Dengue Virus Vaccine Development · 2020-03-13 · Dengue virus (DENV) is a significant cause of morbidity and mortality in tropical and subtropical regions, causing hundreds of millions

CHAPTER SEVEN

Dengue Virus VaccineDevelopmentLauren E. Yauch, Sujan Shresta1Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA1Corresponding author: e-mail address: [email protected]

Contents

1.

AdvaISSNhttp:/

Virology and Epidemiology of DENV Infection

nces in Virus Research, Volume 88 # 2014 Elsevier Inc.0065-3527 All rights reserved./dx.doi.org/10.1016/B978-0-12-800098-4.00007-6

316
2. Adaptive Immune Response to DENV 317 3. Dengue Vaccine Objectives and Challenges 320 4. Animal Models for Testing Dengue Vaccine Candidates 323 5. Dengue Vaccine Approaches 325
5.1
Recombinant subunit protein vaccines/subviral particles 325 6. DNA Vaccines 329 7. Viral Vectored Vaccines 333
7.1
Vaccinia 333 7.2 Adenovirus vectors 334 7.3 Alphavirus replicon particles 335
8.
Inactivated Whole Virus 336 9. Live Attenuated 339
9.1
University of Hawaii/WRAIR 340 9.2 Mahidol University 343 9.3 CDC/Inviragen 345 9.4 NIAID/NIH 346 9.5 DENV Chimeras 350 9.6 Acambis/Sanofi Pasteur (ChimeriVax) 351
10.
Moving Forward 355 References 357
Abstract

Dengue virus (DENV) is a significant cause of morbidity and mortality in tropical andsubtropical regions, causing hundreds of millions of infections each year. Infectionsrange from asymptomatic to a self-limited febrile illness, dengue fever (DF), to thelife-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Theexpanding of the habitat of DENV-transmitting mosquitoes has resulted in dramaticincreases in the number of cases over the past 50 years, and recent outbreaks haveoccurred in the United States. Developing a dengue vaccine is a global health priority.DENV vaccine development is challenging due to the existence of four serotypes of the

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http://dx.doi.org/10.1016/B978-0-12-800098-4.00007-6

316 Lauren E. Yauch and Sujan Shresta

virus (DENV1–4), which a vaccine must protect against. Additionally, the adaptiveimmune response to DENV may be both protective and pathogenic upon subsequentinfection, and the precise features of protective versus pathogenic immune responsesto DENV are unknown, complicating vaccine development. Numerous vaccinecandidates, including live attenuated, inactivated, recombinant subunit, DNA, and viralvectored vaccines, are in various stages of clinical development, from preclinical tophase 3. This review will discuss the adaptive immune response to DENV, dengue vac-cine challenges, animal models used to test dengue vaccine candidates, and historicaland current dengue vaccine approaches.

1. VIROLOGY AND EPIDEMIOLOGY OF DENV INFECTION

Dengue virus (DENV) is the etiologic agent of dengue fever (DF), the

most prevalent arthropod-borne viral illness in humans. DENV belongs to

the Flaviviridae family and is related yellow fever virus (YFV), hepatitis

C virus, West Nile virus, Japanese encephalitis virus (JEV), and St. Louis

encephalitis virus. DENV is an enveloped virus with a single-stranded,

positive-sense RNA genome. The DENV genome is 10.7 kb and contains

a 50methyl guanosine cap, 50untranslated region (UTR), single open reading

frame, and a 30UTR (Clyde, Kyle, & Harris, 2006). The RNA genome is

translated as a single polyprotein that is then cleaved into three structural

proteins (capsid (C), premembrane (prM), and envelope (E)) and seven non-

structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5)

by both viral and host proteases. prM likely functions as a chaperone for

E during virion assembly (Mukhopadhyay, Kuhn, & Rossmann, 2005).

prM is cleaved by furin to M in the trans-Golgi resulting in the formation

of mature virions containing E andM (Murphy &Whitehead, 2011). How-

ever, this cleavage is incomplete (especially in mosquito cells), so many

immature virions that contain prM are released (van der Schaar et al.,

2007). The E protein is structurally conserved among flaviviruses and con-

sists of three domains (EDI, EDII, and EDIII) (Kuhn et al., 2002; Rey,

Heinz, Mandl, Kunz, & Harrison, 1995). The E protein interacts with a cel-

lular receptor(s) and viral uptake occurs via receptor-mediated endocytosis

followed by fusion of the viral and endosomal membranes and release of the

nucleocapsid into the cytoplasm (Heinz et al., 1994; Mukhopadhyay et al.,

2005). Translation and replication of the viral genome occurs in the cyto-

plasm in association with intracellular membranous structures. Virus assem-

bly takes place at intracellular membranes, and viral particles pass through the

Golgi and are exocytosed via secretory vesicles (Heinz et al., 1994).

317Dengue Vaccines

The four serotypes of DENV (DENV1–4) are transmitted to humans

primarily by the mosquitoes Aedes aegypti and Aedes albopictus. The habitat

of DENV-transmitting mosquitoes has expanded, and in the last 50 years,

the incidence of infections has increased 30-fold (WHO, 2009). Infections

with DENV can be asymptomatic or cause a spectrum of clinical disease

ranging from an acute, debilitating, febrile illness (DF) to the more life-

threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/

DSS). Typical symptoms of DF consist of fever, retro-orbital headache,

myalgia, rash, nausea, and vomiting. DHF is characterized by increased vas-

cular permeability, hemorrhagic manifestations, thrombocytopenia, and, in

the case of DSS, shock (WHO, 2009). Epidemiological observations have

revealed that secondary infection with a different dengue serotype is the sin-

gle greatest risk factor for manifestations of severe disease. In addition to the

individual’s immune status, genetic host factors and viral virulence have also

been postulated to affect disease severity (Halstead, 2007; Rico-Hesse,

2007). Thus, the development of dengue disease likely depends on complex

interplays between host and viral factors.

DENV is endemic in Southeast Asia, the Western Pacific, Central and

South America, the Caribbean, and Africa. Recent outbreaks have occurred

in the United States in Hawaii (2001), Texas (2005), and Florida

(2009–2011) (Adalja, Sell, Bouri, & Franco, 2012). Based on a recent pub-

lication reporting new, evidence-based estimates of the global burden of

dengue, 3.6 billion people live in dengue-endemic areas and the virus causes

approximately 400 million infections and 100 million symptomatic cases

annually (Bhatt et al., 2013). Over 2 million cases of severe dengue disease

and over 20,000 deaths are estimated to occur each year (Gubler, 2012).

Despite these high numbers of global morbidity and mortality associated

with DENV infection, no effective antiviral therapy or vaccine exists at pre-

sent and treatment is largely supportive in nature.

2. ADAPTIVE IMMUNE RESPONSE TO DENV

The adaptive immune response presumably affords a lifelong immu-

nity against challenge with the same DENV serotype, but only transient

cross-protection against a heterologous DENV serotype, after which the

memory response may play a pathological role during a secondary infection

(Kyle & Harris, 2008). An early study in human volunteers found homol-

ogous immunity lasted as long as 18 months, and heterologous immunity

for 2–3 months (Sabin, 1952). Epidemiological studies in Thailand and

Cuba support a role for the immune system in disease enhancement, as


most cases of DHF/DSS occur during secondary infections with a heterol-

ogous DENV serotype (Burke, Nisalak, Johnson, & Scott, 1988; Guzman

et al., 1987; Guzman et al., 2000; Halstead, Nimmannitya, & Cohen,

1970; Sangkawibha et al., 1984; Vaughn et al., 2000). Infants born to

dengue-immune mothers are also at greater risk for DHF/DSS, during

the period of time (between 6 and 9 months of age) when circulating

maternal antibodies levels wane to subprotective levels (Halstead, 1988;

Kliks, Nimmanitya, Nisalak, & Burke, 1988). Thus, both actively and

passively acquired DENV-specific antibodies are associated with severe

dengue disease. Consequently, the immunologic investigation of DENV

infection has been dominated by studies examining the role of adaptive

immunity in DENV pathogenesis. Subneutralizing concentrations of

DENV-specific antibodies may contribute to viral replication and disease

severity via a phenomenon known as “antibody-dependent-enhancement”

