46. Thus, a recombinant MV expressing a wild-type H protein with aa481 reversed to that found in attenu-ated strains failed to activate TLR2 signaling (Bieback et al. 2002). Although the TLR2 does not support MV entry, interaction of MV wild-type strains may, besides CD150 induction, contribute to MV-induced maturation of immature DCs. MV wild-type-dependent activation of TLR2 presumably accounts for the induc-tion of CD150, CD86, and cytokines such as IL-6, IL-12p40, and IL-1α/β (Bieback et al. 2002). Other consequences of CD150 ligation by MV on DCs remain specu-lative, but may include modulation of TLR4 but not TLR2 or TLR9 signaling and thereby production of IL-12, TNF-α, NO, and IL-6, as shown in macrophages (Wang et al. 2004).

MV entry into DCs apparently also involves surface molecules other than CD150. Thus, MV uptake into these cells correlated with enhanced fusion rather than binding (Ohgimoto et al. 2001). Though the C-type lectin, CD209 (DC-SIGN)

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does not support MV entry upon transgenic expression in CHO cells, it enhances uptake into immature DCs (de Witte et al. 2006) by an as yet unknown mechanism. Possibly, ligation of this highly abundant surface receptor locally concentrates CD150, thereby favoring viral binding and fusion. For HIV, CD209 has been shown to strongly enhance a process referred to as transinfection, i.e., transfer of virus by DCs to conjugating T cells via infectious synapses (Geijtenbeek et al. 2000; Geijtenbeek and van Kooyk 2003a), which, early after viral exposure, occurs inde-pendently of DC infection. Transmission of MV to T cells in DC/T cell conjugates has been found to be rather inefficient, yet greatly enhanced, if T cells are preacti-vated prior to conjugate formation (Fugier-Vivier et al. 1997; Grosjean et al. 1997). This possibly results from both enhancement of viral replication after CD40 liga-tion (as proposed) and induction of CD150 on T cells enabling viral fusion and entry. Whether this also affects formation of infectious synapses and what role CD209 play in this setting is currently unknown.

MV Infection and DC Viability and Maturation

The efficiency of MV infection in human DCs in vitro varies between strains. In addition to their preferential uptake, wild-type strains replicate faster in DCs than attenuated strains (Schnorr et al. 1997b; Ohgimoto et al. 2001), indicating that MV proteins other than the glycoproteins (gps) favor intracellular replication in DCs. Recently, the stability of the M protein has been directly related to the ability of

MV

CD46

CD150 TLR2

CD209 (DC-SIGN)

MV entry and replicationSignaling

(interference withIL-12 or promotion of IL-10 induction?)

No entry receptors, yet enhance uptake (DC-SIGN)Endocytosis or virus capture for trans-infection?

Maturation signals (TLR2) and/or differentiation signals(TLR2/CD209)

Fig. 12.3 Interaction of MV with cell surface receptors on DCs. Immature DCs express CD46, TR2, and CD209 to high levels and upregulate CD150 upon maturation signals. As for other cell types, CD150 is the major determinant of DC infection by all MV strains, and uptake into DCs is enhanced by CD209 (which could also involve capture and concentration of MV for transinfection of T cells). CD209 and TLR2 do not serve as entry receptors but actively trigger (TLR2) or modulate (CD209) signaling pathways in DCs. In addition to their role in entry, CD150 and CD46 may also modulate signaling pathways in DCs

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attenuated rather than wild-type strains to replicate in DCs (Ohgimoto et al. 2007), and the ability of induce formation of multinucleated giant cells in mature, but not immature DCs (and concomitantly, amplification of type I IFN production) was found restricted to a CD46-adapted strain (Herschke et al. 2007). This is in contrast to earlier findings where wild-type MV (or recombinants expressing wild-type H protein) preferentially caused fusion in DCs (Ohgimoto et al. 2001). Although viral proteins efficiently accumulate, virus production is low from immature or almost absent from LPS-DCs (Fugier-Vivier et al. 1997; Servet-Delprat et al. 2000), indi-cating that, as shown for monocytes (Helin et al. 1999), their differentiation stage imposes particular constraints on MV release. Ligation of CD40 on DCs caused enhancement of MV protein production and virus release (Fugier-Vivier et al. 1997; Servet-Delprat et al. 2000), yet did not affect viral replication or syncytium forma-tion in another study (Klagge et al. 2004). Due to the accumulation of the viral gp complex on the surface of infected DCs, DC fusion with co-cultured T cells was observed (Grosjean et al. 1997). In this stud,y DC apoptosis also occurred late after infection. When cultured in the presence of a peptide inhibiting cellular fusion, DCs remained viable for at least 48 h (Schnorr et al. 1997b; Klagge et al. 2000; Dubois et al. 2001). Early after infection, MV-infected DCs strongly resemble LPS-matured cells morphologically, as determined by formation of leading edges and uropods as well as veiled protrusions (Shishkova et al. 2007; Fig. 12.2A). Indicating that rear-rangements of the actin cytoskeleton were not generally affected, integrin-depend-ent adherence and migration on fibronectin were maintained in MV-infected DCs (Shishkova et al. 2007).

Immature DCs exposed to MV rapidly mature phenotypically, as indicated by upregulation of MHC class I and II molecules, CD40 and costimulatory molecules such as CD80, CD83, and CD86 (Schnorr et al. 1997b; Klagge et al. 2000; Servet-Delprat et al. 2000; Dubois et al. 2001) and cytokine production (summarized in Fig. 12. 4 ). Infection with attenuated MV strains caused induction of IL-12p35, IL-12p40, IL-23p19, IL-10, IL-1α/β, IL-1RA, and IL-6-specific transcripts in mono-cyte-derived DCs (Servet-Delprat et al. 2000; Klagge et al. 2004). On the protein level, IL-12p70 and IL-10 were not detectable after infection with either wild-type or attenuated strains (Klagge et al. 2004). IL-10 (both mRNA and protein) was, however, produced in MV ED-infected DCs generated from CD34 + progenitor cells (Dubois et al. 2001). More recently, induction of IL-10 transcripts in imma-ture DCs exposed to wild-type MV was related to Raf-1 activation in response to CD209 ligation (Gringhuis et al. 2007). Interestingly, for other CD209 ligands analyzed in this study, this only occurred after TLR activation, and that MV was able to mediate this on its own possibly reflects its ability to ligate TLR2 as well (Bieback et al. 2002).

Mechanisms accounting for the induction of cytokine-specific transcripts, par-ticularly by attenuated MV strains that cannot activate TLR2 signaling, are only partially known. There is ample evidence that attenuated strains cause induction of type I IFNs in DCs, and recently this has been associated with accumulation of defective interfering RNAs (DI RNAs), the detection of which relied on RIG-I/MDA-5 rather than onTLR3-TICAM-1-driven activation of IRF-3 (Shingai et al.

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2007). Type I IFNs were identified as important in DC maturation in terms of upregulation of CD80 and CD86, TRAIL, and TLR3 (Klagge et al. 2000; Vidalain et al. 2000; Dubois et al. 2001; Tanabe et al. 2003) while upregulation of NKG2D ligands (UL-16-binding protein 2 and retinoic acid early transcript 1G) can also occur independently of these cytokines (Ebihara et al. 2007). Moreover, acquisition of DC cytolytic activity has also been linked to IFN induction (Vidalain et al. 2000, 2001a). However, MV wild-type strains very inefficiently induced type I IFN in PBMC and DCs from which DI RNAs could not been amplified (Naniche et al. 2000; Shingai et al. 2007).

Impact of MV on External Maturation/Stimulation Signals in DCs

External signals triggered by TLRs, cytokines, and finally CD40 are important for activation, mobilization, and induction of terminal maturation of DCs. The ability of MV-infected DCs to switch their chemokine receptor repertoire and to migrate in response to chemokines has not been addressed in detail. Impairment of chemo-tactic responses to MIP3α were reported, yet were not addressed mechanistically (Dubois et al. 2001). TLR signaling can be modulated by MV infection, as evi-denced in monocytes where MV exposure caused inhibition of LPS- or SACS-stimulated IL-12p70 production by ligation of CD46 (Karp et al. 1996). Long-term suppression of SACS-stimulated IL-12 release in PBMCs isolated from measles

MV contact /uptake orreplication

developmentand expansion

(IFN production?)

maturation

DC viability(by fusion, CD95L

induced apoptosis)

Inhibition of T cell expansionby surface contact mediated signals

Induction of T cell apoptosis,T cell depletion by transfer of virus

or cell fusion

DC function

Differentiation of Th2 cellsor regulatory T cells (eg. by IL-

10 or IFN production?)

Infection (CD150, CD46)FcγRII interactionTLR2 interaction?

upregulation of costimulatory and MHC molecules chemotaxis? T cell attractants?IL-6, IL-1α/β, IL-12low, IL-10low, IL-18 and type I IFNT cell clustering, antigen processing?

Fig. 12.4 Impact of MV on DC maturation and differentiation. MV (dependent and independent of infection) has been found to modulate DC viability, differentiation, and functions at multiple levels ranging from development from precursors and maturation signals (which may be partially incomplete) to depletion of DCs by apoptosis or fusion. DCs can also contribute to T cell loss by fusion or transmission of infectious MV (see also Fig. 12.1), but can also actively silence T cell expansion by contact-mediated signals (as mostly evidenced by loss of allostimulatory properties). Hypothetically, MV-infected DCs could also promote expansion of regulatory T cells by produc-tion of type I IFN and/or IL-10 (this and all other mechanisms indicated which are not yet experimentally proven to occur are listed in italics )

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patients supports an impaired production of this cytokine (Atabani et al. 2001). In DCs, MV interference with TLR-dependent IL-12 production in vitro is less clear. Infection of DCs isolated from peripheral blood with wild-type or attenuated strains did not interfere with release of bioactive IL-12 in response to LPS or SACS (Schnorr et al. 1997b), while inhibition of IL-12 production in MV-infected DC cultures was also reported, which correlated in a timely manner with the accumula-tion of apoptotic DCs (Fugier-Vivier et al. 1997). MV can also promote—rather than interfere with—TLR signaling, as shown by induction of IL-10 transcripts upon co-ligation of CD209 (Gringhuis et al. 2007). MV interference with signaling via TLR7/9 occurs evidently in pDCs that did not produce type I IFN upon infection, yet were also compromised to do so in response to TLR7 or TLR9 trig-gering (Schlender et al. 2005).

Modulation of CD40 signaling by MV infection is very likely to be of functional importance, but this remains controversial. Enhancement of viral replication fol-lowing CD40 ligation by activated T cells or antibodies suggested that CD40 sign-aling pathways are at least partially active. In contrast, downregulation of tyrosine-phosphorylation of cellular proteins, IL-12p40 transcripts, and IL12p70 production, induction of IL-10 transcripts, and failure of MV-infected DCs to drive proliferation of CD8 + T cells after the same stimuli suggested that CD40 signaling is blocked (Servet-Delprat et al. 2000). Enhanced production of IL-12p70 and IL-10 proteins from MV-infected DCs after CD40 ligation were also reported (Dubois et al. 2001; Klagge et al. 2004). Thus, MV-mediated modulation of CD40 signaling, its targets and consequences are far from being understood and require reevaluation with virus strain, timing of stimulation, and probably the source of DCs being the critical components.

The DC/T Cell Conjugate: From Attraction to Transmission and/or T Cell Activation

Little is known about MV modulation of DC chemotaxis on protein or functional level, e.g., the switch from CCR5 to CCR7 expression as required for tissue exit and homing and associated signaling pathways. It is also largely unknown if the chemokine profile released by MV-infected DCs (as analyzed so far only at the transcriptional level Zilliox et al. 2006) is compatible with successful recruitment of T cells (and particular subsets thereof). In vitro, DCs infected with an attenuated MV strain can recruit co-cultured T cells into conjugates and there, both fusion and, albeit to a limited extent, transmission of virus to T cells occurs (Grosjean et al. 1997) (Fig. 12.4). For wild-type strains, transmission to T cells has not been analyzed as yet. For HIV, infection of T cells is greatly enhanced if virus is pro-vided by DCs (Pope et al. 1995; Cameron et al. 1996), and concentration of virus and/or receptors by CD209 and transfer within an infectious synapse at the DC–T cell interface is believed to play an important role. Formation of an infectious syn-apse occurs independently of antigen recognition, yet relies on actin-dependent receptor clustering on the surface of the acceptor cell (Nejmeddine et al. 2005; Jolly et al. 2007). CD209-dependent trapping of MV on the DC surface or within

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endosomal compartments has not been analyzed, though there is evidence that MV proteins are present in stable DC/T cell conjugates at the interface (Shishkova et al. 2007). Receptor concentration on the conjugating T cell may be difficult to achieve since firstly, these cells, unless they are preactivated, do not express CD150, and secondly, they reveal a very limited ability to reorganize their actin cytoskeleton once they have encountered the MV gp complex (Muller et al. 2006). If following transfer impairment of T cell viability and function relies on viral replication, T cell activation would be an essential prerequisite for this to efficiently occur (Borrow and Oldstone 1995). T cell activation is also required for upregulation of FasL and sensitivity to TRAIL-dependent killing has been suggested to contribute to T cell and or DC depletion (Vidalain et al. 2000, 2001b). Thus, the ability infected DCs to promote T cell activation, which requires formation of functional immunological synapses (IS), is crucial. Interfaces formed in stable conjugates between MV-infected DCs and T cells reveal a pattern of CD3, LFA-1, or talin dis-tribution and consistent with an activatory phenotype (Shishkova et al. 2007) (Fig. 12. 5 A), which is surprising given the inability of MV-infected DCs to stimu-late expansion of allogenic T cells in mixed leukocyte reactions (Fugier-Vivier et al. 1997; Grosjean et al. 1997; Kaiserlian et al. 1997; Schnorr et al. 1997b; Steineur et al. 1998; Klagge et al. 2000; Servet-Delprat et al. 2000; Dubois et al. 2001; Klagge et al. 2004). As revealed by live cell imaging, the majority of conjugates between MV DCs and T cells is, however, highly unstable and does not promote T cell activation (Shishkova et al. 2007). Moreover, it appears unlikely that viral transmission occurs there since these conjugates dissolved within less than 2 min. Currently, it is unclear what determines synapse instability, yet it appears that the viral gp complex contributes to this phenomenon. This is because MGV-infected DCs (which express VSV G instead of the MV F/H proteins on their surface) at least partially retain their ability to recruit T cells into stable, stimulatory conju-gates (Shishkova et al. 2007) (Fig. 12.5B).

Viral Proteins Effective at T Cell Silencing and Modulation

MV infection is known to cause cell cycle arrest and to interfere with differentiation of effector functions in infected lymphocytes, including T cells. Infected PBMCs in vivo are very limited in frequency, yet these cells resist signals promoting expan-sion ex vivo, and thus mechanisms other than direct infection are evidently involved. These are most likely provided in vivo by contact with or factors released from infected cells which are of low abundance.

