flavivirus vaccination - globe network · there are currently >200,000 cases and 30,000 death...

Flavivirus Vaccination

Fondation Mérieux Conference Centre‘Les Pensières’

Veyrier-Du-Lac, France

December 6-8, 2010 Steering Committee:

• Farshad GUIRAKHOO

• Franz HEINZ

• Joachim HOMBACH

• Jacques LOUIS

• John ROEHRIG

Coordination: Valentina PICOT

Welcome letter

December 6, 2010

Dear Participant,

It is our pleasure to welcome you to the symposium entitled:

‘Flavivirus Vaccination’

in Fondation Mérieux’s Conference Centre “Les Pensières.” We hope you will enjoy this meeting, which brings together some of the world’s foremost experts.

The format of the meeting is intended to generate discussion and interaction among participants and to foster the dissemination of new information on this topic. The conference will provide an opportunity for specialists to exchange their knowledge and experience through collaboration with researchers from around the world. Over the next three days, the team at Les Pensières will be on hand to help you with any questions you may have and to make your stay and conference as comfortable and valuable as possible.

Yours sincerely,

Benoît MiribelDirector GeneralFondation Mérieux

For more information: www.fondation-merieux.org

Background and objectives

Flaviviruses comprise a family of more than 70 single-stranded RNA viruses, the majority of which are transmitted to their hosts by vectors such as mosquitoes or ticks.

Flavivirions are spherical and composed of a lipid membrane surrounding a capsid (C) protein bound to a viral RNA (positive)-sense, which together form the nucleocapsid core. The surface proteins are the glycosylated envelope (E) and membrane (M) proteins. The M protein is a mature form of the pre-membrane (prM) protein, important for infectivity and pathogenicity.The E protein is responsible for viral attachment to cellular receptors and specific membrane fusion. The E protein is the major target for virus-neutralizing and hemagglutination-inhibiting antibodies.

A single serotype has been identified for Japanese encephalitis (JE) yellow fever (YF), and tickborne encephalitis (TBE) virus, whereas 4 serotypes exist for dengue virus, complicating its vaccine development. The prototype flavivirus is YF virus, which was first isolated in 1927 from “Asibi”, a resident of Ghana. Despite the effective vaccines available against YF, JE, and TBE since many years, the numbers of human cases have increased in the last 2 decades, mainly due to changes in climate conditions and increased travel and urbanization. These diseases have become a major public health problem in a number of countries once again. There are currently >200,000 cases and 30,000 death for YF, and it is believed that JE is responsible for more than 50,000 cases of encephalitis annually, with at least 10,000 deaths. Some 2.5 billion people – two fifths of the world’s population – are now at risk for dengue, and despite 50 years of efforts, there is no dengue vaccine available on the market. WHO currently estimates that there may be 50 million dengue infections worldwide every year. In 2007 alone, there were more than 890,000 reported cases of dengue in the Americas, of which 26,000 cases were diagnosed as Dengue Hemorrhagic Fever (DHF). There are almost 100 asymptomatic infections for every reported flavivirus case, and the case fatality rates vary from 1 to 30% depending on the infecting flavivirus.

The pathogenesis of flaviviruses is not completely understood. The first steps of infection involve the interaction of the virus with the host cells. The virus transmitted to the human body by mosquito or tick bite proliferates locally and in regional lymph nodes, and enters the blood vessels causing short-lived viremia lasting 5 to 15 days. The infection may be asymptomatic or spread to visceral organs or the central nervous system, triggering innate and adaptive immune responses by the host with the outcome of recovery, neurological sequelea, or death, depending on the virus species and host factors which affect susceptibility to the infections.

There is no specific treatment for flavivirus infections and the viruses cannot be eradicated given their animal reservoir. Hence, vaccination is the most effective approach to disease control.

Currently, there are effective vaccines against TBE, JE and YF available for travellers who will spend time in endemic or epidemic regions, as well as the population residing in these areas. These vaccines have been shown to be successful in combating the disease and reducing the number of human cases around the world.

Farshad Guirakhoo, Ph.D.

The meeting’s objectives:

1. To focus on epidemiology, antigenic and molecular structure, as well as on pathogenesis, and host immune responses against flaviviruses;

2. To review the safety and effectiveness of current and future vaccines such as TBE, JE, YF and dengue.

Scientific Programme

Monday 6 december 2010

Tuesday 7 december 2010

Session 1 Infection with flaviviruses: disease burden, epidemiology08.30 - 11.00 Chaired by John Roehrig

17.30 - 18.30 Registration

18.30 - 18.45 Welcome Address Christophe LONGUET

18.45 - 19.30Keynote lecture: Perspective in deve-lopment of vaccines againstFlaviviruses

Thomas MONATH

19.45 Welcome Dinner

08.30 - 08.50 Epidemiology and disease burden of infection with dengue viruses Duane GUBLER

08.50 - 09.10 Discussion

09.10 - 09.30Epidemiology and disease burden of infections with encephalitic flavi-viruses

John ROEHRIG


09.50 - 10.20 Coffee break

10.20 - 10.40Epidemiology and disease burden of infection with the yellow fever virus

Alan BARRETT



Session 2 Molecular structure of flaviviruses; cellular receptors of flaviviruses; flaviviruses cell entry mechanisms11.00 - 15.10 Chaired by Franz Heinz

11.00 - 11.20 Molecular and antigenic structure of flaviviruses Franz HEINZ


11.40 - 12.00 Immature dengue, a veiled pathogen Jolanda SMIT


12.20 - 12.40 Structural basis and mechanisms of neutralization Kim DOWD


13.00 - 14.30 Lunch

14.30 - 14.50 Structural basis and mechanism of ADE Juthathip MONGKOLSAPAYA


Session 3 Pathogenesis and immune response; mechanisms underlying in vivo protection; neutralization and enhancement of infections15.10 - 18.30 Chaired by Joachim HOMBACH

15.10 - 15.30 Clinical aspects of dengue: the potential impact of vaccines Jeremy FARRAR



