vaccination strategy to protect against flavivirus ...€¦ · solution to complement the current...
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Vaccination Strategy To Protect Against Flavivirus Infection Based On Recombinant
Measles Vaccine
by
Rowida Abdelgalel
A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree
Doctor of Philosophy
Approved November 2016 by the Graduate Supervisory Committee:
Jorge Reyes del Valle, Co-Chair
Hugh Mason, Co-Chair Douglas Lake Valerie Stout Wayne Frasch
ARIZONA STATE UNIVERSITY
December 2016
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ABSTRACT
Despite the approval of a Dengue virus (DV) vaccine in five endemic countries,
dengue prevention would benefit from an immunization strategy highly immunogenic in
young infants and not curtailed by viral interference. Problematically, infants younger
than 9 year of age, whom are particularly prone to Dengue severe infection and death,
cannot be immunized using current approved DV vaccine. The most important issues
documented so far are the lack of efficiency and enhancement of the disease in young
seronegative recipients, as well as uneven protection against the four DV serotypes.
Based on data from clinical trials that showed enhanced performance of dengue vaccines
when the host has previous anti-flaviviral immunity, I proposed here an attractive
solution to complement the current vaccine: a recombinant measles vaccine vectoring
dengue protective antigens to be administered to young infants. I hypothesized that
recombinant measles virus expressing Dengue 2 and 4 antigens would successfully
induce neutralizing responses against DV2 and 4 and the vaccine cocktail of this
recombinant measles can prime anti-flaviviral neutralizing immunity. For this
dissertation, I generated and performed preclinical immune assessment for four novel
Measles-Dengue (MV-DV) vaccine candidates. I generated four MVs expressing the pre
membrane (prM) and full length or truncated (90%) forms of the major envelope (E)
from DV2 and DV4. Two virus, MVvac2-DV2(prME)N and MVvac2-DV4(prME),
expressed high levels of membrane associated full-length E, while the other two viruses,
MVvac2-DV2(prMEsol)N and MVvac2-DV4(prMEsol)N, expressed and secreted
truncated, soluble E protein to its extracellular environment. The last two vectored
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vaccines proved superior anti-dengue neutralizing responses comparing to its
corresponding full length vectors. Remarkably, when MVvac2-DV2/4(prMEsol)N
recombinant vaccines were combined, the vaccine cocktail was able to prime cross-
neutralizing responses against DV 1 and the relatively distant 17D yellow fever virus
attenuated strain. Thus, I identify a promising DV vaccination strategy, MVvac2-
DV2/4(prMEsol)N, which can prime broad neutralizing immune responses by using only
two of the four available DV serotypes. The current MV immunization scheme can be
advantageus to prime broad anti-flaviviral neutralizing immunity status, which will be
majorly boosted by subsequent chimeric Dengue vaccine approaches.
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Didicated to the joy of my life, my son: Saleem Mousa
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ACKNOWLEDGMENTS
Foremost, I would like to express my sincere gratitude to my mintor Dr. Jorge
Reyes del Valle for his help during this journey. The work that I have done during my
PhD was mainly done because of his guidance and support. He showed a great deal of
confidence in my abilities and he belives in me. He has been a professional model for me
and a great lifelong firend. I also want to thank my committee members, Dr. Hugh
Mason, Dr. Douglas Lake, Dr. Valerie Stout, and Dr. Wayne Frasch, for their support and
feedback. It is because of all of them that I was able to grow as a scientist, and to learn to
challenge my knowledge and expand my limits.
During my time as a PhD student I was lucky enough to share my experience with
special friends, Ivonne, Emily, Indera, Francisca and Radwa. They gave me great support
and helped me to get over the hard times, esspacially, Ivonne gave me great technical
support.
Last but not the least, I would like to thank a dear friend Wijdan Al Omari for her
help, she gave me lots of support and helped me through the hard times, she is not just a
friend she is my sister as well. I would like to thanks my family members, my joy of life,
my son, Saleem and my husband Mohsen, my parents Zainab and Abdelhameed who
belived on me and gave me love and support.
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TABLE OF CONTENTS
Page
LIST OF FIGURES…………………………………………………………………..…...x
CHAPTER
1 INTRODUCTION…………………………….…………………………………..1
The Measles Virus Reverses Genetics System In The Development Of
Multivalent Vaccine Options.…………………...………………………...…….1
The Measles Virus Biology…….…………………………………………1
The Rescue Of Measles Virus From Cloned DNA..……………………...3
Genomic Modification Of MV, The Insertion Of Foreign Coding
Sequence…………………………………………………………………..4
Measles Candidate Multivalent Recombinant Vaccines…………………..5
The Quest For An Optimal, Pediatric Dengue Virus Vaccine.…………………..7
Epidemiology Of Dengue Virus (DV)…………………………………….7
The Pathogenesis Of Dengue Infection...…………………………………9
Dengue Virus Biology……………………………………………...……10
Immune Response And Immunopathogenesis Of Dengue Virus
Infection.…………………………………………………………………13
Dengue Vaccine Candidates.…………………………………………….16
Central Hypothesis.…………………………………………………………….20
Chapter Organization…………………………………………………………..21
References……………………………………………………………………..25
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CHAPTER Page
2 BROAD NEUTRALIZING RESPONSES ELICITED BY FLAVIVIRUS
INFECTION IN CD46 INFarKO MICE……….…….………………………….32
Abstract………………………………………………………………………32
Importance……………………………………………………………………33
Introduction…………………………………………………………………..34
Materials And Methods………………………………………………………36
Cells And Viruses………………………………………………………..36
Mice Inoculations………………………………………………….……..36
DV And YfV Neutralization Assay……………………………….……..36
Correlation Of PRNT50 And LNI…………………………………….…37
Results…………………………………………………………………….…..38
Monotypic Dengue Virus Immunity………………………………….….38
Heterologous Cross-Neutralization Immunity To DV1 And YFV………39
Discussion…………………………………………………………………….40
References……………………………………………………………………..44
3 GENERATION OF A COMPLEMENTARY ANTI-DENGUE
IMMUNIZATION STRATEGY BASED ON A RECOMBINANT MEASLES
VIRUS VACCINE COCKTAIL……………………………..……………………….46
Abstract………………………………………………………………..……..46
Importance……………………………………………………………………48
Introduction......................................................................................................49
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CHAPTER Page
Materials And Methods…………………………………………………..…..51
Cells And Viruses………………………………………………………..51
Plasmids And Plasmids Constructions..…………………………………52
Indirect Immunofluorescence Microscopy..……………………………..53
Western Blot Assay.……………………………………………………..54
Glycosidase Treatment.………………………………….........................54
Mice Inoculations………………………………………………………..55
Mv Neutralization Assay...………………………………………………55
DV And YFV Neutralization Assay……………………………………..56
Results………………………………………………………………………..57
Generation And Characterization Of Recombinant Mvs Expressing
Dengue 2 Envelope Antigens…………………………………………….57
Expression Of Dengue 2 E Glycoprotein By Recombinant Viruses…….58
Immunogenicity Of Mv-Dengue 2 Recombinant Viruses……………….59
Generation, Characterization And Immunogenicity Of Mvvac2-
D4(PrMEsol)N Expressing A Soluble Version Of Dengue 4 E
Antigen.......................................................................................................60
Immunogenicity Of Recombinant Mv Vectors Expressing Esol Antigens
From Dengue 2 And 4……………………………………………………62
Cross-Neutralizing Immune Responses In Mv-Susceptible Mice
Inoculated With Mvvac2-D2(PrMEsol)N And Mvvac2-D4(PrMEsol)N..63
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CHAPTER Page
Discussion…………………………………………………………………….65
References…………………………………………………………………….81
4 GENERATION OF A RECOMBINANT MEASLES VIRUS EXPRESSING
FULL-LENGTH DENGUE 4 ENVELOPE GLYCOPROTEINS…....……………...85
Abstract………………………………………………………………………85
Introduction………………………………………………………………….87
Materilas And Methods.…………………………………………………..…89
Cells And Viruses.……………………………………………………….89
Plasmids And Plasmids Constructions…………………………………..90
Indirect Immunofluorescence Microscopy………………………………91
Mice Inoculations………………………………………………………..91
MV Neutralization Assay………………………………………………..92
DV Neutralization Assay………………………………………………..92
Results…………………………………………............................................93
Generation Of Recombinant Divalent MV-DV4 Vaccine Vector………93
Replication Fitness Of Recombinant MV-DV4 Is Equivalent To Parental
MV……………………………………………………………………….94
Recombinant MV-DV4 Induces Anti-Measles And Anti- Dengue 4
Immune Responses…………………………………………………..…..95
Discussion……………………………………………………………………95
References…………………………………………………………………..104
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CHAPTER Page
5 SUMMARY AND FUTURE DIRECTIONS………………………..................106
References…………………………………………………………………....111
COMPREHENSIVE LIST OF REFERENCES…………………………………..........113
x
LIST OF FIGURES
Figure Page
1.1. Dengue Vaccine Mode Of Action. ……………………………………...................23
1.2. Schematic Representation Of Central Hypothesis...………………………...…......24
2.1. Immunogenicity Of Monotypic Immunity To Dengue 2 And 4……..….………....42
2.2. Cross-Neutralizing Immune Responses In Mice Inoculated With DV1, DV2, DV4,
And YFV ……………………………………………..…….....……………………......43
3.1. Generation And Characterization Of Recombinant Mvs Expressing Dengue 2
Envelope Antigens. ……………………………………………………..……….……..70
3.2. Expression Of Dengue 2 E Glycoprotein By Recombinant Viruses………….……72
3.3. Immunogenicity Of MV-Dengue 2 Recombinant Viruses. ..……………….…...…74
3.4. Generation, Characterization And Immunogenicity Of Mvvac2-D4(Prmesol)N…..76
3.5. Immunogenicity Of Recombinant MV Vectors Expressing Esol Antigens From
Dengue 2 And 4……………………………………………………………….………...78
3.6. Cross-Neutralizing Immune Responses In MV-Susceptible Mice Inoculated With
Mvvac2-D2(Prmesol)N And Mvvac2-D4(Prmesol)N …………………...……………..80
4.1. Generation And Characterization Of Recombinant Mvs Expressing Dengue 4 PrM
And E Antigens……………………………………………………………...…………..99
4.2. Replication Finesses Of Recombinant MV-DV4………………………………….101
4.3. Immunogenicity Of The MV-DV4 Recombinant Virus.……………………….....102
4.4. Sequance Alignment Of TM Regions Of E Protein From DV1-4 And YFV……..103
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CHAPTER 1
INTRODUCTION
I-The Measles virus reverse genetics system in the development of multivalent
vaccine options.
Measles virus biology.
Measles virus (MV) is a negative-sense single-stranded RNA virus that belongs to
the Paramyxoviridae family. It is an enveloped, pleomorphic virus with two surface viral
glycoproteins, the Hemagglutinin (H) and Fusion (F) proteins arranged as dimer of
dimers and trimmers, respectively [3]. MV H protein is a type II integral membrane
protein and it is responsible for the viral attachment to cellular receptors. Three proteins
have been indentified as MV cellular receptors, which explain MV tropism. The
complement regulatory protein CD 46 (membrane cofactor protein) was identified as a
cell receptor exclusive for attenuated strains, its role in vivo is unknown. CD 150 or
SLAM (Signaling Lymphocyte Activation Molecule), and Nectin-4 explain viral tropism
for immune competent cells and lower-tract epithelium [4-6]. The fusion protein is a type
I integral membrane protein which is expressed initially in an immature form called F0
and mature after portease cleavage into a disulfide bridged protein with two subunits
called F1 and F2. It is responsible for the triggering of membrane fusion when MV enters
into host cells and when infected cells fuses with neighboring cells to induce synctium
formation [7]. Both MV H and F glycoproteins bind to the matrix (M) protein on the
inner side of the viral membrane through their cytoplasmic tail. M protein thus lines the
inner surface of the viral envelope and plays a role in viral assebly. The MV genomic
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RNA is covered by a nucleocapsid (N) protein in tight association with the large RNA-
dependent RNA polymerase (L) and its co-factor, the phosphoprotein (P). MV genome is
infectious only when present in this complex. Full-length MV genomic RNA should be a
multiple of six nucleotides (hexamers) in order to be encapsidated into this RNP
complex[8].
MV replication and transciption. MV N protein is the most abundantly
exppressed protein among measles proteins and plays a critical role in viral replication, as
only encapsidated genomes and antigenomes can replicate. MV L protein is responsible
for the transcription and replication of the genomic RNA and is biologically active in the
presence of the P protein, an essential polymerase cofactor. Replication of MV genomic
RNA is a two-step event. First, the RdRp bind to the leader region at the 3' end of the
genome and synthesize a positive stranded, full-length RNA (antigenomic RNA).
Second, The RdRp uses this anti-genomic RNA as a template to create multiple copies of
the genomic RNA with negative polarity. MV genome transcription is also carried out by
the RdRP and creates transcription gradients of the measles cistrons. MV genes are
organized in independent transcription units with their own cis -active sequences located
at gene boundaries. Short intergenic regions, where the polymerase stops and
reinitializes at intergenic regions, separate each gene. Transcription of cistrons closer to
the genomic 3' end are more abundant; thus the N protein has the higher expression level
while L protein has the lowest. In this way the RdRp modulates the amount of each gene
product. MV replication and transcription is cytoplasmic. MV H and F proteins will be
directed to the endoplasmic reticulum and Golgi to be glycosylated and cleaved to
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generate mature forms and then transported to the plasma membrane. The H-F
glycoproteins complex will be assembled on the infected cell surface resulting in fusion
between neighboring cells. Most of the new synthesized virus particles are not released
into the extracellular space, as they transmit to neighboring cells due to fusion. This
fusion of infected cells results in syncytia formation which a sign of measles infection in
cell culture[9].
The rescue of Measles virus from cloned DNA.
MV antigenomic cDNA were obtained from a vial of the Schwarz vaccine using
RT-PCR in order to generate a plasmid called pB(+)MVvac2[10, 11]. The backbone of
this plasmid is PBluescript. Afterwards, a reverse genetic system using either helper virus
or helper cells was developed to rescue (generate) MV from this infectious
complementary antigenomic DNA[12]. The first attempt to rescue MV was attempted
using a helper virus, Vaccinia virus expressing T7 RNA polymerase, T7 driven
transcription at a high levels is necessary to generate a full-length antigenomic copy from
the cloned cDNA. Additionally, infected cells were transfected with the cDNA clonal
copy of the MV genome along with expression plasmids encoding N, P, and L genes also
under the control of the T7 promoter. MV N, P, and L proteins will be used in the
replication of the T7 transcribed genome. However, although the helper virus method is
an effective approach to rescue MVs, it is not the most straightforward strategy to recover
recombinant MV since further intensive purification steps are required. Indeed the
chemical and physical stress used to eliminate vaccinia infectivity can inactivate MV. A
modified method was introduced by Radecke to rescue MV using Helper cells (293-3-
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46). Those engineered cells expresses P, N proteins, and T7 polymerase in a constitutive
manner. In this approach, Helper cells are transfected with the RDRP expressing plasmid
pEMC-La and the full-length cDNA infectious clone. Thus, expressesion of the L
protein and MV antigenomes flanked by T7 promoter and a precise terminator will start
MV replication and transcription. In helper cells cytoplasm T7 polymerase will transcript
MV anti-genomic RNA that will form the RNP complex with N, P, and L protein. The
RdRp will then generate the MV genomic RNA that will be used the express all of MV
proteins. The progeny of rescued MV is propagated by overlaying the transfected rescue
cells with a cell line that support MV replication such as Vero cells expressing the
receptor SLAM[13].
Genomic modification of MV- insertion of foreign coding sequences-
To facilitate the cloning of foreign coding sequence into MV vectors, additional
transcription units (ATU) need to be inserted into the intergenic regions of the MV
antigenomic sequence. Inserting a multicloning cassette of unique restriction sites is
convenient to facilitate further cloning. The insertion of the ATUs in intragenic regions
allows the direct the transcription by RdRp of the foreign coding sequence. ATUs were
first inserted downstream of the P, H or L genes, in order toallow the possibiity of
modulating expression levels. Afterwards, a second generation of MV vectors directing
the expression of genes inserted downstream the N gene (in position 1710) was generated
to drive protein expression at almost maximum levels. The insertion of open reading
frames coding fluorescent markers have been used to test the functionality of the inserted
ATUs and also track the viral infection in vivo [13].
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Multivalent recombinant measles vaccines.