Ex vivo analyses do not support deficiencies in IL-2 production or a lack of IL-2R expression (Griffin and Ward 1993; Moss et al. 2002). Inhibitory soluble media-tors released from in vitro infected cells may include IL-10, yet IL-10 production in vivo has not consistently been seen (Okada et al. 2001; Moss et al. 2002). Though attenuated MV strains induce type I IFN release from PBMCs, it is still unclear whether this accumulates to significant levels in vivo. Moreover, there is no evidence for type I IFN being responsible for lymphocyte arrest after mitogenic

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stimulation in vitro, and as yet unidentified inhibitory factor(s) released from infected T cells were insensitive to IL-10 or type I IFN specific antibodies (Fujinami et al. 1998; Sun et al. 1998). In contrast, polyclonal MV-specific antibodies were effective at neutralizing the inability of MV-infected T cell cultures to proliferate in response to mitogenic stimulation (Sanchez-Lanier et al. 1988; Yanagi et al. 1992), indicating that viral proteins were directly involved.

The N Protein

The nucleocapsid (N) protein is the major constituent of the virion, and in addition to complex the viral genome and the P protein, it interacts via its C-terminus with cellular proteins such as Hsp72 and IRF-3, thereby modulating viral replication and

DCs seeded onto PLL

T cells loaded withCa-sensitive fluorophore Duration and efficiency

of conjugates

0102030405060708090

100

MOCK LPS W TF

% in

cat

egor

yI II III IV

MOC MVLPS I LPS

a

b

Fig. 12.5 A, B Conjugates formed between MV-infected DCs and T cells are mostly unstable and nonstimulatory. A Conjugates formed between Mock- (immature), LPS-, or MV-infected superan-tigen loaded DCs with allogenic T cells after 20 min were stained for the expression of f-actin ( red ) and CD3 ( green ). As evident in LPS-DC conjugates, CD3 is almost entirely recruited to the DC–T cell interface, sometimes centrally located (exemplied as LPS-II). The majority of LPS-DCs reveal, however, a multifocal distribution of CD3 (LPS-I), and this is also predominantly observed in stable MV-DC–T cell conjugates. Contact with immature DCs (Mock) barely induces CD3 relocalization. B Duration and efficiency of conjugates formed between LPS-, mock- or MV-DCs and T cells were determined by live cell imaging ( left , experimental setup; the red color in some T cells indicates changes in emission of fluo-4 upon calcium fluxing). LPS DCs mainly conjugate stably with and sustain calcium mobilization in T cells ( red bars ), while with mock DCs, conjugates are largely unstable and do not elicit calcium fluxing ( white bars ). Stable, effi-cient conjugates were rare with MV-DCs ( red bars ), which mainly were dissolved within 2 min and unable to sustain calcium mobilization (which was initially successfully induced) ( black bars ). The last contact category ( grey bar ), seen almost exclusively with MV DCs, was durable, yet not associated with efficient calcium fluxing

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IFN-induction, respectively (tenOever et al. 2002; Zhang et al. 2002). Surprisingly, soluble N protein was found to act as a ligand for Fcγ RII on B cells to cause inhibi-tion of antibody production and to reduce T cell rather than B cell responses in mice (Ravanel et al. 1997; Marie et al. 2001). As detailed in the chapter by C.I. Sellin and B. Horvat, this volume, N protein-mediated inhibition of T cells was indirect since the protein prevented IL-12 production and the generation of inflammatory reactions by modulating DC functions in a Fcγ RII-dependent manner (Marie et al. 2002, 2004). In vitro, soluble N protein induced calcium flux in thymic epithelial cells and interfered with proliferation of a variety of cell types, including activated, but not resting human primary T cells (Laine et al. 2003). While the ability of N protein to induce apoptosis relied on binding its core domain to the Fcγ RII, the ability to induce G

1 arrest was attributed to the interaction of its carboxyterminal

tail domain with an as yet unknown receptor (Laine et al. 2003, 2005). Infected cells dying from apoptosis and/or secondary necrosis such as thymic

epithelial cells are considered as a source of extracellular N protein (Laine et al. 2003, 2005). In infected cells, N protein may gain access to late endosomal com-partments, where it recruits Fcγ RII, and is co-transported to the cell surface (where receptor bound N protein could act in neighboring cells) and/or released into the extracellular medium (Marie et al. 2004). Whether there is a role of N protein for T cell silencing in vivo remains questionable. Firstly, the putative receptor for N protein is only found on T cells that are already activated (Laine et al. 2003) and thus would confine inhibitory signals to this particular population. Secondly, the C-terminal domain of N protein is the most variable within all MV proteins and is therefore used for MV strain genotype assignment. Indicating that this domain has significant functional activity, a single amino acid exchange at position 522 (N in the Edmonston and D prevailing in wild-type strains) caused loss of hsp72 binding and at the same time conferred fitness in vivo (Carsillo et al. 2006). It would there-fore be important to determine whether particularly MV wild-type strain-derived N proteins would also efficiently inhibit T cell proliferation via their C-termini. Thirdly, a recombinant MV that expressed VSV G protein instead of the authentic MV F/H gp complex failed to induce T cell arrest in vitro and in vivo, indicating that N protein may not be essential, at least for T cell silencing.

The Glycoproteins

Evidence for the ability of the MV glycoprotein (gp) complex, consisting of the hemagglutinin (H) and the proteolytically activated F

1/2 heterodimer, to signal T

cells came from a number of in vitro and in vivo experiments. In vitro, MV virions, but not those produced by a recombinant MV expressing VSV G as a surface pro-tein, prevented expansion of both T cell lines and mitogen- or anti-CD3/CD28-stimulated primary human or rodent T cells in a dose- and contact-dependent manner. In addition, T cell expansion in vitro was inhibited upon coculture with cells transfected to co-express F and H protein (both not with those expressing F or

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H protein alone), and transfer of these doubly transgenic cells into cotton rats sig-nificantly impaired stimulated T cell proliferation ex vivo (Schlender et al. 1996; Niewiesk et al. 1997; Schnorr et al. 1997a; Niewiesk 1999; Weidmann et al. 2000a; Avota et al. 2001). The requirement of the F/H gp complex as effector structure for T cell inhibition was confirmed for related viruses such as Rinderpest and PPRV (Heaney et al. 2002).

Whereas complex glycosylation and fusogenic activity of the gp complex are not required for contact-mediated T cell arrest, proteolytic processing of the F protein is (Weidmann et al. 2000a, 2000b). Exogenous cleavage of the F

0 /H complex restored

its inhibitory activity, indicating that the effector domain most likely resides within the F protein and is conformation-dependent. The H protein may strengthen the interaction of the complex with the target cell surface by binding to its receptor(s) or stabilizing the conformation of the F

1/2 protein. T cell silencing induced by viral gps

does not involve induction of apoptosis or interfere with stimulated upregulation of surface markers (including the IL-2R α chain) or cytokine release, but rather pre-vented S-phase entry of these cells (Schnorr et al. 1997a; Engelking et al. 1999; Niewiesk et al. 1999; Avota et al. 2001; Schneider-Schaulies and ter Meulen 2002).

Surface Receptors Involved in T Cell Silencing

Though contact-dependent T cell arrest requires co-expression of the H protein, transmission of this particular signal does not rely on the known MV entry recep-tors, because murine T cells, which do not express human CD46 and CD150, are sensitive to inhibition (Dorig et al. 1993; Tatsuo et al. 2000; Erlenhoefer et al. 2001), which in turn, for human T cells, is insensitive to CD46- or CD150-specific antibodies (Erlenhoefer et al. 2001). Both molecules can modulate CD3 signals on T cells upon co-ligation in vitro; however, they have co-stimulatory rather than inhibitory action (Astier et al. 2000; Zaffran et al. 2001; Sidorenko and Clark 2003), though CD150 ligation can also confer sensitivity to CD95-mediated apop-tosis in some B and T cell lines (Mikhalap et al. 2004). Most studies addressing the effects of CD46 ligation on leukocyte functions have focused on APCs rather than T cells and include modulation of production of inflammatory cytokines and enhanced sensitivity to complement-mediated lysis after downregulation from the cell surface (Schnorr et al. 1995; Karp et al. 1996; Schneider-Schaulies et al. 1996; Hirano et al. 1999; Marie et al. 2001, 2002). Since, however, firm interaction with CD46 is a property of attenuated rather than wild-type MV strains (Hsu et al. 2001; Ono et al. 2001; Yanagi et al. 2002), the relevance of these findings for immunosup-pression in vivo remains to be determined.

For immunosuppression, CD150 would be expected to be more important, not at least because its expression is crucial for cell and tissue targeting of wild-type MV. CD150 expression is, however, confined to activated T cells (Aversa et al. 1997), and this, together with its co-stimulatory properties, argues against a direct role in contact-dependent T cell silencing. Moreover, it does not interact with morbilliviral F proteins, which are likely to harbor the effector domains. Cell surface molecules

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interacting with MV include TLR2 (Bieback et al. 2002), CD209 (de Witte et al. 2006), and substance P receptor (Makhortova et al. 2007). These are, however, either not expressed on human B and T cells (CD209, TLR2) or not further analyzed in terms of their ligands (substance P receptors). Moesin was also found to enhance MV uptake (Dunster et al. 1994; Schneider-Schaulies et al. 1995), although it is not supposed to act as a MV binding partner. It may instead have an indirect role in coupling surface receptors to the actin cytoskeleton following MV interaction. This could be important for viral uptake, but also for clustering of signaling receptors or T cell plasticity. Present data support the view that an unknown receptor mediates signals induced by the gp complex at the cell surface of lymphoid cells. Notably, B and monocytic cell lines were also sensitive to gp-mediated inhibition, indicating that the receptor structure involved is not T cell-specific (Schlender et al. 1996).

Signaling Pathways Targeted by MV

The unknown receptor for gp-dependent T cell inhibition apparently locates or is recruited into detergent resistant membrane microdomains (DRMs), which appear as extended clusters, co-staining for MV H protein at binding conditions on resting T cells (Avota et al. 2004). It is very likely that signaling interfering with T cell activation is initiated there. Apparently, activation of the phosphatidyl-inositol-3- (PI3K)/Akt kinase pathway is a, if not the central target for MV interference in T cells. MV is so far the only pathogen shown to interfere with activation of this par-ticular pathway; given its important role in conveying survival and mito- if not oncogenic signals, some viruses rather activate this particular pathway (Yu and Alwine 2002; Yuan et al. 2002; Dawson et al. 2003; Cacciotti et al. 2005). In contrast, activation of PI3/Akt kinases after IL-2R or CD3/CD28 ligation was effi-ciently blocked shortly after MV exposure in vivo and in vitro (Avota et al. 2001, 2004) (Fig. 12. 6 A).

Downstream effectors of this kinase include subunits of cyclin-dependent kinases essentially involved in S-phase entry, and consequently, these were found deregulated in T cell cultures exposed to or infected with MV (Engelking et al. 1999; Naniche et al. 1999). The importance of interruption of Akt kinase activation for MV-induced T cell silencing was directly documented as transgenic expression of a catalytically membrane targeted Akt kinase largely abolished the inhibitory signal (Avota et al. 2001). The regulatory subunit of the PI3K, p85, which acts upstream Akt kinase activation, was tyrosine-phosphorylated shortly after TCR ligation in MV-exposed T cells, yet failed to redistribute to cholesterol-rich DRMs, and this correlated with a lack of TCR-stimulated degradation of Cbl-b protein (Avota et al. 2004). More recently, it has been documented that downmodulation of tonic PI3K activity (as measured under standard growth conditions of primary T cells) targets activation of cellular splice accessory proteins (both members of the STAR and the SR proteins), and causes production of an isoform of the dual phos-phatase SHIP145, SIP110 (Avota et al. 2006). This protein is translated from an alternatively spliced mRNA containing intron-derived sequences (Geier et al.

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1997). Importantly, SIP110, when expressed in Jurkat or primary T cells, was a constitutively membrane-associated, active lipid phosphatase, which continuously depleted the concentration of phosphatidyl-inositol-3,4,5-phosphates, thereby rais-ing the threshold for activation signals. Most likely also as a consequence of its ability to block PI3K activation, MV signaling interferes with activation of the gua-nosine exchange factor Vav and its downstream substrates, the small GTPases Rac and Cdc42. Consequently, cytoskeletal rearrangement in response to TCR ligation and formation of filopodia and lamellipodia is severely impaired (Muller et al. 2006). It is already prior to stimulation that MV causes an almost complete collapse of filopodia protrusions on T cells and this, together with its ability of induce activa-tion of RhoA, implies that interaction of MV with T cells induces membrane signal-ing directly (Muller et al. 2006) (Fig. 12.6B). Although it cannot be sustained, calcium mobilization or overall activity of membrane proximal tyrosine kinases after TCR ligation or contact with MV-infected DCs is unaffected within the first 2 min (Avota et al. 2004; Shishkova et al. 2007). Thus, second messengers acting in close association with the lipid bilayer might be operative in this scenario.

Fig. 12.6 A, B MV interaction with T cells interferes with activation of the PI3/Akt kinase path-way and causes cell collapse. A Activation and membrane recruitment of the PI3K in response to TCR ligation is a central target for MV gp contact-mediated signaling. As a consequence polyi-nositol-polyphosphates do not efficiently accumulate in the plasma membrane and thus, recruit-ment of pleckstrin-homology containing proteins such as the Akt kinase, but also the guanosine exchange factor Vav (not shown). The importance of this pathway is illustrated by the finding that MV contacted T cells are unable to enter the cell cycle and to rearrange their actin cytoskeleton in response to stimulation. B Resting T cells when exposed to Mock cell extracts reveal a pro-nounced front-rear polarization when seeded onto fibronectin ( left panel , Mock). Moreover, microvillar protrusions are clearly discernible by scanning EM. If exposed to MV, T cells fail to polarize on fibronectin and reveal an almost complete collapse of membrane extension ( right panel , MV)

P85 p110

PI-4,5-P2 PI-3,4,5-P3

PTEN SHIP

PI-4-P, PI-4,5-P2 PI-3,4-P2

Akt SurvivalActivation

Cell cycle entryCytoskeletal rearrangements

TCR activation

MOCK MV

a

b

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Shaping of T Cell Responses by Receptor Interactions

Apparently, expression of the viral gp complex on the surface of DCs would be a potent effector mediating contact-dependent T cell inhibition, which, can be con-sidered a physical and functional paralysis. The MV gps, in particular H proteins, could, however, also shape ensuing T cell subset differentiation by interacting with their respective receptor(s) by providing co-stimulatory signals.

When cross-linked by antibodies on T cells, CD150 was suggested to act as a co-stimulator strongly enhancing IFN-γ production and thereby a Th1 response (for review see Sidorenko and Clark 2003). Studies in CD150 −/− mice, however, failed to support a critical role of this molecule in IFN-γ production but rather indicated that CD150 may enhance TCR-stimulated IL-4 release (Wang et al. 2004). DCs infected in vitro with either wild-type or vaccine MV efficiently trigger expansion of IFN-γ-producing T cells from CD45RO − T cell populations, and this even if DCs were generated in a way that expectedly would promote expansion of IL-4-produc-ing T cells (Klagge et al. 2004). Preferential expansion of IFN-γ-producing cells in these cultures relied, however, on soluble mediators released from infected DC cultures rather from direct ligation of CD150.