(ADE). According to the ADE hypothesis, DENV-antibody complexes are

formed and bind to Fc receptors (FcR) on cells such as macrophages, facil-

itating viral entry and replication. Increased viral loads resulting from

ADE then drive the production of inflammatory mediators that increase

vascular permeability. Supporting the ADE hypothesis, nonneutralizing

DENV-specific antibodies increased viral replication in peripheral

blood leukocytes in vitro (Halstead & O’Rourke, 1977; Halstead,

O’Rourke, & Allison, 1977), and studies using a variety of monoclonal anti-

bodies have since shown that neutralizing antibodies can promote ADE

in vitro when present at subneutralizing concentrations (Morens,

Halstead, & Marchette, 1987; Pierson et al., 2007). Studies with monkeys

have confirmed ADE of DENV replication in vivo. Specifically, monkeys

receiving passive transfer of DENV-immune human sera (Halstead, 1979)

or a humanized DENV-specific IgG1 monoclonal antibody (Goncalvez,

Engle, St Claire, Purcell, & Lai, 2007) had higher viral loads than control

monkeys. ADE resulting in disease enhancement was recently demonst-

rated using a mouse model of DENV infection: infection in the

presence of DENV-reactive monoclonal antibodies or immune sera resulted

in increased disease severity and turned a nonlethal illness into a lethal

disease resembling human DHF/DSS (Balsitis et al., 2010; Zellweger,

Prestwood, & Shresta, 2010).

In addition to a pathogenic role for antibodies in severe dengue disease,

altered T-cell responses during secondary infections with heterologous sero-

types have been postulated to contribute to cytokine storm and immuno-

pathogenesis of DHF/DSS. Studies with human samples have shown that

319Dengue Vaccines

serotype cross-reactive T cells are preferentially activated during secondary

infection, and these cross-reactive T cells exhibit suboptimal degranulation

and enhanced TNF and IFN-g production (Mangada & Rothman, 2005;

Mongkolsapaya et al., 2003, 2006). TNF is suspected to cause endothelial

cell dysfunction or damage, leading to plasma leakage, a hallmark of

DHF/DSS. At present, despite several decades of research investigating

the role of T cells in the context of DENV pathogenesis, direct evidence

demonstrating a pathogenic role for DENV-specific T cells is not yet avail-

able. In fact, one study of DENV-infected adults found the breadth and

magnitude of the T-cell response during secondary DENV infection was

not significantly associated with disease severity (Simmons et al., 2005),

and a recent study of T-cell responses in donors in a DENV hyperendemic

area supports an HLA-linked protective role for CD8þ T cells (Weiskopf

et al., 2013). An important protective role for CD8þT cells during primary

DENV2 infection was also identified using a mouse model (Yauch et al.,

2009). These recent studies are beginning to examine the role of T cells

in the context of protection, and are starting to implicate a key role for

T cells, in particular CD8þ T cells, in anti-DENV immunity.

In addition to T cells, virus-specific antibodies are likely to play a pro-

tective role against DENV infection in humans. Sera from infected individ-

uals or anti-DENV monoclonal antibodies can neutralize epitopes that

are required for viral entry (Crill & Roehrig, 2001) and can mediate

antibody-dependent cell-mediated cytotoxicity (ADCC) (Garcia et al.,

2006; Laoprasopwattana et al., 2007). In addition, the amounts of pre-

existing, heterologous neutralizing antibodies and ADCC activity in

presecondary infection plasma samples negatively correlate with plasma vire-

mia levels and disease severity (Endy et al., 2004; Laoprasopwattana et al.,

2007). Studies with mouse models have shown that passive transfer of neu-

tralizing monoclonal antibodies can confer protection from lethal challenge

(Kaufman et al., 1989; Kaufman, Summers, Dubois, & Eckels, 1987) and

antibody-mediated control of flavivirus infection in vivo correlates with neu-

tralizing activity in vitro (Diamond, Shrestha, Marri, Mahan, & Engle, 2003;

Kaufman et al., 1987; Oliphant et al., 2005). The majority of neutralizing

antibodies against DENV are directed against the E protein, and the most

potently neutralizing bind EDIII (Crill & Roehrig, 2001; Megret et al.,

1992; Roehrig, 2003; Shrestha et al., 2010; Sukupolvi-Petty et al., 2010,

2007; Wahala et al., 2010). Although not part of the virion, NS1 is also a

target of the host antibody response, as the protein is expressed on the

surface of infected cells and is also secreted (Muller & Young, 2013).


NS1 is a complement-fixing antigen, and NS1-specific antibodies can pro-

tect via complement-dependent killing of infected cells. Recent studies

examining the human DENV-specific antibody response have identified

neutralizing antibodies that bind EDIII (Beltramello et al., 2010; de Alwis

et al., 2011), as well as neutralizing antibodies that recognize a complex

epitope present on the virion but not on soluble E protein (de Alwis et al.,

2012). prM/Mis also a dominant targetof thehumanDENV-specific antibody

response, however prM/M-specific antibodies were shown to be broadly

cross-reactive and weakly or nonneutralizing (Beltramello et al., 2010;

de Alwis et al., 2011; Dejnirattisai et al., 2010). These studies have begun to

decipher features of a protective anti-DENV antibody response in humans.

Collectively, studies to date demonstrate that DENV-specific antibodies

can both protect against infection and, under certain conditions, enhance

infection and disease severity, whereas the role of T cells remains to be fully

elucidated. Thus, the adaptive immune response to dengue can be both

protective and pathogenic, which complicates vaccine development, as dis-

cussed in the succeeding text.

3. DENGUE VACCINE OBJECTIVES AND CHALLENGES

Several DENV vaccines are currently under development, including

some in phase 3 safety and efficacy testing (Table 7.1). These include

inactivated, live attenuated, recombinant subunit, viral vectored, and

DNA vaccines. Dengue vaccine development has focused on eliciting a

neutralizing antibody response, as T cells are assumed to play a minor or

secondary role in dengue vaccine-mediated protection. The WHO has

published guidelines on the clinical evaluation of dengue vaccines in

endemic areas (WHO Initiative for Vaccine Research, & World Health

Organization. Dept. of Immunization Vaccines and Biologicals, 2008)

and on the quality, safety, and efficacy of live attenuated dengue vaccines

(WHO, 2011).

The successful development of live attenuated vaccines for the

flaviviruses YFV and JEV suggest a DENV vaccine is feasible. However,

DENV vaccine development is more complicated due to the existence of

four serotypes of DENV that a vaccine must induce protection against. Viral

interference, which is when one or more serotype(s) replicates better than

the others and the immune response against that serotype dominates, has

been an issue in tetravalent DENV vaccine development. Another signifi-

cant challenge to dengue vaccine development is the potential for

Table 7.1 Vaccines in developmentType Approach Developer Status

Recombinant

subunit

Affinity-purified E protein Hawaii Biotec/

Merck

Phase 1

Recombinant

subunit

EDIII protein fused to carrier

protein

Preclinical

DNA

monovalent

prM and E of DENV1 NMRC Phase 1

DNA

tetravalent

prM and E of DENV1–4 NMRC Phase 1

DNA

tetravalent

EDIII from DENV1–4, synthetic

consensus (SynCon™) human

codon optimized

Inovio Preclinical

DNA shuffle DNA shuffling of codon-

optimized DENV1–4 E to

generate single chimeric antigen

NMRC/Maxygen Preclinical

DNA NS1 Various Preclinical

Adenoviral

vector

Recombinant adenoviral vector

expressing DENV1–4 prM and E

NMRC/GenPhar Preclinical

Alphavirus

replicon

particles

VRP expressing prM and E or

soluble E dimers from DENV1–4

Global Vaccines Preclinical

Inactivated

monovalent

Purified, inactivated DENV1 WRAIR Phase 1

Inactivated

tetravalent

Purified, inactivated DENV1–4 WRAIR Phase 1

Live

attenuated

tetravalent

Tissue culture-passaged WRAIR/GSK Phase 2

Live

attenuated

tetravalent

chimeric

Tissue culture-passaged DENV2

backbone and prM/E from

DENV1–4

CDC/Inviragen Phase 2

Live

attenuated

tetravalent

chimeric

Gene deletion (D30 30UTR

deletion mutations)

NIAID/NIH Phase 1

Live

attenuated

tetravalent

chimeric

YFV/DENV chimera Acambis/Sanofi

Pasteur

Phase 3


nonneutralizing antibody responses to enhance DENV infection and dis-

ease. A dengue vaccine must induce antibody responses to all four serotypes

simultaneously, and must provide long-lasting immunity to avoid the risk of

ADE. Long-term studies are needed to evaluate the duration of vaccine-

induced immunity, as epidemiological studies of sequential outbreaks in

Cuba (DENV1 followed by DENV2 and DENV3) revealed that 20 years

or more between DENV infections resulted in DHF/DSS, and the risk

of severe disease was actually greater at longer intervals (Alvarez et al.,

2006; Guzman et al., 2000, 2002).