16.20 - 16.40Innate immune responses to infection with flaviviruses: role in protection and pathogenesis

Michael DIAMOND



08.30 - 08.50IMOJEVTM a new single dose recombinant vaccine for Japanese encephalitis

Farshad GUIRAKHOO


09.05 - 09.25 RepliVax vaccine for tick borne encephalitis Harry KLEANTHOUS


09.40 - 10.00 A new cell culture inactivated vaccine for yellow fever Dennis W. TRENT



Session 4 Vaccination against flaviviruses: vaccines in development08.30 - 16.30 Chaired by Farshad Guirakhoo

Wednesday 8 december 2010

17.00 - 17.20

Cellular immunity to dengue virus immunity and pathogenesis: humanized mice as a new animal model

Anuja MATHEW


17.40 - 18.00 Innate and adaptive immunity to vaccines in development Bruno GUY


18.20 - 18.30

ANRS EP46 - NOVAA the yellow fever vaccine immunity in HIV infected patients: development of a new assays for virological and immunological monitoring in HIV infected patient

François SIMON

19.00 Dinner

Scientific Programme 10.45 - 11.05 Recombinant live attenuated dengue

vaccine based on YF 17D virus Jean LANG


11.20 - 11.40 Live attenuated and inactivated approach for dengue vaccine Bruno INNIS


11.55 - 12..15Recombinant live attenuated dengue vaccine candidates based on the delta-30 mutation

Stephen WHITEHEAD


12.30 - 14.00 Lunch

14.00 - 14.20 Recombinant live attenuated dengue vaccine based Dengue2 ( DENVax) Dan STINCHCOMB



15.00 - 15.20 Recombinant subunit vaccine Beth Anne COLLER


15.35 - 15.55Closing lecture: implementation of Dengue vaccination: role of public-private partnerships

Scott HALSTEAD

15.55 - 16.30 Discussion, conclusions and end of meeting

Keynote lecture

Perspective in development of vaccines against flaviviruses

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Perspective in development of vaccines against flaviviruses

Thomas MONATHKleiner Perkins Caufield & Byers and Adjunct Professor Harvard School of Public Health - USA

Vaccine development has naturally followed the medical and veterinary public health importance of members of the virus genus. Some vaccines have been developed in response to dramatic and unpredicted disease emergences, such as Kyasanur Forest disease (1957), Rocio encephalitis (1975), and West Nile (1999), whereas others were developed in response to the recognition of the long-standing burden of endemic (and periodically epidemic) disease (e.g. yellow fever, Japanese encephalitis, tick-borne encephalitis, dengue). The approach to vaccine development has evolved in concert with improvements in virological methods and with the advent of molecular definition of antigenic determinants and cloning techniques. Yellow fever was the first Flavivirus vaccine to be developed, and since the history of that development illustrates many important tenets of vaccinology, it will be briefly reviewed. The remarkable self-adjuvanting activation of innate immunity appears to explain the very high and durable adaptive response to this vaccine. Other classical live, attenuated Flavivirus vaccines have been developed, some successful, some not. The current focus for replicating vaccines is the use rationally designed viral vectors, and some of these approaches will be described. Such vectors provide a plug-and-play approach that could be used to rapidly develop a vaccine against a new, emerging Flavivirus threat. A remaining challenge in the development of vaccine strategies against this group of viruses is a safe and effective vaccine against dengue, which is complicated by the occurrence of four serotypes in the species and by the unique pathogenesis of enhanced disease on heterologous reinfection. With the introduction and deployment of each new effective vaccine, the risk:benefit equation ultimately shifts the emphasis from efficacy and disease control to safety and avoidance of risk. This is driving the development of some new vaccines with improved safety profiles.

Session 1

Infection with flaviviruses: disease burden, epidemiology

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Epidemiology and disease burden of Dengue

Duane GUBLERResearch Program in Emerging Infectious DiseasesDuke-NUS Graduate Medical School, Singapore, and Asia-Pacific Institute for Tropical Medicine and Infectious Diseases, John A. Burns School of Medicine, University of Hawai‘i, Honolulu, Hawaii

The global spread of dengue viruses has resulted in a dramatic increase in the frequency and magnitude of epidemic dengue/dengue hemorrhagic fever. With an estimated 50 to 100 million infections annually, dengue is the most important vector-borne viral disease of humans and is one of the most important emerging tropical diseases globally. Efforts to control the disease have been largely unsuccessful. The changing epidemiology and disease burden of dengue, and challenges for reversing the trend of increasing epidemic disease will be discussed.

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Epidemiology and disease Burden of infections with encephalitic flaviviruses

John T. ROEHRIGCenters for Disease Control and Prevention - USA

Arthropod-borne flaviviruses cause a wide variety of human illness ranging from hemorrhagic fever to encephalitis. The flaviviruses that cause human encephalitis or neuroinvasive disease (ND) be-long to two antigenically-related serocomplexes: the tick-borne encephalitis virus (TBEV) complex and the Japanese encephalitis virus (JEV) complex. Medically important flaviviruses that are mem-bers of these serocomplexes cause disease throughout the world: TBEV and TBEV-related viruses, e.g., Powassan virus (Europe, Asia, and North America); JEV (Asia, northern Australia); West Nile virus, WNV (Africa, Asia, Europe, North and South America, and Australia as Kunjin virus); St. Louis encephalitis virus, SLEV (North and South America), and Murray Valley encephalitis virus, MVEV (Australia). Of these viruses only TBEV, JEV, and WNV currently cause significant numbers of human infections, with JEV and WNV being responsible for human epidemics. The last major epidemic of SLEV occurred in the late 1970’s in North America. For all ence-phalitic flaviviruses the human is a dead-end host. Identification of laboratory-confirmed human cases of JEV and WNV encephalitis is confounded by the serological cross-reactivity of human infec-tion-immune sera with other flaviviruses that might co-circulate with these viruses (e.g., SLEV and WNV in North America, and JEV and dengue viruses in Asia). Identification of either virus or viral RNA in clinical specimens is the surest way to confirm infections with these viruses. When diagnosis requires serological testing, identifi-cation of virus-specific IgM or virus-neutralizing antibody is needed for confirmation of infection. Because most human infections with TBEVs occur through the bite of an infected Ixodid tick, these viru-ses primarily cause endemic disease. TBEV infection of humans is controlled through vaccination with inactivated TBEV vaccines that have been in use for many years. Both JEV and WNV are trans-mitted through the bite of an infected Culex sp. mosquito. JEV is still a major cause of encephalitis in children throughout Asia. Multiple JEV vaccines are used to interdict and prevent human out-breaks, however their use is not yet universal. Both inactivated and attenuated JEV vaccines are used.