It is also possible, if not likely, that particularly those DCs infected with attenu-ated strains induce expansion or differentiation of regulatory T cells (natural or adaptive, respectively). Thus, CD3/CD46 co-ligation by antibodies can induce Tr1 cell in vitro (Kemper et al. 2003, 2005), and this scenario could be envisaged for DCs expressing H proteins of CD46-adapted MV strains, although this would be limited to CD46-interacting MV strains. Moreover, incomplete or aberrant matura-tion and/or production of type I IFN and IL-10 rather than IL-12, as seen in infected DCs, could provide a favorable environment. So far, however, regulatory T cells have not been directly shown to be induced or expanded during measles or after vaccination.

Conclusions and Perspectives

Given the role of DCs to induce and shape immune responses, it is evident that interaction with these cells is central for immunomodulation by MV, although their interaction has not been extensively studied in vivo. From in vitro data, it is clear that modulation of DC viability and function is induced by MV independ-ently of or as a result of infection, with its pattern and efficiency being partially virus strain-dependent. DCs most likely serve as primary target cells during infec-tion and receive their maturation signals such as PRR signaling or other as yet unknown stimuli. MV can enter into DCs via its cognate receptors, and it is not yet known whether other modes of uptake such as endocytosis (for instance via DC-SIGN (Geijtenbeek and van Kooyk 2003b; van Kooyk and Geijtenbeek 2003) are also operative. Early after infection (or, if occurring, endocytosis), presenta-tion or of MV peptides generated by processing for subsequent MHC loading and

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presentation certainly occurs, as does cross-presentation, and this results in the efficient induction of MV-specific immune responses. Initial activation of DCs and their ability to stimulate T cells are not grossly affected early after MV encounter, since they retain phagocytic activity, reveal a normal phenotypic matu-ration pattern, and are perfectly able to stimulate allogenic T cell proliferation in vitro (Klagge et al. 2000, 2004; Shishkova et al. 2007), and, given the efficiency of induction of an MV-specific immunity, may reach the secondary lymphatic tis-sues. The extent to which loss of DC or T cells by fusion occurs needs to be determined. In lymph node material of experimentally infected rhesus macaques, giant cells were very infrequent (McChesney et al. 1997). In contrast, these Warthin-Finkeldey cells were described in hyperplastic lymphoid tissue in a mea-sles patient at autopsy, although evidence for MV there was provided only by immunohistology, but not by electron microscopy (Nozawa et al. 1994).

Incomplete T cell activation or active inhibition of T cell expansion probably mark late phases in MV DC interaction. Then these cells may transmit virus to T cells expressing CD150 (and this may also include activated CD25 + regulatory T cells (Browning et al. 2004)). Then, apoptosis, the inability to respond to external maturation signals and, in addition, accumulation of viral proteins on the cell sur-face, may collectively act in T cell silencing. For this, direct negative signaling of infected DCs to scanning T cells, by expression of MV N or gps, is an attractive hypothesis. Obviously, T cells do not die as a consequence of this inhibitory signal and, at least in vitro, recover and regain their mitogenic response. In agreement with this, recall responses that are suppressed during and after measles, also return to normal in vivo.

As regulatory T cells can control persistent infections (reviewed in Mills 2004; Schneider-Schaulies and Dittmer 2006), they also may have a role in the establish-ment and/or maintenance of the persistent MV CNS infection SSPE, where no deficiencies of effector T cell functions have been found to date. Lastly, MV can trigger postinfectious encephalitis, which is generally believed to be a virus-induced autoimmune disease in which Treg cells might have a role to play. It is certainly that this hypothesis will be experimentally addressed in the near future and that the frequency and potential activation of this particular T cell subset will be evaluated ex vivo.

Acknowledgements We thank The Deutsche Forschungsgemeinschaft and the Interdisciplinary Center for Clinical Research Würzburg for financial support of our work.

References

Addae MM, Komada Y, Zhang XL, Sakurai M (1995) Immunological unresponsiveness and apop-totic cell death of T cells in measles virus infection. Acta Paediatr Jpn 37:308–314

Addae MM, Komada Y, Taniguchi K, Kamiya T, Osei-Kwasi M et al (1998) Surface marker pat-terns of T cells and expression of interleukin-2 receptor in measles infection. Acta Paediatr Jpn 40:7–13

Arneborn P, Biberfeld G, Forsgren M, von Stedingk LV (1983) Specific and non-specific B cell activation in measles and varicella. Clin Exp Immunol 51:165–172

b12325337-0012ztc.indd 262 10/3/2008 11:08:34 AM


Arrieumerlou C, Meyer T (2005) A local coupling model and compass parameter for eukaryotic chemotaxis. Dev Cell 8:215–227

Asselin-Paturel C, Trinchieri G (2005) Production of type I interferons: plasmacytoid dendritic cells and beyond. J Exp Med 202:461–465

Astier A, Trescol-Biemont MC, Azocar O, Lamouille B, Rabourdin-Combe C (2000) Cutting edge: CD46, a new costimulatory molecule for T cells, that induces p120CBL and LAT phos-phorylation. J Immunol 164:6091–6095

Atabani SF, Byrnes AA, Jaye A, Kidd IM, Magnusen AF et al (2001) Natural measles causes pro-longed suppression of interleukin-12 production. J Infect Dis 184:1–9

Aversa G, Carballido J, Punnonen J, Chang CC, Hauser T et al (1997) SLAM and its role in T cell activation and Th cell responses. Immunol Cell Biol 75:202–205

Avota E, Avots A, Niewiesk S, Kane LP, Bommhardt U et al (2001) Disruption of Akt kinase acti-vation is important for immunosuppression induced by measles virus. Nat Med 7:725–731

Avota E, Muller N, Klett M, Schneider-Schaulies S (2004) Measles virus interacts with and alters signal transduction in T-cell lipid rafts. J Virol 78:9552–9559

Avota E, Harms H, Schneider-Schaulies S (2006) Measles virus induces expression of SIP110, a constitutively membrane clustered lipid phosphatase, which inhibits T cell proliferation. Cell Microbiol 8:1826–1839

Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392(6673):245–252

Bieback K, Lien E, Klagge IM, Avota E, Schneider-Schaulies J et al (2002) Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signaling. J Virol 76:8729–8736

Black FL, Berman LL, Borgono JM, Capper RA, Carvalho AA et al (1986) Geographic variation in infant loss of maternal measles antibody and in prevalence of rubella antibody. Am J Epidemiol 124:442–452

Borrow P, Oldstone MB (1995) Measles virus-mononuclear cell interactions. Curr Top Microbiol Immunol 191:85–100

Browning MB, Woodliff JE, Konkol MC, Pati NT, Ghosh S et al (2004) The T cell activation marker CD150 can be used to identify alloantigen-activated CD4(+)25+ regulatory T cells. Cell Immunol 227:129–139

Burns S, Hardy SJ, Buddle J, Yong KL, Jones GE et al (2004) Maturation of DC is associated with changes in motile characteristics and adherence. Cell Motil Cytoskeleton 57:118–132

Cacciotti P, Barbone D, Porta C, Altomare DA, Testa JR et al (2005) SV40-dependent AKT activ-ity drives mesothelial cell transformation after asbestos exposure. Cancer Res 65:5256–5262

Cameron P, Pope M, Granelli-Piperno A, Steinman RM (1996) Dendritic cells and the replication of HIV-1. J Leukoc Biol 59:158–171

Carsillo T, Zhang X, Vasconcelos D, Niewiesk S, Oglesbee M (2006) A single codon in the nucleocapsid protein C terminus contributes to in vitro and in vivo fitness of Edmonston mea-sles virus. J Virol 80:2904–2912

Condack C, Grivel JC, Devaux P, Margolis L, Cattaneo R (2007) Measles virus vaccine attenuation: suboptimal infection of lymphatic tissue and tropism alteration. J Infect Dis 196:541–549

Dawson CW, Tramountanis G, Eliopoulos AG, Young LS (2003) Epstein-Barr virus latent mem-brane protein 1 (LMP1) activates the phosphatidylinositol 3-kinase/Akt pathway to promote cell survival and induce actin filament remodeling. J Biol Chem 278:3694–3704

de Swart RL, Ludlow M, de Witte L, Yanagi Y, van Amerongen G et al (2007) Predominant infec-tion of CD150(+) lymphocytes and dendritic cells during measles virus infection of macaques. PLoS Pathog 3:e178

de Witte L, Abt M, Schneider-Schaulies S, van Kooyk Y, Geijtenbeek TB (2006) Measles virus targets DC-SIGN to enhance dendritic cell infection. J Virol 80:3477–3486

Dollimore N, Cutts F, Binka FN, Ross DA, Morris SS et al (1997) Measles incidence, case fatality, and delayed mortality in children with or without vitamin A supplementation in rural Ghana. Am J Epidemiol 146:646–654


b12325337-0012ztc.indd 263 10/3/2008 11:08:34 AM


Dubois B, Lamy PJ, Chemin K, Lachaux A, Kaiserlian D (2001) Measles virus exploits dendritic cells to suppress CD4+ T-cell proliferation via expression of surface viral glycoproteins inde-pendently of T-cell trans-infection. Cell Immunol 214:173–183

Dunster LM, Schneider-Schaulies J, Loffler S, Lankes W, Schwartz-Albiez R et al (1994) Moesin: a cell membrane protein linked with susceptibility to measles virus infection. Virology 198:265–274

Ebihara T, Masuda H, Akazawa T, Shingai M, Kikuta H et al (2007) Induction of NKG2D ligands on human dendritic cells by TLR ligand stimulation and RNA virus infection. Int Immunol 19:1145–1155

Engelking O, Fedorov LM, Lilischkis R, ter Meulen V, Schneider-Schaulies S (1999) Measles virus-induced immunosuppression in vitro is associated with deregulation of G1 cell cycle control proteins. J Gen Virol 80:1599–1608

Erlenhoefer C, Wurzer WJ, Loffler S, Schneider-Schaulies S, ter Meulen V et al (2001) CD150 (SLAM) is a receptor for measles virus but is not involved in viral contact-mediated prolifera-tion inhibition. J Virol 75:4499–4505

Esolen LM, Ward BJ, Moench TR, Griffin DE (1993) Infection of monocytes during measles. J Infect Dis 168:47–52

Forthal DN, Aarnaes S, Blanding J, de la Maza L, Tilles JG (1992) Degree and length of viremia in adults with measles. J Infect Dis 166:421–424

Fugier-Vivier I, Servet-Delprat C, Rivailler P, Rissoan MC, Liu YJ et al (1997) Measles virus suppresses cell-mediated immunity by interfering with the survival and functions of dendritic and T cells. J Exp Med 186:813–823

Fujinami RS, Sun X, Howell JM, Jenkin JC, Burns JB (1998) Modulation of immune system function by measles virus infection: role of soluble factor and direct infection. J Virol 72:9421–9427

Geier SJ, Algate PA, Carlberg K, Flowers D, Friedman C et al (1997) The human SHIP gene is differentially expressed in cell lineages of the bone marrow and blood. Blood 89:1876–1885

Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC et al (2000) DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 100:587–597

Geijtenbeek TB, van Kooyk Y (2003a) DC-SIGN: a novel HIV receptor on DCs that mediates HIV-1 transmission. Curr Top Microbiol Immunol 276:31–54

Geijtenbeek TB, van Kooyk Y (2003b) Pathogens target DC-SIGN to influence their fate DC-SIGN functions as a pathogen receptor with broad specificity. Apmis 111:698–714

Griffin DE (1995) Immune responses during measles virus infection. Curr Top Microbiol Immunol 191:117–134

Griffin DE, Ward BJ (1993) Differential CD4 T cell activation in measles. J Infect Dis 168:275–281

Gringhuis SI, den Dunnen J, Litjens M, van Het Hof B, van Kooyk Y et al (2007) C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB. Immunity 26:605–616

Grosjean I, Caux C, Bella C, Berger I, Wild F et al (1997) Measles virus infects human dendritic cells and blocks their allostimulatory properties for CD4+ T cells. J Exp Med 186:801–812

Hahm B, Arbour N, Naniche D, Homann D, Manchester M et al (2003) Measles virus infects and suppresses proliferation of T lymphocytes from transgenic mice bearing human signaling lymphocytic activation molecule. J Virol 77:3505–3515


Heaney J, Barrett T, Cosby SL (2002) Inhibition of in vitro leukocyte proliferation by morbillivi-ruses. J Virol 76:3579–3584

Helin E, Salmi AA, Vanharanta R, Vainionpaa (1999) Measles virus replication in cells of myelo-monocytic lineage is dependent on cellular differentiation stage. Virology 253:35–42

Herschke F, Plumet S, Duhen T, Azocar O, Druelle J et al (2007) Cell–cell fusion induced by measles virus amplifies the type I interferon response. J Virol 81:12859–12871

b12325337-0012ztc.indd 264 10/3/2008 11:08:34 AM


Hirano A, Yang Z, Katayama Y, Korte-Sarfaty J, Wong TC (1999) Human CD46 enhances nitric oxide production in mouse macrophages in response to measles virus infection in the presence of gamma interferon: dependence on the CD46 cytoplasmic domains. J Virol 73:4776–4785

Hsu EC, Iorio C, Sarangi F, Khine AA, Richardson CD (2001) CDw150 (SLAM) is a receptor for a lymphotropic strain of measles virus and may account for the immunosuppressive prop-erties of this virus. Virology 279:9–21

Hussey GD, Goddard EA, Hughes J, Ryon JJ, Kerran M et al (1996) The effect of Edmonston-Zagreb and Schwarz measles vaccines on immune response in infants. J Infect Dis 173:1320–1326

Jolly C, Mitar I, Sattentau QJ (2007) Adhesion molecule interactions facilitate human immunode-ficiency virus type 1-induced virological synapse formation between T cells. J Virol 81:13916–13921

Kaiserlian D, Grosjean I, Caux C (1997) Infection of human dendritic cells by measles virus induces immune suppression. Adv Exp Med Biol 417:421–423

Karp CL, Wysocka M, Wahl LM, Ahearn JM, Cuomo PJ et al (1996) Mechanism of suppression of cell-mediated immunity by measles virus. Science 273(5272):228–231

Katz M (1995) Clinical spectrum of measles. Curr Top Microbiol Immunol 191:1–12 Kemper C, Chan AC, Green JM, Brett KA, Murphy KM et al (2003) Activation of human CD4+

cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype. Nature 421(6921):388–392

Kemper C, Verbsky JW, Price JD, Atkinson JP (2005) T-cell stimulation and regulation: with complements from CD46. Immunol Res 32:31–44

Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C et al (2005) SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121:1109–1121

Klagge IM, ter Meulen V, Schneider-Schaulies S (2000) Measles virus-induced promotion of dendritic cell maturation by soluble mediators does not overcome the immunosuppressive activity of viral glycoproteins on the cell surface. Eur J Immunol 30:2741–2750