Another challenge of DENV vaccine development is that the correlates

of protection, that is, the immune functions responsible for protection, are

presently unknown. Therefore, vaccine efficacy must be measured as pro-

tection from infection in human vaccinees. Neutralizing antibodies are

thought to be best surrogate for vaccine-induced protection, and high

DENV neutralizing antibody titers (measured by plaque-reduction neutral-

ization tests (PRNT)) in monkeys have been correlated with protection

(Clements et al., 2010; Guirakhoo et al., 2004). However, there is no proof

that neutralizing antibodies are absolutely required to protect. In fact,

numerous studies in monkeys found a lack of correlation between neutral-

izing antibody titers and protection (Blaney, Matro, Murphy, &Whitehead,

2005; Raviprakash, Porter, et al., 2000; Robert Putnak et al., 2005; Scott

et al., 1980; Simmons, Porter, Hayes, Vaughn, & Putnak, 2006; White

et al., 2013). Similarly, studies with mouse models have revealed a lack of

correlation between neutralizing antibody titers and protection (Brien

et al., 2010; Zellweger, Miller, Eddy et al., 2013). Additionally, a live atten-

uated vaccine candidate recently tested in a phase 2b trial induced high titers

of neutralizing antibodies against DENV2 but was ineffective at preventing

DENV2 infection (Sabchareon et al., 2012). Thus, different features of the

anti-DENV antibody response, such as ADCC and complement-fixation,

or PRNT assays using cell types other than the standard epithelial cell lines

for measurement of neutralization activity may correlate with antibody-

mediated protection against DENV in vivo. Based on recent studies impli-

cating a role for CD8þ T cells in protection against DENV in humans

and mouse models, certain T-cell-mediated functions may also correlate

with protection in vivo.

As DENV is a significant public health problem in many resource-poor

countries, a dengue vaccine must be manufactured economically, which is

difficult, as the vaccine needs to include viruses or antigens from all four

serotypes. The vaccine must be safe and not cause DF-like disease. Both

323Dengue Vaccines

safety and efficacy must be tested in different ethnicities, and the vaccines

must be safe and immunogenic in children and adults. DENV cocirculates

in areas with other flaviviruses, including YFV and JEV, and therefore a den-

gue vaccine needs to be effective in inducing an immune response in

flavivirus-immune individuals. Studies have found preexisting immunity

to YFV resulted in enhanced DENV-specific antibody responses following

DENV vaccination or infection (Bancroft et al., 1984; Carey, Myers, &

Rodrigues, 1965; Dorrance et al., 1956; Guirakhoo et al., 2006; Poo

et al., 2010; Scott et al., 1983). Thus, it appears dengue vaccination in

flavivirus-immune individuals is feasible, albeit the precise features of the

anti-DENV antibody response (in terms of specificity, isotype, avidity,

and in vivo protective capacity) in flavivirus-immune versus flavivirus-naive

individuals are as yet unknown, and none of these published studies exam-

ined the anti-DENV T-cell responses.

Finally, live attenuated vaccines must be evaluated for neurovirulence in

nonhuman primates (NHP) although testing a rodent model may be suffi-

cient in the future (Monath et al., 2005;WHO, 2011). Neurovirulence test-

ing is particularly important for vaccines created using the YF 17D

backbone, as that vaccine has been associated with neurotropic disease

(Khromava et al., 2005).

4. ANIMAL MODELS FOR TESTING DENGUE VACCINECANDIDATES

Although the natural hosts for DENV are humans and mosquitoes, a

sylvatic cycle involving NHP has been observed in Africa and Southeast Asia

(Diallo et al., 2003; Wolfe et al., 2001). NHP used in dengue vaccine

research include rhesus monkeys (Macaca mulatta), cynomolgus monkeys

(Macaca fascicularis), and owl monkeys (Aotus nancymaae). NHP develop vire-

mia and an antibody response upon DENV infection but show very few

clinical signs of disease observed in humans (Halstead, Casals, Shotwell, &

Palumbo, 1973; Scherer, Russell, Rosen, Casals, & Dickerman, 1978).

Rhesus monkeys infected with DENV develop transient viremia lasting

3–6 days (Blaney et al., 2005, 2007; Guirakhoo et al., 2001). After subcu-

taneous (s.c.) infection, the virus quickly spreads to the regional lymph nodes

and can be isolated from the skin and distant lymph nodes, and rarely from

the spleen, thymus, liver, lungs, and bone marrow (Marchette, Halstead,

Falkler, Stenhouse, &Nash, 1973). Some hallmarks of human clinical disease

have been observed in NHP after s.c. infection, including leukopenia and


thrombocytopenia (Halstead & Palumbo, 1973; Marchette et al., 1973). In

one study, infection of rhesus monkeys with DENV via the intravenous

(i.v.) route resulted in hemorrhage and petechiae (Onlamoon et al.,

2010). In NHP, vaccine efficacy and safety have typically been measured

by changes in the duration of viremia, peak viral titer, and magnitude of

the antibody response. Important differences have been noted between

vaccination of humans and NHP; in particular, shorter immunization pro-

tocols are effective in NHP. Twomonths between doses of a live attenuated

vaccine protected rhesus monkeys (Simmons, Burgess, Lynch, & Putnak,

2010), whereas in humans, 3 months between doses of a live attenuated

vaccine did not significantly enhance immunity (Sun et al., 2003). In addi-

tion, a live attenuated tetravalent vaccine protected monkeys from DENV

challenge but was not protective in a human phase 2b trial, although the

reasons for the lack of efficacy remain to be determined (Guirakhoo

et al., 2004; Sabchareon et al., 2012).

Wild-type mice are highly resistant to infection with DENV clinical iso-

lates. Mouse models that have been developed for studying dengue patho-

genesis and testing vaccine and antiviral candidates include intracerebral

(i.c.) inoculation with mouse brain-adapted virus, infection of immuno-

compromised mice (including mice lacking components of the interferon

(IFN) response), and mouse–human chimeras (Zompi & Harris, 2012).

DENV infection of suckling mice via the i.c. route causes encephalitis

and death and has been used to test the efficacy of DENV vaccines

(Blaney et al., 2001; Bray et al., 1989; Eckels et al., 1984; Falgout, Bray,

Schlesinger, & Lai, 1990; van Der Most, Murali-Krishna, Ahmed, &

Strauss, 2000). However, both the route of infection and outcome are

not relevant to human dengue disease. The WHO guidelines suggest the

suckling mouse/encephalitis model is not useful for testing the safety and

efficacy of dengue vaccine candidates but could be used to test vaccine

lot consistency (WHO, 2011).

Some of the immunocompromised mice and mouse–human chimeras

develop signs of dengue disease observed in humans, including fever,

increased vascular permeability, and thrombocytopenia after DENV infec-

tion. Severe combined immunodeficiency (SCID) mice transplanted

with human liver cells (SCID-HuH-7) have been used to test the attenua-

tion of live attenuated dengue vaccines by measuring viral titers (Blaney,

Hanson, Hanley, Murphy, & Whitehead, 2004; Blaney et al., 2005).