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Suite

No vaccine is currently available for WNV, even though this virus has been responsible for a recent major human epidemic in the Western hemisphere. In 1999 WNV was discovered in New York City, and since then the virus has moved throughout the United States (U.S.) and Canada, and southward into Mexico, Central and South America. WNV is currently the leading cause of viral en-cephalitis in the U.S., with 20% of human infections resulting in clinical disease, and less than 1% of human infections resulting in WNND. The overall case-fatality rate for individuals with WNND is 9-10%. Prior to the approval of an equine WNV vaccine, the equine case-fatality rate was 30% for horses in the U.S. There are no ap-proved prophylactic or therapeutic agents that have been shown to be effective for flaviviral infections. Both human and humanized monoclonal antibodies (MAbs) are currently undergoing intensive study in this regard. A humanized MAb that has been shown to be both protective and therapeutic in animal models for WNV infection is currently in human clinical trials in the U.S.

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Epidemiology and disease burden of infection with yellow fever virus

Alan D. T. BARRETTSealy Center for Vaccine Development and Depart. of Pathology, University of Texas Medical Branch at Galveston - USA

The disease yellow fever (YF) is caused by yellow fever virus (YFV) and is found in sub-Saharan Africa and tropical regions of South America where it is endemic and intermittently epidemic. The majority of cases are reported in sub-Saharan Africa. Only a small proportion of yellow fever cases are officially reported because of the occurrence of the disease in remote areas and lack of specific diagnostic facilities. The virus is transmitted between primates by mosquitoes (Aedes species in Africa, and Hemagogus and Sabethes species in South America). The normal vertebrate hosts are non-human primates but humans can get the disease if bitten by a virus-infected mosquito. In the last 25 years there has been an increase in the number of cases of YF and accordingly it has been classified as a reemerging disease.The virus exists as at least seven genotypes; two in West Africa, three in Central and East Africa, and two in South America. Evolutionary studies indicate the virus originated in Central or East Africa, moved to West Africa, and then spread to South America during the slave trade era. There is no antiviral therapy to treat the disease and the disease is prevented by the use of a live attenuated vaccine, strain 17D, which is considered to be one of the most effective and safe vaccines. The vaccine was developed in the 1930s and has been administered to over 550 million people. It is effective against all known genotypes of the virus despite genetic and antigenic variation. Despite the availability of an effective vaccine there are still estimated to be 200,000 cases of wild-type YF each year, including 30,000 deaths. Without the availability of a vaccine, YF would be considered a biosafety level-4 pathogen. Recently, reports of serious adverse events, including fatal outcomes, in temporal association with 17D yellow fever vaccination have highlighted the need for good surveillance and assessment of the benefit/risk of receiving the vaccine.

Session 2

Molecular structure of flaviviruses; cellular receptors of flaviviruses; flaviviruses cell entry mechanisms

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Molecular and antigenic structure of flaviviruses

Franz X. HEINZMedical University of Vienna - Austria

Flaviviruses are isometric lipid-enveloped particles with a diameter of about 50 nm. Virus assembly occurs in the endoplasmic reticulum and first leads to the formation of non-infectious immature virions, composed of a spherical core (containing the genomic RNA and multiple copies of the capsid protein) and a lipid bilayer with two membrane-associated proteins, prM and E. These proteins form an icosahedral scaffold of 60 spikes of heterodimers. The generation of infectious virions requires the proteolytic cleavage of prM by furin or similar proteases which occurs in the trans-Golgi-network during exocytosis of virus particles, shortly before their release from infected cells. Fully mature virions (in which all of the prM proteins have been cleaved) have a smooth surface and the E proteins are re-organized to form a closed shell of densely packed E-dimers. Parallel sets of three such homodimers form rafts that are arranged in a ‘herringbone’ pattern at the virion surface. The acquisition of infectivity does not require the cleavage of all prM-molecules in a virion and the release of partially mature but infectious particles is a hallmark of cells infected with certain flaviviruses.

The E protein has a double membrane-spanning anchor, an alpha-helical ‘stem’ region and an ectodomain - composed of three distinct domains (DI, DII, DIII) - that is oriented parallel to the membrane in mature virions. It mediates interactions with the cellular surface as well as membrane fusion in endosomes after uptake by receptor-mediated endocytosis and is the major target of virus-neutralizing antibodies. Epitopes involved in virus neutralization have been mapped to each of the three domains. The avidity of antibodies to these epitopes and their accessibility determine the degree of occupancy at any given concentration and are the most important determinants of neutralization potency. So far, the highest neutralizing activity was described for monoclonal antibodies directed to DIII. The extent of cross-reactivity between flaviviruses and cross-neutralization between and within the flavivirus serocomplexes is a reflection of amino acid sequence divergence in E. Broadly flavivirus cross-reactive antibodies are primarily directed to the highly conserved fusion peptide loop at the tip of domain II and their contribution to virus neutralization varies among different flaviviruses.