Klagge IM, Abt M, Fries B, Schneider-Schaulies S (2004) Impact of measles virus dendritic-cell infection on Th-cell polarization in vitro. J Gen Virol 85:3239–3247

Kobune F, Takahashi H, Terao K, Ohkawa T, Ami Y et al (1996) Nonhuman primate models of measles. Lab Anim Sci 46:315–320

Kruse M, Meinl E, Henning G, Kuhnt C, Berchtold S et al (2001) Signaling lymphocytic activa-tion molecule is expressed on mature CD83+ dendritic cells and is up-regulated by IL-1 beta. J Immunol 167:1989–1995

Laine D, Trescol-Biemont MC, Longhi S, Libeau G, Marie JC et al (2003) Measles virus (MV) nucleoprotein binds to a novel cell surface receptor distinct from FcgammaRII via its C-termi-nal domain: role in MV-induced immunosuppression. J Virol 77:11332–11346

Laine D, Bourhis JM, Longhi S, Flacher M, Cassard L et al (2005) Measles virus nucleoprotein induces cell-proliferation arrest and apoptosis through NTAIL-NR and NCORE-FcgammaRIIB1 interactions, respectively. J Gen Virol 86:1771–1784

Lennon JL, Black FL (1986) Maternally derived measles immunity in era of vaccine-protected mothers. J Pediatr 108:671–676

Makhortova NR, Askovich P, Patterson CE, Gechman LA, Gerard NP et al (2007) Neurokinin-1 enables measles virus trans-synaptic spread in neurons. Virology 362:235–244

Manchester M, Smith KA, Eto DS, Perkin HB, Torbett BE (2002) Targeting and hematopoietic suppression of human CD34+ cells by measles virus. J Virol 76:6636–6642

Marie JC, Kehren J, Trescol-Biemont MC, Evlashev A, Valentin H et al (2001) Mechanism of measles virus-induced suppression of inflammatory immune responses. Immunity 14:69–79

Marie JC, Astier AL, Rivailler P, Rabourdin-Combe C, Wild TF et al (2002) Linking innate and acquired immunity: divergent role of CD46 cytoplasmic domains in T cell induced inflamma-tion. Nat Immunol 3:659–666

Marie JC, Saltel F, Escola JM, Jurdic P, Wild TF et al (2004) Cell surface delivery of the measles virus nucleoprotein: a viral strategy to induce immunosuppression. J Virol 78:11952–11961

b12325337-0012ztc.indd 265 10/3/2008 11:08:34 AM


McChesney MB, Fujinami RS, Lerche NW, Marx PA, Oldstone MB (1989) Virus-induced immu-nosuppression: infection of peripheral blood mononuclear cells and suppression of immu-noglobulin synthesis during natural measles virus infection of rhesus monkeys. J Infect Dis 159:757–760

McChesney MB, Miller CJ, Rota PA, Zhu YD, Antipa L et al (1997) Experimental measles. I. Pathogenesis in the normal and the immunized host. Virology 233:74–84

Mikhalap SV, Shlapatska LM, Yurchenko OV, Yurchenko MY, Berdova GG et al (2004) The adaptor protein SH2D1A regulates signaling through CD150 (SLAM) in B cells. Blood 104:4063–4070

Mills KH (2004) Regulatory T cells: friend or foe in immunity to infection? Nat Rev Immunol 4:841–855

Minagawa H, Tanaka K, Ono N, Tatsuo H, Yanagi Y (2001) Induction of the measles virus recep-tor SLAM (CD150) on monocytes. J Gen Virol 82:2913–2917


Mrkic B, Pavlovic J, Rulicke T, Volpe P, Buchholz CJ et al (1998) Measles virus spread and pathogenesis in genetically modified mice. J Virol 72:7420–7427

Mrkic B, Odermatt B, Klein MA, Billeter MA, Pavlovic J et al (2000) Lymphatic dissemination and comparative pathology of recombinant measles viruses in genetically modified mice. J Virol 74:1364–1372

Muller N, Avota E, Schneider-Schaulies J, Harms H, Krohne G et al (2006) Measles virus contact with T cells impedes cytoskeletal remodeling associated with spreading, polarization, and CD3 clustering. Traffic 7:849–858

Nanan R, Chittka B, Hadam M, Kreth HW (1999) Measles virus infection causes transient deple-tion of activated T cells from peripheral circulation. J Clin Virol 12:201–210

Naniche D, Reed SI, Oldstone MB (1999) Cell cycle arrest during measles virus infection: a G0-like block leads to suppression of retinoblastoma protein expression. J Virol 73:1894–1901

Naniche D, Yeh A, Eto D, Manchester M, Friedman RM et al (2000) Evasion of host defenses by measles virus: wild-type measles virus infection interferes with induction of alpha/beta inter-feron production. J Virol 74:7478–7484

Nejmeddine M, Barnard AL, Tanaka Y, Taylor GP, Bangham CR (2005) Human T-lymphotropic virus, type 1, tax protein triggers microtubule reorientation in the virological synapse. J Biol Chem 280:29653–29660

Niewiesk S (1999) Cotton rats ( Sigmodon hispidus ): an animal model to study the pathogenesis of measles virus infection. Immunol Lett 65:47–50

Niewiesk S, Eisenhuth I, Fooks A, Clegg JC, Schnorr JJ et al (1997) Measles virus-induced immune suppression in the cotton rat ( Sigmodon hispidus ) model depends on viral glycopro-teins. J Virol 71:7214–7219

Niewiesk S, Ohnimus H, Schnorr JJ, Gotzelmann M, Schneider-Schaulies S et al (1999) Measles virus-induced immunosuppression in cotton rats is associated with cell cycle retardation in uninfected lymphocytes. J Gen Virol 80:2023–2029

Nozawa Y, Ono N, Abe M, Sakuma H, Wakasa H (1994) An immunohistochemical study of Warthin-Finkeldey cells in measles. Pathol Int 44:442–447

Ohgimoto S, Ohgimoto K, Niewiesk S, Klagge IM, Pfeuffer J et al (2001) The haemagglutinin protein is an important determinant of measles virus tropism for dendritic cells in vitro. J Gen Virol 82:1835–1844

Ohgimoto K, Ohgimoto S, Ihara T, Mizuta H, Ishido S et al (2007) Difference in production of infectious wild-type measles and vaccine viruses in monocyte-derived dendritic cells. Virus Res 123:1–8

Ohno S, Ono N, Seki F, Takeda M, Kura S et al (2007) Measles virus infection of SLAM (CD150) knockin mice reproduces tropism and immunosuppression in human infection. J Virol 81:1650–1659

Okada H, Kobune F, Sato TA, Kohama T, Takeuchi Y et al (2000) Extensive lymphopenia due to apoptosis of uninfected lymphocytes in acute measles patients. Arch Virol 145:905–920

b12325337-0012ztc.indd 266 10/3/2008 11:08:34 AM


Okada H, Sato TA, Katayama A, Higuchi K, Shichijo K et al (2001) Comparative analysis of host responses related to immunosuppression between measles patients and vaccine recipients with live attenuated measles vaccines. Arch Virol 146:859–874

Oldstone MB, Dales S, Tishon A, Lewicki H, Martin L (2005) A role for dual viral hits in causa-tion of subacute sclerosing panencephalitis. J Exp Med 202:1185–1190

Ono N, Tatsuo H, Hidaka Y, Aoki T, Minagawa H et al (2001) Measles viruses on throat swabs from measles patients use signaling lymphocytic activation molecule (CDw150) but not CD46 as a cellular receptor. J Virol 75:4399–4401

Permar SR, Moss WJ, Ryon JJ, Douek DC, Monze M et al (2003) Increased thymic output during acute measles virus infection. J Virol 77:7872–7879

Pfeuffer J, Puschel K, Meulen V, Schneider-Schaulies J, Niewiesk S (2003) Extent of measles virus spread and immune suppression differentiates between wild-type and vaccine strains in the cotton rat model ( Sigmodon hispidus ). J Virol 77:150–158

Pope M, Betjes MG, Romani N, Hirmand H, Hoffman L et al (1995) Dendritic cell–T cell conju-gates that migrate from normal human skin are an explosive site of infection for HIV-1. Adv Exp Med Biol 378:457–460

Ravanel K, Castelle C, Defrance T, Wild TF, Charron D et al (1997) Measles virus nucleocapsid protein binds to FcgammaRII and inhibits human B cell antibody production. J Exp Med 186:269–278

Rethi B, Gogolak P, Szatmari I, Veres A, Erdos E et al (2006) SLAM/SLAM interactions inhibit CD40-induced production of inflammatory cytokines in monocyte-derived dendritic cells. Blood 107:2821–2829

Ryon JJ, Moss WJ, Monze M, Griffin DE (2002) Functional and phenotypic changes in circulating lymphocytes from hospitalized Zambian children with measles. Clin Diagn Lab Immunol 9:994–1003

Sanchez-Lanier M, Guerin P, McLaren LC, Bankhurst AD (1988) Measles virus-induced suppres-sion of lymphocyte proliferation. Cell Immunol 116:367–381

Sanchez-Madrid F, del Pozo MA (1999) Leukocyte polarization in cell migration and immune interactions. EMBO J 18:501–511

Schlender J, Schnorr JJ, Spielhoffer P, Cathomen T, Cattaneo R et al (1996) Interaction of measles virus glycoproteins with the surface of uninfected peripheral blood lymphocytes induces immunosuppression in vitro. Proc Natl Acad Sci U S A 93:13194–13199

Schlender J, Hornung V, Finke S, Gunthner-Biller M, Marozin S et al (2005) Inhibition of toll-like receptor 7- and 9-mediated alpha/beta interferon production in human plasmacytoid dendritic cells by respiratory syncytial virus and measles virus. J Virol 79:5507–5515

Schneider-Schaulies J, Dunster LM, Schwartz-Albiez R, Krohne G, ter Meulen V (1995) Physical association of moesin and CD46 as a receptor complex for measles virus. J Virol 69:2248–2256

Schneider-Schaulies J, Schnorr JJ, Schlender J, Dunster LM, Schneider-Schaulies S et al (1996) Receptor (CD46) modulation and complement-mediated lysis of uninfected cells after contact with measles virus-infected cells. J Virol 70:255–263

Schneider-Schaulies J, ter Meulen V, Schneider-Schaulies S (2001) Measles virus interactions with cellular receptors: consequences for viral pathogenesis. J Neurovirol 7:391–399

Schneider-Schaulies S, Dittmer U (2006) Silencing T cells or T-cell silencing: concepts in virus-induced immunosuppression. J Gen Virol 87:1423–1438

Schneider-Schaulies S, ter Meulen V (2002) Measles virus and immunomodulation: molecular bases and perspectives. Expert Rev Mol Med 4:1–18

Schneider-Schaulies S, Kreth HW, Hofmann G, Billeter M, Ter Meulen V (1991) Expression of measles virus RNA in peripheral blood mononuclear cells of patients with measles, SSPE, and autoimmune diseases. Virology 182:703–711

Schnorr JJ, Dunster LM, Nanan R, Schneider-Schaulies J, Schneider-Schaulies S et al (1995) Measles virus-induced down-regulation of CD46 is associated with enhanced sensitivity to complement-mediated lysis of infected cells. Eur J Immunol 25:976–984

b12325337-0012ztc.indd 267 10/3/2008 11:08:34 AM


Schnorr JJ, Seufert M, Schlender J, Borst J, Johnston IC et al (1997a) Cell cycle arrest rather than apoptosis is associated with measles virus contact-mediated immunosuppression in vitro. J Gen Virol 78:3217–3226

Schnorr JJ, Xanthakos S, Keikavoussi P, Kampgen E, ter Meulen V et al (1997b) Induction of maturation of human blood dendritic cell precursors by measles virus is associated with immu-nosuppression. Proc Natl Acad Sci U S A 94:5326–5331

Servet-Delprat C, Vidalain PO, Bausinger H, Manie S, Le Deist F et al (2000) Measles virus induces abnormal differentiation of CD40 ligand-activated human dendritic cells. J Immunol 164:1753–1760

Shingai M, Inoue N, Okuno T, Okabe M, Akazawa T et al (2005) Wild-type measles virus infec-tion in human CD46/CD150-transgenic mice: CD11c-positive dendritic cells establish sys-temic viral infection. J Immunol 175:3252–3261

Shingai M, Ebihara T, Begum NA, Kato A, Honma T et al (2007) Differential type I IFN-inducing abilities of wild-type versus vaccine strains of measles virus. J Immunol 179:6123–6133

Shishkova Y, Harms H, Krohne G, Avota E, Schneider-Schaulies S (2007) Immune synapses formed with measles virus-infected dendritic cells are unstable and fail to sustain T cell activa-tion. Cell Microbiol 9:1974–1986

Shutt DC, Daniels KJ, Carolan EJ, Hill AC, Soll DR (2000) Changes in the motility, morphology, and F-actin architecture of human dendritic cells in an in vitro model of dendritic cell develop-ment. Cell Motil Cytoskeleton 46:200–221

Sidorenko SP, Clark EA (2003) The dual-function CD150 receptor subfamily: the viral attraction. Nat Immunol 4:19–24

Steineur MP, Grosjean I, Bella C, Kaiserlian D (1998) Langerhans cells are susceptible to measles virus infection and actively suppress T cell proliferation. Eur J Dermatol 8:413–420

Steinman RM (2003) The control of immunity and tolerance by dendritic cell. Pathol Biol (Paris) 51:59–60

Steinman RM, Pack M, Inaba K (1997) Dendritic cell development and maturation. Adv Exp Med Biol 417:1–6

Sun X, Burns JB, Howell JM, Fujinami RS (1998) Suppression of antigen-specific T cell prolifer-ation by measles virus infection: role of a soluble factor in suppression. Virology 246:24–33

Tamashiro VG, Perez HH, Griffin DE (1987) Prospective study of the magnitude and duration of changes in tuberculin reactivity during uncomplicated and complicated measles. Pediatr Infect Dis J 6:451–454

Tanabe M, Kurita-Taniguchi M, Takeuchi K, Takeda M, Ayata M et al (2003) Mechanism of up-regulation of human Toll-like receptor 3 secondary to infection of measles virus-attenuated strains. Biochem Biophys Res Commun 311:39–48

Tatsuo H, Ono N, Tanaka K, Yanagi Y (2000) SLAM (CDw150) is a cellular receptor for measles virus. Nature 406(6798):893–897

tenOever BR, Servant MJ, Grandvaux N, Lin R, Hiscott J (2002) Recognition of the measles virus nucleocapsid as a mechanism of IRF-3 activation. J Virol 76:3659–3669

Valentin H, Azocar O, Horvat B, Williems R, Garrone R et al (1999) Measles virus infection induces terminal differentiation of human thymic epithelial cells. J Virol 73:2212–2221

Valsamakis A, Auwaerter PG, Rima BK, Kaneshima H, Griffin DE (1999) Altered virulence of vaccine strains of measles virus after prolonged replication in human tissue. J Virol 73:8791–8797

van Kooyk Y, Geijtenbeek TB (2003) DC-SIGN: escape mechanism for pathogens. Nat Rev Immunol 3:697–709