The attenuated MV vaccine is one of the most stable, safe, and effective human
vaccines. Vaccination using measles vaccine induces life-long protection against MV
infection. This strong and long-term protection could be extended if recombinant MV
expressing of other pathogens’ antigens are obtained and amplified. The MV vaccine has
been used for more than five decades, with a well-established production and distribution
network, therefore the cost of production of recombinant multivalent MV vaccine would
be relatively cheaper than other vaccines candidates. The MV vaccine platform is a safe
choice to deliver foreign antigens, as it replicates exclusively in the cytoplasm of the
infeted cells, thus the danger of integration into human genome is excluded. Also, MV
vaccine vectors have the capacity to carry and express foreign inserts without restriction
due to its helical capsid structure.
The capacity of MV genome to carry and expresses large inserts and the stability
of the expression make it a target of intense research to generate a variety of recombinant
multivalent vaccines[14-18]. The first multivalent MV-based vaccine was generated to
induce protective MV and hepatitis B virus (HBV) immune responses by expressing the
small surface antigen (HBsAg)[19]. The coding sequence was inserted downstream P, H,
and L genes. In HuCD46Ge-IFNarKO mice, the three recombinants MV induced
antibodies responses against both MV and HBV in which the vectors with the insertion
downstream P elicited the highest and the vectors with the insertion downstream L
elicited the lowest humoral immune responses against HBV. At that time, the P cassette
vector was the strongest expressing and generated an optimal immune response against
6
HBV. Rhesus monkeys immunized with the high-expression vector, with the HBsAg
insertion downstream of the P gene, had the most robust immune response against HBV
and remain protexted against a mealses virus challenge, providing proof -of-principle for
the divalent vaccine strategy, where a pediatric measles virus vaccine vector.
Furthermore, AIDS is arguably one of the most important infectious disease worldwide
against which a vaccination option has not been approved yet. HIV and MV efficiently
infect monocytes, dendritic cells, and macrophages and induce immune suppression.
Thus, an MV-derived vector has the ability to deliver HIV protein antigens to the same
target cells than HIV does, resulting in strong humoral and cellular immune responses
against HIV and MV. To test the immunogenicity of MV encoding both an HIV Gag
antigen inserted downstream the MV P gene, as well as an Env (gp 140) antigen
downstream H gene, macaques were immunized twice, two and a half months apart[20].
All vaccinated animals developed immune responses against measles and HIV. This
finding makes the MV-based HIV vaccine a promising candidate to be tested in human to
determine its safety and immunogenicity[21]. Chikungunya virus is a mosquito-
transmitted alphavirus causing millions of cases of severe polyarthralgia with no vaccine
currently available. Recently, a recombinant live attenuated measles vaccine (MV)
expressing Chikungunya virus (CHIKV) virus-like particles by inserting capsid and
envelope structural proteins coding sequences downstream the P position[22] was tested
in HuCD46Ge-IFNarKO mice with two doses of MV-CHIKV, ranging from 10^3 to
10^5 TCID50, given one month apart. High neutralizing antibodies titers were induced
(PRNT50= 450–4050 and PRNT90= 50–450). The MV- based CHIKV has been the first
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recombinant MV multivalent vaccine to progress to phase I trial based on
immunogenicity studies in mice. In this phase I clinical trial, healthy men and women
aged 18–45 years received either one of three doses of the measles-virus-based candidate
vaccine 1•5 × 10³, 7•5 × 10³ or 3•0 × 10⁰ TCID50 and then received a booster injection
on either day 28 or day 90 after the first vaccination. The primary endpoint was the
presence of neutralizing anti-chikungunya antibodies on day 28, as assessed by 50%
plaque reduction neutralization test. The candidate vaccine raised neutralizing antibodies
in all dose cohorts after the second vaccination dose resulting in a 100% seroconversion
for all participants. The immunogenicity of the candidate vaccine was not affected by
pre-existing anti-measles immunity which make it a promising measles-virus-based
candidate vaccine for use in human [23].
II- The quest for an optimal, Pediatric Dengue virus vaccine.
Epidemiology of dengue virus (DV).
In the last 60 years, the incidence of clinical cases of dengue infections reported
to WHO has increased 30-fold, with a much-increased geographic range, including the
expansion from predominantly urban to rural settings with a tremendous medical
importance in tropical and sub-tropical areas[24]. Dengue infections are endemic in large
parts of Africa, Asia, and Latin America with approximately 3.5 billion people living in
endemic countries. With over 500,000 estimated hospitalizations annually due to dengue
infections, of which 12000 cases are fatal, the tremendous burden of health care
represented by dengue virus has a high impact on health costs[25]. Most frequently,
8
Dengue infection results in a spectrum of clinical manifestation ranging from
asymptomatic to dengue fever (DF), which is characterized by flu-like-symptoms, the
patient will fully recover with no hospitalizations. Severe dengue infections might cause
life-threatening dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS)[26].
In DHF, patients experience hemorrhagic tendencies evidenced by spontaneous bleeding
from the mucosa, gastrointestinal tract, and other locations, as weel as thrombocytopenia
and evidence of plasma leakage due to increased vascular permeability. As proof for
capillary hemorrhage, patients with a high tendency to hemorrhage present a positive
tourniquet sign. DSS is characterized by a rapid or a weak pulse and narrow pulse
pressure. Hemodynamically, the shock syndrome is associated mainly with a generalized
vasodilation that causes a reduction in peripheral resistance. This severe dengue infection
is life threatening primarily in patients younger than 15 years old[27, 28].
Dengue virus is the most important arbovirus disease that is transmitted to human
by mosquito bites. Two mosquito vectors transmit dengue virus, Aedes.aegypti is the
predominant vector and Aedes.albopictus can sustain transmission in some endemic
areas[29]. Mosquitos are infected after the ingestion of infectious blood meal. DV infects
and multiplies in midgut epithelial cells. The viral infection gets amplified by infected
other mosquitoes’ cells, when it reaches the epithelial cells of the salivary gland the
mosquitos will be capable of transmitting the virus to human when feeding. The period
comprehended between the transmission of dengue virus from an infected human to the
mosquito and the subsequent amplification of dengue virus in mosquito’s cells is called
the extrinsic incubation period. Once the infected mosquitoes transmit the virus to a
9
human host, the virus will replicate into resident cells at the site of the mosquito bite. The
period between human infection and the onset of symptoms is known as intrinsic
incubation period[30].
The pathogenesis of Dengue infection.
As mentioned, DV infects human through the cutaneous route. After the mosquito
bites, the virus crosses skin epithelial cells and reaches the subepithelial tissues[30, 31].
Epidermal dendritic cells, known as Langerhans cells, are the primary targets of
infection. DV will be amplified in dendritic cells. Upon infection, dendritic cells get
activated and migrate to neighbor lymph nodes where monocytes and macrophages are
recruited and they get infected in a process called lymphatic spread of DV[32]. The
infected macrophages and monocytes will circulate through the blood stream, which
constitutes the primary viremia. Once the virus gets into the blood stream the viral
infection will be spread to other organs. Intense DV replication in targeted host cells
results in secondary viremia with an intensity of 10^5 to 10^6 dengue-infectious units per
ml at its peak. This period includes infection of blood-derived monocytes, myeloid
dendritic cells and tissular macrophages[33]. Infected macrophages will be major hosts in
the liver, spleen, and bone marrow stromal cells, while kidney, lung, and the central
nervous system infection by dengue is one of the main events associated with DHF and
DSS, where the viremia is 10 to 100 fold greater than in DF[26]. In DHF, infected
macrophages infect both hepatocytes and Kupffer cells resulting in necrosis and
apoptosis in liver. As a result, decreases in the liver function include the release of toxic
products, reducing the synthesis of coagulation factors, declining the level of platelets
10
and the development of coagulopathy disorder[34]. The replication of DV in bone
marrow stromal cells causes suppression of haemopoiesis that may cause lymphopenia
and leukopenia. In conjunction, severe thrombocytopenia and platelet dysfunction,
associated with eventual loss of vascular stability, can occur. Thus, increasing capillary
fragility and gastrointestinal mucosal bleeding is a characteristic of DHF. Furthermore, a
severe infection of endothelial cells causes plasma leakage as a feature of DSS[33].
Dengue virus biology.
Dengue is an enveloped, positive sense, single-stranded RNA virus of the
Flaviviridae family with an icosahedral scaffold of 90 glycoproteins envelope (E) dimers.
The DV complex has four serotypes (DV 1-4), phylogenetically the four DV serotypes
are diatintive and always cluster conspicuosly within the mosquito-borne flaviviruses
branch. The amino acid sequence of the envelope protein from the four serotypes are
homologous by 63-68%, DV 3 and 1 sequence is more related to each other than to the
other two serotypes[35]. The positive polarity genomic RNA is about 10.6 kbp with a
type I 5’ cap and a 3’ end lacking a polyadenylate tail. The genome encodes a single
long open reading frame (ORF) flanked by 5’ and 3’ noncoding regions including
structural and non-structural proteins which includes, capsid (C), precursor membrane
(PrM), envelope (E), nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and
NS5)[31]. The capsid (C) protein is a highly basic alpha-helical protein. The nascent C
contains a hydrophobic anchor at their C-terminal that serves as a signal peptide for
endoplasmic reticulum (ER) translocation of PrM and the first signal sequence to be
translated by cellular ribosomes. This hydrophobic domain is cleaved by viral proteases
11
to release mature C proteins, which fold into compact dimers with each monomer
containing alpha helices. These will then bind to the genomic RNA and interact with the
membrane surface to form the DV nucleocapsid or the viral core. The glycoprotein
precursor of M protein contains glycosylation sites at the N –terminal region while the C-
terminal has the transmembrane domain (TM) that act as ER- retention signals to keep
the protein associated with ER membrane. The PrM protein is about 26 KDa present on
the surface of the immature virus and responsible of forming a cap to cover the fusion
loop of the E protein to prevent this from fusion during transit through the secretory
pathway. During DV maturation, the PrM will be cleaved by cellular proteases to release
the glycosylated, N-terminal Pr fragment from the small, membrane anchored M protein
which is about 9 KDa and function as a scaffold to maintain E protein dimers folded
correctly[36, 37]. The envelope protein is type I integral glycoprotein and the major
protein on the surface of Dengue virions, This glycoprotein of about 53 KDa mediates
host cell receptor binding and membrane fusion[38]. Each E protein monomer contains
two helix transmembrane regions, which are about 50 a.a called stem and anchor regions
and contain an ER retention signal, which is responsible for association of the E protein
with ER membrane. The ectodomain is structurally divided into three domains that are
joined by a flexible junction. Domain I is the middle domain and participate in the
conformational changes induced during viral fusion. Domain II contains a hydrophobic
fusion loop that mediates the viral- cell membrane fusion. Domain III has an
immunoglobulin-like folding pattern and is responsible for attachment and receptor
binding onto host cells[39, 40]. Non-structural protein 1 (NS1) is present on the surface
12
of infected cells in form of highly stable homodimers or is secreted into the extracellular
space. The function of this protein in viral replication is not known. NS2A can act as an
interferon antagonist and assist DV evasion of the interferon inhibition of viral
replication. One of the most important nonstructural proteins is NS3 which carries out
multiple functions including serine-like protese, RNA helicase- NTPase and RTPase
enzymatic activities. The protease activity of NS3 depends on the NS2B that is a cofactor
forming an NS2B-NS3 serine protease complex. NS4A and B are small hydrophobic
proteins which acts as antagonist to block type I interferon-signaling pathways that are
essential to enhance DV replication. The largest (104 KDa) and most conserved protein
among DV serotypes is the NS5 protein that is the RNA-dependent RNA polymerase
(RdRp)[36, 41-44].
The basic steps of DV replication are attachment to cell surface; internalization;
release of the viral nucleocapsid and transfer of viral RNA into the cytoplasm; translation
of the viral polyprotein; replication of genomic RNA; assembly, maturation of virions,
and ultimately the release of mature viruses from the cells. Receptor recognition and
attachment of E glycoprotein and concentration of dengue virions on the surface of host
cells initiate viral infection by receptor-mediated endocytosis of the mature DV. The
plasma membrane vesicles bud into the cytoplasm and deliver the virus particle to early
endosomes in which the viral membrane- endosome membrane fusion occurs as the pH
dropps. The fusion is mediated by the hydrophobic sequence present in the fusion peptide
resulting in the generation of a pore in the endosome membrane that allows the release of
nucleocapsid into the cytoplasm. The RNA is then translated into a polyprotein and
13
directed into the endoplasmic reticulum (ER). Signal sequences within polyprotein
translocate the E, PrM, and NS1 proteins into lumen of the ER, while the C protein, NS3
and NS5 are released into the cytoplasm. The RdRp is used to replicate the genomic RNA
in the cytoplasm which then migrate to the ER where the assembly of the virus take place
once there are enough glycoprotein copies. The assembled immature virus particles
transit from ER through trans-Golgi network to the cell surface. During the maturation,
PrM protein is cleaved by cellular furin protease to Pr and M and will remain associated
with the virions, once the virions are released into the extracellular space the Pr peptide
will be released and virions will be on its mature infectious form where the E protein is
arranged in a 90 homodimer lattice liying upon the virus membrane and completely
covering the small M protein. DV replication can generate subviral particles that contain
only PrM and E, these particles are less dense than the virions and have significant
importance in vaccine development[31, 38, 40, 45, 46].
Immune response and immunopathogenesis of Dengue virus infection.
After the initial bite of an infected mosquito, host cells are the first line to mount
innate immune response to DV infection by ultimately activating type I interferon.
Infected cells posses cytoplasmic sensors for the replication of DV genomic RNA such as
retinoic acid inducible gene I (RIG-I), which triggers the anti-viral interferon responses in
infected cells, transfering signals to neighborhood cells to clear the infection[47]. DV has
evolved to escape the first line of innate defense through blocking the interferon system
signaling pathways using NS2A, NS4A, NS4B, and NS45[48, 49]. The cytokine release
set up a state of tissue inflammation that is associated with dilation of local blood vessels
14
and permeability increase in endothelial cells, allowing the recruitment of other immune
cells such as neutrophils and monocytes out of the blood vessel and into infected tissue
and active phagocytosis of DV present in the extracellular space. The most important
event of the innate immune response is the recruitment of natural killer (NK) cells that
are responsible for recognizing and killing infected cells. At this point, DV infection may
progress and spread into innate immune cells which will migrate to local lymph nodes for
subsequent presentation of DV antigen to naïve T and B-lymphocytes. Activated
lymphocytes coordinate the adaptive immune response which has two goals; the blocking
of viral entry by antibodies secreted by plasma cells (from mature B cells); and the
elimination of infected cells by cytotoxic T cells (CTL). CTL recognize DV antigenic
epitopes in complex with MHC class I molecules on the surface of infected cells where
CTL can be activated to secrete cytotoxic protein products including granzymes and
perforin to induce apoptosis of infected cells [50-53]. The activity of CTL is part of the
cellular immune response to clear DV in infected patients, however the most effective
immune response to curtail dengue infection is the humoral response mediated by
secreted antibodies. Effector functions of anti-DV antibodies includes neutralization of
DV; opsonization of DV coated by antibodies; complement activation; and antibody
dependent cellular cytotoxicity[54, 55]. The primary role of the antibodies is to block the
viral entry into target cells by recognizing and binding DV glycoproteins on the surface
of virions. Anti-E antibodies are responsible for neutralization and inhibition of DV
infection by binding to E glycoprotein and blocking DV attachment and entry. It has
been shown that the recognition and binding of neutralizing monoclonal antibodies to
15
exposed regions in the viral envelope causes the destabilization of the infectious particles
and interfere with viral entry[56]. Thus, the elicitation of neutralizing antibodies through
vaccination has been the most desired strategy to curtail the spread of mosquito-born
flavivirus[57]. For instance, the attenuated Yellow Fever virus (YFV) strain 17D has
been in use for more than 60 years. A single dose elicits seroconversion in 100% of
vaccinees. Therefore, neutralizing immunity is the most clear and documented correlate
of protection against dengue virus. Protective immunity against secondary infection of
DV is one of the most significant consequences of adaptive immunity by generating a
population of memory B-lymphocytes that produce rapid and vigorous immune responses
in secondary infection due to the same serotype. Individuals who have been infected with
one dengue serotype have long-term protection against that specific serotype and short-
term protection against the other three serotypes of DV.