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Suite

Non-neutralizing antibodies or antibodies at sub-neutralizing concentrations can lead to the phenomenon of antibody-dependent enhancement of infection of Fc-gamma receptor-bearing cells, implicated in the exacerbation of disease during secondary dengue infections or in infants born to seropositive mothers. Such enhancement phenomena can also be observed with barely neutralizing antibodies to prM in combination with partially immature viruses, as is characteristic especially for dengue viruses. Even completely immature virions can be rendered infectious by prM-specific antibodies that mediate virus uptake into Fc-receptor positive cells followed by prM cleavage at a post-entry stage. The possible biological implications of the induction of antibodies to prM are an important issue in the context of dengue vaccines.

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Immature dengue virus: a veiled pathogen?

Jolanda M. SMITand Izabela A. Rodenhuis-Zybert, Julia Da-Silva Voorham, Bastiaan Moesker, Va-nesa Ayala-Nuñez, Heidi van der Ende-Metselaar, Jan Wilschut

Department of Medical Microbiology, Molecular Virology Section, University Medical Center Groningen, University of Groningen, Groningen - The Netherlands

Dengue virus (DENV) is a major emerging pathogen which causes disease symptoms ranging from febrile illness to devastating he-morhagic manifestations. Increased disease severity is associated with pre-existing DENV antibodies and high circulating virus titers, which suggests that antibodies directly influence the infectious pro-perties of the virus. The molecular mechanism by which antibodies enhance DENV infection however remains elusive.Cells infected with DENV release a high proportion of prM-contai-ning immature virions. It is generally believed that immature par-ticles are irrelevant by-products of infected cells since numerous functional studies have demonstrated that fully immature particles lack the ability to infect cells. On the other hand, dengue-positive patients secrete substantial levels of prM antibodies, which suggest that immature particles are involved in disease pathogenesis. In attempt to unravel these contrasting paradigms, we investigated the infectious properties of anti-prM antibody-opsonized immature DENV in various FcR- expressing cell lines and human primary PB-MCs (peripheral blood mononuclear cells). We found that immature DENV particles become virtually as infectious as wild type virus par-ticles in presence of antibodies. We observed that prM antibodies facilitate efficient binding and cell entry of immature particles into Fc-receptor expressing cells. In addition, enzymatic activity of furin is critical to render the internalized immature virus infectious. Fur-thermore, we found that multiple E antibodies interact with imma-ture particles and some of these antibodies stimulate the infectious properties of immature virions in a furin-dependent manner. Also, we investigated the cell entry chacteristics of prM-opsonized im-mature DENV particles in macrophages by single particle tracking analysis and revealed that the dynamics and route of viral entry is different compared to that of wild-type DENV particles. Altogether, our data suggests that in presence of antibodies, immature DENV particles have the potential to be highly infectious and hence may contribute to DENV pathogenesis. During the conference the criti-cal determinants in immature DENV cell entry will be discussed.

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Structural basis and mechanisms of neutralization

Kimberly DOWDNIH - USA

Neutralizing antibodies are a significant component of the host’s protective response against flavivirus infection, and are directed primarily against the envelope (E) protein. Neutralization of flaviviruses occurs when individual virions are engaged by antibodies with a stoichiometry that exceeds a required threshold. From this «multiple-hit» perspective, the neutralizing activity of antibodies is governed by the affinity with which it binds its epitope and the number of times this determinant is displayed on the surface of the virion. If the number of times an epitope is displayed on the virion surface does not exceed the stoichiometric threshold, neutralization will not occur, regardless of antibody affinity. At least two factors modulate epitope accessibility. Several classes of epitopes are bound by antibodies whose neutralization potency is sensitive to the maturation state of the virion. In these cases, the process of maturation reduces the accessibility of epitopes on the mature virion below the threshold required for neutralization. Epitope accessibility is also modulated by the dynamic motion of the E proteins on the virion that occur at equilibrium. Experiments utilizing monoclonal antibodies (MAbs) known to recognize relatively inaccessible epitopes on mature virions identified a role of virus «breathing» in regulating neutralization activity. Both time- and temperature-dependent increases in neutralization were observed that could be explained by exposure of inaccessible epitopes through dynamic movement of E proteins on the virion surface. This phenomenon was observed in studies with a large panel of MAbs specific for epitopes in each domain of the WNV envelope protein, with sera from recipients of a live attenuated WNV vaccine, and in experiments with dengue virus. Our findings suggest that the structural dynamics of flaviviruses has a widespread impact on antibody-mediated neutralization via changes in epitope accessibility; given enough time, even poorly neutralizing antibodies can dock on the virion with a stoichiometry sufficient for neutralization. Taken together, epitope accessibility is a critical yet dynamic factor that governs the functional properties of anti-flavivirus antibodies.

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Structural basis and mechanism of ADE

Juthathip MONGKOLSAPAYAImperial College - UK

Abstract not provided

Session 3

Pathogenesis and immune response; mechanisms underlying in vivo protection; neutralization and enhancement of infections

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Clinical aspects of dengue: the potential impact of vaccines

Jeremy FARRAROxford University Clinical Research Unit - Vietnam


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Innate immune responses to infection with flaviviruses: role in protection and pathogenesis

Michael DIAMONDand Stephane Daffis, Kristy J. Szretter, Jianqing Li, Jill Schriewer, Roland Zust, Hongping Dong, Volker Thiel, R. Mark Buller, Michael Gale, Jr., and Pei-Yong Shi

Washington University School of Medicine - USA

Type I interferon (IFN) cell-intrinsic antiviral defenses protect against many virus infections by signaling host blockade of viral translation, transcription, and replication, thus limiting spread and pathogenesis. Cellular mRNA of higher eukaryotes and many viral RNA are methylated at the N-7 and 2’O positions of the 5’ guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability, the function of 2’O methylation and its role in virus infection has remained uncertain since its discovery 35 years ago. We show that a West Nile virus (WNV) mutant that lacks 2’O MTase activity was attenuated in wild type primary cells and mice but was pathogenic in the absence of IFN signaling. 2’O methylation of viral RNA did not affect IFN induction in WNV-infected cells but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISG) implicated in regulation of protein translation. The first part of this talk will focus on recent results demonstrating that the 2’O methylation of the 5’ cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression. Thus, differential methylation of cytoplasmic RNA serves as a paradigm for pattern recognition and restriction of propagation of foreign viral RNA in host cells. The second part of the talk will discuss recent progress on using high-throughput shRNA screens in mouse and human cells to identify novel interferon stimulated genes that restrict flavivirus infection.