Vidalain PO, Azocar O, Lamouille B, Astier A, Rabourdin-Combe C et al (2000) Measles virus induces functional TRAIL production by human dendritic cells. J Virol 74:556–559

Vidalain PO, Azocar O, Rabourdin-Combe C, Servet-Delprat C (2001a) Measle virus-infected dendritic cells develop immunosuppressive and cytotoxic activities. Immunobiology 204:629–638

Vidalain PO, Azocar O, Yagita H, Rabourdin-Combe C, Servet-Delprat C (2001b) Cytotoxic activity of human dendritic cells is differentially regulated by double-stranded RNA and CD40 ligand. J Immunol 167:3765–3772

b12325337-0012ztc.indd 268 10/3/2008 11:08:35 AM


Wang N, Satoskar A, Faubion W, Howie D, Okamoto S et al (2004) The cell surface receptor SLAM controls T cell and macrophage functions. J Exp Med 199:1255–1264

Ward BJ, Griffin DE (1993) Changes in cytokine production after measles virus vaccination: pre-dominant production of IL-4 suggests induction of a Th2 response. Clin Immunol Immunopathol 67:171–177

Weidmann A, Fischer C, Ohgimoto S, Ruth C, ter Meulen V et al (2000a) Measles virus-induced immunosuppression in vitro is independent of complex glycosylation of viral glycoproteins and of hemifusion. J Virol 74:7548–7553

Weidmann A, Maisner A, Garten W, Seufert M, ter Meulen V et al (2000b) Proteolytic cleavage of the fusion protein but not membrane fusion is required for measles virus-induced immuno-suppression in vitro. J Virol 74:1985–1993

Welstead GG, Iorio C, Draker R, Bayani J, Squire J et al (2005) Measles virus replication in lym-phatic cells and organs of CD150 (SLAM) transgenic mice. Proc Natl Acad Sci U S A 102:16415–16420

Wilson NS, Villadangos JA (2005) Regulation of antigen presentation and cross-presentation in the dendritic cell network: facts, hypothesis, and immunological implications. Adv Immunol 86:241–305

Yanagi Y, Cubitt BA, Oldstone MB (1992) Measles virus inhibits mitogen-induced T cell prolif-eration but does not directly perturb the T cell activation process inside the cell. Virology 187:280–289

Yanagi Y, Ono N, Tatsuo H, Hashimoto K, Minagawa H (2002) Measles virus receptor SLAM (CD150). Virology 299:155–161

Yu Y, Alwine JC (2002) Human cytomegalovirus major immediate-early proteins and simian virus 40 large T antigen can inhibit apoptosis through activation of the phosphatidylinositide 3′-OH kinase pathway and the cellular kinase Akt. J Virol 76:3731–3738

Yuan H, Veldman T, Rundell K, Schlegel R (2002) Simian virus 40 small tumor antigen activates AKT and telomerase and induces anchorage-independent growth of human epithelial cells. J Virol 76:10685–10691

Zaffran Y, Destaing O, Roux A, Ory S, Nheu T et al (2001) CD46/CD3 costimulation induces morphological changes of human T cells and activation of Vav, Rac, and extracellular signal-regulated kinase mitogen-activated protein kinase. J Immunol 167:6780–6785

Zhang X, Glendening C, Linke H, Parks CL, Brooks C et al (2002) Identification and characteriza-tion of a regulatory domain on the carboxyl terminus of the measles virus nucleocapsid pro-tein. J Virol 76:8737–8746


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Chapter 13 Hostile Communication of Measles Virus with Host Innate Immunity and Dendritic Cells

B. Hahm

Abstract Following measles virus (MV) infection, host innate immune responses promptly operate to purge the virus. Detection of alerting measles viral components or replication intermediates by pattern-recognizing host machinery of Toll-like receptors and RNA helicases triggers signaling to synthesize array of anti-viral and immunoregulatory molecules, including type I interferon (IFN). Diverse subtypes of dendritic cells (DCs) play pivotal roles in both host innate immunity on the pri-mary MV-infected site and initiating adaptive immune responses on secondary lymphoid tissues. Responding to the predictable host immune responses, MV appears to have devised multiple strategies to evade, suppress, or even utilize host innate immunity and DC responses. This review focuses on versatile actions of MV-induced type I IFNs causing beneficial or deleterious influence on host immu-nity and the interplay between MV and heterogeneous DCs at distinct locations.

B. Hahm Departments of Surgery and Molecular Microbiology and Immunology, Center for Cellular and Molecular Immunology , University of Missouri-Columbia School of Medicine , One Hospital Dr., Columbia , MO 65212 , USA , e-mail: [email protected]

Contents

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272Host Innate Immunity Following Measles Virus Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

Measles Virus Regulation of Type I IFN-Mediated Innate Immunity . . . . . . . . . . . . . . . . . 273Type I IFN as an Immunoregulator During Measles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

TLR System Targeted by Measles Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277TLR Signaling Pathway Influenced by Measles Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277Measles Viral Regulation of TLR-Mediated Cytokine Synthesis . . . . . . . . . . . . . . . . . . . . 279

Interplay Between Measles Virus and Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280Measles Virus Suppression of Dendritic Cell Development . . . . . . . . . . . . . . . . . . . . . . . . 280Measles Virus-Induced Dendritic Cell Maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281Measles Virus Interaction with Dendritic Cells at Lymphoid Organs . . . . . . . . . . . . . . . . . 281Modulation of Dendritic Cell Subtypes by Measles Virus . . . . . . . . . . . . . . . . . . . . . . . . . 282

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283


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272 B. Hahm

Abbreviations

MV Measles virus IFN Interferon DC Dendritic cell PDC Plasmacytoid dendritic cell iDC Immature dendritic cell TLR Toll-like receptor NK Natural killer ISGF3 IFN-stimulated gene factor 3 ISRE IFN-stimulated response element CTL Cytotoxic T lymphocyte BM Bone marrow HSC Hematopoietic stem cell GM-CSF Granulocyte macrophage colony-stimulating factor Flt3-L FMS-like tyrosine kinase 3 ligand ADAR Adenosine deaminase acting on RNA

Introduction

Host immunity has evolved along with continuous pathogenic invasions. During the last decade, great scientific interests have arisen in the field of host innate immu-nity, especially of type I interferon (IFN) synthesis and toll-like receptor (TLR) signaling, as well as diverse dendritic cell (DC) responses. DCs not only regulate host innate immune response, but also control initiation of host adaptive immunity by stimulating virus-specific naïve T lymphocytes. Upon measles virus (MV) infec-tion, the host immune system is activated to eventually clear the virus. However, MV in return eludes or counterattacks the first line of host defense, the critical innate immune system of the type I IFN network and T cell stimulatory capacity of DCs as well. Consequently, MV could induce profound suppression of host immu-nity, predisposing MV-infected individuals to other microbial pathogens. Recent studies on MV immunobiology yielded interesting and astonishing results and helped us better understand how host immunity has evolved to combat pathogenic MV and more importantly how immunosuppressive MV contends with protective host innate immune response and DCs.

Host Innate Immunity Following Measles Virus Infection

MV is transmitted via aerosol and thus thought to initially infect cells located in respiratory tissues, including epithelial cells and mucosal DCs. On the primary sites infected by MV, multiple host innate immune responses are swiftly operated by

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13 Hostile Communication of Measles Virus 273

cellular machineries like macrophages, DCs, and natural killer (NK) cells. Pulmonary macrophages have phagocytotic activity engulfing viruses and apoptotic bodies to destroy them into pieces. Viral components released via cell lysis could act as specific signatures, recognizable by cellular sensors, to induce inflammatory cytokines and chemokines and type I IFN amplification. Type I IFNs secreted from infected cells activate NK cells. Subsequently, NK cells release type II interferon (IFN), IFN-γ to directly kill virus-infected cells. Although severe lymphopenia accompanies MV infection with a significant decrease in the number of T and B lymphocytes, the quantity of NK cells did not seem to decrease (Okada et al. 2001). MV was reported to induce expression of a NKG2D-ligand, UL16-binding proteins 2 on infected monocyte-derived human DCs, resulting in enhanced secretion of IFN-γ from NK cells (Ebihara et al. 2007). However, MV could infect and disturb the activity of DCs to regulate host innate and adaptive immunity.

Measles Virus Regulation of Type I IFN-Mediated Innate Immunity

Following MV infection, immunoregulatory proteins are synthesized from innate immune cells to induce antiviral status. The best-known soluble antiviral protein is type I IFN (Vilcek 2006), which is a multi-gene cytokine family consisting of six classes in humans: IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, and IFN-ν. Among them, IFN-α has diverse subtypes yielding over 12 separate IFN-α subtype pro-teins. IFN-α and IFN-β have been extensively characterized for their antiviral activities in numerous systems, ever since they were discovered by Isaacs and Lindenmann in 1957 (Isaacs and Lindenmann 1957). Type I IFN is induced through the recognition of viral components or replication intermediates by cellu-lar sensors such as TLRs and the family of DExD/H box RNA helicases of retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA-5) (Fig. 13. 1 A) (Takeda and Akira 2004; Thompson and Locarnini 2007). Binding of type I IFNs to cognate receptors on the cell surface triggers JAK/STAT signaling pathway leading to a formation of IFN-stimulated gene factor 3 (ISGF3) complex comprised of STAT1, STAT2, and IRF9 (Fig. 13.1B) (Aaronson and Horvath 2002; Darnell et al. 1994). Subsequently, the ISGF3 complex translocates into the nucleus and activates the IFN-stimulated response element (ISRE)-mediated gene transcription, resulting in synthesis of numerous proteins to establish the antiviral state (Grandvaux et al. 2002).

MV was reported to inhibit cellular signaling pathways for both type I IFN induction and JAK/STAT signaling (Fig. 13.1A, B). Wild-type MV was shown to induce type I IFN to a far lesser extent than vaccine strains of attenuated MV (Naniche et al. 2000). MV maintained on epithelial cells attained the ability to gen-erate an elevated level of defective interfering RNA, which is an intense stimulator of type I IFN, through the cell culture adaptation, thereby inducing large quantities of type I IFN (Shingai et al. 2007). In addition, MV C protein appeared to inhibit

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Fig. 13.1 A–D MV induces and regulates type I IFN responses. (A) MV infection is detected by cellular sensors of RNA helicases RIG-I/MDA-5 or TLRs to induce type I IFNs via IRF3 or IRF7 activation. (B) Secreted type I IFNs bind to cognate receptors IFNAR1 and 2 complex activating tyrosine kinases JAK1/Tyk2. ISGF3 complex consisting of STAT1, STAT2, and IRF9 recognizes ISRE to activate transcription for ISGs leading to antiviral state. However, MV could inhibit sig-naling for both type I IFN induction (A) and JAK/STAT-mediated ISG induction (B) . (C) Type I IFNs were reported to increase protective host immunity by activating NK cells to produce IFN-γ, and enhancing CTL response and DC terminal maturation. It is rarely investigated if and how MV regulates these IFN-mediated defensive host immunity, although MV-induced type I IFNs were shown to facilitate MV-induced DC maturation. (D) MV-induced type I IFNs could suppress the host immune system by disrupting lymphopoiesis of T lymphocytes in thymus and inhibiting DC development. Blockade of GM-CSF or Flt3-L-mediated DC development occurs via MV-induced type I IFN-mediated unique STAT2-specific signaling

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the production of type I IFNs, as C gene-deficient MV induced a higher level of IFNs (Nakatsu et al. 2006). Nevertheless, regardless of the MV strains, infection of humans by MV should induce synthesis of type I IFN through viral components, including 5'-triphosphate-ended viral leader transcript (Plumet et al. 2007) gener-ated in infected cells (Berghall et al. 2006; Helin et al. 2001). Also, it is likely that viral products released from cells destroyed by MV-induced apoptosis or cellular phagocytosis stimulate type I IFN synthesis from neighboring cells. Further, cell-to-cell fusion caused by MV drastically enhanced synthesis of type I IFNs (Herschke et al. 2007).

As a second line of strategy to elude type I IFN-mediated host defense, MV instructs its own viral proteins to directly inhibit JAK/STAT signaling of type I IFN. Thus, even in the presence of type I IFN, MV sought a way to multiply and spread. The role of MV V, C, and P proteins in suppressing type I IFN signaling is reported (Caignard et al. 2007; Devaux et al. 2007; Palosaari et al. 2003; Takeuchi et al. 2003).

Whether MV regulates antiviral activity of specific type I IFN-induced proteins as a third level of strategy for MV evasion or suppression of the type I IFN system remains elusive. Interestingly, numerous IFN-stimulated genes (ISGs) such as 2'5' oligoadenylate synthetase and Mx protein were significantly upregulated on MV-infected DCs, whereas a well-characterized antiviral gene, double-stranded RNA-dependent protein kinase (PKR) was not induced on human DCs by MV infection (Zilliox et al. 2006). Among IFN-stimulated proteins, double-stranded RNA bind-ing protein, Adenosine Deaminase Acting on RNA (ADAR) 1a is thought to dis-play a critical activity during MV persistence (Bass et al. 1989; Cattaneo et al. 1988; Patterson et al. 2001). MV genome isolated from subacute sclerosing panen-cephalitis (SSPE) patients featured hypermutation of A to G and U to C in the matrix gene. The mutation is postulated to be mediated by ADAR 1a enzymatic activity. Also, ADAR1a was recently reported to bind and inhibit the kinase activity of PKR and consequently enhance host susceptibility to vesicular stomatitis virus (VSV) infection (Nie et al. 2007). The role of IFN-induced ADAR1a and PKR in MV persistence and pathogenesis requires further investigation.

Type I IFN as an Immunoregulator During Measles

Blockade of virus spread by type I IFN appears to be mediated by antiproliferative or apoptotic activity concerted by IFN-induced proteins. Apoptosis of virus-infected cells or disruption of cellular proliferation before virus amplification leads to the antiviral state of neighboring cells. However, type I IFN was recently reported to exhibit pro-proliferative or anti-apoptotic actions, demonstrating the complexity of the IFN network (Gimeno et al. 2005; Tanabe et al. 2005; Yang et al. 2001). Furthermore, diverse subtypes of type I IFNs seem to retain distinctly different physiological functions (Cull et al. 2003), representing myriad IFN systems. How the complex IFN system influences host immune responses during measles is poorly understood. As a potent immunomodulator, type I IFN activates NK cells,

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enhances the expansion of cytotoxic T lymphocytes (CTLs), and prolongs the sur-vival of stimulated T cells (Fig. 13.1C) (Biron 2001; Brinkmann et al. 1993). Uniquely, type I IFN regulates DCs in opposite paradoxical directions at two dis-parate stages of DCs. Type I IFN stimulates terminal maturation of committed DCs (Luft et al. 1998), which is important for stimulation of T cells. Indeed, concomi-tant treatment of anti-IFN antibody to MV-infected DCs, at least in part, suppressed MV-induced DC maturation consistent with the type I IFN’s capacity to stimulate DC maturation (Dubois et al. 2001). In contrast, MV-induced type I IFN severely suppressed development of DCs from bone marrow (BM) hematopoietic stem cells (HSCs) (Fig. 13.1D) (Hahm et al. 2005). The experiment was conducted by using transgenic mice bearing human SLAM receptor on DCs. MV-induced inhibition of DC generation via type I IFNs occurred in DC culture systems in vitro or ex vivo using either a granulocyte macrophage colony-stimulating factor (GM-CSF) or a fms-like tyrosine kinase 3 ligand (Flt3-L), which are decisive cytokines for DC development. Dual contradictory activities of MV-induced type I IFN on two DC stages were recapitulated using recombinant IFN-β. For instance, simultaneous administration of recombinant IFN-β into mice with Flt3-L blocked Flt3-L-medi-ated DC expansion in vivo. However, terminal maturation of committed DCs was enhanced by recombinant IFN-β treatment. Intriguingly, suppression of DC devel-opment caused by MV-induced type I IFN required the expression of STAT2, but not of STAT1, STAT4, or STAT6. This florid STAT1-independent but STAT2-spe-cific type I IFN signaling resulting in DC inhibition was also reproduced in vitro and in vivo using either recombinant IFN-β or an immunosuppressive strain of lymphocytic choriomeningitis virus (LCMV) Cl 13. Currently, it is unclear why the host immune system is engineered to have type I IFN display contradictory activi-ties on two DC stages. It is conceivable that MV utilizes the immunosuppressive function of type I IFN to inhibit DC development to survive insidiously in the host. Inhibition of DC replenishment following MV infection could enhance susceptibil-ity of the MV-infected individual to secondary microbial invasion.