The immune response to secondary heterotypic dengue infection can result in
unfavorable clinical outcomes such as DHF and DSS, which is known as
immunopathogenesis of DV infection. There is some evidence suggesting that specific
antibodies play a role in viral pathogenicity, such as the increased risk to develop DHF
and DSS during primary infection in infants whom are born to mothers with established
herteotypic immunity to DV. The tropism of DV for monocytes and dendritic cells, both
expressing Fc receptor for the heavy of IgG creates the opportunity for DV specific
antibodies to enhance the viral entry in a phenomenon called antibody- dependent
enhancement (ADE). In ADE, cells covered by Fc receptor will up take the virion-
antibody complex more efficiently than Dengue viral entry through specific cell-
16
receptors. This results in increasing of the viral loads and enhancement of the disease.
Therefore, development of dengue vaccine requires the generation of tetravalent vaccine
formulations to avoid the risk of ADE development in vaccinees[34, 55, 58-61].
Dengue vaccine candidates.
The first attempt to generate a Dengue vaccine dates from 1945 by A.Sabin who
tried to produce an attenuated DV bypassing the virus in mouse brain; the virus was
reactogenic and caused a rash in human volunteers[62]. Since this, there have been
efforts to develop a dengue vaccine; the reason why dengue is one of the most difficult
pathogens to develop a vaccine against is the difficulty to induce a balanced protection
without viral interference following the simultaneous vaccination against the four
serotypes[54]. Several vaccines candidates are under different stages of development
including six advanced candidates that are currently in various stages of clinical
development with vaccine trials in phases I–III[63]. Three candidates are in phase I, a
recombinant subunit vaccine based on the DV premembrane and truncated envelope
proteins (DV-PrM- 80% E) via antigenic expression in Drosophilla S2 cell system
(V180) by Merck[64]. A monovalent plasmid DNA expressing DV 1 PrM and E proteins
under control of the human cytomegalovirus promoter (D1ME 100) by Naval Medical
Research Center (NMRC)[65, 66]. And a tetravalent purified inactivated vaccine (TDEN
PIV) evaluated jointly by GSK and Walter Reed Army Institute of Research (WRAIR)
and tested in flavivirus naïve adults (USA) and dengue primed population (Puerto Rican)
with no adjuvants or with different type of adjuvants respectively[67, 68]. Two
17
candidates have advanced to the phase II trials, TV003/TV005, and DENVax that show
promising results to progress to phase III.
The National Institute of Allergy and Infectious Disease (NIAID) developed a
tetravalent vaccine based on wild-type strains with targeted mutation in the 3’
untranslated region to attenuate the virus. Vaccine virus serotypes 1, 3, and 4 are based
on whole attenuated virus, while serotype 2 is a recombinant virus based on attenuated
serotype 4 psuedotyped with serotype 2 PrM and E glycoproteins. TV003 and TV005 are
identical vaccine candidates except for the dosage of DV2 component, where each
component was given at a dose of 3 log10 PFU as a single subcutaneous dose with the
exception of the DENV2 component of TV005, which was given at a dose of 4 log10
PFU. To determine the safety and the immunogenicity of TV003 and TV005 in 168
flavivirus-naive adults, a single dose of TV003 induced seroconversion rates to DENV1–
4 of 92%, 76%, 97%, and 100%, respectively. Seventy-four percent of TV003 recipients
mounted a tetravalent response. A single dose of TV005 induced seroconversion rates to
DENV1–4 of 92%, 97%, 97%, and 97% , respectively and an overall tetravalent response
in 90% of recipients. A phase II study in Thailand started in early 2015 to examine safety
and immune responses of two doses of TV003 at 0 and 6 months in adults and children.
In a phase II, TV005 is currently being evaluated in an age de-escalation trial in Thailand.
A phase II study of TV003 in Brazil started in October 2013 in collaboration with the
Butantan Institute. Safety and immunogenicity of one dose of TV003 in dengue naïve and
dengue primed individuals will be evaluated with results completed by December
2018[69-73].
18
Takeda is also developing a live recombinant tetravalent vaccine DENvax (TDV)
including a mixture of a live attenuated DV2 and chimeric DV1, 3, 4 based on the
attenuated DV2 backbone[74]. The wild type DV2 was attenuated by passaging in
primary dog kidney (PDK) cells, the DEN-2-PDK-53 vaccine, which is further attenuated
by a mutation in NS3 gene and used as backbone for the rest of the vaccine component
by replacing the PrM and E of DV2 with ortholog genes from the respective virus wild
type serotypes. There have been several ongoing and completed phases II and I trails in
both endemic and dengue naïve populations. An ongoing phase II study of 344 children
and adults assessing the safety and immunogenicity of the vaccine in Colombia, Puerto
Rico, Singapore and Thailand started in 2011. In late 2014 a phase II study in 1800
children in Asia and Latin America commenced to examine immunogenicity of a 3 dose
schedules[75-77].
CYD-TDV (Dengvaxia®) is a tetravalent, live attenuated viral vaccine developed
by Sanofi Pasteur and licensed in five endemic countries: México, Philippines, Brazil, El
Salvador, and Paraguay. CYC-TDV is composed of 4 live attenuated chimeric viral
recombinants representing dengue 1, 2, 3, and 4 each one obtained by replacing the genes
encoding prM and E proteins in the attenuated YFV 17D genome with the corresponding
genes from the 4 serotypes of wild type dengue[78]. It is approved to prevent severe
dengue disease caused by DV in individuals 9 through 45 or 60 years of age, with a clear
contraindication to be used in children aged 2-5 years old due to safety concerns, as
evidenced after phase 3 clinical trials. The efficacy of the CYD14 included all children of
all ages where vaccine efficacy (VE) in young children was 33.7% compared to VE in
19
adult 74.3%[79, 80]. Vaccine efficacy measures the proportionate reduction in cases
among vaccinated person by calculating the risk of disease among vaccinated and
unvaccinated persons and determining the percentage reduction in risk of disease among
vaccinated persons relative to unvaccinated persons. Data from endemic and non-
endemic countries show an increase risk of hospitalization and severe dengue at later
follow up periods as a result of vaccination at age younger than 6 years (58
hospitalization cases in vaccinees in comparison with 23 among control recipients). Thus,
it is documented the possibility that vaccination may be not sufficient or even increase
the risk of hospitalization and severe dengue outcome in young children who are
seronegative at the time of first vaccination if this occurs at 2-6 years of age[81, 82].
Several hypotheses have been suggested to explain this result in seronegative young
children[83], including that the vaccine act as a silent natural infection that prime
seronegative vaccinees to experience a secondary-like infection upon their first exposure
to dengue virus (Figure 1.1)[2]. Following vaccination of individuals with no pre-existing
immunity, primary infection acts like secondary infection while in persons who were
vaccinated after experiencing one natural infection behave like a natural tertiary
infection. Under these conditions, very young individuals who were vaccinated while
seronegative are at higher risk for dengue disease upon infection.
20
Central Hypothesis
The recent phase III and II of the CYD-TDV, DENvax, and TV003/5 show
efficacy in terms of protection against severe dengue disease in old children and adult.
However, it has been documented the possibility that vaccination may be not sufficient or
even increase the risk of hospitalization and severe dengue in young children who are
seronegative at the time of first vaccination when this occurs at 2-6 years of age. The
need to protect young infants from dengue is imperative. The disease is more severe at
this age, and the intense virus replication can be the reservoir to sustain an epidemic
outbreak. Therefore, closing the gap between the timing of the current vaccine and risk of
severing Dengue infection is critical. The enhanced efficacy of the CYD-TDV vaccine in
a seropositive recipient with average vaccine efficacy of 74.6% in seropositive and only
34.5% in seronegative, proves that DV immune responses and protection efficacy is
majorly boosted when the recipient’s immune system is primed against flaviviruses due
to prior exposure to DV or other flavivirus. In this dissertation, we explored an
alternative pediatric vaccine strategy based on multivalent Dengue-Measles
immunization, to prime DV or other representative flaviviral neutralizing immune
responses, reducing the window of DV infection risk. We specifically hypothesized that
recombinant measles virus expressing Dengue 2 and 4 antigens would successfully
induce neutralizing responses against DV2 and 4 and prime cross-neutralizing responses
against other closely related or distance flaviviruses (Figure1.2). The goal of this
dissertation is to develop the concept for a measles vaccine viral vector (MVvac2) to
express the pre-membrane (prM) and the most immunogenic form of the major envelope
21
(E) from DV2 and DV4. Also to test homologous neutralizing responses and cross-
neutralizing immune responses elicited by single-component or combined recombinant
MV vaccines in MV-susceptible mice. I expect that this research would lay the
foundation for future experimentation in more relevant primate models.
Chapter Organization
Chapter 2 of this Dissertation is entitled “Broad heterologous protection in
flavivirus infections." describes homologous neutralizing immune responses to DV2 or
DV4 and heterologous neutralizing responses to DV1 and YFV 17 D (attenuated).
Additionally, in this chapter we show the correlation between titrating neutralizing
antibodies by 50% plaque reduction neutralization titer (PRNT50) and the logarithmic
neutralization index (LNI).
Chapter 3 of the dissertation is entitled "Generation of a complementary anti-
dengue immunization strategy based on a recombinant measles virus vaccine cocktail." It
describes the generation of three recombinant measles. Two of which expresses PrM and
two forms of DV2 E protein: full length or truncated (90% of E) and one recombinant
measles expressing PrM and truncated E of DV4. The two recombinant measles
expressing DV2 antigens replicate with equal efficiency to their parental strain. The
expression of DV2 antigens is evident by indirect immunofluorescence and western blot.
While both vectors are immunogenic, the virus expressing truncated E showed a
distinctive more immunogenic profile in MV-susceptible small animal model. Based on
that, we generated the recombinant measles expressing truncated E protein of DV4 and
22
showed that this vector is immunogenic as well. Finally, we combined the recombinant
measles expressing truncated E protein of DV2 and DV4. The cocktail of recombinant
measles generates homologous and heterologous immune responses against DV2 and 4,
and DV1 and YFV, respectively.
Chapter 4 of the dissertation is entitled "Generation of a recombinant measles
virus expressing full-length Dengue 4 envelope glycoproteins”. It describes the
generation of recombinant measles expressing full-length DV4 E protein. This virus
replicates with equal efficiency the parental strain in two cell lines. The expression of
DV4 antigens is evident by indirect immunofluorescence. Finally, the immunogenicity of
the virus is tested in MV susceptible mice. We documented that, despite clear differences
in DV4 E transmembrane region, immunogenicity against vectored DV4 Esol antigen
followed the same principle as the MV-vectored DV2 Esol.
Chapter 5 of the dissertation is entitled "Summary and future directions." It
discusses the implications of my findings presented in this dissertation as well as avenues
for future research.
23
Figure 1.1. Hypothesized mode of action of the current Dengue vaccine. Red arrow indicates exposure to dengue infection. The height of the boxes illustrates the chance of severe dengue of occurring. The syringe symbol shows the timing of vaccination. Upper panel shows the outcome of multiple dengue infections in naïve, unvaccinated individuals over their lifetime. Middle panel shows the impact of the vaccine on increasing the risk of severe disease in seronegative vaccinees. In this model, vaccine acts as natural silent primary infection, which put the vaccinated individuals at high risk to develop severe dengue upon exposure to wild type dengue virus. Lower panel shows vaccine-induced protection in seropositive individuals. Vaccination confers long-lived high-level protection against disease in individual who are vaccinated after a single flavivirus infection[2]
24
Figure 1.2. Schematic representation of our central hypothesis. A. Cartoon representation of a divalent measles-dengue vaccine concept and the expected neutralizing antibody responses. B. Proposed mode of action for a MV-Dengue vaccine. The syringe symbol shows the timing of vaccination. Seronegative infants receive a cocktail of MV-DV vaccine at one year old to prime their immune systems. At 18 month of age they receive the CYD-TDV, by this time all the vaccinees will be seropositive, closing the gap of opportunity for a wild type infection to cause severe Dengue.
Dengue2'Glycoproteins''
Measles'glycoproteins'
An56Measles'an5body''
An56Dengue'an5body'
Dengue4'Glycoproteins''
A'
B'!"#$%&'%()*%+",$%&-*."*/&
01231&$"44-*%&4)45/"-6&
'%()7)8,$-/9&
:%/("$"6%*/&31&1"44-*%&
;<&=)*/>&&)6?&
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8-A&=)*/>8&
B(-="(9&-*.%4,)*&!)&8%$%(%&3%*+C%& D)*+&6-.%&7()/%4,)*&
25
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DENVax given intradermally by needle or needle free PharmaJet injector. 2015 [cited 2016 10-14-2016]; Available from: http://clinicaltrials.gov/show/NCT01765426.
76. ClinicalTrials.gov. Safety and immunogenicity of different schedules of Takeda's
tetravalent dengue vaccine candidate (TDV) in healthy participants. 2015 [cited 2016 10-14-2016]; Available from: https://clinicaltrials.gov/show/NCT02302066.
77. Guy, B., et al., From research to phase III: preclinical, industrial and clinical
development of the Sanofi Pasteur tetravalent dengue vaccine. Vaccine, 2011. 29(42): p. 7229-7241.
78. Sin Leo, Y., et al., Immunogenicity and safety of recombinant tetravalent dengue
vaccine (CYD-TDV) in individuals aged 2–45 years: Phase II randomized controlled trial in Singapore. Human vaccines & immunotherapeutics, 2012. 8(9): p. 1259-1271.
79. Amar-Singh, H., et al., Safety and immunogenicity of a tetravalent dengue
vaccine in healthy children aged 2–11 years in Malaysia: A randomized, placebo-controlled, Phase III study. Vaccine, 2013. 31(49): p. 5814-5821.
80. ClinicalTrails.gov. Study of ChimeriVax™ tetravalent dengue vaccine in healthy subjects. 2015 [cited 2016 10-14-2016]; Available from: http://clinicaltrials.gov/show/NCT00875524.
81. ClinicalTrials.gov. Study of a novel tetravalent dengue vaccine in healthy children
aged 2 to 14 years in Asia. 2015 [cited 2016 10-14-2016]; Available from: http://clinicaltrials.gov/show/NCT01373281.
82. Guy, B. and N. Jackson, Dengue vaccine: hypotheses to understand CYD-TDV-
induced protection. Nature Reviews Microbiology, 2015.
32
CHAPTER 2
BROAD NEUTRALIZING RESPONSES ELICITED BY FLAVIVIRUS
INFECTION IN CD46 INFarKO MICE
ABSTRACT Arthropod-borne flaviviruses are a very conservative group of viruses that share
the envelope glycoprotein protein structure that determines its rigid eicosahedral
symmetry. As such, antibodies directed against conserved regions of this envelope
glycoprotein lattice can precipitate cross-reaction, cross neutralization and cross
protection among diverse flavivirus depending on antibody concentration, specificity and
avidity. We showed here that the inoculation of CD46-IFNarko mice with one dengue
serotype could generate neutralizing antibodies against homologous and heterologous
dengue virus serotypes. This animal host is relevant because it will be use for subsequent
immunogenicity experiments. As such this chapter is relevant because it gives substrate
to the search of cross-neutralizing respnses elicited by vectored viruses. Animals were
inoculated with DV2 or DV4 elicited anti-DV1 antibodies at 28 days postinfection with a
titer of 1:140 and 1:160 PRNT50, respectively. Most interestingly, we show that serum
from animals inoculated with dengue 2 virus generates a more robust cross-neutralizing
immune response to Yellow fever virus when compared to serum from animals
inoculated with Dengue 4 virus: 1:853 and 1:20 PRNT50, respectively. Furthermore, in
this chapter, we documented the correlation between two different methods to quantify
neutralizing antibodies in serum, the classic plaque reduction neutralization titer method
at an end point of 50 (PRNT50) and the log neutralization index (LNI).
33
IMPORTANCE
Dengue infections have been a major public heath concern. The data from the
recent clinical trial of CYD vaccine showed a new challenge for dengue vaccine
development. Naïve vaccinees with no previous exposure to any flavivirus infection were
at risk of generating severe dengue disease after receiving the tetravalent dengue vaccine
DengvaxiaMR. Here, we show that monotypic dengue infections generate a neutralizing
immune response to the homologous serotype as well as other dengue serotypes and
yellow fever. The titers of the neutralizing antibodies were different based on how closely
related the tested virus is. This finding is important because supports the notion of cross-
neutralizing protection given an adequate antigenic stimulus for vaccination.
34
INTRODUCTION Clinical responses to dengue infections are determined by the previous flaviviral
exposure; individuals who experienced primary dengue infection are at high risk in
generating a severe dengue disease upon a secondary, heterologous dengue infection than
dengue naïve people. Therefore, tetravalency has been a sine-equa-non requirement to in
dengue vaccine development to prevent the enhancement of secondary dengue
infection[84]. In monotypic dengue infections, young children are 6–7 times more likely
to be hospitalized with a secondary DV infection than are adults[85]. Furthermore, the
sequence of DV infection determined the clinical outcomes of the secondary infection.