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Improved dengue-specific adaptive immune responses in humanized BLT mice

Anuja MATHEWand Smita Jaiswal, Alan L. Rothman, Leonard D. Shultz, Dale L. Greiner, Michael BrehmCenter for Infectious Disease and Vaccine Research - USA

The lack of a suitable animal model to study viral and immunological mechanisms of human dengue disease has been a constraint to dengue research. We recently established an animal model for dengue virus (DENV) infection and immunity using non-obese diabetic/severe combined immunodeficiency interleukin-2 receptor γ-chain knockout (NOD-scid IL2rγnull) mice engrafted with human hematopoietic stem cells. We sought to further improve this model by using humanized BLT mice which involves the cotransplantation of human fetal thymus and liver tissues and CD34+ fetal liver cells into NOD-scid IL2rγnull mice. The education and maturation of human T cells on autologous human thymic tissue in the BLT model was a key factor which led us to hypothesize that infection with DENV would lead to improved virus-specific cellular and humoral immune responses. Our data show consistent DENV replication in the bone marrow of engrafted mice. We detected significantly higher anti-dengue specific antibody titers in the sera of BLT mice compared to the previously tested standard and HLA-A2-transgenic NOD-scid IL2rγnull engrafted mice. Antibody responses were sustained for up to 6 weeks in the sera of infected BLT mice. Human T cells that developed following engraftment of BLT mice with HLA-A2+ human cord blood hematopoietic stem cells secreted IFN-γ in response to stimulation with overlapping peptides that spanned the entire DENV genome. Furthermore, four A2-restricted dengue peptides- NS4b 2353 (111-119), NS4b 2423 (181-189), NS4a 2148 (56-64) and a novel epitope on NS5 specifically stimulated IFN-γ secretion in human T cells. Overall, BLT mice will provide a much needed platform to assess human immune responses to DENV vaccines and the effects of prior immunity on subsequent DENV infections.

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Innate and adaptive immunity to dengue vaccines in development

Bruno GUYand Claire Balas, Anke Harenberg, Audrey Kennel, Florence Deauvieau, Regis Sodoyer, Nadege Arnaud-Barbe, Melanie Saville, Jean LangSanofi Pasteur - France

Dengue infection is a major and growing public health issue worldwide. Different vaccine candidates are being developed, including YFV 17D vaccine-based chimeric dengue virus vaccines (CYDs). These vaccine candidates have been characterized from the early stages of research through to clinical development, to assess in particular their safety and immunogenicity. Firstly, innate responses were investigated in vitro in human monocyte-derived dendritic cells (mDCs), upon infection by each of the 4 CYDs and their tetravalent combination. This was first done by flow cytometry and ELISA on a limited set of parameters, and then in a second step by using Agilent DNA microarrays. In this latter study, CYDs were also compared to wt serotype 3 virus, or to a classically attenuated serotype 3 virus (VDV3) shown to be reactogenic in a clinical trial. The results of the second study confirmed and expanded upon the first: we observed a very reproducible signature for each of the 4 CYDs, involving stimulation of Type I IFN genes and associated ISGs, together with genes encoding chemokines and other mediators involved in the initiation of adaptive responses. In contrast, the wt virus induced a predominantly inflammatory profile, while VDV3 appeared to induce a blunted response, which may have been insufficient to trigger early immune responses and prevent initial viral replication. This could have contributed to VDV3 symptomatic outcome in clinical trials. These studies contributed to documenting the safety and immunogenicity of the 4 CYD candidate vaccine viruses, which are currently in evaluation in large scale efficacy trials.Secondly, adaptive immune responses induced by the vaccine candidates were monitored in Phase 1 and Phase 2 clinical trials. Th1 and CD8 responses were induced with an IFN-γ/TNF-α ratio favouring IFN-γ in both cases, regardless of whether the vaccine recipients were flavivirus naive or not. There was an absence of Th2 response in all cases. The Th1 response was dominated by serotype 4 in flavivirus naive individuals after initial vaccination but broadened to include all serotypes after second vaccination. This broadened response was also observed after primary dengue TV vaccination in subjects previously administered monovalent dengue 1 and dengue 2 vaccines.

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Suite

Extending these analyses to children will be essential in the dengue TV development program, and the techniques we used so far are being modified in an ongoing trial, in order to monitor cellular responses from only 3–5 ml of blood. In conclusion, pre-clinical and clinical results support the favorable immunogenicity and short-term safety of the dengue TV. Ongoing and future studies will establish the longevity of the vaccine-induced immunity and requirements for boosters.

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The yellow fever vaccine immunity in HIV infected pa-tients: development of new assays for virological and im-munological monitoring in HIV infected patient(Essai EP46 ANRS NOVAA)

François SIMONand N Colin de Verdière, S Delarue, V Melfreidy, B Labrosse, B Autran, JP Aboulker, JM MolinaCHU Saint LOUIS, INSERM U 941 - France

In order to compare the virological and immune responses in HIV-positive and HIV-negative individuals YF naive-vaccine populations, we initiated a clinical trial phase III multicentric protocol in Paris, France. Specific tools for the evaluation of the cell-mediated and humoral immunity after YFV and virological responses were developed: - an ELISPOT technology for YF - a pseudotype -based assay for quantifying the anti-YF virus neutralizing antibodies

Thirty HIV-negative subjects and 40 HIV-positive voluntary subjects (CD4 > 350/mm3 under HAART for at least one year, with a viral load < 50 copies/mL since at least 6 months) will be included. HIV positive subjects will be matched according to age (18-40 years and 40-55 years) and sex, with the HIV negative subjects. Subjects will be vaccinated at J0 with STAMARIL® and followed over one year.