Type I IFN was initially regarded as a magic bullet to purge viruses and cure multiple diseases, and was thought to be devoid of deleterious function. This ideal-istic concept was challenged by scientific and medical observations:

1. Neutralizing antibodies against type I IFN inhibited liver cell necrosis and growth retardation, reducing the mortality of suckling mice caused by LCMV infection (Riviere et al. 1977). The data indicates that virus-induced IFN could contribute to the exacerbation of disease and death.

2. A significantly elevated level of type I IFN (IFN-α) was detected in the sera of patients with multiple autoimmune diseases such as systemic lupus erythemato-sus (SLE) and insulin-dependent diabetes mellitus (IDDM) (Theofilopoulos et al. 2005). The therapeutic approach with type I IFN was reported to cause or exacerbate the autoimmune disorders of SLE, thyroiditis, arthritis, and IDDM.

3. Direct injection of experimental mice with type I IFN could cause growth inhibition, glomerulonephritis later in life, and death. Administration of IFN to humans also induces harmful effects such as fever, fatigue, myalgia, and anemia (Vilcek 2006).

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4. Highly virulent influenza viruses such as the reconstructed 1918 pandemic influ-enza virus induce a cytokine storm, which includes hyperactivation of the type I IFN system (Kash et al. 2006; Kobasa et al. 2007), suggesting possible roles of type I IFN in pathogenic immune response, i.e., viral immunopathology.

It is possible that MV-induced type I IFNs exhibit harmful immune responses in certain locations, e.g., intense suppression of DC development at the site of DC commitment. Also, MV caused terminal differentiation and apoptosis of thymic epithelial cells (Auwaerter et al. 1996) via MV-induced type I IFNs, which could lead to disruption of T cell lymphopoiesis in the thymus (Fig. 13.1D) (Vidalain et al. 2002). The data were confirmed by the direct application of IFNs. While the ordinary host immune response is turned off effectively after its purpose is accom-plished, perhaps via a host regulatory circuit mechanism, excessive stimulation of the immune system at specific locations might be too strong to be controlled and could consequently turn into a detrimental and pathogenic response. It is also plau-sible that MV is clever enough to utilize the pathogenic function of host immunity by causing a more hostile environment.

TLR System Targeted by Measles Virus

TLR Signaling Pathway Influenced by Measles Virus

TLRs belong to pattern-recognizing receptors detecting molecular signatures with specificity (Bowie and Haga 2005; Reis e Sousa 2004). Triggering the TLR system activates molecules for two main pathways of (1) type I IFN induction and (2) syn-thesis of inflammatory cytokines. While TBK-1/IKK-e-induced IRF-7/IRF-3 acti-vation is crucial for TLR-mediated type I IFN induction (Honda et al. 2005), IRF-5 activation of NF-κB and AP-1 signaling operates the production of inflammatory cytokines, including IL-12, IL-6, and tumor necrosis factor (TNF)-α (Takaoka et al. 2005). It remains unclear how MV induces or regulates TLR-mediated expression of inflammatory cytokines and chemokines. Stimulation of immature DCs with ligands for TLR2 and/or TLR4 significantly increased MV amplification in DCs, which directly correlates with upregulation of SLAM, the MV receptor (Murabayashi et al. 2002). Wild-type MV hemagglutinin (H) protein was reported to activate TLR2, leading to induction of proinflammatory cytokines such as IL-6 and enhancement of surface expression level of SLAM (Fig. 13. 2 A) (Bieback et al. 2002). The increase in its own receptor expression via TLR2 attachment of MV might be an effective viral immunoregulatory strategy for the spread of the virus (Bieback et al., 2002). Laboratory-adapted MV upregulated TLR3 expression via type I IFN regulation, which could contribute to host innate immunity (Berghall et al. 2006; Tanabe et al. 2003). However, MV displayed inhibitory activity on type I IFN induction signaling mediated by TLR7 or TLR9 on plasmacytoid DCs (PDCs) (Schlender et al. 2005).

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Fig. 13.2 Schematic model for the interplay between MV and diverse DCs. MV infects DCs via cellular receptor SLAM or CD46. Attachment proteins of TLR2 and DC-SIGN are known to interact with MV to influence DC phenotype or function. MV HA binding to TLR2 enhances the level of SLAM receptor on DCs [A] . MV inhibits TLR4-mediated IL-12 synthesis, which could enhance permissiveness of individuals to the infection by secondary microbe Y activating TLR4 [B] . Cross-talk of DCs with other immune cells such as NK cells affects host immunity to MV infection [C] . MV-infected DCs could express inflammatory cytokines including type I IFNs, TNF-α, IL-6, and IL-10 on the infected site. Such cytokines and chemokines may display crucial roles in either protective host immunity or MV-induced immunosuppression [D] . MV interferes with GM-CSF or Flt3-L-dependent DC development from BM HSCs via type I IFN-mediated STAT2-specific signaling. Inhibition of DC amplification and replenishment contributes to the suppression of host immunity [E] . MV infection or attachment to DCs leads to phenotypic matu-ration of DCs [F] , which may cause migration of MV-infected DCs to secondary lymphoid tissues such as mediastinal lymph node [G] . MV-infected DCs could prevent T cells from proliferating via diverse mechanisms, including direct contact of MV glycoproteins to T cells, inhibition of

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Measles Viral Regulation of TLR-Mediated Cytokine Synthesis

Among inflammatory cytokines induced by the TLR system, IL-12 is a crucial cytokine for eliciting cellular immunity by skewing cytokine responses toward Th1 phenotype, whereas IL-10 displays immune suppressive activity and one of the core Th2 type cytokines. During measles, Th2 form cytokine response prevailed, con-tributing to the suppression of cellular immunity (Moss et al. 2002); these phenom-ena could increase host susceptibility to other microbial infections. Indeed, prolonged detection of upregulated IL-10 was observed in the plasma of measles patients for several weeks following measles-specific rash (Moss et al. 2002). MV was reported to stimulate the Raf-1-acetylation-dependent pathway to enhance IL-10 production with or without TLR4 ligation (Gringhuis et al. 2007). The role of IL-10 in measles pathogenesis and mechanism for viral regulation of this immuno-suppressive cytokine remains to be determined.

In multiple experimental settings, IL-12 inhibition was observed in human mono-cytes (Karp et al. 1996) and DCs (Servet-Delprat et al. 2000b) and murine DCs (Hahm et al. 2007; Marie et al. 2001) when they were infected with MV followed by stimulation of TLR4 (Fig. 13.2B) or CD40 ligand. In contrast, TLR4-mediated IL-12 synthesis was not inhibited on epithelial cells infected by MV, suggesting the exist-ence of cell type specificity (Indoh et al. 2007). On the virus side, MV nucleoprotein was shown to cause IL-12 inhibition (Marie et al. 2001). Also, the presence of human SLAM or CD46 on murine cells facilitated MV-induced IL-12 inhibition, indicating that HA interaction with CD46 or SLAM facilitates IL-12 interference (Hahm et al. 2007; Marie et al. 2001). However, whether the receptors for MV simply acted as a door for efficient MV entrance or MV HA interaction with the receptor affected spe-cific CD46 or SLAM signaling cascade to influence IL-12 synthesis remains unde-termined. It is likely that MV HA binding to the receptors in part mediates IL-12 suppression, as this inhibition does not necessitate direct MV infection of DCs (Hahm et al. 2007; Karp et al. 1996). Notably, SLAM was reported to affect TLR4 signaling and subsequent cytokine secretion, including IL-12 in an animal model of SLAM-deficient mice (Wang et al. 2004), providing evidence for the involvement of SLAM signaling in TLR-mediated cytokine generation. MV P protein upregulated ubiquitin-modifying enzyme A20, which is a host negative feedback regulator of NF-κB in infected monocytic cells to suppress NF-κB-mediated TLR4 signaling (Yokota et al., 2008). However, MV V and C proteins were not responsible for TLR4-mediated IL-12 inhibition, as recombinant MV deficient in V or C gene dis-played potent IL-12 inhibitory activity, as did wild-type MV (Hahm et al. 2007).

Fig. 13.2 (Continued) CD40–CD40L signaling, or induction of apoptosis of DCs and T cells by FAS or TRAIL synthesized in DCs [H] . In contrast, certain DCs stimulated by viral components may display ordinary DC activity to stimulate virus-specific T cells leading to eventual clearance of MV. Transfer of antigenic stimuli from migrated DCs to lymph node-resident DCs could be blocked by MV regulation of infected DCs, but not studied yet [I]

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280 B. Hahm

On the other hand, MV appears to inhibit IL-12 specifically upon TLR4 stimula-tion, as other cytokines of IL-6 and TNF-α were not diminished on monocytes and DCs upon TLR4 stimulation (Hahm et al. 2007; Karp et al. 1996). Additionally, IL-12 inhibition was barely observed when MV-infected murine DCs expressing SLAM were treated with other TLR ligand such as peptidoglycan (PGN, TLR2L), the synthetic analog of double-stranded RNA poly(I:C) (TLR3L), unmethylated CpG DNA (CpG, TLR9L) or guanosine analog loxoribine (TLR7L) (Hahm et al. 2007). The data indicate that MV targets the signaling of TLR4-mediated IL-12 synthesis selectively. MV suppressed production of IL-12 after TLR4 engagement in DCs generated by two different DC culture systems utilizing either Flt3-L or GM-CSF. Moreover, MV-induced IL-12 inhibition via TLR4 activation is domi-nantly intensive. Indeed, TLR9-mediated IL-12 synthesis in MV-infected DCs was suppressed by simultaneous treatment with TLR4 agonist LPS. Thus, MV could specifically target TLR4 signaling to inhibit IL-12 expression even in the presence of other molecular signatures stimulating diverse types of TLR signaling.

Interplay Between Measles Virus and Dendritic Cells

DCs are ideally scattered throughout the entire body as a cellular detector of patho-gens invading the host. With multiple subtypes at different locations, DCs have diverse activities, including type I IFN synthesis, cross-talk with various immune cells (Fig. 13.2C), inflammatory response (Fig. 13.2D), antigen presentation to stimulate naïve T cells, and immune tolerance. Investigation on the MV–DC inter-action revealed that MV could modulate multiple functions of DCs.

Measles Virus Suppression of Dendritic Cell Development

DCs are initially thought to be nondividing end-stage cells (half-life 1.5–2.9 days) (Kamath et al. 2002). However, recent studies revealed that DCs could undergo a limited number of cell divisions over a 10- to 14-day period in peripheral lymphoid organs in situ and replenished from blood-borne precursors (Liu et al. 2007). Synthesized GM-CSF or Flt3-L following virus infection in tissues should increase the number of DCs for effective host immunity. However, MV was shown to sup-press GM-CSF or Flt3-L-dependent DC amplification, which is a strategy of immunosuppression employed by MV infection (Fig. 13.2E). As described above, MV-induced type I IFN triggers STAT2-specific signaling to suppress DC develop-ment. In addition, MV was reported to inhibit hematopoiesis of HSCs indirectly by infecting BM stromal cells (Manchester et al. 2002), which provides an explana-tion of the long-term immunosuppression seen in measles patients. MV might dis-turb regeneration of certain types of immune cells, including DCs, leading to long-term suppression of host immune system.

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Measles Virus-Induced Dendritic Cell Maturation

DCs are developed from BM HSCs and exist as an immature form in the periphery. MV-infected immature DCs (iDC) might mature phenotypically, which could facil-itate migration of MV-infected DCs to lymphoid tissues. MV-induced phenotypic maturation of iDCs was judged by an increased level of DC marker proteins, including co-stimulatory molecules CD80, CD86, and CD40 (Fig. 13.2F) (Klagge et al. 2000; Schnorr et al. 1997). It is unclear whether MV-induced DC maturation simply represents prompt host immune response or is a strategy designed by the virus for its effective spread. It is conceivable that MV wants to quickly move toward lymphoid tissues (Fig. 13.2G), where major cellular receptor SLAM is widely expressed, before intense host innate immunity occurs on initial target tis-sues. The idea is supported by reports demonstrating that MV interacts with DC-SIGN (de Witte et al. 2006) or TLR2, which activates the cells by enhancing the synthesis of inflammatory cytokines and SLAM receptor (Bieback et al. 2002) (Fig. 13.2A). Efficient transport of HIV bound to DC-SIGN from the infected place to the lymphoid tissues, where viral target CD4 + T cells reside, was well docu-mented (Geijtenbeek et al. 2000; Kwon et al. 2002). It is also possible that matured DCs via the attachment of MV to TLR2 or DC-SIGN express an increased amount of SLAM receptor, as SLAM is highly expressed upon maturation, and thereby become more permissive to MV infection. How does MV infection cause pheno-typic DC maturation? MV-induced type I IFNs appear to play a role in enhancing phenotypic maturation of iDCs, as described earlier. However, type I IFN-deficient DCs from mice bearing human SLAM receptor on DCs reached a similar level of maturation to the wild-type DCs expressing SLAM when infected by MV (Hahm et al., unpublished data), suggesting that type I IFN is not an essential component for the phenotypic DC maturation induced by MV.

Measles Virus Interaction with Dendritic Cells at Lymphoid Organs

Upon maturation, DCs migrate to secondary lymphoid tissues such as mediastinal lymph node, a major site for naïve T cell stimulation upon pulmonary infection, trans-mitting the danger signal to T cells and inducing adaptive immunity. We do not clearly understand how MV, following respiratory infection, induces and regulates DC responses in the lung as well as LN in vivo. Another respiratory pathogen of influenza virus was reported to activate DCs in the lung and certain subtype, but not all, of these mature myeloid DCs migrate efficiently to MLN not only to directly induce virus-specific T cells (Fig. 13.2H), but also to stimulate lymph node-resident CD8α + DCs (Fig. 13.2I) (Belz et al. 2004). A murine model study revealed that when MV-infected BM-derived DCs were adoptively transferred, the virus was found in secondary lym-phoid organs, suggesting that DCs could disseminate MV into secondary lymphoid

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tissues (Shingai et al. 2005). Studies with proper animal models for MV infection should further clarify host DC responses to MV infection at distinct locations.