For instance infections in the series DV1 then DV 2 or DV 3 are more likely to be severe
in nature than DV 2 then DV 3[86-88]. The enhancement of dengue disease in secondary
infection is explained by the presence of neutralizing antibodies at sub-protective levels.
However, such an effect does not occur in flavivirus seropositive patients. Patients with
Japanese encephalitis virus (JEV) immunity, either due to prior wild-type JEV infection
or immunization with the corresponding attenuated or inactivated vaccine, may
experience dengue infection that range in outcome from in apparent to mild disease, but
very rarely progress to severe forms of dengue disease[89]. The same observation is
verified among multitype DV immune individuals, where most of third DV infections
result in mild-moderate febrile disease that rarely requires hospitalization depite the fact
of the presence of antibodies against three DV serotypes[90]. The evaluation of clinical
responses to Sanofi Pasteur dengue vaccine in naïve, younger than five years of age
recipients, provided evidence for vaccine enhanced dengue disease, which is dependent
35
upon the serological status of vaccine recipients[83, 91]. Despite the presence of
neutralizing antibodies against the four DV serotypes, this group of vaccinees
experienced an enhancement of dengue disease. In seronegative children of all ages,
severe forms of dengue disease occurred more often in vaccinated (18/65) than in non-
vaccinated groups (6/39). In the Asian phase III trial (CYD14), vaccine efficacy in
seropositive children was somewhat lower in <9-year-old than ≥9-year-old seropositive,
70.1%, and 79.2%, respectively. However, in seronegative recipients, vaccine efficacy
was modest, in <9 year-olds as low as 14.4%. As a result, vaccinated seronegative
individuals presumably will retain risk to enhanced DV disease for many years[92, 93].
Therefore, it is imperative to consider improving the recipient’s serological status in
order to complement the use of the Sanofi vaccine to prevent severe forms of DV disease.
In this chapter, we explore viral neutralization among flaviviruses in CD46-
IFNarko mice immunized with DV2 or DV4. We demonstrated that dengue infection
could generate cross-neutralizing antibodies responses to heterologous dengue serotypes
(DV1) as well as relatively distance flavivirus (YFV 17). In addition we showed the
correlation between two different methods to quantify neutralizing antibodies: the classic
method, PRNT50, dependent on serial dillutions of tested serum to measure the dilution
that causes a 50% reduction in a determined number of plaques, and the LNI method that
depends on mixing equal volume of the virus with serum and thus measures serum
impact on viral infectivity. We observed that LNI gaves us a larger dynamic range of
neutralization measurements, furthermore LNI represents the only vaccine correlate of
protection for a flavivirus, YFV.
36
MATERIALS & METHODS
Cells and viruses.
BHK 21 cells were maintained in DMEM (DMEM, Sigma-Aldrich, St. Louis,
MO, USA) supplemented with 10% FBS (FBS, Atlanta Biologicals, Flowery Branch,
GA, USA), and 1% penicillin-streptomycin (Sigma-Aldrich). DV1, DV2, and DV4
viruses were obtained from the ATCC (VR-1586 strain TH Soman, VR-1584 strain New
Guinea C and VR-1257 strain H241, respectively) and propagated in Vero cells. The
viruses were then amplified in BHK21 cells for up to two or three passages. YFV 17D
viral strain was obtained using reverse genetic techniques in BHK-21 cells from the full-
length plasmid pACNR-FLYF\17 [94]and propagated in a similar way as DV.
Mice inoculations.
All experimental procedures were performed according to a protocol approved by
the Arizona State University Institutional Animal Care and Use Committee. Mice were
inoculated by the intraperitoneal (i.p.) route with the indicated doses of 10^5 PFU/ml
diluted in 100 uL of Opti-MEM. Five weeks after the inoculation, mice were
exsanguinated. Sera were separated and heat-inactivated at 56°C for 1 h[95].
DV and YFV neutralization assay.
To determine DV2, DV4, DV1, and YFV Log10 Neutralization Index titer the
corresponding viral prep was mixed with heat-inactivated serum in a 1:1 proportion in a
25 uL volume of Opti-MEM and incubated for 1 h at 37 °C. Infectivity after serum
incubation was determined using a plaque assay in BHK-21 cells. Briefly, ten-fold serial
dilutions of the virus/serum mixtures were generated using Opti-MEM and added to 2.4 ×
37
106 BHK-21 cells in a 24-well plate and then incubated for 4 h at 37 °C. At this time, 500
uL of 3% carboxymethyl cellulose was added to each well and plates were incubated for
5 days at 37 °C. The wells were then washed; the monolayer was fixed and stained with
acetic acid/naphtol blue-black and plaques were counted. The neutralization index was
expressed as the logarithmic difference between the averaged DV2, DV4, DV1 or YFV
titers when incubated with control, namely, that of animals inoculated with MVvac2, and
preimmune. Neutralization indexes thus determined were graphed as Log10NI.
In another approach, we performed a standard 50% plaque reduction
neutralization assay (PRNT50) using pooled sera from each experimental group. Briefly,
50 DV2, DV4, DV1 or YFV PFU were incubated with two-fold serum dilutions (from
1:5 to 1:320) for 1 h at 37 °C. Remaining infectivity was determined by plaque assay, as
described above. The neutralization titer was defined as the dilution that resulted in a
50% reduction in plaque numbers, as compared to unrelated serum.
Correlation of PRNT50 and LNI
Serum obtained from single animal inoculated by either DV2 or DV4 was used in
this experiment. Two fold serum dilution (from 1:5 to 1: 12560) was incubated with 50
PFU of DV2 or DV4 to perform a standard 50% plaque reduction neutralization assay
(PRNT50), as described above. The same dilution was used to perform a Log10
Neutralization Index titer by mixing the corresponding virus in a ration of 1:1 to the
diluted serum in 25 ul volume and incubated at 37C for 1 hr. Infectivity after serum
incubation was determined as described above.
38
RESULTS
Monotypic Dengue virus immunity
To rank the immunogenicity of homologous dengue serotypes, we immunized
groups of three huCD46Ge-IFNarKO mice with one 105 PFU/ml intraperitoneal dose of
DV2 or DV4, and analyze neutralizing immune responses siw weeks postinoculation. We
assessed DV2 or DV4 neutralization using two related approaches. First, a Log10
neutralization index (LNI), where the neutralizing effects of individual 1:2 serum
dilutions upon a high titer viral DV2 or DV4 prep infectivity was calculated using
averaged preimmune measurements to determine basal, non-neutralizing conditions. Thus
preimmune, or non-reactive samples are set for an LNI of zero. Additionally, we
determined the classic PRNT50 titer. As shown in figure 2.1A, serum samples from a
group of animals inoculated by DV2 reduced the infectivity of a 105.48 PFU/ml DV2 viral
prep by 4.2 orders of magnitude after one dose, which is equivalent to a PRNT50 of
1:1280. In figure 2.1C, serum samples from a group of animals inoculated by DV4
reduced the infectivity of DV4 (106 PFU/ml) by 4.63 orders of magnitude after one dose,
which is equivalent to PRNT50 of 1: 2133.3.
Subsequently, we explored the correlation between the titer of neutralizing anti-
DV2 or DV4 expressed by LNI and PRNT50. As shown in figure 2.1B and 2.1D, there is
a consistent correlation between the titer of neutralizing antibodies expressed either by
LNI or PRNT50.
39
Heterologous cross-neutralization immunity to DV1 and YFV
To explore the heterologous immune response of DV2 and DV4 we performed
viral neutralization assays against DV1 and the more phylogenetically distant YFV 17D.
Figure 2.2 documents that serum samples from four groups of the experimental animals
received one dose of DV1, DV2, DV4 and YFV measurably neutralize these two
flaviviruses. The infectivity of a high titer (104.84 PFU/ml) DV1 viral prep was
neutralized for an averaged LNI of 4.2 equivalent to 1:960 PFU after one dose DV1
vaccination. A lower titer was obtained after DV2 or DV4 one-dose inoculation scheme.
The documented DV1 LNI neutralization titer for DV2 and DV4 experimental groups
was 2.2 and 1.9, respectively, which equivalent to 1:426 and 1: 480 PRNT50 (Fig 2.2A).
In a comparable result, all of the animals neutralized YFV. An YFV 17D high
titer viral prep (106.78 PFU/ml) infectivity was neutralized by an LNI average of 3.86 and
0.9) equivalent to a PRNT50 of 1:853 and 1:20 after DV2 or DV4 inoculation,
respectively. Of note, an LNI of 0.7 is the reported correlate of protection for YFV
(equivalent approximately to a PRNT50 of 1:10)[96]. The YFV LNI neutralization titer
of YFV experimental group was 4.13, which equal to 1:1706 PRNT50 (Figure2.2B).
40
DISCUSSION
In this chapter, we have shown that immunization by DV2 or DV4 can elicit
neutralizing immune responses in protective ranges against the homologous DV2, or
DV4 and also against the heterologous DV1 and the relatively phylogenetically distant
YFV 17D attenuated strain. It has been known that primary infection with one-dengue
serotypes can generate a long-term protection against that serotype and a short term
(around one month) protection against any of the other three DV serotypes[97]. The
efficacy of a tetravalent dengue vaccine depends on the presence of flavivirus
immunity[91, 93]. Therefore, immunization strategy using a cocktail of different
flavivirus can be used to protect against a wide range of flavivirus infection including the
four dengue serotypes[98]. There are two important factors when we consider cross
protection against flaviviruses, the level of the cross-neutralizing antibodies, and the type
of flaviviruses that circulate in that endemic area[99]. Human volunteers inoculated seven
years previously with 17D yellow fever responded with a rapid production of neutralizing
antibodies to yellow fever and dengue type 1 and 2 when inoculated with an attenuated
DV2. The ability of these individuals to respond with anamnestic neutralizing antibodies
when infected with an heterologous flavivirus obeys to their previous exposure to
relatively closely flavivirus due to conserved artgropod-borne flaviral structures[100].
Such a concept is very critical when considering the development of dengue vaccine, as it
can be used as a novel strategy to design a vaccination strategy using a cocktail of closely
related flaviviruses with a combination of different serotypes of dengue. Multiple
observations of cross protection among flavivirus using different animal hosts have been
41
reported. Sequential immunization of spider monkeys with 17D YFV, DV2, and JEV
protected animals from challenges by DV1, 3, and 4[99]. Inoculation of hamsters with
DV1 or DV2 protect against a West Nile (WN) virus or St. Louis encephalitis virus
challenge[101]. In a remarkable work by Winston Price, spider monkeys immunized
sequential with 17D YFV, live attenuated Langat E5 virus, and live attenuated DV2 were
protected against viral challenges with more than 14 different flavivirus including
DV1,2,3,4, JEV, WNV, and Zika. Importantly, this sequential immunization did not only
protect monkeys against all four dengue serotypes but also generated a solid immunity
against the four serotypes as determined by no dengue antibody booster HI responses,
which supports the notion that the sequential immunization with 3 related flaviviruses
would not cause enhancement of dengue disease[98]. This strategy depends on the
sequence of the immunization and type f flavivirus election; the virus chosen for the use
in vaccination must give the broadest heterologous protection[102].
In sum, we showed that immunization with type 4 or 2 dengue virus generates
cross-neutralizing immune responses to 17D YFV. Unexpectedly, animals inoculated by
DV2 had a more robust neutralizing response to YFV than animals immunized by DV4,
despite the fact that DV4 is more closely related to YFV phylogenetically. More work is
needed to test the perfect combination of viruses to generate the broadest neutralizing
immune response among different flaviviruses. In the following chapter, we documented
the homologous immune responses and heterologous cross immune response of animals
inoculated by recombinant measles expressing monovalent dengue antigens or a cocktail
of recombinant measles expressing divalent dengue antigens.
42
Figure2.1. Monotypic immunogenicity after Dengue 2 and 4 inoculation. Mice received one IP dose of DV2 or DV4. Neutralizing immune responses were analyzed 32 days later. (A) and (B) Analysis of neutralization against Dengue 2; and (C) and (D) against Dengue 4 viruses. Anti-dengue neutralizing immune responses are assessed by LNI (left panel) and PRNT50 (right panel) in A and C. Each symbol represents one animal; horizontal lines and vertical brackets indicate group average and standard deviation, respectively. In B and D the correlation between anti-dengue LNI (Y axes) and PRNT50 (X axes) were determined.
43
Figure 2.2. Cross-neutralizing immune responses in mice inoculated with DV1, DV2, DV4, and YFV (A) Analysis of neutralization against Dengue 1 and (B) against attenuated YFV. Anti-dengue and anti-YFV neutralizing immune responses are assessed by LNI (left panel) and PRNT50 (right panel). Averages of anti-Dengue and Anti-YFV LNI and PRNT50 titers determined in triplicate of pooled serum samples are indicated.
44
REFERENCES
1. Wilder-Smith, A. and D.J. Gubler, Dengue vaccines at a crossroad. Science, 2015. 350(6261): p. 626-627.
2. Guzman, M.a.G., et al., Effect of age on outcome of secondary dengue 2
infections. International journal of infectious diseases, 2002. 6(2): p. 118-124. 3. Guzman, M.G., et al., Epidemiologic studies on Dengue in Santiago de Cuba,
1997. American journal of epidemiology, 2000. 152(9): p. 793-799. 4. Guzman, M.G., et al., Epidemiological studies on dengue virus type 3 in Playa
municipality, Havana, Cuba, 2001–2002. International journal of infectious diseases, 2012. 16(3): p. e198-e203.
5. OhAinle, M., et al., Dynamics of dengue disease severity determined by the
interplay between viral genetics and serotype-specific immunity. Science translational medicine, 2011. 3(114): p. 114ra128-114ra128.
6. Anderson, K.B., et al., Preexisting Japanese encephalitis virus neutralizing
antibodies and increased symptomatic dengue illness in a school-based cohort in Thailand. PLoS Negl Trop Dis, 2011. 5(10): p. e1311.
7. Kosasih, H., et al., Report of four volunteers with primary, secondary and tertiary
dengue infections during a prospective cohort study. Dengue Bulletin, 2006. 30: p. 87.
8. Capeding, M.R., et al., Clinical efficacy and safety of a novel tetravalent dengue
vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. The Lancet, 2014. 384(9951): p. 1358-1365.
9. Guy, B. and N. Jackson, Dengue vaccine: hypotheses to understand CYD-TDV-
induced protection. Nature Reviews Microbiology, 2015. 10. Hadinegoro, S.R., et al., Efficacy and long-term safety of a dengue vaccine in
regions of endemic disease. New England Journal of Medicine, 2015. 373(13): p. 1195-1206.
11. Villar, L., et al., Efficacy of a tetravalent dengue vaccine in children in Latin
America. New England Journal of Medicine, 2015. 372(2): p. 113-123. 12. Bredenbeek, P.J., et al., A stable full-length yellow fever virus cDNA clone and
the role of conserved RNA elements in flavivirus replication. Journal of general virology, 2003. 84(5): p. 1261-1268.
45
13. Mrkic, B., et al., Measles virus spread and pathogenesis in genetically modified
mice. Journal of virology, 1998. 72(9): p. 7420-7427. 14. Mason, R.A., et al., Yellow fever vaccine: direct challenge of monkeys given
graded doses of 17D vaccine. Applied microbiology, 1973. 25(4): p. 539-544. 15. SANGKAWIBHA, N., et al., Risk factors in dengue shock syndrome: a
prospective epidemiologic study in Rayong, Thailand I. The 1980 outbreak. American journal of epidemiology, 1984. 120(5): p. 653-669.
16. Price, W.H., et al., Sequential immunization procedure against group B
arboviruses using living attenuated 17D yellow fever virus, living attenuated Langat E5 virus, and living attenuated dengue 2 virus (New Guinea C isolate). The American journal of tropical medicine and hygiene, 1973. 22(4): p. 509-523.
17. PRICE, W.H., Sequential immunization as a vaccination procedure against
dengue viruses. American journal of epidemiology, 1968. 88(3): p. 392-397. 18. Schlesinger, R.W., et al., Clinical and serologic response of man to immunization
with attenuated dengue and yellow fever viruses. The Journal of Immunology, 1956. 77(5): p. 352-364.
19. PRICE, W.H. and I.S. THIND, Protection against West Nile virus induced by a
previous injection with dengue virus. American journal of epidemiology, 1971. 94(6): p. 596-607.
20. Price, W.H., et al., A sequential immunization procedure against certain group B
arboviruses. The American journal of tropical medicine and hygiene, 1963. 12(4): p. 624-638.