The responses will be analysed within 7, 14, 28, 90 and 365 days of administration of YFV in terms of : (1) seroconversion by EIA, (2) cytotoxic response in ELISPOT, (3) neutralizing antibody levels in PRNT (reference method) and a new pseudotype based method, (4) post-vaccination viremia and (5) diversity of viral quasi-species.The titles and kinetics of viremia, neutralizing antibodies and ELISPOT will be considered as surrogate markers of response in terms of groups.

Impact of YFV on the T-lymphocyte response against HIV by ELISPOT and HIV-1 RNA viral load will be assessed. Subjects with previous vaccination against YF or YF anti-IgG positive, recent immunoglobulins therapy, HCV infection, history of thymic dysfunction (including thymoma and thymectomy) or others immunosuppression, whether congenital, idiopathic will not be included in the study.

Clinical and biological tolerance: At all follows up will be measured the incidence of CDC classification events (for HIV+) and general and local reactions of degree ≥ 2 in the setting of the injection of STAMARIL®

Schedule : Date of first enrolment: December 2010 Inclusion period: 18 months

Session 4

Vaccination against flaviviruses: vaccines in development

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IMOJEVTM a new single dose recombinant vaccine for Japa-nese encephalitis

Farshad GUIRAKHOOSanofi Pasteur - USA

Flaviviruses are a global public health concern affecting nearly half of the world’s population. Japanese encephalitis (JE) is a mosquito-borne infection with high epidemic potential, ~30% case-fatality rate, and high severe sequelae among survivors long after their acute illness. Although several types of JE vaccines are available, disadvantages including reactogenicity, production shortages, and need for multiple doses hinder their effectiveness to combat JE.

IMOJEV™ is a live attenuated single dose vaccine developed to provide convenient immunization against JE. It was constructed by inserting coding sequences of the premembrane and envelope structural proteins of the JE vaccine strain SA14 14 2 virus into the core and non-structural genes of yellow fever (YF) vaccine strain 17D virus.

Nonclinical studies showed that IMOJEV™ was significantly less neurovirulent than an YF 17D vaccine (YF VAX®) in both mouse and monkey models inoculated by the intracerebral (IC) routes. A single subcutaneous low dose of IMOJEV™ conferred 100% protection in monkeys against a lethal IC challenge with a highly virulent JE virus. Environmental safety testing included vector transmission, biodistribution and recombination studies showed that the vaccine virus is genetically stable in vitro and in vivo, does not shed to the environment from vaccinees, is unable to infect mosquitoes, and when artificially recombined with a wild type virus, its neurovirulence phenotype is lower than that of the wild type parental virus.

Nine clinical studies (Phase I/II to Phase III) in healthy adult populations in the USA and Australia and three clinical studies (Phase II and Phase III) in healthy children, toddlers and infants were conducted with IMOJEV™ which demonstrated a profile of a highly safe and effective vaccine. Further trials are underway in pediatric populations.

IMOJEV™ is currently licensed in Australia as a single dose vaccine.

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RepliVAx vaccine for tick borne encephalitis

Harry KLEANTHOUSSanofi Pasteur - USA


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A new cell culture inactivated vaccine for yellow fever

Dennis W. TRENTUniversity Texas Medical Branch - USA

Live, attenuated yellow fever (YF) 17D vaccine is highly efficacious but causes rare, serious adverse events resulting from active replication of the virus in the host resulting in direct injury to vital organs. We have reported development of a potentially safer β-propiolactone-inactivated whole virion YF vaccine (XRX-001) that is highly immunogenic in mice, hamsters, and monkeys (Vaccine 2010; 28:3827-3840). Immunization of hamsters with two doses of the XRX-001 stimulated YF virus neutralizing antibody titers that were significantly higher than a single dose of YF-VAX® vaccine. Hamsters given a single dose or two doses of XRX-001 or a single dose of YF-VAX® vaccine were fully protected against hepatitis, viremia, weight loss and death when challenged with the virulent Jimenez strain of YF virus.

To determinate protective efficacy of neutralizing antibodies stimulated by the inactivated XRX-001 YF virus vaccine, graded doses of serum from hamsters immunized with the inactivated vaccine or live attenuated YF-VAX® were given to hamsters 24 hours prior to virulent YF virus challenge. Passive antibody neutralizing antibody (PRNT50) titers in sera of treated hamsters were determined 4 hours before virus challenge and at 4 and 21 days after virus challenge. Neutralizing antibodies mediated protection from YF virus infection and related disease. Animals that had PRNT50 antibody titers of ≥ 20 four hours before virus challenge were protected from disease as evidenced by lower viremia, liver enzyme evels and infection associated illness (weight change). Passive PRNT50 antibody at a titer ≥ 40 produced sterilizing immunity in most of the hamsters, shown byt he absence of an immune response to the challenge YF virus. Immunization with the XRX-001 vaccine stimulated YF neutralizing antibodies equally effective (based on dose response) as antibodies stimulated by the live attenuated YF-VAX® vaccine.

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Sanofi Pasteur Phase III stage recombinant live attenuated dengue vaccine based on YF 17D virus

Jean LANGand Melanie Saville, Bruno Guy

Sanofi Pasteur - France

The development of the Dengue tetravalent (TV) recombinant YF17D-based chimeric vaccine has been continuously driven by a benefit/ risk evaluation and mitigation strategy. Earlier Pre-clinical studies have demonstrated that the sanofi pasteur dengue vaccine is genetically and phenotypically stable, non-hepatotropic, less neu-rovirulent than YF17D and does not infect mosquitoes by the oral route. In vitro and in vivo preclinical studies also showed that the TV dengue vaccine induced controlled stimulation in human den-dritic cells, and a significant immunogenicity in monkeys. Results of Phase II trials in the USA, the Philippines and Mexico showed that the majority of adverse events were mild to moderate and transient in nature. Viraemia was transient and low, and was not increased after initial dengue TV administration, even in the case of incom-plete responses. PRNT50 seroconversion ranged between 80 and 100% for all four serotypes in subjects injected with 2 to 3 doses of TV dengue vaccine.Responses were also monitored at the cellular level in humans: Th1 and CD8 responses were induced with an IFN-γ/TNF-α ratio favoring IFN-γ. A worldwide clinical development program for dengue TV is underway including completion of the enrollment of 4000 4-11 years old children in an efficacy trial in Thailand .The phase III clinical program is already started since October 2010 with the evaluation of industrial scale lots in Austra-lia. Assuming continued successful outcomes, initial submissions to national regulatory authorities in dengue high disease burden countries could now been envisaged within a 5-year period.