Mice lacking both type I and type II IFN receptors (AG129) are highly

susceptible to DENV and were developed to test dengue vaccine candidates

325Dengue Vaccines

(Johnson & Roehrig, 1999). The AG129 mice develop paralysis even when

inoculated via a peripheral route, although infection with certain DENV

strains or infection in the presence of DENV-specific antibodies leads to

a severe disease mimicking DHF/DSS (Balsitis et al., 2010; Jelinek et al.,

2002; Prestwood, Prigozhin, Sharar, Zellweger, & Shresta, 2008; Tan

et al., 2010; Zellweger et al., 2010). A live attenuated vaccine candidate

(DENVax) has been tested in AG129 mice (Brewoo et al., 2012; Huang

et al., 2003); however, results assessing dengue vaccine-induced immune

responses in these mice with compromised or altered immune system should

be interpreted with caution. Due to the limitations of the animal models and

the lack of known correlates of protection, protection mediated by DENV

vaccine candidates, in particular live attenuated vaccines that replicate

poorly in animal models, will ultimately be defined by the ability to protect

humans from DF and DHF/DSS (WHO, 2011).

5. DENGUE VACCINE APPROACHES

5.1. Recombinant subunit protein vaccines/subviral
particles
Recombinant subunit vaccines have several advantages for DENV vaccina-

tion compared with live attenuated vaccines. Protein vaccines are safe,

inducing a balanced immune response to the four serotypes should be fea-

sible, and the immunization schedule can be accelerated, which reduces the

risk of incomplete immunity and the potential for ADE. The disadvantages

of these vaccines include the requirement for adjuvant and multiple doses to

achieve optimal immunogenicity, and they may not be as efficient at induc-

ing long-lasting immunity as live attenuated vaccines.

The target of subunit vaccine development for dengue has been the

E glycoprotein, as the majority of neutralizing epitopes on the DENV virion

are in the E protein. Recombinant E protein has been produced using

Escherichia coli, baculovirus/insect cells, yeast, and mammalian cells.

E. coli has been used to express truncated versions of E that are fused to

other carrier proteins. EDIII, which is believed to be the receptor-binding

domain, has been the focus of these E. coli-expressed fusion proteins. EDIIIs

fromDENV1–4 fused to theE. coli trpE protein and expressed in E. coliwere

recognized by immune ascites fluid from mice infected with the homolo-

gous, but not heterologous, serotypes (Fonseca, Khoshnood, Shope, &

Mason, 1991; Mason, Zugel, Semproni, Fournier, & Mason, 1990). EDIII

from DENV2 fused to the maltose-binding protein (MBP) from E. coli


induced neutralizing antibodies in immunized mice and partially protected

against lethal DENV2 i.c. challenge (Simmons, Nelson, Wu, & Hayes,

1998). Tetravalent immunization of mice (intramuscular (i.m.), in alum)

with EDIII-MBP fusion proteins from each of the four serotypes resulted

in neutralizing antibody responses against all four serotypes (Simmons,

Murphy, & Hayes, 2001). Immunization of mice with the recombinant

E protein together with a DENV2 DNA vaccine encoding prM and

E induced high titer antibody and neutralizing antibody responses, as mea-

sured by enzyme-linked immunosorbent assay (ELISA) and PRNT50,

respectively (Simmons, Murphy, Kochel, Raviprakash, & Hayes, 2001).

The DENV2 EDIII-MBP fusion protein, along with the prM/E DNA vac-

cine and a purified inactivated virus (PIV), was tested in rhesus monkeys in

various combinations of prime–boost vaccination (Simmons et al., 2006).

The highest neutralizing antibody titers were observed following combina-

tion DNA and recombinant protein vaccination; however, only PIV vacci-

nation protected monkeys from viremia after challenge with DENV2.

Immunization of mice with DENV2 EDIII fused to the meningococcal

P64k protein induced neutralizing antibodies and partial protection from

lethal i.c. DENV2 challenge (Hermida et al., 2004). Vaccination of

cynomolgus monkeys with this recombinant protein in Freund’s adjuvant

protected from DENV2 challenge (Hermida et al., 2006), and green mon-

keys vaccinated with the fusion protein formulated with serogroup

A capsular polysaccharide from Neisseria meningitidis (adsorbed on alum)

developed neutralizing antibody titers against DENV2 and were partially

protected from DENV2 challenge (Valdes, Hermida, et al., 2009). Finally,

an EDIII-C chimeric protein expressed in E. coli induced neutralizing anti-

bodies in mice (Valdes, Bernardo, et al., 2009). When aggregated with

oligodeoxynucleotides, the protein also induced a stronger cell-mediated

immune (CMI) response and protected 70% of mice from i.c. DENV2 chal-

lenge. Thus, a wide variety of E. coli-expressed EDIII containing fusion pro-

teins have been generated and tested in both mouse and NHP models.

The yeast Pichia pastoris has been used to generate recombinant E protein

from DENV4 (Guzman et al., 2003). To improve secretion, the E protein

was truncated at the C-terminus to remove the hydrophobic membrane

anchor. Cynomolgus monkeys immunized with recombinant E plus alum

developed neutralizing antibodies but were only partially protected against

DENV4 challenge.

Advantages of the baculovirus and insect cell expression system include

high yields and proper processing and glycosylation of the expressed protein.

327Dengue Vaccines

DENV E produced by baculovirus has been shown to be in its native con-

formation and immunogenic. Recombinant baculovirus encoding DENV4

C-M-E-NS1-NS2A was expressed in Spodoptera frugiperda (Sf )-derived Sf9

cells (Zhang et al., 1988). Rabbits immunized with infected Sf9 cell lysate

developed a low titer antibody response against prM, E, and NS1, and

immunized mice did not develop virus-neutralizing antibodies but were

protected from i.c. lethal challenge. Rhesus monkeys were then immunized

with the lysate, which induced low levels of antivirion antibodies, but vac-

cination did not significantly protect monkeys from DENV4 challenge

(Eckels et al., 1994). C-terminally truncated E and part of the M protein

fromDENV1 were expressed in Sf cells (Putnak et al., 1991). Immunization

of BALB/c mice with the recombinant protein in complete and incomplete

Freund’s adjuvant induced neutralizing antibodies and protected some mice

from DENV1 i.c. challenge. Similarly, C-terminally truncated DENV2 and

DENV3 E proteins expressed in Sf9 cells induced neutralizing antibodies in

mice (Delenda, Staropoli, Frenkiel, Cabanie, & Deubel, 1994). Recombi-

nant DENV2 E protein protected against lethal DENV2 i.c. challenge, and

immunization with DENV3 E protein was partially protective against het-

erologous DENV2 infection. Vaccination of cynomolgus monkeys with the

recombinant E protein only partially protected from viral challenge (Velzing

et al., 1999). A hybrid E protein containing 36 amino acids fromM, EDI and

EDII from DENV2 E, and EDIII from DENV3 was constructed and

expressed in Sf21 cells (Bielefeldt-Ohmann, Beasley, Fitzpatrick, &

Aaskov, 1997). The recombinant protein was recognized by a panel of

DENV-reactive monoclonal antibodies and inhibited binding of DENV2

and DENV3 to human cells. Immunization of mice induced DENV2-

and DENV3-specific antibody and cross-reactive T-cell responses.

Expression of E along with prM allows for the secretion of E from cells,

and the integrity of the neutralizing epitopes on E are maintained (Fonseca,

Pincus, Shope, Paoletti, & Mason, 1994). Expression of prM and E DENV

proteins in cells can generate virus-like particles (VLP), which contain the

glycosylated viral proteins in a lipid membrane. DENVVLP have been gen-

erated from E and prM constructs expressed in yeast (Sugrue, Fu, Howe, &

Chan, 1997), insect (Kelly, Greene, King, & Innis, 2000; Kuwahara &

Konishi, 2010), and mammalian (Konishi & Fujii, 2002; Zhang et al.,

2011) cells. The VLP are similar to infectious virions in terms of structure

but are safer as they are noninfectious. The E contained in the VLP was

shown to be equivalent to E produced in infected cells (Konishi & Fujii,

2002; Kuwahara & Konishi, 2010), and immunization of rabbits and mice


with the VLP induced neutralizing antibodies (Kelly et al., 2000; Konishi &

Fujii, 2002; Sugrue et al., 1997; Zhang et al., 1988).