46
CHAPTER 3
GENERATION OF A COMPLEMENTARY ANTI-DENGUE IMMUNIZATION
STRATEGY BASED ON A RECOMBINANT MEASLES VIRUS VACCINE
COCKTAIL
ABSTRACT
Despite the approval of a Dengue virus (DV) vaccine in five endemic countries,
dengue prevention would benefit from an immunization strategy highly immunogenic in
young infants and not curtailed by viral interference. Vaccine efficiency is majorly
improved when the recipient’ immune system has been previously primed against related
flaviviruses. To create a pediatric vaccination strategy with the potential to prime
neutralizing anti-flaviviral responses in one-year-old infants, we modified a measles
vaccine viral vector (MVvac2) to express the pre membrane (prM) and a truncated (90%)
form of the major envelope (E) from DV2 and DV4. Single-component recombinant MV
vaccines elicited robust homologous neutralizing responses in all MV-susceptible mice
(1:214 and 1:640 PRNT50 against DV2 and DV4 in average). When MV-DV 2/4
recombinant vaccines were combined, we documented an enhancement in homologous
neutralizing responses and cross-neutralizing immune responses against DV 1 and YFV
17D in all experimental animals. Thus, we can prime broad neutralizing immune
responses using only two of the four available DV serotypes. Using the current MV
immunization scheme to prime anti-flaviviral neutralizing immunity in young infants
47
may be a highly convenient and cost-effective strategy to robustly enhance the
immunogenicity and efficiency of subsequent chimeric Dengue vaccine approaches.
48
IMPORTANCE
The four dengue virus serotypes are the cause of the most important arthropod-
borne viral disease worldwide. After six decades of research, a dengue vaccine has been
approved in five countries and another two candidates are approximating approval. The
two most important issues documented so far are the lack of efficiency in young
recipients and an uneven protection against the four DV serotypes. Since the dengue
vaccine performs better when the host has previous anti-flaviviral immunity, we proposed
here an attractive solution to both issues by using a recombinant measles vaccine
vectoring dengue protective antigens to be administered to young infants. We have
generated recombinant measles virus expressing Dengue 2 and 4 antigens that
successfully induce neutralizing responses in experimental animals. Interestingly, when
combined, the vaccine cocktail is able to prime cross-neutralizing responses against DV 1
and the relatively distant 17D yellow fever virus attenuated strain. The vaccine
equivalency of our MVvac2 viral vector suggests usage of a recombinant measles-dengue
cocktail immunization to prime a broad, anti-flaviviral response in one year-old recipients
that would be majorly boosted when the chimeric Dengue vaccine approach is used later.
49
INTRODUCTION
In terms of human suffering and economic impact, infections caused by any of the
four existing Dengue virus (DV) serotypes cause the most important arthropod-borne
viral infectious disease in the world. Currently, it is estimated that almost half of the
globe population is at risk of contracting Dengue, mainly people living in Asia, Africa
and Latin America, in this order. With an annual incidence of Dengue clinical cases
estimated at 100 million and a case fatality rate of 1%, the introduction of a vaccine
strategy for countries with a high prevalence of dengue has been an important advance to
control this devastating infectious disease[103, 104].
As for other arthropod-borne flaviviruses such as Yellow Fever virus (YFV)
Japanese encephalitis virus or Thick-borne encephalitis virus, protective immunity
against DV is associated with the development of neutralizing antibodies[105]. These are
majorly directed against the envelope (E) glycoprotein[106, 107]. DV envelope displays
an icosahedral array of ninety E dimers held structurally in place by a subjacent
membrane (M) protein network[35]. After a natural DV infection, a serotype-specific and
long-lived neutralizing immunity is developed, thus the formulation of a tetravalent
vaccine with the capability of eliciting viral neutralization has been the goal for more
than six decades[55, 63]. The currently approved DV vaccine (DengvaxiaTM) consist of a
tetravalent formulation of attenuated chimeric viruses where the envelope glycoproteins
from the relevant DV serotype enclose the replication machinery of the attenuated YFV
17D vaccine strain. After phase three clinical trials and a careful analysis of
immunogenicity and vaccine efficiency, Dengvaxia was approved for use in highly
50
dengue endemic countries targeting 9-45 years-old population, exclusively. This
indication represents the main limitation for this vaccination strategy, namely an
individual with a high probability of having pre-existent immunity against Dengue [91,
108-111].
In contrast, measles virus (MV) vaccination has been available for more than six
decades with excellent records of safety and efficiency. The current two-dose vaccine
schedule recommended by the WHO is credited to be responsible of eliminating
indigenous MV transmission in the American continent and is the spearheading strategic
effort to eradicate this infectious disease[112].Ours and other groups have proposed to
use viral vectors with MV vaccine equivalency to generate divalent and multivalent
vaccine options[14-16]. To give substrate to this overarching aim we have documented
that recombinant MV vaccines remain protective against a measles challenge [113].
In this paper, we showed that vectored MV-DV2 and MV-DV4 vaccines expressing
relevant DV envelope proteins and administered simultaneously, are able to elicit broad
neutralizing immune responses in MV-susceptible mice. Using MV vaccination to
purposefully enhance DV seroprevalence in the very young has the potential to expand
the indications of the current DV vaccine.
51
MATERIALS AND METHODS
Cells and Viruses
Vero/hSLAM cells[114, 115] were maintained in Dulbecco’s modified Eagle’s
medium with high glucose concentration (DMEM, Sigma-Aldrich, St. Louis, MO, USA)
supplemented with 5% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch,
GA, USA), 1% penicillin–streptomycin (Sigma-Aldrich) and 0.5 mg/mL G418 (Enzo
Life Sciences, Farmingdale, NY, USA) to maintain hSLAM expression selection. BHK
21 cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-
streptomycin. Helper 293-3-46 cells [116]a derivative from HEK 293 cells were
maintained in DMEM with 10% FBS, 1% PS, and 1.2 mg/mL G418.
DV1, DV2 and DV4 viruses were obtained from the ATCC (VR-1586 strain TH
Soman, VR-1584 strain New Guinea C and VR-1257 strain H241, respectively) and
propagated in Vero cells. The viruses were then amplified in BHK21 cells for up to two
or three passages. YFV 17D viral strain was obtained using reverse genetic techniques in
BHK-21 cells from the full length-plasmid pACNR-FLYF\17[94] and propagated in a
similar way as DV.
Recombinant MVs were generated using the method of Radecke et al. [117]
modified by Parks et al.[118]. After detection of their cytopathic effect on mixed cultures
of Helper 293-3-46 and Vero/hSLAM cells, individual syncytia were transferred to and
propagated in Vero/hSLAM cells. Viral clones stocks were amplified by infecting
Vero/hSLAM cells at a multiplicity of infection (MOI) of 0.03 and incubated at 37°C.
When approximately 80% cytopathic effect was observed, cells were scraped in Opti-
52
MEM (Life Technologies, Grand Island, NY) and viral particles were released by two
freeze-thaw cycles.
Multi-step growth kinetics of the viruses were determined by infecting
Vero/hSLAM cells at an MOI of 0.03 and incubating them at 37°C. Infected cells and
supernatants were collected and lysed by a single cycle of freeze-thaw at prescribed times
post-infection, and the 50% tissue culture infectious dose (TCID50) was determined in
Vero/hSLAM using the Spearman-Kärber end-point dilution method [119]. Primary
chick embryo fibroblast (CEF) were obtained from ATCC (CRL-1590 F2) and grown in
Dulbecco’s modified Eagle’s medium with high glucose concentration (DMEM, Sigma-
Aldrich, St. Louis, MO, USA) supplemented with 10% FBS, 1% penicillin–streptomycin
and 10% tryptose phosphate broth (Gibco, Life Technologies). Kinetics of infection of
CEF were performed by infecting CEF at MOI of 0.03 with MV and DV-expressing
recombinant MV and collecting cell-associated and supernatant at the indicated time
post-infection.
Plasmids and plasmids constructions
The plasmid pB(+)MVvac2(HBsAg)N was used to construct the recombinant
MVs expressing DV antigens. This plasmid contains an infectious MV cDNA
corresponding to the antigenome of the Schwarz measles vaccine strain [113, 120] with
an additional transcription unit (ATU) inserted downstream of the nucleocapsid (N)
cistron to direct the expression of an inserted foreign gene. To generate the recombinant
measles expressing DV2 or DV4 prM- E or prM-Esol glycoproteins, MluI and AatII
digestion fragments containing the desired coding sequence were obtained by PCR
53
(primer sequence available upon request) and swapped with the HBsAg insert. DV2
prME coding sequences were obtained from the full-length PD2/I/C/30P/A plasmid
(strain 16681[121, 122], and DV4 prME coding sequences were obtained from the full-
length pR424-D4 plasmid (H241 strain). The recombinant full-length plasmids were
termed pB(+)MVvac2-DV2(prME)N, pB(+)MVvac2-DV2(prMEsol)N, pB(+)MVvac2-
DV4(prME)N and pB(+)MVvac2-DV4(prME)N. Correct insertions and relevant gene
boundaries were corroborated by sequencing.
Indirect Immunofluorescence Microscopy
Vero/hSLAM cells were seeded into 4 well glass Millicell EZ slides
(MILLPORE) and infected at MOI of 0.06, 0.1 and 1 with MV, recombinant MV,
DENV2 and 4, respectively. After 48 hours of incubation at 37 C, the cells were fixed
with 3% paraformaldehyde and incubated at 37 °C overnight. The cells were
permeabilized with 0.1% Triton X-100 in phosphate- buffer saline (PBS). Both primary
and secondary antibodies dilution were prepared in 0.1% triton X-100 in 10% FBS serum
in 1 X PBS. Two Mixes of primary antibodies were used, monoclonal antibody directed
against DENV 1,2,3,4 E protein at 1:50 dilution (a mouse IgG2a cat. # 76 ab9202 from
abcam) with the polyclonal antibodies directed against MV N protein at 1:300 dilution (a
rabbit IgG antibody provided from R. Cattaneo). The primary antibodies incubations
were carried out overnight at 4 °C. A mix of goat anti-mouse IgG coupled to Fitc (Green
fluorescence), and a goat anti-rabbit IgG coupled to PE (Red fluorescence) were used as a
secondary antibody with dilution of 1:300 and incubated at room temperature for 2 hours.
The coverslips were mounted on slides by using flouromount G with DAPI (Electron
54
Microscopy Science). The slides examined and images were taken by inverted
microscope with epifluorescence illumination (EVOSFL).
Western Blot Assay
Vero/hSLAM cells were seeded in 100-mm-diameter dishes and infected at an
MOI of 0.3 with MV or recombinant MVs, or were mock-infected. Forty h after
infection, cells were washed three times with phosphate-buffered saline (PBS) then lysed
with RSB-NP40 buffer (1.5 mM MgCl2, 10 mM Tris-HCl, 10 mM NaCl, and 1%
Nonidet P-40, Sigma-Aldrich, St. Louis, MO) plus protease inhibitors (cOmplete Mini
Protease Inhibitor Tablets, Roche Diagnostics, Manheim, Germany). The protein extracts
were mixed with Laemmli buffer (Bio-Rad Laboratories, Hercules, CA) containing β
mercaptoethanol and denatured at 96C.
25 ul of protein extraction loaded and separated by polyacrylamide gel
electrophoresis (PAGE) in a 4-15% or 10% acrylamide gel. Following electrophoretic
separation, proteins were transferred to nitrocellulose for immunoblotting using a
1:20,000 dilution of rabbit polyclonal anti-MV N, a 1:100 dilution of a mouse
monoclonal antibody directed against DV2 E DIII (GTX29202, GeneTex, Irvine, CA,
USA) or 1:100 dilution of a mouse monoclonal antibody directed against DV4 E protein
(clone E62, VR-1726, ATCC, VA, USA) , or a 1:10,000 dilution of mouse anti-actin
(Sigma-181 Aldrich, St. Louis, MO), followed by the appropriate horseradish peroxidase
(HRP) conjugated secondary antibody (GE Healthcare, Little Chalfont, United
Kingdom). Reactions were developed using a chemiluminescence kit (SuperSignal West
Pico Chemiluminescent Substrate, Pierce Biotechnology, Rockford, IL, USA).
55
Glycosidase treatment.
MVvac2DV2(PrME)N or MVvac2DV2(PrMEsol)N infected cell lysates were
treated with endoglycosidase H (endo-H) and peptide N-glycosidase F (PNGase-F) (both
from NEB, Ipswich, MA) as per the manufacturer’s instructions for 1 h at 37°C. Protein
material was analyzed by immunoblotting using anti-H- or anti-E-specific antibodies.
Mice inoculations.
All experimental procedures were performed according to a protocol approved by
the Arizona State University Institutional Animal Care and Use Committee. To determine
the immunogenicity of recombinant MVs in a MV susceptible small animal host, groups
of HuCD46Ge-IFNarKO mice [123] were inoculated by the intraperitoneal (i.p.) route
with the indicated doses diluted in 100 uL of Opti-MEM. Three weeks after the initial
inoculation, the mice received a similar booster dose. Two weeks after the second dose,
mice were exsanguinated. Sera were separated and heat-inactivated at 56°C for 1 h.
MV neutralization assay.
To determine MV neutralization titer, serial two-fold dilutions of heat-inactivated
sera in Opti-MEM were incubated with 100 TCID50 of MVvac2 for 1 h at 37 °C.
Vero/hSLAM cells were added to the serum–virus mixtures and the plates were then
incubated at 37 °C for three days, at which time neutralization titer was assessed as the
highest dilution of serum capable of complete neutralization of infectivity, defined as the
56
absence of MV cytopathic effect[124]. Neutralization titers were reported as the average
of a determination made in triplicate.
DV and YFV neutralization assay.
To determine DV2, DV4, DV1, and YFV Log10 Neutralization Index titer the
corresponding viral prep was mixed with heat-inactivated serum in a 1:1 proportion in a
25 uL volume of Opti-MEM and incubated for 1 h at 37 °C. Infectivity after serum
incubation was determined using a plaque assay in BHK-21 cells. Briefly, ten-fold serial
dilutions of the virus/serum mixtures were generated using Opti-MEM and added to 2.4 ×
106 BHK-21 cells in a 24-well plate and then incubated for 4 h at 37 °C. At this time, 500
uL of 3% carboxymethyl cellulose was added to each well and plates were incubated for
5 days at 37 °C. The wells were then washed, the monolayer was fixed and stained with
acetic acid/naphtol blue-black and plaques were counted. The neutralization index was
expressed as the logarithmic difference between the averaged DV2, DV4, DV1 or YFV
titers when incubated with control, namely, that of animals inoculated with MVvac2, and
preimmune. Neutralization indexes thus determined were graphed as Log10NI. In another
approach, we performed a standard 50% plaque reduction neutralization assay (PRNT50)
using pooled sera from each experimental group. Briefly, 50 DV2, DV4, DV1 or YFV
PFU were incubated with two-fold serum dilutions (from 1:5 to 1:320) for 1 h at 37 °C.
Remaining infectivity was determined by plaque assay, as described above. The
neutralization titer was defined as the dilution that resulted in a 50% reduction in plaque
numbers, as compared to unrelated serum.
57
RESULTS
Generation and characterization of recombinant MVs expressing Dengue 2 envelope
antigens
Initially, to generate a MV-DV2 divalent vaccine vector, we modified a full-
length infectious cDNA plasmid named p(B(+)MVvac2. This plasmid and its derivatives
can be used to recover recombinant MVs by well-established reverse genetic techniques
[125, 126]. Two forms DV2 prME coding sequences were inserted downstream MV N
gene. The first one consisted of prM and E full-length coding sequences preceded by the
signal sequence of the carboxyl terminus of the capsid protein (C anchor). The recovered
virus was termed MVvac2-DV2(prME)N and expresses intact full length prM-E
glycoproteins. The second insertion consisted of similar C-anch, prM and a truncated
version of DV2 E. We deleted 45 amino acids from its carboxyl terminus corresponding
to E cell membrane anchor and cytoplasmic tail. We named the virus derived from this
construction MVvac2-DV2(prMEsol)N because this virus expresses an unattached
version of the DV2 E glycoprotein (Fig 3.1A). To corroborate the effect of DV2 prME
insertions upon recombinant MV replication fitness, we performed multistep growth
analysis using two cell substrates. Initially, viral replication in Vero/hSLAM did not
demonstrate a major deleterious effect because of the genetic modification on MV
backbone. Intracellular MVvac2-DV2(prME)N viral yields reached 6.6 Log10 TCID50/ml
at 48h PI, which were slightly higher than but not significantly different of the parental
strain MVvac2. MVvac2-DV2(prMEsol)N had a measurable negative effect on viral
replication in Vero/hSLAM cells, reaching intracellular titers of 5.8 Log10 TCID50/ml at
58
the same time point and a slightly delayed viral growth kinetics. The extracellular
fractions for both viruses had similar patterns of replication with viral yields diminished
by one log10 approximately in comparison to the intracellular fraction (Fig3.1B). Since
both recombinants are attenuated MV strain (Moraten/Schwarz) derivatives, we analyzed
their replication in a cell line used for commercial production, primary chick embryo
fibroblasts. In these cells we observed a very similar replication profile for both
recombinants when compared to the parental MVvac2, reaching maximum intracellular
titers of around 5 log10 TCID50/ml at 6 days PI and extracellular titers of 102.5 TCID50/ml
(Fig 3.1C). Furthermore, plaque morphology analysis showed that MVvac2-
DV2(prME)N and MVvac2-DV2(prMEsol)N had circular plaques of around 1.075 and
1.045 mm in diameter respectively and not majorly different from MVvac2 ones (1.078
mm, Fig 3.1D).