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Live attenuated and inactivated approach for dengue

Bruno INNISGlaxoSmithKline - USA


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A recombinant live attenuated dengue vaccine based on the delta-30 mutation

Stephen WHITEHEADNIAID - NIH - USA

The goal of the National Institutes of Allergy and Infectious Diseases (NIAID) intramural DENV vaccine program is to produce a minimally reactogenic, highly immunogenic, genetically stable, live attenuated DEN vaccine that is cost-effective and safe for the community. Over the past ten years, the NIH has developed numerous live attenuated candidate vaccines against the four individual DENV serotypes using a variety of techniques based on the delta-30 mutation in the 3’ untranslated region of the genome. We have tested 8 monovalent vaccines in 16 separate Phase I clinical trials to identify DEN1, DEN2, DEN3, and DEN4 candidate vaccine viruses that are safe and maintain optimal infectivity and immunogenicity profiles for inclusion in a tetravalent formulation. Each monovalent candidate was well tolerated by volunteers with no volunteer experiencing a dengue-like illness. Following a single subcutaneous injection of 1,000 PFU, each of the selected candidate vaccines induced seroconversion rates of 80 – 100% to wildtype DENV. Factors such as infectivity, immunogenicity, and reactogenicity were used to determine which vaccine candidates were appropriate to include in a tetravalent formulation. A Phase I Clinical trial evaluating 3 different tetravalent admixtures given as a single dose of 1000 PFU/serotype was initiated in 2010 in flavivirus-naïve subjects. Safety and immunogenicity results from this Phase I clinical trial will be presented.

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Phase 1 Clinical Trials of DENVax - A Live Attenuated, Tetravalent Dengue Vaccine

Dan STINCHCOMBand Jorge Osorio, Aurelia A. Haller, Joseph Brewoo, Charalambos Partidos, Shawn Silengo, John Arguello, Tim Powell, Jill Livengood, Yuping Ambuel, Cynthia Thom-son, Joseph Santangelo, Claire Huang, Sarah George, Ivan Velez

Inviragen, Inc. - USA

Inviragen’s DENVax tetravalent dengue vaccine consists of a mixture of the live, attenuated DEN-2PDK-53 virus and three chimeric recombinant viruses. Each chimera contains the attenuating mutations in the DEN-2 PDK-53 non-structural gene backbone while expressing DEN-1, DEN-3 or DEN-4 structural genes. The four DENVax strains have been shown to be attenuated, immunogenic and protective in mice and in nonhuman primate dengue models. Tetravalent formulations generate neutralizing antibodies to all four dengue serotypes and preclinical data suggest that intradermal administration of DENVax improves vaccine immunogenicity. Currently, two Phase 1 studies are ongoing to evaluate the safety, tolerability and immunogenicity of DENVax in healthy adults. One study is sponsored by the National Institute of Allergy and Infectious Diseases in the United States (St. Louis, MO) and the second study is sponsored by Inviragen in Rionegro, Colombia, a high altitude area with no Aedes aegypti and no dengue exposure. The clinical trial design is similar for both studies: two different formulations of DENVax are administered by intradermal or subcutaneous injection in two doses, three months apart. Initially, volunteers receive a low dose of DENVax and safety data collected for 21 days is reviewed prior to administrating the booster or immunizing the next cohort with the high dose formulation of DENVax. These trials will allow direct comparison of subcutaneous and intradermal delivery in placebo controlled trials in two different continents. A third Phase 1 trial is planned to directly compare needle and syringe to needle free delivery of DENVax. Results obtained to date indicate that the low dose of DENVax is well-tolerated in both U.S. and Colombian healthy adult populations; additional data will be summarized as they become available.

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Development of a recombinant subunit vaccine for Dengue

Beth-Anne COLLERHawaii Biotech, Inc., Merck Research Laboratories - USA

Dengue viruses are a major cause of morbidity and mortality throughout the tropics and subtropics with an estimated 50-100 million cases of dengue annually. To date no specific vaccine or therapy has been licensed to combat this important disease. To address this unmet medical need, and to avoid issues with viral interference seen with live virus vaccines, Merck is evaluating a recombinant subunit vaccine originally developed by Hawaii Biotech. The tetravalent subunit vaccine is based on the envelope glycoprotein of the dengue viruses and designed to protect individuals against dengue virus induced disease. Preclinical studies conducted in mice, rats, or rabbits have demonstrated the immunogenicity of both monovalent and tetravalent formulations adjuvanted with alum or ISCOMATRIX™ adjuvant and formal toxicology studies have demonstrated acceptable safety of all formulations. An alum-based monovalent DEN1 formulation and a tetravalent ISCOMATRIX™ adjuvant-based formulation were shown to be immunogenic and protective in non-human primates. A Phase 1 clinical study of monovalent DEN1 recombinant envelope protein adjuvanted with alum has been conducted in healthy volunteers demonstrating the immunogenic potential of the recombinant antigens in human subjects.