To avoid the drawbacks of expressing the E protein in E. coli, yeast, and

baculovirus/insect cell systems, including expression of the protein in a non-

native conformation, low yields, and modest immunogenicity, theDrosoph-

ila melanogaster Schneider 2 (S2) cell expression system has been utilized by

Hawaii Biotech to express the E protein (Coller, Clements, Bett, Sagar, &

Ter Meulen, 2011). S2 cells were stably transformed with constructs

expressing full-length prM and 80% of the E protein (C-terminally trun-

cated; 80E) from the four DENV serotypes (strains DENV1 258848,

DENV2 PR159/S1, DENV3 CH53489, and DENV4 H241) (Clements

et al., 2010; Robert Putnak et al., 2005). Glycosylated recombinant 80E

proteins were produced at high levels (10–40 mg/L) in native-like confor-

mation. Immunogenicity of the DENV2-80E recombinant protein was

tested in rhesus monkeys (Robert Putnak et al., 2005). DENV2-80E was

given with five different adjuvant formulations, including AS04-OH,

AS04-PO, AS05, AS08 (all produced by GlaxoSmithKline (GSK)), and

alum. Monkeys were immunized at 0 and 3 months, and all animals

seroconverted after the second dose. The highest neutralizing antibody titers

were observed when DENV2-80E was given with AS04, AS05, or AS08.

The booster immunization increased neutralizing antibody titers, which

then dropped before challenge. DENV2-80E partially protected monkeys

fromwild-typeDENV2 challenge; most vaccinatedmonkeys had no detect-

able live virus but some had DENV RNA in the sera as measured by real-

time RT-PCR. Immunization of BALB/c mice with 80E subunits from the

four serotypes in ISCOMATRIX® adjuvant induced long-lasting neutral-

izing antibody titers against all serotypes (Clements et al., 2010). The neu-

tralizing antibody titers were similar when the antigens were given as

tetravalent or monovalent immunization, implying no antigenic interfer-

ence with the tetravalent formulation. Rhesus monkeys immunized with

low doses (1 or 5 mg) of each of the four DENV-80E proteins (along with

the DENV2NS1 protein to enhance immunogenicity) induced neutralizing

antibodies against all four serotypes, and monkeys were protected against

challenge with DENV2 or DENV4. These vaccine candidates were recently

transferred from Hawaii Biotech to Merck. A phase 1 trial of the DENV1-

80E vaccine candidate (three doses of 10 or 50 mg in alum) has been

completed (Coller et al., 2011), and a phase 1 trial of a tetravalent formula-

tion (V180) with ISCOMATRIX® began in 2012 (Clinicaltrials.gov

NCT01477580).

329Dengue Vaccines

6. DNA VACCINES

DNA vaccination involves cloning the gene(s) of interest into a plas-

mid backbone and delivering the DNA intradermally (i.d.), s.c., or i.m. The

DNA is taken up by cells, the protein of interest is expressed, and antigen-

presenting cells take the antigen to the draining lymph nodes (Gurunathan,

Klinman, & Seder, 2000). DNA vaccination results in antigen expressed by

both MHC class I and class II, leading to activation of CD8þ and CD4þT cells, as well as antibody responses. Other advantages include low cost,

ease of production, and temperature stability. DNA vaccines are non-

replicating, are therefore safer than live attenuated vaccines, and have low

reactogenicity. However, DNA vaccines are not highly immunogenic,

and require multiple doses and coimmunization with adjuvants.

Research done at the Naval Medical Research Center (NMRC) has led

to the first dengue DNA vaccine tested in a clinical trial. In initial studies, the

prM protein and 92% of the E protein from DENV2 (strain New Guinea C,

NGC) (C-terminally truncated) were cloned into eukaryotic expression

vectors (Kochel et al., 1997). E protein was expressed by transfected cells

in vitro, and immunization of mice (i.d.) resulted in DENV2 neutralizing

antibodies. Coimmunization with a plasmid expressing immunostimulatory

CpG motifs improved the neutralizing antibody response, and mice vacci-

nated with the DENV2 prM/E vaccine and CpG-containing plasmid were

significantly protected from lethal i.c. DENV2 challenge (Porter et al.,

1998). The DENV2 prM/EDNA vaccine (D) was tested in mice along with

the recombinant fusion protein containing DENV2 EDIII and MBP (R) as

part of various prime–boost strategies (Simmons, Murphy, Kochel, et al.,

2001). Mice received three doses of the vaccines alone or together:

R/R/R, D/R/R, D/D/D, R/D/D, or RD/RD/RD. Modest levels of

neutralizing antibody were induced by the DNA vaccine alone, whereas

immunization with the DNA vaccine together with the recombinant pro-

tein induced high titer antibody and neutralizing antibody responses. The

highest antibody titers (measured by ELISA) were observed following

D/D/D or RD/RD/RD vaccination, whereas the highest neutralizing

antibody responses (measured by PRNT) were induced by RD/RD/RD

and R/R/R, and the lowest were induced by D/D/D and R/D/D. The

DNA and protein vaccines were then tested in rhesus monkeys, along with

a PIV (P) (Simmons et al., 2006). After the third dose, all monkeys had

equivalent antibody titers by ELISA; the highest neutralizing antibody titers


were observed following DR/DR/DR, P/P/P, and DP/DP/DP vaccina-

tion, and the lowest neutralizing antibody titers were observed in D/D/D-

immunized monkeys. Monkeys were challenged with DENV2 (strain

S16803) 5 months after the last dose, and protection from DENV2 viremia

was only seen with PIV alone. DNA vaccination alone or in combination

with recombinant protein or PIV did not significantly reduce viremia.

To increase the immunogenicity of the DENV2 prM/E DNA vaccine,

antigen was targeted to lysosomes in an attempt to increase antigen presen-

tation on MHC class II, thereby enhancing CD4þ T-cell and antibody

responses (Raviprakash et al., 2001). The transmembrane and cytoplasmic

regions of E were replaced with carboxy-terminal sequence of lysosome-

associated membrane protein (LAMP), which contains the endosomal/

lysosomal targeting sequences of LAMP. The modification resulted in DENV

antigens colocalized with endogenous LAMP in transfected cells and signifi-

cantly increased neutralizing antibody titers in mice (Lu et al., 2003;

Raviprakash et al., 2001).

DENV1 DNA vaccine candidates were created using truncated or full-

length E with or without prM from strain Western Pacific 74 (West Pac 74)

(Raviprakash, Kochel, et al., 2000). Cells transfected with prM and full-

length E formed VLP in transfected cells and induced long-lasting neutral-

izing antibody responses in mice; therefore, this construct was selected for

further study. Rhesus monkeys were vaccinated i.d. or i.m. with three or

four doses of the DENV1 DNA vaccine (D1ME100) (Raviprakash,

Porter, et al., 2000). I.m. immunization resulted in higher antibody levels

than i.d., and protection from DENV1 challenge 4 months after the last

immunization. Four of eight monkeys vaccinated i.m. were completely

protected and four partially protected, despite very low neutralizing anti-

body titers. In contrast, i.d. vaccination did not protect. The D1ME100 vac-

cine was also tested in Aotus monkeys (Kochel et al., 2000). The monkeys

received three doses i.d. or i.m., and all developed neutralizing antibodies

and were partially or completely protected from viremia after DENV1 chal-

lenge 6 months after the third dose. To enhance the neutralizing antibody

response, Aotus monkeys were coimmunized with the D1ME100 vaccine

and plasmids expressing human immunostimulatory sequences (ISS) and/or

Aotus GM-CSF (Raviprakash et al., 2003). In addition, delivery of the vac-

cine using the needle-free Biojector®was tested. Coimmunization with ISS

or GM-CSF did not increase neutralizing antibody titers; however,

Biojector® vaccination resulted in significantly higher neutralizing antibody

titers for immunization with D1ME100 plus ISS and GM-CSF than needle

331Dengue Vaccines

injection (i.d.). D1ME100 given with the GM-CSF gene and ISS (whether

via Biojector® or needle) induced stable neutralizing antibody responses that

protected 87% of monkeys challenged with DENV1 6 months after a third

vaccination. D1ME100 was compared with a candidate vaccine (D1ME-

VRP) expressing DENV1 prM and E in a Venezuelan equine encephalitis

(VEE) virus replicon particle (VRP) (Chen et al., 2007). Cynomolgus mon-

keys were vaccinated with three doses of the DNA vaccine (DDD) or the

VRP (VVV) or given two doses of the DNA vaccine followed by a dose of

the VRP (DDV). All regimens were immunogenic and protective, but the

heterologous prime–boost of DDV induced the highest DENV1-specific

IgG and neutralizing antibody titers and complete protection from DENV1

challenge.