Expression of Dengue 2 E glycoprotein by recombinant viruses
To corroborate the expression of DV2 vectored antigens, we performed initially
an indirect immunofluorescence assays using antibodies specific for MV-N (stained with
secondary Rhodamine conjugated antibodies), DV2 prM and E glycoproteins (stained
with FITC conjugated antibodies). To track the viral cytopathic effect, cell nuclei were
counter stained with DAPI. As evident in Figure 3.2A, merged epifluorescence images
from cells infected with either MVvac2-DV2(prME)N or MVvac2-DV2(prMEsol)N had
a strong yellow epifluorescence resulting from co-localization of green and red signals
coming from bona-fide cell syncitia induced by viral infection. In contrast control cells
infected with MVvac2 or DV-2 and treated in parallel similarly, showed only red and
59
green epifluorescence, respectively. A similar result was obtained with a specific anti-
prM antibody. Thus, MVvac2-DV2(prME)N and MVvac2-DV2(prMEsol)N express the
expected DV-2 glycoproteins and the insertion of full-length or 90% DV2 prME coding
sequences did not have a major effect on MV replication in two different cell substrates.
Then, we analyzed biochemically the vectored DV2 E antigen. As shown in the left panel
of Figure 3.2B a specific monoclonal anti-DV2 E antibody detected protein bands of 52
and 48 kDa in lysates obtained from cells infected with MVvac2-DV2(prME)N and
MVvac2-DV2(prMEsol)N respectively. The approximately 4 kDa difference in the
vectored DV2 E molecular weight certainly obeys to the 45 amino acid carboxyl terminus
truncation of E as expressed by MVvac2-DV2(prMEsol)N. To demonstrate that such
modification of DV2 E promote its routing towards cell secretion, an analysis with
EndoH and PNGaseF glycosidases was performed. The right panel of Figure 3.2C
showed that and endoH-resistant DV2E form was detected in treated lysates from cells
infected with MVvac2-DV2(prMEsol)N in a clear contrast with MVvac2-DV2(prME)N
infected cell lysates treated similarly. To control this experiment, the same infected cell
lysates were probed with an anti-H antibody. Endo-H resistant MV H were detected in
both MVvac2-DV2(prME)N and MVvac2-DV2(prMEsol)N infected cell extracts. Thus,
the expression of a carboxyl terminus truncated from of DV2 E promotes its routing to
cell secretion pathways.
Immunogenicity of MV-Dengue 2 recombinant viruses
To rank the immunogenicity of MVvac2-DV2(prME)N and MVvac2-
DV2(prMEsol)N, we immunized huCD46Ge-IFNarKO [123] mice with one or two 105
60
TCID50 intraperitoneal doses separated by four weeks. As shown in figure 3.3A, measles
neutralization titers of around 1:420 were obtained after one dose of MVvac2 or the two
DV2 E expressing vectors. MV- specific immunity was importantly boosted for MVvac2-
DV2(prMEsol)N which reached neutralizing titers of 1:1,500 14d after the booster dose,
while MVvac2-DV2(prME)N induced only 1:1000 in average. We assessed DV2
neutralization using two related approaches. First, a Log10 neutralization index (LNI),
where the neutralizing effect of individual 1:2 serum dilutions upon a high titer viral DV2
prep infectivity was calculated using averaged preimmune and MVvac2 immunized
serum to determine basal, non-neutralizing conditions. Thus preimmune, MVvac2-
immunized or non-reactive samples tend to have an LNI of zero. Additionally we
determined the classic PRNT50 titer per experimental group using pooled serum samples.
As shown in figure 3.3B, regardless of the immunization schedule, MVvac2-
DV2(prME)N only elicited DV2 neutralization titers just above background. In a
remarkably contrast, MVvac2-DV2(prMEsol)N serum samples reduced the infectivity of
a 105.48 PFU/ml DV2 viral prep by 2.78 and 3.76 orders of magnitude after one or two
doses (equivalent to PRNT50 of 1:160 and 1:214), respectively. Thus, the expression of a
soluble DV2 E glycoprotein and a two-dose schedule outperformed the full-length E
insertion and, expectedly, a single dose immunization scheme.
Generation, characterization and immunogenicity of MVvac2-D4(prMEsol)N
expressing a soluble version of dengue 4 E antigen
Since dengue immunization must elicit a broad neutralizing immunity encompassing
all DV variability, we build upon our DV2-MV findings to generate and characterize an
61
MV expressing equivalent antigens from DV4. Figure 3.4A depicts the construction of
an MV vectored vaccine expressing a carboxyl-terminus truncated DV4 E glycoprotein.
The recombinant virus, termed MVvac2-DV4(prMEsol)N, replication was assayed by
multistep growth analysis and found to be more similar to MVvac2 than its DV2 Esol
expressing counterpart. DV4 Esol expressing MV reached maximum intracellular titers
of 106.5 TCID50/ml at 48 h post-infection (Fig 3.4B). Similarly, the expression of DV4 E
was corroborated by immunoblot (Fig 3.4C) using specific antibodies against DV4 E and
the expression of MV N as control. The concentration of polyacrylamide (10%) used for
the blot shown did not allow observing a clearer shift in electrophoretic migration of DV4
Esol in comparison with the full-length DV4 E reference. As shown in figure 4E, we
documented DV4 immunogenicity elicited by MVvac2-DV4(prMEsol)N by immunizing
MV-susceptible mice with a two dose IP scheme, as before. As expected, measles
neutralizing immunity was very robust 14 days after the booster dose, reaching titers of
around 1:1,500 (Fig. 3.4E), whereas DV4 neutralizing immunity was also very robust.
Serum samples from immunized animals neutralized (reduced) the infectivity of a high
titer DV4 viral prep (106 PFU/ml) by 4.63 orders of magnitude (DV4 LNI) in average.
This DV4neutralization is equivalent to a 1:640 PRNT50 titer as obtained from pooled
samples. Thus, we were capable of reproducing our MV-DV2 observations using a
similar DV4 vectored antigens.
62
Immunogenicity of recombinant MV vectors expressing Esol antigens from Dengue
2 and 4
Subsequently, we decided to explore DV2 and DV4 immunogenicity after the
simultaneous administration of MV-DV2 and MV-DV4 Esol vectored vaccines. To this
effect, we immunized MV-susceptible mice groups with either two 105 TCID50 IP doses
of each component for a total measles vaccine dosage of 105.3 per dose (a vaccine scheme
termed “full”) or a 50% reduced dose of each component, 104.7 TCID50 per dose (a
vaccine scheme termed “half”) in order to directly compare the obtained results with our
previous single-component immunizations. Figure 3.5A documents DV2 neutralizing
immunity in mice 14d after two doses of the MV-DV2/4 vectored cocktail. Individual
serum samples had in average a neutralization index of 3.89 and 3.6 Log10 upon a 105.08
PFU/ml DV2 preparation infectivity (equivalent to PRNT50 of 1:430 and 1:240 PRNT50
in pooled serum samples) after full or half dose vaccination schemes, respectively. In a
similar result than the single-component experiment, DV4 neutralizing immunity was
quantitatively higher than DV2 by at least an order of magnitude (Fig 3.5B). The
experimental group had in average an LNI of 5 and 4.38 Log10 upon the DV4 infectivity
of a 106 PFU/ml viral prep (equivalent to PRNT50 of 1:1066 and 1:640 PRNT50 in pooled
serum samples) after full or half dose vaccination schemes, respectively. In average, the
documented measles neutralization immunity was slightly higher than the obtained after
two single-component doses, measles neutralizing titers averaging 1:2,169 and 1:2072,
for full and half vaccination dosage regimes, respectively were documented (data not
shown).
63
Cross-neutralizing immune responses in MV-susceptible mice inoculated with
MVvac2-D2(prMEsol)N and MVvac2-D4(prMEsol)N
Finally, we explored the neutralizing immunity breadth of our approach by performing
neutralization assays against DV1 and the more phylogenetically distant YFV 17D.
Figure 3.6 documents that serum samples from all of the experimental animals
measurably neutralize these two flaviviruses. The infectivity of a high titer (104.84
PFU/ml) DV1 viral prep was neutralized between 10 to 300 times for an averaged LNI of
2.03 after a full dose vaccination scheme. An almost three times lower titer was obtained
after a half dose vaccination scheme in average. The documented DV1 PRNT50
neutralization titer for full and half dose experimental groups was 1:130 and 1:90,
respectively (Fig 3.6A). Of note, the obtained DV1 immunogenicity after the DV2/4
vaccination cocktail was contrastingly higher than the one obtained after a similar, DV2
single component vaccination (LNI 0.74, PRNT50 1:15) or DV4 single component
vaccination (LNI 1.14, PRNT50 1:36). In a comparable result all of the animals
neutralized YFV. An YFV 17D high titer viral prep (106.78 PFU/ml) infectivity was
neutralized by a factor of 15 to 10 (LNI average of 1.1625 and 0.98) equivalent to a
PRNT50 of 1:60 and 1:40 after full or half vaccination schemes, respectively. Of note, an
LNI of 0.7 is the reported correlate of protection for YFV (equivalent approximately to a
PRNT50 of 1:10) [96]. Again, the obtained YFV 17D immunogenicity after the DV2/4
vaccination cocktail was higher than the one obtained after a similar, DV2 single
component vaccination (LNI 0.056, PRNT50 below detection level) or DV4 single
component vaccination (LNI 0.83, PRNT50 1:12). Thus, by combining DV2 and DV4
64
insertions we were able to enhance the homologous and heterologous neutralizing
immunity. This effect is not due to transient cross reactivity since single component
immunization did not reach the observed neutralizing titers.
65
DISCUSSION
In this report we have shown that a fully equivalent, pediatric measles vaccine strain
engineered to express relevant DV2 and DV4 antigens can elicit neutralizing immune
responses in protective ranges against the homologous vectored viral components by the
vaccine cocktail but also against the heterologous DV1 and the relatively distant YFV
17D attenuated strain. We selected the more optimal DV2 antigen to include in our
cocktail. Other studies have suggested the presence of a strong endoplasmic reticulum
retention signal on the transmembrane region of DV2 E protein [127]. This finding
coincides with our observation that a truncated (soluble) DV2 E is significantly more
immunogenic than its full-length counterpart. We have reproduced this observation for
DV4 E immunogenicity also (data not shown). We have tested in-vitro the stability of our
recombinants through passage five, however their genetic stability needs to be assessed
after an in vivo passage. Previous recombinants using the same platform have shown a
robust stability [128].
The enhanced DV neutralizing immunity resulting from the simultaneous
administration of only two serotypes highly suggests that a cocktail formed by the four
DV serotypes will elicit an even more potent DV neutralization. Our selection contains
the representative and significant DV2 and a more distant DV4. The latter is the more
ancient DV serotype, with an E protein sequence closer to other flaviviruses such as JEV
and YFV [129]. A study done by Fu et al., reported that the homology of E-proteins of
DV -1 to -4 share approximately 65%-75% homology at the amino acid level [130, 131].
It remains to be tested experimentally whether the observed broad response is long
66
lasting, however studies performed in non-human primates with MV vaccine derivatives
point to a long-lived immunity against the vectored antigen [113, 120, 132]. It is
significant, that a robust and simultaneous neutralizing response against two DV
serotypes is usually not associated with enhancement of DV entry and severe forms of
infection. Indeed, hemorrhagic forms associated with a tertiary DV infection are an
extremely rare event [90].
From the practical point of view, the usage of current MV vaccination practices to
deliver DV or flaviviral antigens offer several advantages over other approaches seeking
to supplement the efficiency of current dengue vaccination platforms. First, the individual
pediatric measles dosage is stipulated to contain at least 103 and not more than 104
TCID50 per administered dose[133]. Second, measles protective responses observed in
primates vaccinated with recombinant MV demonstrated vaccine potency equivalence
with the Moraten vaccine strain. Thus, arguably it would be feasible to compose a four
DV serotypes MV vaccine cocktail containing the WHO mandated measles vaccine
dosage to protect one-year-old infants against measles. It would be interesting to explore
the possibility of supplementing the observed MV-DV2 and -DV4 immunogenicity with
prME antigens from other representative mosquito-borne flaviviruses, such as those from
YFV 17D, or Japanese encephalitis virus SA 14-14-2. And thirdly, up-scale production
methods and distribution networks are in place. Vero/hSLAM cells have been the gold
standard to amplify recombinant MV vaccines in the laboratory, however commercial
production requires of the robust fermentation using CEF cells. It is significant then that
we did not observe any effect upon MV-DV2 replication in those cells. Also positive are
67
the results from the first phase I clinical trial of a MV-Chinkungunya vaccine
documenting the safety and immunogenicity of measles vaccine derivatives [134, 135].
Indeed, Themis Bioscience GmbH has declared intentions for preclinical development of
a MV-Dengue vaccine [136].
There are aspects of our MV-DV approach that need to be experimentally explored.
Importantly, although YFV 17D and measles vaccination does not interfere in African
children that need both vaccinations at the same time[137], DV immune interference
needs to be determined experimentally.
As mentioned, the current dengue vaccination option (DengvaxiaTM, Sanofi Pateur) is
based on chimeric YFV17D virus replication machineries wrapped with the relevant
dengue virus envelope glycoproteins. DV immune responses and protection efficacy after
vaccination is majorly boosted when the recipient’s immune system is primed against
flaviviruses due to a prior exposure to DV, or potentially vaccination against YFV or JEV
in South America or Asia, respectively. Thus, Dengvaxia has been recommended for use
in older than 9 year-old recipients in countries where DV endemicity is close to 80%.
Flavivirus naïve and younger recipients (two to nine-year-old) failed to mount protective
responses against the four dengue serotypes and retrospectively they had a significant
increased risk of hospitalization because of severe dengue infection, [110, 138] . In
highly endemic countries, this vaccination is expected to be safe regardless of the
possibility of having been exposed or not. Indeed, the need to protect young infants from
dengue is very important. The disease is more severe at this age and the intense virus
replication can be the reservoir to sustain an epidemic outbreak [139]. Thus, it would be
68
very practical to take advantage of the pediatric measles immunization to artificially
prime globally the population against DV or other representative flaviviruses. MV-DV
vaccination would take the place of the primal infection accelerating the start for
immunization with other options and closing the time-gap that would represent the higher
risk. When the time or indication to administer DV, YFV or JEV vaccination presents,
the resulting immune response would be greatly enhanced. Another highly advanced
candidate, spearheaded by Takeda pharmaceuticals, consist of chimeric viruses where the
replication machinery is based on an attenuated DV2. Phase II clinical trial of this
candidate also demonstrated an unbalanced neutralizing immunity against the four DV
serotypes, and most probably will have the same indications upon approval[140]. Thus,
we believe that priming flaviviral immunity at an early age may prove convenient and
complementary to many other flavivirus vaccine approaches.
69
70
Figure 3.1. Generation and characterization of recombinant MVs expressing Dengue 2
envelope antigens. (A) Maps of recombinants MVs derived from MVvac2, the parental
strain; MVvac2-D2(prME)N, vectoring prM and a full-length Dengue 2 E glycoprotein
(top) and MVvac2-D2(prMEsol)N expressing a soluble version of the same antigen
(bottom). Both recombinant vectors were obtained from their respective full-length
infectious plasmids by reverse genetics. The native MV coding cistrons are indicated in
gray and the added Dengue 2 antigens in colored boxes. Relevant Dengue 2 E amino
acids and their numbers are indicated. (B) Time-course of cell-associated (top) and cell-
free (bottom) virus production at the indicated time points in Vero/hSLAM cells infected
at an MOI of 0.03 with either the parental strain MVvac2 (continuous line, circles)
MVvac2-D2(prME)N (interrupted line, squares) and MVvac2-D2(prMEsol)N
(interrupted line triangles). Averages and standard deviations of three independent
experiments are shown. (C) Time-course of cell-associated (top) and cell-free (bottom)
virus production at the indicated time points in primary Chick embryo fibroblasts. CEF
cells infected at an MOI of 0.03 with either the parental strain MVvac2 (black bars)
MVvac2-D2(prME)N (grey bars) and MVvac2-D2(prMEsol)N (light grey bars). Average
of a representative experiment performed in triplicate are shown. (D) Viral plaques
morphology in Vero/hSLAM cells obtained 96h post-infection and stained with naphtol
blue-black.