MV-infected DCs fail to induce expansion of T cells (Fig. 13.2H) (Dubois et al. 2001; Fugier-Vivier et al. 1997; Grosjean et al. 1997; Hahm et al. 2004; Schnorr et al. 1997). Moreover, DCs infected by MV could actively inhibit uninfected T cell proliferation by cell-to-cell contact or TRAIL-mediated T cell apoptosis. Indeed, replication-incompetent UV-inactivated MV or MV glycoproteins expressed on DCs inhibited proliferation of uninfected T cells upon antigenic or mitogenic stim-ulation (Dubois et al. 2001; Klagge et al. 2000; Schlender et al. 1996). Co-culture of MV-infected DCs with T cells induced Fas-mediated DC apoptosis with enhanced viral replication (Servet-Delprat et al. 2000a). MV interfered with CD40-ligand-dependent differentiation of DCs upon cross-talk with T cells (Servet-Delprat et al. 2000b). Consequently, blockade of CD40-to-CD40L interaction could perturb DC-mediated signal transmission to T cells. Mouse genetics studies showed that individual expression of human SLAM MV receptor, type I IFN receptor, TNF-α, lymphotoxin (LT)-α or LT-β from T cells was not required for MV-infected DCs to inhibit the proliferation of T cells (Hahm et al. 2004). Additionally, antibodies against IL-10 or type I IFN could not block MV-infected human DC-mediated T cell suppression (Dubois et al. 2001).

Modulation of Dendritic Cell Subtypes by Measles Virus

PDCs were identified as a major cell type producing type I IFNs. MV was reported to retain the capability of inhibiting TLR-dependent or -independent type I IFN productions from PDCs (Schlender et al. 2005). Yet, there are multiple questions that remain unanswered regarding the protective role of PDCs in host innate immu-nity to MV, expression of MV receptors on PDCs, and the mechanism of PDC regu-lation by MV. On the other hand, several reports showed that type I IFNs are released from MV-infected conventional myeloid DCs including, monocyte-derived DCs, although controversy exists.

Accumulated data indicates that CD8α + draining lymph node-resident lymphoid DCs are the most efficient in priming naïve CD8 + T cells (Allan et al. 2003) upon pulmonary infection of mouse with influenza virus (Belz et al. 2004). Currently, no data are available to address the specific interaction mode of lymphoid DCs in MLNs with MV. Most research on the interplay between MV and human DCs was conducted using myeloid DCs, perhaps due to the availability of DCs derived from human blood in the GM-CSF-dependent culture system and limited sources of human DCs isolated from tissues. Recent development of animal models might help to advance the studies on MV-DC subtype interplay. Interestingly, MV sup-pressed the upregulation of co-stimulatory molecules and MHC proteins on splenic DCs from mice expressing human SLAM receptor (Hahm et al. 2004), whereas the virus enhanced the phenotypic maturation of DCs derived from the BM of the mice. The data suggest that MV may regulate DC maturation differentially depending on the locations and subtypes of DCs.

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Conclusions

Because it is highly contagious and immunosuppressive, MV continues to cause outbreaks and threaten humans. Recent studies demonstrate that MV modulates host immune signaling pathways involving DC activation by targeting specific host molecules. Surprisingly, research on measles virus immunobiology uncovered novel mechanisms for host immunity–virus interaction, which were recurrently observed in a situation of another pathogenic invasion. Identification of both gen-eralized and MV-specific immune signaling mechanisms would advance our under-standing of relationship between viruses and the host immune system. Consequently, mechanistic studies to reveal how MV regulates host innate immunity and DC responses should help us develop novel therapeutics for the treatment of diseases caused by immunosuppressive viruses like MV.

Acknowledgements I thank the editors for giving me the opportunity to contribute to the CTMI book on measles virus. I greatly appreciate Drs. Sung Key Jang (Pohang University of Science and Technology) and Michael B.A. Oldstone (The Scripps Research Institute) for their excellent sup-port and mentorship. My current measles virus research is supported by the Departments of Surgery and Molecular Microbiology and Immunology and Center for Cellular and Molecular Immunology at University of Missouri-Columbia School of Medicine.

References

Aaronson DS, Horvath CM (2002) A road map for those who don’t know JAK-STAT. Science 296:1653–1655

Allan RS, Smith CM, Belz GT, van Lint AL, Wakim LM, Heath WR, Carbone FR (2003) Epidermal viral immunity induced by CD8alpha+ dendritic cells but not by Langerhans cells. Science 301:1925–1928

Auwaerter PG, Kaneshima H, McCune JM, Wiegand G, Griffin DE (1996) Measles virus infec-tion of thymic epithelium in the SCID-hu mouse leads to thymocyte apoptosis. J Virol 70:3734–3740

Bass BL, Weintraub H, Cattaneo R, Billeter MA (1989) Biased hypermutation of viral RNA genomes could be due to unwinding/modification of double-stranded RNA. Cell 56:331

Belz GT, Smith CM, Kleinert L, Reading P, Brooks A, Shortman K, Carbone FR, Heath WR (2004) Distinct migrating and nonmigrating dendritic cell populations are involved in MHC class I-restricted antigen presentation after lung infection with virus. Proc Natl Acad Sci U S A 101:8670–8675

Berghall H, Siren J, Sarkar D, Julkunen I, Fisher PB, Vainionpaa R, Matikainen S (2006) The interferon-inducible RNA helicase, mda-5, is involved in measles virus-induced expression of antiviral cytokines. Microbes Infect 8:2138–2144

Bieback K, Lien E, Klagge IM, Avota E, Schneider-Schaulies J, Duprex WP, Wagner H, Kirschning CJ, Ter Meulen V, Schneider-Schaulies S (2002) Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signaling. J Virol 76:8729–8736

Biron CA (2001) Interferons alpha and beta as immune regulators—a new look. Immunity 14:661–664

Bowie AG, Haga IR (2005) The role of Toll-like receptors in the host response to viruses. Mol Immunol 42:859–867

Brinkmann V, Geiger T, Alkan S, Heusser CH (1993) Interferon alpha increases the frequency of interferon gamma-producing human CD4+ T cells. J Exp Med 178:1655–1663

b12325337-0013ztc.indd 283 10/3/2008 11:10:37 AM

284 B. Hahm

Caignard G, Guerbois M, Labernardiere JL, Jacob Y, Jones LM, Wild F, Tangy F, Vidalain PO (2007) Measles virus V protein blocks Jak1-mediated phosphorylation of STAT1 to escape IFN-alpha/beta signaling. Virology 368:351–362


Cull VS, Tilbrook PA, Bartlett EJ, Brekalo NL, James CM (2003) Type I interferon differential therapy for erythroleukemia: specificity of STAT activation. Blood 101:2727–2735

Darnell JE Jr, Kerr IM, Stark GR (1994) Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415–1421

de Witte L, Abt M, Schneider-Schaulies S, van Kooyk Y, Geijtenbeek TB (2006) Measles virus targets DC-SIGN to enhance dendritic cell infection. J Virol 80:3477–3486


Dubois B, Lamy PJ, Chemin K, Lachaux A, Kaiserlian D (2001) Measles virus exploits dendritic cells to suppress CD4+ T-cell proliferation via expression of surface viral glycoproteins inde-pendently of T-cell trans-infection. Cell Immunol 214:173–183

Ebihara T, Masuda H, Akazawa T, Shingai M, Kikuta H, Ariga T, Matsumoto M, Seya T (2007) Induction of NKG2D ligands on human dendritic cells by TLR ligand stimulation and RNA virus infection. Int Immunol 19:1145–1155

Fugier-Vivier I, Servet-Delprat C, Rivailler P, Rissoan MC, Liu YJ, Rabourdin-Combe C (1997) Measles virus suppresses cell-mediated immunity by interfering with the survival and func-tions of dendritic and T cells. J Exp Med 186:813–823

Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, Middel J, Cornelissen IL, Nottet HS, KewalRamani VN, Littman DR, Figdor CG, van Kooyk Y (2000) DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 100:587–597

Gimeno R, Lee CK, Schindler C, Levy DE (2005) Stat1 and Stat2 but not Stat3 arbitrate contradic-tory growth signals elicited by alpha/beta interferon in T lymphocytes. Mol Cell Biol 25:5456–5465

Grandvaux N, tenOever BR, Servant MJ, Hiscott J (2002) The interferon antiviral response: from viral invasion to evasion. Curr Opin Infect Dis 15:259–267

Gringhuis SI, den Dunnen J, Litjens M, van Het Hof B, van Kooyk Y, Geijtenbeek TB (2007) C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB. Immunity 26:605–616

Grosjean I, Caux C, Bella C, Berger I, Wild F, Banchereau J, Kaiserlian D (1997) Measles virus infects human dendritic cells and blocks their allostimulatory properties for CD4+ T cells. J Exp Med 186:801–812


Hahm B, Trifilo MJ, Zuniga EI, Oldstone MB (2005) Viruses evade the immune system through type I interferon-mediated STAT2-dependent, but STAT1-independent, signaling. Immunity 22:247–257


Helin E, Vainionpaa R, Hyypia T, Julkunen I, Matikainen S (2001) Measles virus activates NF-kappa B and STAT transcription factors and production of IFN-alpha/beta and IL-6 in the human lung epithelial cell line A549. Virology 290:1–10

Herschke F, Plumet S, Duhen T, Azocar O, Druelle J, Laine D, Wild TF, Rabourdin-Combe C, Gerlier D, Valentin H (2007) Cell-cell fusion induced by measles virus amplifies the type I interferon response. J Virol 81:12859–12871

b12325337-0013ztc.indd 284 10/3/2008 11:10:37 AM


Honda K, Yanai H, Negishi H, Asagiri M, Sato M, Mizutani T, Shimada N, Ohba Y, Takaoka A, Yoshida N, Taniguchi T (2005) IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 434:772–777

Indoh T, Yokota S, Okabayashi T, Yokosawa N, Fujii N (2007) Suppression of NF-kappaB and AP-1 activation in monocytic cells persistently infected with measles virus. Virology 361:294–303

Isaacs A, Lindenmann J (1957) Virus interference. I. The interferon. By A. Isaacs and J. Lindenmann, J Interferon Res 7:429–438

Kamath AT, Henri S, Battye F, Tough DF, Shortman K (2002) Developmental kinetics and lifespan of dendritic cells in mouse lymphoid organs. Blood 100:1734–1741


Kash JC, Tumpey TM, Proll SC, Carter V, Perwitasari O, Thomas MJ, Basler CF, Palese P, Taubenberger JK, Garcia-Sastre A, Swayne DE, Katze MG (2006) Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus. Nature 443:578–581

Klagge IM, ter Meulen V, Schneider-Schaulies S (2000) Measles virus-induced promotion of dendritic cell maturation by soluble mediators does not overcome the immunosuppressive activity of viral glycoproteins on the cell surface. Eur J Immunol 30:2741–2750

Kobasa D, Jones SM, Shinya K, Kash JC, Copps J, Ebihara H, Hatta Y, Kim JH, Halfmann P, Hatta M, Feldmann F, Alimonti JB, Fernando L, Li Y, Katze MG, Feldmann H, Kawaoka Y (2007) Aberrant innate immune response in lethal infection of macaques with the 1918 influ-enza virus. Nature 445:319–323

Kwon DS, Gregorio G, Bitton N, Hendrickson WA, Littman DR (2002) DC-SIGN-mediated internalization of HIV is required for trans-enhancement of T cell infection. Immunity 16:135–144

Liu K, Waskow C, Liu X, Yao K, Hoh J, Nussenzweig M (2007) Origin of dendritic cells in peripheral lymphoid organs of mice. Nat Immunol 8:578–583

Luft T, Pang KC, Thomas E, Hertzog P, Hart DN, Trapani J, Cebon J (1998) Type I IFNs enhance the terminal differentiation of dendritic cells. J Immunol 161:1947–1953

Manchester M, Smith KA, Eto DS, Perkin HB, Torbett BE (2002) Targeting and hematopoietic suppression of human CD34+ cells by measles virus. J Virol 76:6636–6642

Marie JC, Kehren J, Trescol-Biemont MC, Evlashev A, Valentin H, Walzer T, Tedone R, Loveland B, Nicolas JF, Rabourdin-Combe C, Horvat B (2001) Mechanism of measles virus-induced suppression of inflammatory immune responses. Immunity 14:69–79


Murabayashi N, Kurita-Taniguchi M, Ayata M, Matsumoto M, Ogura H, Seya T (2002) Susceptibility of human dendritic cells (DCs) to measles virus (MV) depends on their activa-tion stages in conjunction with the level of CDw150: role of Toll stimulators in DC maturation and MV amplification. Microbes Infect 4:785–794

Nakatsu Y, Takeda M, Ohno S, Koga R, Yanagi Y (2006) Translational inhibition and increased interferon induction in cells infected with C protein-deficient measles virus. J Virol 80:11861–11867

Naniche D, Yeh A, Eto D, Manchester M, Friedman RM, Oldstone MB (2000) Evasion of host defenses by measles virus: wild-type measles virus infection interferes with induction of alpha/beta interferon production. J Virol 74:7478–7484

Nie Y, Hammond GL, Yang JH (2007) Double-stranded RNA deaminase ADAR1 increases host susceptibility to virus infection. J Virol 81:917–923

Okada H, Sato TA, Katayama A, Higuchi K, Shichijo K, Tsuchiya T, Takayama N, Takeuchi Y, Abe T, Okabe N, Tashiro M (2001) Comparative analysis of host responses related to immu-nosuppression between measles patients and vaccine recipients with live attenuated measles vaccines. Arch Virol 146:859–874

b12325337-0013ztc.indd 285 10/3/2008 11:10:37 AM

286 B. Hahm


Patterson JB, Cornu TI, Redwine J, Dales S, Lewicki H, Holz A, Thomas D, Billeter MA, Oldstone MB (2001) Evidence that the hypermutated M protein of a subacute sclerosing panencephalitis measles virus actively contributes to the chronic progressive CNS disease. Virology 291:215–225

Plumet S, Herschke F, Bourhis JM, Valentin H, Longhi S, Gerlier D (2007) Cytosolic 5'-triphos-phate ended viral leader transcript of measles virus as activator of the RIG I-mediated inter-feron response. PLoS ONE 2:e279

Reis e Sousa C (2004) Toll-like receptors and dendritic cells: for whom the bug tolls. Semin Immunol 16:27–34

Riviere Y, Gresser I, Guillon JC, Tovey MG (1977) Inhibition by anti-interferon serum of lym-phocytic choriomeningitis virus disease in suckling mice. Proc Natl Acad Sci U S A 74:2135–2139