A tetravalent DNA (TDNA) vaccine was made and tested in rhesus

monkeys as part of a prime–boost vaccination strategy with a tetravalent live

attenuated vaccine (TLAV) boost (Simmons et al., 2010). The DNA con-

structs contained prM and full-length E from West Pac 74 (DENV1) and

near wild-type Philippine strains fromDENV2, 3, and 4. The DENV2 con-

struct contained the LAMP sequences. Monkeys were primed with TDNA

(1.25 mg of each serotype i.m. using Biojector®) or tetravalent PIV (TPIV)

in alum, boosted 2 months later with TLAV, and challenged with DENV3

(strain CH53489) 8 months later. Monkeys immunized with TDNA/

TDNA/TLAV were partially protected, whereas TPIV/TLAV monkeys

were completely protected from viremia.

A phase 1 study of the monovalent D1ME100 has been completed

(Beckett et al., 2011). Twenty-two flavivirus-naive adults received a high

or low dose (5 or 1 mg) of the DNA vaccine using the Biojector®

needle-free system at 0, 1.5, and 5 months. The vaccine was safe and well

tolerated; the most commonly reported side effect was mild pain or tender-

ness at the injection site. However, the vaccine was poorly immunogenic.

Of those receiving the high dose, only 41.6% (5/12) developed DENV1

neutralizing antibodies, and no neutralizing antibody responses were

detected in the low dose group. E protein-specific T-cell IFN-g responses

were detected in 50% and 83.3% of individuals in the low and high dose

groups, respectively. Various approaches are being explored to enhance

the immunogenicity of the DENV DNA vaccine, including alternative

delivery strategies, plasmid modifications, testing as part of prime–boost

strategies, and coimmunization with adjuvants (Danko, Beckett, &

Porter, 2011). Danko et al. found formulation with the adjuvant Vaxfectin®

enhanced the neutralizing antibody response in monkeys immunized with a


tetravalent DNA vaccine (Danko, Beckett, & Porter, 2011), and a phase 1

study of the tetravalent DNA vaccine (TVDV) given with Vaxfectin® began

in 2011 (Clinicaltrials.gov NCT01502358).

In parallel, DNA shuffling and screening technologies were utilized to

develop a single recombinant antigen containing epitopes from all four

DENV serotypes (Apt et al., 2006). Three chimeric clones (one containing

truncated E and two expressing full-length prM/E) induced neutralizing

antibodies against all four serotypes and protected mice from lethal i.c.

DENV2 challenge. The three clones were then used to immunize rhesus

monkeys; some monkeys vaccinated with the constructs expressing prM/E

developed neutralizing antibodies against all four serotypes, but only partial

protection against DENV1 challenge and no protection against DENV2was

observed (Raviprakash et al., 2006).

Konishi et al. developed a tetravalent DENV DNA vaccine containing

constructs expressing prM and E from DENV1–4 (Konishi, Kosugi, &

Imoto, 2006; Konishi, Terazawa, & Fujii, 2003; Konishi, Yamaoka,

Kurane, & Mason, 2000). Mice immunized with 25 mg of each of the four

constructs using a needle-free jet injector developed neutralizing antibodies

against all four serotypes (Konishi et al., 2006). Simultaneous immunization

with protein, in the form of DENV2 extraviral particles or inactivated JEV

vaccine, enhanced the immunogenicity of the DNA vaccine (Imoto &

Konishi, 2007).

A synthetic consensus (SynCon™) human codon optimized DNA

vaccine has been developed by Inovio Pharmaceuticals. A single plasmid was

constructed containing consensus EDIII sequences from DENV1–4

(Ramanathan et al., 2009). In vivo electroporation of mice with the DNA vac-

cine induced neutralizing antibodies against the four serotypes.

DNA vaccines based on the NS1 protein have also been created and

tested in mice (Costa et al., 2007, 2006; Timofeev, Butenko, &

Stephenson, 2004; Wu et al., 2003). As mentioned earlier, anti-NS1 anti-

bodies can mediate complement-dependent killing of infected cells, and

as the protein is not expressed on the virion, antibodies against NS1 cannot

mediate ADE. A DNA vaccine expressing DENV2 NS1 induced moderate

antibody responses and T-cell responses in mice and provided partial pro-

tection against i.v. DENV2 challenge (Wu et al., 2003). Coimmunization

with a plasmid expressing IL-12 enhanced the protective efficacy. Vaccina-

tion with a plasmid containing the DENV2 NS1 gene fused to the secretory

signal sequence of human tissue plasminogen activator (t-PA) was also found

to be immunogenic and protective in mice challenged with DENV2 i.c.

333Dengue Vaccines

(Costa et al., 2007, 2006). A DNA vaccine expressing the DENV1 prM-E-

NS1 proteins induced greater ADCC and cytotoxic T-lymphocyte activity

and better protection from lethal DENV1 i.c. challenge than a DNA vaccine

expressing prM and E without NS1 (Zheng et al., 2011).

Altogether, most of the DNA vaccine-based approaches for develop-

ment of dengue vaccines have focused on eliciting immune responses to

the prM and E proteins, and similarly to the recombinant E protein-based

vaccines, these vaccine-induced immune responses are mainly evaluated for

induction of anti-DENV antibodies. Results of the phase 1 trial of TVDV

given with Vaxfectin® will be informative. A few candidates generate NS1-

specific B-cell and T-cell responses. Further advances in DNA vaccination

technology that overcome the poor immunogenicity may lead to a success-

ful DENV DNA vaccine in the future.

7. VIRAL VECTORED VACCINES

Several viral vector platforms have been explored as delivery vehicles

for DENV antigens, including vaccinia virus, adenovirus, and alphavirus

vectors.

7.1. VacciniaAdvantages of poxviruses, including vaccinia virus, as vaccine vectors

include the ability to insert large pieces of DNA, high levels of gene expres-

sion, lack of persistence or viral integration into the host genome, high

immunogenicity, and relative ease of vaccine production (Drexler,

Staib, & Sutter, 2004). However, early attempts using vaccinia virus as a vac-

cine vector for DENV antigens were disappointing. The vaccinia Western

Reserve (WR) strain was used to express prM, E, NS1, and NS2A from

DENV4 (Zhao et al., 1987). CV-1 monkey kidney cells infected with

the recombinant virus expressed the structural proteins and NS1; however,

infection of cotton rats did not result in an antibody response to prM or E,

and only 1/11 animals had an antibody response to NS1, likely due to low

level of gene expression. Mice immunized with recombinant viruses con-

taining the structural proteins (with or without NS1 and NS2A) were

protected from lethal DENV4 i.c. challenge despite a low titer antibody

response to E (Bray et al., 1989). Immunization with recombinant viruses

expressing DENV4NS1 completely protected mice from i.c. DENV4 chal-

lenge, whereas vaccination with DENV2 NS1 resulted in only partial pro-

tection from DENV2 challenge (Falgout et al., 1990). To improve the


immunogenicity of recombinant DNA-expressed E, various recombinant

vaccinia virus strains were constructed that expressed full-length or

C-terminally truncated E from DENV4 (Men, Bray, & Lai, 1991). Full-

length E was not secreted from recombinant virus-infected CV-1 cells,

but several C-terminally truncated mutants were secreted extracellularly

or expressed on the cell surface. Immunization of mice with vaccinia virus

recombinants expressing the truncated proteins that were recognized by

dengue hyperimmune ascitic fluid (i.e., were expressed in native conforma-

tion) protected from lethal encephalitis. Passive transfer of immune sera

suggested anti-E antibodies mediated the protection.