71
72
Figure 3.2. Expression of Dengue 2 E glycoprotein by recombinant viruses. (A).
Vero/hSLAM cells infected at an MOI of 0.5 were mock- infected or either with the
parental strain MVvac2 , MVvac2-D2(prME)N and MVvac2-D2(prMEsol)N. At 48h
post-infection, infected cells were fixed and stained with fluorescent labeled antibodies
and nuclei counterstained with DAPI (blue channel). Single channel and overlaid images
after epifluorescence microscopy are shown, red channel (anti-MV reactivity) and green
channel (anti dengue 2 prM and E), white bar represents 200nm. (B) Analysis of
expression by Western blot of proteins extracted from MVvac2- , MVvac2-D2(prME)N-
and MVvac2-D2(prMEsol)N-, or mock-infected cells. Proteins were detected using
antibodies against dengue 2 E, MV N, and actin, and the positions of molecular weight
standards are indicated to the left. Left panel, analysis of the glycosylation of the vectored
Dengue 2 E glycoprotein. Lysates from cells infected with MVvac2, MVvac2-
D2(prME)N and MVvac2-D2(prMEsol)N were treated or not with the indicated
glycosidase and analyzed by western blot using anti MV-H (top panel) and anti Dengue 2
E (bottom panel) antibodies. Endo-H resistant forms are indicated with asterisks.
Molecular weight standard positions are indicated to the left.
73
74
Figure 3.3. Immunogenicity of MV-Dengue 2 recombinant viruses. MV-susceptible mice
received one or two IP doses of MVvac2 , MVvac2-D2(prME)N and MVvac2-
D2(prMEsol)N. Neutralizing immune responses were analyzed six weeks after the first
dose. (A) Anti MV neutralization assessed by PRNT90. (B) Anti-Dengue neutralization
assessed by Log10 Neutralization Index (LNI, left panel) or PRNT50 performed in
triplicate using pooled serum samples. Each symbol represents one animal; horizontal
lines and vertical brackets indicate group average and standard deviation, respectively.
Anti-Dengue 2 PRNT50 titers are indicated.
75
76
Figure 3.4. Generation, characterization and immunogenicity of MVvac2-D4(prMEsol)N
expressing a soluble version of dengue 4 E antigen. (A) Map of MVvac2-D4(prMEsol)N.
Native MV coding cistrons are indicated in gray and the added Dengue 4 prM and Esol
antigens in colored boxes. Relevant Dengue 4 E amino acids and their numbers are
indicated. (B) Time-course of cell-associated virus production at the indicated time points
in Vero/hSLAM cells infected at an MOI of 0.03 with either the parental strain MVvac2
(continuous line, circles) and MVvac2-D4(prMEsol)N (interrupted line, diamonds).
Averages and standard deviations of three independent experiments are shown. (C)
Expression of Dengue E protein demonstrated by Western blot. Cell lysates of infected
cells (inoculum indicated on top) were probed with anti MV N, anti Dengue E or anti-
actin antibodies. The relative migration of molecular weight markers is indicated on left.
(D). MV-susceptible mice received two IP doses of MVvac2 and MVvac2-
D4(prMEsol)N. Neutralizing immune responses were analyzed two weeks after the
booster dose. (A) Anti MV neutralization assessed by PRNT90. (B) Anti-Dengue
neutralization assessed by Log10 Neutralization Index (LNI, left panel) or PRNT50
performed in triplicate using pooled serum samples (right panel). Each symbol represents
one animal; horizontal lines and vertical brackets indicate group average and standard
deviation, respectively. Anti-Dengue 4 PRNT50 titers are indicated.
77
78
Figure 3.5. Immunogenicity of recombinant MV vectors expressing Esol antigens from
Dengue 2 and 4. MV-susceptible mice received two IP doses of MVvac2-D2(prMEsol)N
and MVvac2-D4(prMEsol)N in a 28 day interval. Neutralizing immune responses were
analyzed two weeks after the booster dose. (A) Analysis of neutralization against Dengue
2 and (B) against Dengue 4 viruses. Anti dengue neutralizing immune responses were
assessed by LNI (left panel) and PRNT50 (right panel). Each symbol represents one
animal; horizontal lines and vertical brackets indicate group average and standard
deviation, respectively. Averages of anti-Dengue PRNT50 titers determined in triplicate of
pooled serum samples are indicated.
79
80
Figure 3.6. Cross-neutralizing immune responses in MV-susceptible mice inoculated
with MVvac2-D2(prMEsol)N and MVvac2-D4(prMEsol)N (A) Analysis of
neutralization against Dengue 1 and (B) against attenuated YFV. Anti dengue and anti-
YFV neutralizing immune responses were assessed by LNI (left panel) and PRNT50 (right
panel). Each symbol represents one animal; horizontal lines and vertical brackets indicate
group average and standard deviation, respectively. Averages of anti-Dengue PRNT50
titers determined in triplicate of pooled serum samples are indicated.
81
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CHAPTER 4 GENERATION OF A RECOMBINANT MEASLES VIRUS EXPRESSING FULL-
LENGTH DENGUE 4 ENVELOPE GLYCOPROTEINS
ABSTRACT
Dengue virus (DV), an arthropod-borne viral disease, causes major public health
concern in the world due to a large number of people infected and the economic impact
for endemic countries. There have been multiple attempts to generate a vaccine to prevent
infection by any of the four described DV serotypes. A recently approved vaccine
demonstrated disease enhancement in young children associated with a flavivirus naïve
status. Therefore, a pediatric vaccination strategy with the potential to prime neutralizing
anti-flaviviral responses in one-year-old infants can be used to complement vaccine
immunity to protect young children. In a previous work, we modified a measles vaccine
viral vector (MVvac2) to express the pre membrane (prM) and a full length or a truncated
(90%) form of the major envelope (E) from DV2, and we showed that a truncated DV2 E
isoform has superior immunogenicity to its coserponding full-length construct. Here, we
generated a recombinant measles virus expressing DV4 PrM and full-length E protein.
The recombinant MV-D4-PrME vaccine elicited robust anti-measles neutralizing
responses (1: 1280 TCID50/ml in average) and a very discreye homologous anti-dengue
4 neutralizing responses in all MV-susceptible mice 1:18 PRNT50 in average). In sum,
we were able to reproduce the poor immunogenicity of a DV2 envelope glycoprotein
antigen when expressed at full-length in the more distant DV4 background. It is possible
that soluble versions of flaviviral envelope antigens are more immunogenic despite the
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presence or not of endoplasmic reticulum retention signals in its transmembrane region,
as it is the case for DV2.
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INTRODUCTION
Dengue virus envelope glycoprotein is the major antigen in the surface of the
virion and a biomarker of evolution of the Dengue serogroup. The phylogenetic
difference in E sequence between DV 1 to DV 4 is 25-35%, mainly due to differences in
domain III of its ectodomain. The transmembrane (TM) region of DV glycoproteins E
and PrM is of paramount importance to consider because it may contain endoplasmic
reticulum ER sequestering regions. The presence of the ER localization signals in the
flavivirus envelope proteins is essential for the virion morphogenesis of the flaviviruses.
That occurs in association with ER membranes, suggesting that there should be
accumulation of the virion components in this compartment[129] [141]. Flavivirus
acquire their envelope by budding through ER membranes, therefore the E protein needs
to accumulate in the ER compartment before budding can take place. The secretion of
truncated E protein of flavivirus with its C-terminal transmembrane removed suggested
that there is no ER retention signal in the ectodomain of this protein. Some studies
showed that transmembrane domanine of E protein from YFV and DV2 contain strong
retention signal. When the ectodomain of CD4 fused with the TM domain of E from DV2
or YFV, it was retained in the ER. Indicating that the TM domains of flavivirus envelope
proteins contains ER retention signal.
The envelope E protein is essential for vaccine development as it has many
epitopes that react with neutralizing antibodies. Envelope protein is a glycoprotein, which
has been studied for its antigenicity and immunogenicity[142, 143]. The presence of this
retention signal in the transmembrane region may result in poor immunignicity of viral
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vectors expressing the full length E protein. In the pervious work we showed that
recombinant measles expressing full length E protein generate poor anti-DV2 neutralzing
antibodies. This finding suggested that presence of ER retention signal contributed to the
poor immunity due to inefficient antigen presentation.
The correct folding of E protein is critical determine of immunogenicity. The co-
expression of Pre membrane PrM protein is vital for proper folding as it acts as a scaffold
for the E protein[144]. We constructed a vaccine candidate based on measles vaccine
virus expressing pre-membrane (prM) and envelope (E) proteins of dengue 4 virus (DV-
4) and tested it in mice to evaluate immunogenicity and protection against DV-4
infection. Measles has been successfully used as vaccine vectors for several viruses to
induce strong humoral and cellular immune responses.
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MATERIAL AND METHODS
Cells and Viruses.
Vero/hSLAM cells [114, 115]were maintained in Dulbecco’s modified Eagle’s
medium with high glucose concentration (DMEM, Sigma-Aldrich, St. Louis, MO, USA)
supplemented with 5% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch,
GA, USA), 1% penicillin–streptomycin (Sigma-Aldrich) and 0.5 mg/mL G418 (Enzo
Life Sciences, Farmingdale, NY, USA) to maintain hSLAM expression selection. BHK
21 cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-
streptomycin. Helper 293-3-46 cells [116]a derivative from HEK 293 cells were
maintained in DMEM with 10% FBS, 1% PS, and 1.2 mg/mL G418.
DV4 virus was obtained from the ATCC (VR-1257 strain H241 and propagated in
Vero cells. The virus was then amplified in BHK21 cells for up to two or three passages.
Recombinant MVs were generated using the method of Radecke et al. [145]
modified by Parks et al. [118] After detection of their cytopathic effect on mixed cultures
of Helper 293-3-46 and Vero/hSLAM cells, single syncytia were transferred to and
propagated in Vero/hSLAM cells. Viral clones’ stocks were amplified by infecting
Vero/hSLAM cells at a multiplicity of infection (MOI) of 0.03 and incubated at 37°C.
When approximately 80% cytopathic effect was observed, cells were scraped in Opti-
MEM (Life Technologies, Grand Island, NY) and viral particles were released by two
freeze-thaw cycles.
Multi-step growth kinetics of the viruses were determined by infecting
Vero/hSLAM cells at an MOI of 0.03 and incubating them at 37°C. Infected cells and
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supernatants were collected and lysed by a single cycle of freeze-thaw at prescribed times
post-infection, and the 50% tissue culture infectious dose (TCID50) was determined in
Vero/hSLAM using the Spearman-Kärber end-point dilution method. Primary chick
embryo fibroblast (CEF) were obtained from ATCC (CRL-1590 F2) and grown in
Dulbecco’s modified Eagle’s medium with high glucose concentration (DMEM, Sigma-
Aldrich, St. Louis, MO, USA) supplemented with 10% FBS, 1% penicillin–streptomycin
and 10% tryptose phosphate broth (Gibco, Life Technologies). Kinetics of infection of
CEF were performed by infecting CEF at MOI of 0.03 with MV and DV-expressing
recombinant MV and collecting cell-associated and supernatant at the indicated time
post-infection.
Plasmids and plasmids constructions
The plasmid pB(+)MVvac2(HBsAg)N was used to construct the recombinant
MVs expressing DV4 antigens[120]. This plasmid contains an infectious MV cDNA
corresponding to the antigenome of the Schwarz measles vaccine strain with an
additional transcription unit (ATU) inserted downstream of the nucleocapsid (N) cistron
to direct the expression of an inserted foreign gene. To generate the recombinant measles
expressing DV4 prM- E glycoproteins, MluI and AatII digestion fragments containing the
desired coding sequence were obtained by PCR (primer sequence available upon request)
and swapped with the HBsAg insert. DV4 prME coding sequences have been achieved
from the full-length pR424-D4 plasmid (H241 strain). The full-length recombinant
plasmid was termed pB(+)MVvac2-DV4(prME)N. Correct insertions and relevant gene
boundaries were corroborated by sequencing.
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Indirect Immunofluorescence Microscopy
Vero/hSLAM cells were seeded into 4 well glass Millicell EZ slides
(MILLPORE) and infected at MOI of 0.06, 0.1 and 1 with MV, MVvac2DV4(PrME)N,
and DV4, respectively. After 48 hours of incubation at 37 C, the cells were fixed with 3%
paraformaldehyde and incubated at 37 °C overnight. The cells were permeabilized with
0.1% Triton X-100 in phosphate- buffer saline (PBS). Both primary and secondary
antibodies dilution were prepared in 0.1% Triton X-100 in 10% FBS serum in 1 X PBS.
Two Mixes of primary antibodies were used, monoclonal antibody directed against DV
1,2,3,4 E protein at 1:50 dilution (a mouse IgG2a cat. # 76 ab9202 from abcam) or
monoclonal antibody directed against DV1,2,3,4 PrM protein at 1:50 dilution (a mouse
IgG cat.# ab41473 from abcam) with the polyclonal antibodies directed against MV N
protein at 1:300 dilution (a rabbit IgG antibody provided from R. Cattaneo). The primary
antibodies incubations were carried out overnight at 4 °C. A mix of goat anti-mouse IgG
coupled to Fitc (Green fluorescence), and a goat anti-rabbit IgG coupled to PE (Red
fluorescence) were used as a secondary antibody at dilution of 1:300 and incubated at
room temperature for 2 hours. The coverslips were mounted on slides by using
flouromount G with DAPI (Electron Microscopy Science). The slides examined, and
images were taken by an inverted microscope with epifluorescence illumination
(EVOSFL).
Mice inoculations
All experimental procedures were performed according to a protocol approved by
the Arizona State University Institutional Animal Care and Use Committee. To determine
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the immunogenicity of MVvac2DV4(PrME)N in an MV susceptible small animal host,
groups of HuCD46Ge-IFNarKO mice were inoculated by the intraperitoneal (i.p.) route
with the indicated doses diluted in 100 uL of Opti-MEM[120]. Two weeks after the
initial inoculation, the mice received a similar booster dose. One week after the second
dose, mice were exsanguinated. Sera were separated and heat-inactivated at 56°C for 1 h.
MV neutralization assay
To determine MV neutralization titer, serial two-fold dilutions of heat-inactivated
sera in Opti-MEM were incubated with 100 TCID50 of MVvac2 for 1 h at 37 °C.
Vero/hSLAM cells were added to the serum–virus mixtures and the plates were then
incubated at 37 °C for three days, at which time neutralization titer was assessed as the
highest dilution of serum capable of complete neutralization of infectivity, defined as the
absence of MV cytopathic effect. Neutralization titers were reported as the average of a
determination made in triplicate.
DV neutralization assay
To determine DV4 Log10 Neutralization Index titer the corresponding viral prep
was mixed with heat-inactivated serum in a 1:1 proportion in a 25 uL volume of Opti-
MEM and incubated for 1 h at 37 °C. Infectivity after serum incubation was determined
using a plaque assay in BHK-21 cells. Briefly, ten-fold serial dilutions of the virus/serum
mixtures were generated using Opti-MEM and added to 2.4 × 106 BHK-21 cells in a 24-
well plate and then incubated for 4 h at 37 °C. At this time, 500 uL of 3% carboxymethyl
cellulose was added to each well and plates were incubated for 5 days at 37 °C. The wells
were then washed; the monolayer was fixed and stained with acetic acid/naphtol blue-
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black and plaques were counted. The neutralization index was expressed as the
logarithmic difference between the averaged DV4 titer when incubated with control,
namely, that of animals inoculated with MVvac2, and preimmune. Neutralization indexes
thus determined were graphed as Log10NI.
In another approach, we performed a standard 50% plaque reduction
neutralization assay (PRNT50) using sera from each animal. Briefly, 50 DV4 PFU were
incubated with two-fold serum dilutions (from 1:5 to 1:320) for 1 h at 37 °C. Remaining
infectivity was determined by plaque assay, as described above. The neutralization titer
was defined as the dilution that resulted in a 50% reduction in plaque numbers, as
compared to unrelated serum.