Schlender J, Schnorr JJ, Spielhoffer P, Cathomen T, Cattaneo R, Billeter MA, ter Meulen V, Schneider-Schaulies S (1996) Interaction of measles virus glycoproteins with the surface of uninfected peripheral blood lymphocytes induces immunosuppression in vitro. Proc Natl Acad Sci U S A 93:13194–13199

Schlender J, Hornung V, Finke S, Gunthner-Biller M, Marozin S, Brzozka K, Moghim S, Endres S, Hartmann G, Conzelmann KK (2005) Inhibition of toll-like receptor 7- and 9-mediated alpha/beta interferon production in human plasmacytoid dendritic cells by respiratory syncy-tial virus and measles virus. J Virol 79:5507–5515

Schnorr JJ, Xanthakos S, Keikavoussi P, Kampgen E, ter Meulen V, Schneider-Schaulies S (1997) Induction of maturation of human blood dendritic cell precursors by measles virus is associ-ated with immunosuppression. Proc Natl Acad Sci U S A 94:5326–5331

Servet-Delprat C, Vidalain PO, Azocar O, Le Deist F, Fischer A, Rabourdin-Combe C (2000a) Consequences of Fas-mediated human dendritic cell apoptosis induced by measles virus. J Virol 74:4387–4393

Servet-Delprat C, Vidalain PO, Bausinger H, Manie S, Le Deist F, Azocar O, Hanau D, Fischer A, Rabourdin-Combe C (2000b) Measles virus induces abnormal differentiation of CD40 lig-and-activated human dendritic cells. J Immunol 164:1753–1760

Shingai M, Inoue N, Okuno T, Okabe M, Akazawa T, Miyamoto Y, Ayata M, Honda K, Kurita-Taniguchi M, Matsumoto M, Ogura H, Taniguchi T, Seya T (2005) Wild-type measles virus infection in human CD46/CD150-transgenic mice: CD11c-positive dendritic cells establish systemic viral infection. J Immunol 175:3252–3261

Shingai M, Ebihara T, Begum NA, Kato A, Honma T, Matsumoto K, Saito H, Ogura H, Matsumoto M, Seya T (2007) Differential type I IFN-inducing abilities of wild-type versus vaccine strains of measles virus. J Immunol 179:6123–6133

Takaoka A, Yanai H, Kondo S, Duncan G, Negishi H, Mizutani T, Kano S, Honda K, Ohba Y, Mak TW, Taniguchi T (2005) Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors. Nature 434:243–249

Takeda K, Akira S (2004) Microbial recognition by Toll-like receptors. J Dermatol Sci 34:73–82 Takeuchi K, Kadota SI, Takeda M, Miyajima N, Nagata K (2003) Measles virus V protein blocks

interferon (IFN)-alpha/beta but not IFN-gamma signaling by inhibiting STAT1 and STAT2 phosphorylation. FEBS Lett 545:177–182

Tanabe M, Kurita-Taniguchi M, Takeuchi K, Takeda M, Ayata M, Ogura H, Matsumoto M, Seya T (2003) Mechanism of up-regulation of human Toll-like receptor 3 secondary to infection of measles virus-attenuated strains. Biochem Biophys Res Commun 311:39–48

Tanabe Y, Nishibori T, Su L, Arduini RM, Baker DP, David M (2005) Cutting edge: role of STAT1, STAT3, and STAT5 in IFN-alpha beta responses in T lymphocytes. J Immunol 174:609–613

Theofilopoulos AN, Baccala R, Beutler B, Kono DH (2005) Type I interferons (alpha/beta) in immunity and autoimmunity. Annu Rev Immunol 23:307–336

b12325337-0013ztc.indd 286 10/3/2008 11:10:37 AM


Thompson AJ, Locarnini SA (2007) Toll-like receptors, RIG-I-like RNA helicases and the antivi-ral innate immune response. Immunol Cell Biol 85:435–445

Vidalain PO, Laine D, Zaffran Y, Azocar O, Servet-Delprat C, Wild TF, Rabourdin-Combe C, Valentin H (2002) Interferons mediate terminal differentiation of human cortical thymic epi-thelial cells. J Virol 76:6415–6424

Vilcek J (2006) Fifty years of interferon research: aiming at a moving target. Immunity 25:343–348

Wang N, Satoskar A, Faubion W, Howie D, Okamoto S, Feske S, Gullo C, Clarke K, Sosa MR, Sharpe AH, Terhorst C (2004) The cell surface receptor SLAM controls T cell and macro-phage functions. J Exp Med 199:1255–1264

Yang CH, Murti A, Pfeffer SR, Kim JG, Donner DB, Pfeffer LM (2001) Interferon alpha /beta promotes cell survival by activating nuclear factor kappa B through phosphatidylinositol 3-kinase and Akt. J Biol Chem 276:13756–13761


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289

AActin cytoskeleton, 255Activation-induced death, 247ADAR. See Adenosine deaminase

acting on RNAADAR1a knockout mice, 50Adenosine deaminase acting on

RNA, 40, 42, 43, 49, 50, 275Adenovirus, 114Adjuvants, 201Adverse outcomes, 192Aerosol, 57, 60, 61Aerosol administration, 197Age

for measles vaccination, 197of vaccination, 196

Alpha/beta interferon receptor, 118Alphavirus, 201Alphavirus replicon particle

vaccines, 203Altered basolateral targeting

signals, 94Alternate routes of delivery, 197Alu1 hypermutated M genes, 42Anergy, 244Animal model(s), 112, 199Antibody, 156, 193, 200

modulation, 43responses, 198

Antibody to F, 195Antibody to H, 195Antibody to MV, 42, 43, 46Antivirals, 90, 91, 99, 100A to G (U to C) base changes, 49Attenuation, 123Atypical measles, 57–58, 67, 156, 194, 195,

200Autism, 183Avidity, 198

BB95a cells, 61Basic reproductive number R, 181Biased hypermutation, 34, 37–42,

45, 50Bioterrorism, 185Bouteille, 33

CCationic lipids, 201Cbl-b protein, 259CD46, 34, 35, 41, 43, 49, 113CD209 (DC-SIGN), 250CD 150 or SLAM, 119CD40 signaling, 254CD4+ T cells, 118CD8+ T cells, 118, 194Cell cycle retardation, 99Cell targeting, 245Cellular immune response

acute measles, 157IFN-γ, 157IL-12, 157T-helper type 1, 157T-helper type 2, 157vaccination, 157

Cellular immune responses, 196, 200Cellular splice accessory

proteins, 259Challenged, 58, 60Chemokines, 253Cholesterol-rich DRMs, 259CNS complications

MIBE, 8, 9PIE, 7, 8, 13SSPE, 7–9, 12–14, 20, 22

CNS disease, 34, 37–39, 44, 47Collapse, 260Complement fi xing (CF) antibodies, 194

Index

CTMI_330_Index.indd 289 10/6/2008 9:44:58 AM

290 Index

Contact-dependent T cell arrest, 258Contact-dependent T cell silencing, 258Cotton rat(s), 89–105, 199C-type lectin, 250Cynomolgus macaques, 61–63Cytokine

IL-10, 279IL-12, 279–280

Cytoplasmic inclusion bodies in neurons, 33Cytoskeletal rearrangement, 260

DDahlem Conference on Disease

Eradication, 177DC-SIGN, 281DC/T cell conjugate, 254Defi ciencies of cellular immunity, 197Dendritic cell (DC)

development, 276, 277, 280maturation, 276, 281, 282Plasmacytoid DC (PDC), 277, 282

Dendritic cells, 121, 243, 246, 247, 249, 250Differentiation of regulatory T cells, 261Disease elimination, measles, 130DNA vaccines, 200, 201Dose, 198dsRNA adenosine deaminase, 49DTH model, 97Dual-Hit Hypothesis, 44–46Dynein, 13, 15, 17, 19

EEdmonston B virus, 195Effector structure, 258EGFP. See Enhanced green

fl uorescent proteinEndemic transmission, measles, 137, 141Enhanced disease, 57, 58Enhanced green fl uorescent protein

(EGFP), 61, 63–67EPI, 176Epitopes, 200Expanded Programme on

Immunization, 174Extracellular virus, 7, 20

FFcγ receptor, 115Fcγ RII, 257Follicular dendritic cells, 250Formalin-inactivated measles vaccine, 192F Protein, 193Francis Payne, 33Fusion, 5, 16, 17, 20, 21

GGenetic polymorphisms, 159

CD46, 158HLA, 158IL-2, 158IL-10, 158IL-12b, 158SLAM, 158

Global Immunization Vision and Stratergy (GIVS), 178

Glutamate receptor editing, 40, 49, 50Goldberger and Anderson, 32, 41GTPases Rac and Cdc42, 260

HHeat shock protein 72, 95Hemagglutination inhibiting

(HI) antibodies, 194Herpesvirus, 6, 114H glycoprotein, 193High-titer vaccines, 198HIV, 184, 198HIV-1, 197HIV-infected infants, 197Horta-Barbosa, 33H protein, 194, 202Human tonsil slices, 245Humoral immunity, 155

affi nity-maturation, 156avidity, 156IgG3, 156isotype selection, 156

Hydroxymethylglutaryl coenzyme A reductase, 114

IIFN-γ, 118IFN induction, 253IL-10, 252IL-12, 115Immature DCs, 248Immune activation, 154Immune complex, 195Immune suppression, 90, 96, 97, 99Immunity, 56, 59, 60, 63, 67Immunocompromised hosts

children, 161giant-cell pneumonia, 161HIV, 161

Immunopathological, 67Immunosuppression, 6, 10, 11, 41, 44,

45, 61, 63, 64, 67, 115, 154, 244, 245

Inactivated MV vaccines, 194


Index 291

Infant immunitymaternal antibody, 160vaccination, 160vulnerability, 160

Infants, 199Infectious synapses, 251, 254Infi ltration of B and T cells, CNS, 44Infl ammation, 115Innate immune response, 154Innate immunity, 272, 273, 277,

281, 282Interferon-γ, 47Iscoms, 202Isolation, 192

JJohn Enders, 32

KKinesin, 13, 17, 18

LLck promoter, 119LCMV Cl 13, 44–46Live-attenuated vaccines, 57, 59Longevity, 159Louis Pasteur, 32Low-avidity antibody, 195Lymphopenia, 243–247

MMacaques, 6, 12, 200Maternal antibody, 90, 101–105,

193, 196, 199, 200Matrix protein M, 119Mature DCs, 249Measles, 154Measles and Rubella Laboratory

Network (LabNet), 177Measles immunity

CTL, 159vaccination, 159

Measles Initiative, 174Measles rash, 157Measles-specifi c antibody

hemagglutinin, 155hemagglutinin-inhibition, 155IgM, 155neutralization, 155plaque-reduction neutralizing, 155waning, 160

Measles virus, 32–50genome structure, 131genotypes, 131–133, 136, 137, 140, 144

molecular biology, 132molecular epidemiology, 129–144proteins, 131, 136subacute sclerosing panencephalitis,

132, 136–137transmission pathways, 131, 134,

135, 143, 144vaccination programs, 130, 136,

141, 142, 144Mediastinal lymph node (MLN), 281M gene, 34, 37–42, 44–46, 49Mice, 202Microtubules, 13, 17–19Mixed leukocyte reactions, 255MMR (measles, mumps, and rubella), 196Moesin, 259Monkeys, 56, 57, 59Moraten, 195Morbillization, 192More attenuated vaccines, 195Morphological changes associated with DC

maturation, 248Mortality, 192, 193, 198MV antibody-induced antigen modulation, 43MV Glycoproteins, 94, 98MV-infected DCs, 252MV postinfection encephalitis, 33MV receptor, 34, 41, 44MV receptor CD46, 34MV replication, 6, 11, 12, 14, 18, 20–22MV spread, 9, 12, 14–17, 19–22

NNeurokinin, 20, 21Neurokinin 1, 116Neurons, 33–41, 43–47, 49, 50Neuron-specifi c promoter, 115Neutralization, 156Neutralizing antibody, 193, 196, 199–201New-generation vaccines, 57, 59–60NF-κB, 277, 279NK cells, 273, 275Non-human primates, 56, 59, 199N protein, 257NSE-CD46, 35Nucleocapsids, 33, 37, 38, 43, 45

PPan American Health Organization

(PAHO), 176Pasteur-Chamberland fi lters, 32Pathogenesis, 57, 58, 61, 63–65, 67, 112Pathogen recognition receptors, 248Pathology, 56, 64


292 Index

Phenotypic maturation, 262Phosphatidyl-inositol-3-(PI3K)/Akt

kinase pathway, 259PKR. See Protein kinasePlasmacytoid DCs, 249Pneumonitis, 194Polio, 182–184Polioviruses, 181, 185Polymicrobial disease, 90Population immunity, 198Primary neurons, 10, 12, 20Primates, 195Progressive infection, 198Protection, 60, 61, 63, 64Protective immunity, 192, 198, 199Protein kinase, 275

RRash, 194Receptors, 34, 35, 40, 41, 43, 44, 49, 50Recombinant viruses, 119Reverse genetics, 3, 14, 38, 41Rhesus macaques, 58, 62, 63, 199, 201, 202Robert Koch, 32Rodent-adapted MV, 11, 20

SSchwarz, 195Secondary vaccine failure, 198Second dose, 199Seroconversion, 196SHIP145, 259SIA, 180, 183Sigmodon hispidus, 90Silencing, 243, 247, 255, 257–259, 262SLAM, 35

as receptor, 195Smallpox, 181, 186Smallpox virus, 178S-phase entry, 259STAT-1, 121STAT-2, 121Stem cells, 246Subacute sclerosing encephalitis, 116Subacute sclerosing panencephalitis

(SSPE), 31–50Supplementary immunization

activities, 176Supplementary immunization

campaigns, 199Suppression of primary and secondary

humoral and cell-mediated immune responses, 36

Surveillance, 177Synapse instability, 255

TT cell epitopes, 194T cells, 243, 245–247, 249, 251–262Thymocytes, 246TLR (toll-like receptor)

TLR2, 277, 281TLR4, 277, 279, 280

Toll-like receptor 2, 250Toll-like receptors (TLRs), 121Transgenic animals, 113Transgenic mice, 199Transgenic mouse, 3, 10, 11, 19Transgenic (tg) mouse

model, 34, 46Transinfection, 251Two-dose strategy, 198Type I IFN (interferon)

antiviral activity, 275deleterious function, 276ISRE, 273STAT, 273, 275, 276, 280

VVaccine(s), 91, 95, 96, 99, 100,

105, 192Vaccinia virus, 202Viremia, 200Vitamin A, 176von Pirquet, 41V protein, 94

WWindow of vulnerability, 192World Health Assembly, 178World Health Organization

African Region, 130, 139alternative sampling techniques, 143Eastern Mediterranean region, 130Laboratory manual, 130–131Measles and Rubella Laboratory

Network, 130measles nomenclature, 130–132recommended protocols, 132Region of the Americas, 130, 134Southeast Asian Region, 130Western Pacifi c Region, 130

YYAC-CD46, 34–40, 42, 45–50Yeast artifi cial chromosome, 117


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