Due to safety concerns for the nonattenuated WR strain, the highly

attenuated, replication-deficient modified vaccinia Ankara (MVA) was

selected as a vector to express C-terminally truncated E proteins (80%) from

DENV2 and DENV4 (Men et al., 2000). The MVA-DENV2 80%E, but

not MVA-DENV4 80%E, induced neutralizing antibodies in mice after i.

m. inoculation. Two doses of MVA-DENV2 80%E in rhesus monkeys

induced a low antibody response and partial protection against DENV2

challenge, and three doses was completely protective.

7.2. Adenovirus vectorsAdenovirus vectors have a number of advantages as vaccine vectors, includ-

ing the adenovirus genome is well characterized and easy tomanipulate, they

can be rendered replication-defective to increase safety, they have broad tro-

pism that allows for high levels of antigen expression in numerous cell types,

and they are easy to produce and store (Tatsis & Ertl, 2004). Adenoviral vec-

tors have been used for gene replacement therapy and as vaccine vectors and

have been shown to induce robust CD8þ T-cell and antibody responses

against the transgene. Preexisting immunity to adenoviruses can affect

immunization; however, this can be overcome by using adenoviruses from

different species, such as chimpanzees.

A recombinant, replication-deficient adenovirus (rAd) was constructed

expressing the ectodomain of the DENV2 E protein and part of prM

(Jaiswal, Khanna, & Swaminathan, 2003). Immunization of BALB/c mice

(intraperitoneally (i.p.)) elicited DENV2-specific T-cell responses and neu-

tralizing antibodies. A replication-deficient Ad vector was also used to

express a chimeric antigen consisting of the EDIIIs of DENV2 and DENV4

(Khanam, Rajendra, Khanna, & Swaminathan, 2007). The vector was used

as part of a heterologous prime–boost strategy: mice were immunized with

335Dengue Vaccines

the rAd (i.p.), followed by an i.d. boost with a plasmid vector encoding the

EDIIIs. The vaccinations induced neutralizing antibodies and T-cell

responses against DENV2 and DENV4. A tetravalent vaccine expressing

the EDIII sequences from the four DENV serotypes was then created using

the rAdV5 vector (Khanam, Pilankatta, Khanna, & Swaminathan, 2009).

Prime–boost immunization of mice (rAd i.p. followed by plasmid i.d.)

induced neutralizing antibody responses and T-cell responses against the

four serotypes. A homologous prime–boost with the rAd vector encoding

the DENV EDIIIs revealed anti-AdV5 Ab did not interfere with boosting

the anti-DENV antibody response.

The complex rAd-based vaccine platform (cAdVax), developed by

GenPhar Inc., was used to construct a pair of adenoviral vectors that each

express prM and E from two DENV serotypes: cAdVaxD(1-2) and

cAdVaxD(3-4) (Holman et al., 2007; Raja et al., 2007). Vaccination of mice

(i.p.) induced neutralizing antibody titers against all four serotypes and a

broadly reactive T-cell response. Tetravalent vaccinationwas studied in rhesus

monkeys by mixing the two bivalent vectors (Raviprakash et al., 2008). Two

doses of the vaccines (i.m., 8 weeks apart) resulted in high titer neutralizing

antibodies against all four serotypes and significantly protected against live

DENV challenge 4 or 24 weeks after the second immunization. Complete

protection against DENV1 and DENV3 viremia was observed; however,

for DENV4, the duration of viremia after challenge at 24 weeks was reduced

but the viral titers were increased compared with control vaccinated animals.

Despite the induction of anti-Ad antibodies induced by the first dose, the sec-

ond immunization was able to boost anti-DENV antibody titers.

7.3. Alphavirus replicon particlesAlphavirus-derived replicon vaccines have shown promise as a platform for

dengue vaccination. VEE VRP are nonreplicating VLP containing a mod-

ified genome expressing a protein of interest. Vaccination with VRP induces

high levels of antigen expression in a single round of infection, and antigen

presentation is robust due to the adjuvant activity of VRP and the targeting

of the VRP to dendritic cells (DC) in the lymph nodes (MacDonald &

Johnston, 2000; Thompson et al., 2006). A VRP expressing DENV1

prM and E (D1ME-VRP) was shown to be immunogenic and protective

when given in three doses or as part of a heterologous prime–boost with

a DENV1 DNA vaccine to cynomolgus monkeys (Chen et al., 2007).

DENV2 prM and E have also been cloned into a VEE replicon vector


and packaged into VRP (White et al., 2007). Immunization of mice (s.c.)

resulted in DENV2-specific IgG and neutralizing antibodies, and a second

immunization at 12 weeks resulted in increased neutralizing antibody titers

that lasted for 30 weeks. Vaccination was protective: two doses of 1E6 infec-

tious units (IU) in young mice completely protected against lethal i.c.

DENV2 challenge, and lower doses induced partial protection. VRP

expressing two configurations of the E protein (subviral particles (prM/

E), or soluble E dimers (E85)) were compared (White et al., 2013). Immu-

nization of rhesus macaques with E85-VRP resulted in serotype-specific

antibody responses targeting EDIII that developed more rapidly and to a

higher titer than the prM-E-VRP response. Monkeys were then vaccinated

with a tetravalent vaccine containing E85-VRP from the four serotypes.

After 2 doses, all animals had robust neutralizing antibody responses against

all four serotypes, and were partially protected from challenge with DENV1

and DENV2, and completely protected fromDENV3 and DENV4. Impor-

tantly, antivector immunity from the first dose did not seem to reduce the

effectiveness of second dose. The authors believe clinical trials with the tet-

ravalent E85-VRP vaccine candidates are warranted. Overall, similarly to

recombinant protein- and DNA-based vaccine approaches, viral vectored

dengue vaccine candidates are focused on eliciting and evaluating E protein-

specific antibody responses. In contrast with recombinant E protein- and

DNA-based vaccine approaches, no viral vectored vaccine has advanced

to clinical phase 1 testing.

8. INACTIVATED WHOLE VIRUS

Vaccination with inactivated DENV vaccines ideally should induce a

balanced immune response without the viral interference that can occur

with live attenuated vaccines. In addition, with inactivated vaccines, there

is no risk of viral replication or reversion to wild-type virus that could occur

with a live virus vaccine. However, inactivated DENV vaccines contain

only the C, M, E, and NS1 proteins (Putnak, Barvir, et al., 1996;

Putnak, Cassidy, et al., 1996) and therefore the immune response is directed

only against these proteins, and there is no response to the other non-

structural proteins. Inactivated vaccines are less effective than live attenuated

vaccines in inducing long-lasting immunity, and as with other nonliving

vaccines, multiple doses and adjuvants will likely be necessary for optimal

immunogenicity in unprimed individuals. In addition, inactivated vaccines

may not be as efficient at inducing CMI as live vaccines. However, an

337Dengue Vaccines

inactivated vaccine for dengue may be useful as part of heterologous prime–

boost vaccine regimen, for example, with a DNA vaccine.

The Walter Reed Army Institute of Research (WRAIR) has developed

PIV vaccine candidates. The DENV2 strain S16803 was grown in Vero

(African greenmonkey kidney epithelial) cells, purified on sucrose gradients,

and inactivated with formalin (Putnak, Barvir, et al., 1996). Immunization of

mice and rhesus monkeys with PIV (absorbed on alum) induced a high titer

neutralizing antibody response. Immunization was also protective; two

doses protected mice from DENV2 i.c. challenge, and three doses in mon-

keys led to reduced or absent viremia after DENV2 challenge. A PIV was

also made with the DENV2 strain 16681 grown in fetal rhesus lung

(FRhL) cells and inactivated with formalin (Putnak, Cassidy, et al., 1996).

This PIV was also immunogenic, and doses of 100 or 1000 ng (but not

10 ng) adjuvanted with alum significantly protected mice from lethal i.c.

challenge.