RESULTS
Generation of recombinant divalent MV-DV4 vaccine vector.
We modified a full-length infectious cDNA plasmid named pB(+)MVvac2. This
plasmid and its derivatives can be used to recover recombinant MVs by well-established
reverse genetic techniques. DV4 prME coding sequences were inserted downstream MV
N gene. The coding sequence consisted of prM and E full-length coding sequences
preceded by the signal sequence of the carboxyl terminus of the capsid protein (C
anchor). The recovered virus was termed MVvac2-DV4(prME)N and expresses intact
full-length prM-E glycoproteins. (Fig4.1A). To corroborate the expression of DV4
vectored antigens, we performed an indirect immunofluorescence assays using antibodies
specific for MV-N and DV4 PrM or E (stained with secondary RFP and GFP conjugated
antibodies, respectively). To track the viral cytopathic effect, cell nuclei were
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counterstained with DAPI. As evident in Figure 4.1B, merged epifluorescence images
from cells infected with either MVvac2-DV4(prME)N had a strong yellow
epifluorescence resulting from co-localization of green and red signals coming from
bonafide cell syncitia induced by viral infection. In contrast, control mock infected cells
showed no green or red epifluorescence, respectively. Furthermore, plaque morphology
analysis revealed that MVvac2 and MVvac2-DV4(prME)N had circular plaques of
around 1.082 and 1.079 mm in diameter respectively (Figurer 4.1D).
Replication fitness of recombinant MV-DV4 is equivalent to parental MV.
To corroborate the effect of DV4 prME insertion upon recombinant MV
replication fitness, we performed multistep growth analysis using two cell substrates.
Initially, viral replication in Vero/hSLAM did not demonstrate a major deleterious effect
because of the genetic modification on MV backbone. Intracellular MVvac2-
DV4(prME)N viral yields reached 6.8 Log10 TCID50/ml at 48h PI, which was slightly
higher than but not significantly different of the parental strain MVvac2. The
extracellular fractions had similar patterns of replication with viral yields diminished by
one log10 approximately in comparison to the intracellular fraction (Fig4.2A &B). The
recombinant MV-DV4 is attenuated MV strain (Moraten/Schwarz) derivatives; we
analyzed its replication in a cell line used for commercial production, primary chicken
embryo fibroblasts. In these cells we observed a very similar replication profile of Mv-
DV4when compared to the parental MVvac2, reaching maximum intracellular titers of
around 5 log10 TCID50/ml at 120 hours PI and extracellular titers of 2.5 log10
TCID50/ml (Fig4.2C&D).
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Recombinant MV-DV4 induces anti-measles and anti- dengue 4 immune responses.
We immunized huCD46Ge-IFNarKO mice with two 105 TCID50 intraperitoneal
doses separated by three weeks. As shown in figure 4.3A, measles neutralization titers of
around 1:420 were obtained after one dose of MVvac2. MV- specific immunity was
boosted for two doses of MVvac2-DV4(prME)N which reached neutralizing titers of 1:
1280. We assessed DV4 neutralization using two related approaches. First, a Log10
neutralization index (LNI), where the neutralizing effect of individual 1:2 serum dilutions
upon a high titer viral DV4 prep infectivity was calculated using averaged preimmune
and MVvac2 immunized serum to determine basal, non-neutralizing conditions. Thus
preimmune, MVvac2-immunized or non-reactive samples tend to have an LNI of zero.
Additionally, we determined the classic PRNT50 titer per experimental group using
pooled serum samples. As shown in figure 4.3B, MVvac2-DV4(prME)N only elicited
DV4 neutralization titers just above background with average 0.5 LNI and 1: 18
PRNT50.
DISCUSSION
There have been challenges to develop a dengue vaccine. The results of a phase 3
clinical trial of the the Sanofi Pasteur yellow fever/dengue chimeric vaccine “CYD” in
children of the Asia-Pacific and Latin America regions, failed to protect against DV2 and
showed limited protection against DV1, including the enhancement of disease and the
increased risk of hospitalization of children under 9 year old, indicating the need to
pursue new strategies to reach a broader protection against dengue infections[83]. The
recombinant live attenuated viruses are attractive strategies to construct dengue vaccines
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because they can induce strong humoral responses that mimic natural infection and are
safe, low coast vaccine candidates[14-16, 22]. However, it is very critical to expresses the
antigen in forms that will generates a robust immune response.
In this study, we describe, the construction of measles vectoring dengue 4-
glycoprotein candidate vaccine. Our recombinant measles expresses prM and E proteins
of DV-4 as was demonstrated by immunofluorescent. The replication fitness of the
recombinant virus was comparable to the parental strain as it was tested in two different
cell types. Our MV-DV4-prME induces anti- MV and anti-DV4 humoral response in
HuCD46Ge-IFNarKO mice. Inducing neutralizing antibodies is the main target of
vaccine candidates as it blocks virus entry into target cells. Therefore, we explored the
construction of dengue vaccines by expressing full-length E protein to induce
neutralizing antibody which can bind these proteins and prevent it from binding to host
cell receptors. However, our data show weak neutralizing immune response against
dengue 4 virus (average 1:18 PRNT50). In our previous work we generated recombinant
measles expressing both prM and truncated DV4 E protein, which produces strong anti-
DV4 neutralizing response, the difference in the immunogenicity between the two
recombinant measles can be explained by different antigen presentations of the full-
length and truncated E protein. This data suggested that soluble versions of flaviviruses E
could be a better candidate for construction of anti-falvivirus vector vaccines. Flavivirus
envelope proteins TM lack any of the classical ER retrieval signals, KDEL, KKXX, or
RRXX , the two helices TM1 and TM2 in the transmembrane region of the four DV
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serotypes and YFV share identical amino acids resdiues which may play a role in the
retention of the E proteins in the ER(Figure4.4).
In future work, we will compare the enhancement of DV1 and DV2 infection in
macrophage cell line (P388 D1) between the serum of animal inoculated by the truncated
or full-length DV4 recombinant measles.
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99
Figure4.1. Generation and characterization of recombinant MVs expressing Dengue 4 PrM and E antigens. (A) Maps of recombinants MVs derived from MVvac2, the parental strain; MVvac2-D4(prME)N, vectoring prM and a full-length Dengue 4 E glycoprotein. Recombinant MV is obtained from its respective full-length infectious plasmids by reverse genetics. The native MV coding cistrons are indicated in gray and the added Dengue 4 antigens in colored boxes. Relevant Dengue 4 E amino acids and their numbers are reported. (B) Expression of Dengue 4 PrM or E proteins by recombinants MV vectors. Vero/hSLAM cells infected at an MOI of 0.5 are mock- infected or MVvac2-D4(prME)N infected. At 48h post-infection, infected cells were fixed and stained with fluorescent-labeled antibodies and nuclei counterstained with DAPI (blue channel). Overlaid images after epifluorescence microscopy are shown, red channel (anti-MV reactivity) and green channel (anti-dengue 4 prM or E), white bar represent 200nm. (C) Viral plaques morphology in Vero/hSLAM cells obtained 96h post-infection and stained with naphtol blue-black.
100
101
Figure4.2. Replication finesses of recombinant MV-DV4. Time-course of cell-associated (A) and cell-free (B) virus production at the indicated time points in Vero/hSLAM cells infected at an MOI of 0.03 with either the parental strain MVvac2 (continuous line, circles) MVvac2-D4(prME)N (continuous line, squares) Averages and standard deviations of three independent experiments are shown. (C) Time-course of cell-associated and (D) cell-free virus production at the indicated time points in primary Chick embryo fibroblasts. CEF cells infected at an MOI of 0.03 with either the parental strain MVvac2 (dark grey bars) MVvac2-D4(prME)N (grey bars). Averages and standard deviations of three independent experiments are shown.
102
Figure4.3. Immunogenicity of the MV-DV4 recombinant virus. MV-susceptible mice received one IP dose of MVvac2 or two IP doses MVvac2-D4(prME)N. Neutralizing immune responses were analyzed six weeks or 4 weeks after the first dose. (A) Anti MV neutralization assessed by PRNT90. (B) Anti-Dengue neutralization evaluated by Log10 Neutralization Index (LNI, left panel) or PRNT50 performed in triplicate using pooled serum samples. Each symbol represents one animal; horizontal lines and vertical brackets indicate group average and standard deviation, respectively. Anti-Dengue 4 PRNT50 titers are shown.
103
Figure 4.4. Sequance alignment of TM regions of E protein from DV1-4 and YFV. DV1(Genbank accession number FJ687432), DV2(Genbank accession number NC_001474), DV3 (Genbank accession number FJ850055), DV4 (Genbank accession number AY618990), YFV (Genbank accession number AY640589). Identical amino acid residues are highlighted in tallow. The predicted TM1 and TM2 helices are indicated at the bottom. Adapted from [1]
104
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of dengue-4 viruses. Journal of General Virology, 1997. 78(9): p. 2279-2284. 6. Wang, E., et al., Evolutionary relationships of endemic/epidemic and sylvatic
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Caribbean basin, 1981–2000. Virology, 2004. 324(1): p. 48-59. 9. Jenkins, G.M., et al., Evolution of base composition and codon usage bias in the
genus Flavivirus. Journal of molecular evolution, 2001. 52(4): p. 383-390. 10. Kuno, G., et al., Phylogeny of the genus Flavivirus. Journal of virology, 1998.
72(1): p. 73-83. 11. Chen, Y., et al., Dengue virus infectivity depends on envelope protein binding to
target cell heparan sulfate. Nature medicine, 1997. 3(8): p. 866-871. 12. Whitehead, S.S., et al., Prospects for a dengue virus vaccine. Nature Reviews
Microbiology, 2007. 5(7): p. 518-528. 13. Li, L., et al., The flavivirus precursor membrane-envelope protein complex:
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after heat shock. Journal of virology, 1999. 73(5): p. 3560-3566. 19. Reyes-del Valle, J., et al., Protective anti-hepatitis B virus responses in rhesus
monkeys primed with a vectored measles virus and boosted with a single dose of hepatitis B surface antigen. Journal of virology, 2009. 83(17): p. 9013-9017.
20. Guy, B. and N. Jackson, Dengue vaccine: hypotheses to understand CYD-TDV-
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106
CHAPTER 5
SUMMARY AND FUTURE DIRECTIONS
Altogether, this work provides proof of principle for a novel vaccination platform
to induce neutralizing responses in protective range aganist Dengue virus infections,
while maintaining the protective measles vaccine phenotype. In chapter 2, I described
homologous and cross-immunity within the same subgroups of the flavivirus (DV2, DV4,
and DV1) and the related YFV. Cross-immunity between the various flavivirus can be
sufficiently construed in a host as a mean of generating balanced and a broad vaccine
against dengue virus and other closely related arthropod-borne viral pathogens. In chapter
3, I described the generation of a Measles-Dengue pediatric vaccine that induces
neutralizing immune responses against Measles, Dengue type 1,2,4 and YFV. This study
suggests a novel strategy for a pediatric anti-flavivirus vaccine to prime a population’s
immune response against various flavivirus infections. With a focus in immunity against
Dengue virus infections. I generated three recombinant measles expressing DV2 or 4
glycoproteins at high expression levels using a vaccine-equivalent measles genome; I
also described the homologous immunogenicity of single component vector and cocktails
of two vectors expressing DV2 and DV4 antigens. The neutralization potential of the
cocktail of recombinant measles vector was determined against DV1 and YFV. In
chapter 4, I documented that recombinant measles expressing DV4 PrM and full-length
major E protein generates neutralizing immune responses against Measles virus and a
weak anti-DV4 immune responses. This observation is important because it reflects a
correlation between antigenic exposure and neutralizing immune responses. Furthermore,
107
since it also coincides with the lack of anti-DV2 immunity when using a full-length
antigen, it is arguable a consistent event for arthropod-borne flaviviruses or even related
agents like hepatitis C virus, that sequester their envelope proteins in an attempt to escape
native immunity. In sum, it may reflect a trend for flaviviral vaccine responses.
Additionally, I presented an alternative measurement of anti-flavivirus neutralizing
antibodies. I described the correlation between expressing the titer of neutralizing
antibodies by log neutralization index and the PRNT50. The log neutralization index
method depends on the direct measurements of the reduction of the viral titer as it relates
to non-relative serum such as pre-immune.
This project primarily depended upon the well-established measles reverse
genetic system[9,10], which has been used to generate many and different recombinants
and modified MVs[1, 2-6]. The availability of a full-length infectious cDNA encoding a
Moraten equivalent genome with engineered unique restriction sites for insertion of an
additional coding sequence in multiple loci in MV genome is practically convenient, for
this work insertion of foreign coding sequences in high level expressing locus proved
essential to obtain robust immune responses[4]. Immunogenicity studies presented this
dissertation depended on the availability of genetically modified measles susceptible
small animal model, the HuCD46Ge-IFNarKO mouse, which is the most reliable and
relevant small animal model for preclinical evaluation of MV vaccines.
This dissertation contributes to the field of Flavivirus vaccines development in
three clearly discernible ways. First, while previous studies have demonstrated cross
immune responses between different dengue serotypes[10,11], ours is the first study to
108
show that these responses can be obtained using a recombinant vaccine, MV-Dengue
multivalent vaccine developed a homologous and heterologous neutralizing immune
responses against DV1, 2,4 and YFV. However, there have been limited investigations
of cross-protection between different subgroups. YFV and DVs belong to different sero-
complexes. In the present study, we demonstrated cross-immunity against YFV at
protective levels, which was induced by the recombinant Measles-Dengue 2 and 4
cocktail vaccine in the mice. Our results provide valuable information in understanding
the interaction between different flavivirus members, which further assist the
development of a novel multivalent vaccine against DVs serotypes, and multiple
flaviviruses such as YFV and JEV.
Second, and significantly, this dissertation identifies a promising complementary
anti-dengue immunization strategy based on a recombinant measles virus vaccine
cocktail to protect children in the age range from 2 to 9 years, whom may develop severe
forms of DV infection. Recently, the long-term follow-up results for the third year of the
phase III trial of the The Sanofi-Pasteur vaccine, Dengvaxia, conducted in Southeast Asia
(~10,000 children aged 2 to 14 years) result showed that vaccinees in the youngest age
group, younger than 5 years, had a substantially and significantly higher risk of
hospitalization for severing dengue disease than controls[7]. Importantly, seropositive
vaccine recipients had high and sustained antibody levels after the first dose of vaccine;
seronegative recipients had a lower peak of neutralizing antibodies by a factor of 10
times. The reduced efficacy in seronegative recipients especially younger age groups
suggested that the effectiveness of DV vaccine would mainly depend on the serostatus at
109
the date of vaccination[]. In this dissertation, we proposed that a MV-Dengue pediatric
vaccine would induce broad anti-flaviral responses when administered to one-year infants
to prime their immune system. Protective flavivirus neutralization will be substantially
boosted upon administration of the subsequent chimeric Dengue vaccine. This approach
would close the current gap (younger than 9 years of age) and geographical indication
(only for hyperendemic countries), which makes the current dengue vaccine so
unpractical.
In a Third immense contribution to the field, this dissertation presents an idea for
a new generation of flavivirus vaccines which, may provide effective cross-protective
immunity against heterologous flavivirus infection in the endemic countries. Serological
cross-reactivity providing cross-protective immunity between related viruses has been
proven more than 200 years ago, as it was the immunological basis for the first human
vaccine against smallpox[14-16]. Indeed, whatever the nature of the attenuated virus used
as vaccine to eradicate smallpox (most probably camelpox or horsepox), cross-protective
responses proved safe and efficient. Cross protective flaviviral responses explain the
global distribution of mosquito-borne flaviviruses, for instance the practical absence of
yellow fever virus in Asia or the abysmal reduction of St Louis Encephalitis cases in the
USA after the introduction of West Nile virus. This study offers a platform to produce a
multivalent anti-flavivirus vaccine; the cocktail of recombinant vaccine may include
other recombinant measles expressing major E protein of other close flaviviruses
including YFV, JEV, and WNV. The cocktail of recombinant measles expressing
110
multiple flavivirus antigens can be modified based on the co-circulation of the viruses
within different endemic countries.
In conclusion, this dissertation explores the generation of a recombinant measles-
dengue cocktail immunization to prime a broad, anti-flaviviral response in one-year-old
recipients as a complementary immunization strategy to enhance the immunogenicity and
efficiency of subsequent chimeric Dengue vaccine approaches. I sincerely hope that this
work will prove generative to the intelligent design of vaccines against DVs and other
flaviviruses.
111
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