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Implementation of Dengue vaccination: role of public-private partnerships

Scott B. HALSTEADPediatric Dengue Vaccine Initiative - USA

PDVI is a Product Development Public-Private Partnership founded in 2001 with support from the Rockefeller and Gates Foundations. Program goals for PDVI were: 1) to accelerate the development, evaluation and introduction of dengue vaccines; 2) to make safe and effective dengue vaccines available to reduce disease incidence in populations at risk in dengue endemic countries. An enormous challenge confronting the world will be to deploy vaccines to reduce the burden of dengue disease among an at-risk population of between 2.5 and 3 billion persons. Fortunately, each of the four dengue viruses is highly immunogenic; natural infection conveys life-long homologous protective immunity and some degree of heterotypic immunity. There is epidemiological evidence that two different dengue infections protect individuals from severe dengue disease during a third or fourth infection. Applying this strategy, sanofipasteur seeks to protect individuals based by administering two or three doses of a chimeric live-attenuated tetravalent dengue vaccine. New research models - the AG 129 mouse and intrinsic ADE in primary human monocytes/macrophages – now available may permit more accurate assessment of protection afforded by these heterotypic dengue antibodies than has been possible in the past. Dengue vaccines should be deployed strategically to protect the unvaccinated, especially young infants who may be at risk to severe disease accompanying a first dengue infection. This will require use of herd immunity. To prepare for this option, herd immunity to the four dengue viruses should be measured in several at-risk populations. Mathematical models may then be applied to design vaccine programs that achieve herd immunity efficiently. The Dengue Vaccine Initiative (DVI), successor to PDVI, a new Public-Private Partnership is ready to contribute to these and other components of the strategic implementation of new dengue vaccines.

Speakers & Chair

Alan Barrett University of Texas Medical Branch USA

Beth Ann Coller Hawaii Biotech Inc USA

Michael Diamond Washington University School of Medicine USA

Kim Dowd NIH USA

Jeremy Farrar Oxford University Clinical Research Unit Vietman

Duane Gubler Duke-NUS Graduate Medical SchoolUSA

Farshad Guirakhoo Sanofi Pasteur France

Bruno Guy Sanofi Pasteur France

Scott Halstead PDVI USA

Franz Xaver Heinz Medical University of Vienna Austria

Joachim Hombach WHO Switzerland

Bruce L. Innis GlaxoSmithKline USA

Harold Kleanthous Sanofi Pasteur USA

Jean Lang Sanofi Pasteur France

Christophe Longuet Fondation Mérieux France

Jacques Louis Fondation Mérieux France

Anuja Mathew University of MassachusettsUSA

Thomas Monath Kleiner Perkins Caufield & Byiers USA

Juthathip Mongkolsapaya Imperial College London England

Valentina Picot Fondation Mérieux France

John Roehrig U.S. Centers for Disease Control and Prevention USA

François Simon APHP France

Speakers & Chair

Jolanda Smit University Medical Center Groningen The Netherlands

Dan Stinchcomb Inviragen USA

Dennis Trent University Texas Medical Branch USA

Stephen Whitehead NIAID / NIH USA

Participants

Odofin Akintunde AllenFederal Medical CentreNigeria

Mohamed Bangura Bajito Onda Africa Foundation Sierra Leone

Véronique BarbanSanofi Pasteur France

Martine Baudoin Sanofi Pasteur France

Obasuyi Bolaji National Agency for Food and Drug Administration Nigeria

Gabriel Breugelmans AAMP France

Yen-Giang Bui Institut National de Santé Publique du Quebec Canada

Sara Calattini E.N.S France

Germano Leonel Coelho Ferreira Sanofi Pasteur France

Nathalie Colin de VerdièreHôpital Saint-LouisFrance

Pascal Cottin Sanofi Pasteur France

Luis J da Silva IVI Brazil

Júlia M. da Silva Voorham UMCG The Netherlands

Isabelle Delannoy Sanofi Pasteur France

Clarisse DemontSanofi Pasteur France

Katrin Dubischar-Kastner Intercell Austria

Andre Habel Themis Bio Austria

Edward Hayes CRESIB Spain

Julian Hickling PATH England

Rebecca Jones PATH England

Lee Kennhwa Jeju National University School of Medicine South Korea

Jehanara Korimbocus AFSSAPSFrance

Thomas Kreil Baxter Austria

Merita KukucuNational Regulatory Authority of Vaccines & Immunobiological Products Albania

Participants

Michel LandryINSPQ Canada

Thomas Laurent Service des Urgences - CHU Martinique

Dimitri LaviletteINSERM - ENS LyonFrance

Nathalie Mantel Sanofi Pasteur France

Claude Meric Sanofi Pasteur France

Heidi Meyer PEI Germany

Jules Minke Merial France

Srinivasulu MummmadiNOVOK India

Pem Namgyal WHO / IVR Switzerland

Deepack PatelNational Institute of Communicable Disease India

Sandrine Pillon Sanofi Pasteur France

Eric Plennevaux Sanofi PasteurFrance

Katrin Ramsaver Themis Bio Austria

Lanke Madam Ravi SREDIndia

Izabela Rodenhuis-Zybert UMCG The Netherlands

Chantal Rotario Sanofi Pasteur France

Julia Schmitz WHO / IVR Switzerland

Hanneke SchuitemakerCrucell B.V The Netherlands

John Sesay Centre For Peace & Development Sierra Leone

George Sips UMCGThe Netherlands

Jocobus Smit Sanofi Pasteur France

Rai Spier Elsevier England

Karin Stiasny Medical University of Viena Austria

Olaoye Olalekan Sunday Federal Medical CentreNigeria

Participants

Teodóra Szalkai ANTSZ Hungary

Remy Teyssou Sanofi Pasteur France

Sahr Irrahim Thorlie Bajito Onda Africa Foundation Sierra Leone

Alusine Turay Centre For Peace & Development Sierra Leone

Fons Uytdehaag Crucell B.V The Netherlands

Ralf Wagner Paul-Ehrlich Institute Germany

Viktoriia Zadorozhna MOH of Ukraine Ukraine

Nataliya Zubkova MOH of Ukraine Ukraine

This meeting was made possible through unrestricted educational grants from Sanofi Pasteur.

flavivirus vaccination - globe network · there are currently >200,000 cases and 30,000 death...

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