The DENV2 strain S16803 PIV was compared with a live attenuated

vaccine (DENV2 PDK-50) and recombinant subunit protein vaccine

(r80E) in rhesus monkeys (Robert Putnak et al., 2005). Monkeys were

immunized at 0 and 3 months, and five different adjuvants (alum, or

AS04-OH, AS04-PO, AS05, and AS08 from GSK) were tested with the

PIV and r80E vaccines. All monkeys seroconverted after the second dose,

and the highest neutralizing antibody titers were observed after vaccination

with 5 mg of PIV adjuvanted with AS05 or AS08 or 5 mg r80E in AS05 or

AS08. Unlike the live attenuated vaccine, the PIV and r80E vaccines did not

induce stable antibody titers; the titers increased after the boost but declined

before DENV2 challenge 2 months later. In addition, whereas vaccination

with the live attenuated virus resulted in no viremia after challenge, some

PIV-vaccinated monkeys had viremia. A subsequent study compared vacci-

nation of rhesus monkeys with combinations of three nonreplicating

DENV2 vaccine candidates: DNA vaccine expressing prM and E, EDIII-

MBP fusion protein, and PIV (Simmons et al., 2006). After the third dose,

all monkeys had high antibody titers (measured by ELISA) and neutralizing

antibodies (measured by PRNT50). The highest neutralizing antibody titers

were observed after vaccination with the DNA vaccine and fusion protein

together; however, significant protection from DENV2 challenge 5 months

after the last immunization was observed only with PIV vaccination. Pro-

tection correlated with total antibody levels (including antibodies against

NS1) as measured by ELISA and antibody avidity, but not with neutralizing

antibody titers.


A TPIV vaccine was made from wild-type DENV1–4 strains grown

in Vero cells and inactivated with formalin (Simmons et al., 2010). The

TPIV was tested as part of a heterologous prime–boost strategy. Rhesus

monkeys were primed with one dose of TPIV in alum and boosted

2 months later with a TLAV. TPIV immunization resulted in a low titer

neutralizing antibody response, but boosting with TLAV increased titers.

The highest neutralizing antibody titers were against DENV2, and the

lowest were against DENV3. TPIV/TLAV vaccinated monkeys were

completely protected from challenge with DENV1, 2, 3, or 4 at 8 months,

and anamnestic neutralizing antibody responses were detected after the live

viral challenge.

A phase 1 clinical trial of the WRAIR DENV1-PIV began in 2011,

and two phase 1 trials of the tetravalent TDENV-PIV candidate began in

2012 in a dengue-primed population (Clinicaltirals.gov NCT01702857)

and in a nonendemic area (NCT01666652). The tetravalent vaccine

candidates will be tested with three different adjuvants: alum, AS01E,

and AS03B.

As an alternative to formalin inactivation, psoralen-inactivation has been

used to inactivate DENV. Psoralens intercalate between nucleic acids and

covalently cross-link pyrimidines following UVA exposure. This method

inactivates viruses while leaving immunogenic surface epitopes intact

(Groene & Shaw, 1992). A psoralen-inactivated DENV1 vaccine has been

tested in mice (Maves, Castillo Ore, Porter, & Kochel, 2010) and monkeys

(Maves, Ore, Porter, & Kochel, 2011). Aotusmonkeys immunized i.d. with

three doses (10 ng each) of the inactivated DENV1 virus in alum developed

DENV1-specific IgG and neutralizing antibodies and were moderately

protected from DENV1 challenge. The authors suggest alternate routes

of administration, higher or greater number of doses, or different adjuvants

may enhance the immunogenicity.

Thus, similarly to recombinant protein-, DNA-, and viral vector-based

dengue vaccine candidates, studies with inactivated whole virus vaccines

have primarily assessed vaccine-induced antibody responses in terms

of the duration and levels of ELISA-binding and PRNT titers and the

capacity to protect against lethal i.c. challenge of mice and viremia in mon-

keys. Unlike recombinant protein-, DNA-, and viral vector-based dengue

vaccines that induce E (or NS1-)-specific antibody responses, vaccination

with whole virus vaccines induces antibody responses against E, prM,

and NS1.

339Dengue Vaccines

9. LIVE ATTENUATED

Most dengue vaccine efforts have focused on developing live attenu-

ated vaccines, and these are the furthest along in development and clinical

testing. Live attenuated vaccines have a number of advantages including

their ability to induce immune responses that mimic the response to natural

infection, the induction of robust B- and T-cell responses, and the ability to

confer lifelong immune memory (Pulendran & Ahmed, 2011). The most

successful vaccines developed to date, including the smallpox vaccine, are

live attenuated vaccines. Live attenuated vaccines can be produced at rela-

tively low cost and may be effective after one dose. It has been estimated that

a live attenuated DENV vaccine could be produced at an affordable cost in

developing countries (Mahoney et al., 2012).

Live attenuated DENV vaccine candidates must be attenuated for mos-

quitoes as well as humans, to prevent transmission after vaccination. The

vaccine strains must be genetically stable to avoid reversion to wild-type

viruses, and genetic stability must be monitored throughout manufacture.

The major challenges of developing a live attenuated vaccine for DENV

include the need for the vaccine to induce balanced immune responses to

all four serotypes, and be sufficiently attenuated to not cause symptoms of

DF. Viral interference is a key issue in tetravalent live attenuated dengue

vaccine development and has been observed with live attenuated vaccine

candidates in monkeys and human volunteers (Guy et al., 2009; Kanesa-

thasan et al., 2001; Kitchener et al., 2006; Osorio, Brewoo, et al., 2011).

Booster immunizations will likely be required to overcome the interference

and induce immune responses against all four serotypes. If more than one

dose is required, the time between vaccinations must be optimized to allow

replication of all four strains in subsequent immunizations; that is, booster

immunizations must be given after sterilizing immunity has waned.

A study in rhesus monkeys found a second immunization with a live atten-

uated tetravalent vaccine at 4 months, but not 1 month, boosted neutralizing

antibody titers (Blaney et al., 2005). Similarly, a study in humans found a

second immunization 1 or 3 months after the first dose did not significantly

increase neutralizing antibody titers (Sun et al., 2003) so subsequent studies

boosted at 6 months (Simasathien et al., 2008; Sun et al., 2009). Prolonged

immunization schedules seem to be necessary but may be difficult to imple-

ment or track in DENV endemic areas.


Early dengue vaccine research attempted to attenuate the virus by serial

passaging through mice. Passaging DENV through the brain of suckling

mice via i.c. inoculation resulted in increased neurovirulence in mice

(Cole & Wisseman, 1969; Sabin & Schlesinger, 1945) and attenuation in

humans (Hotta, 1952; Sabin, 1952). After 7–10 passages through mice,

the virus was deemed attenuated enough to test as a vaccine (Sabin,

1952). The fifteenth mouse-passaged virus was given to 16 human volun-

teers. The vaccine was safe; all volunteers developed a maculopapular rash,

but systemic symptoms were absent or mild. The vaccine induced protective

immunity, as the vaccinees were immune to exposure to DENV-infected

mosquitoes 21–38 days after vaccination. A mouse-passaged DENV1 vac-

cine was found to protect adults and adolescents in Puerto Rico during a

heterologous DENV outbreak (Bellanti et al., 1966). The heterologous pro-

tection developed in three weeks and lasted for at least 85 days.

In 1971, the US Armed Forces Epidemiological Board initiated efforts to

develop live attenuated DENV vaccines with the strategy of attenuation by

serial tissue culture passage, and passaging began at the University of Hawaii

in 1971 (Halstead & Marchette, 2003). Initial efforts focused on passaging

wild-type DENV strains through various types of primary cells or cell lines,

including primary dog kidney (PDK) and African green monkey kidney

(GMK) cells. Some wild-type and some attenuated strains were sent to

Mahidol University in Thailand. Passaging of DENV in vitro was done

simultaneously in Hawaii, Thailand, and at WRAIR.

9.1. University of Hawaii/WRAIRPR-159/S-1 is a vaccine strain that was derived at WRAIR by passaging a

DENV2 clinical isolate, PR-159, through primary GMK cells and FRhL

cells (Eckels, Brandt, Harrison, McCown, & Russell, 1976; Eckels,

Harrison, Summers, & Russell, 1980; Harrison, Eckels, Sagartz, &

Russell, 1977). PR-159/S-1 has in vitro and in vivo attenuation character-

istics including temperature sensitivity, small plaque size (on rhesus mon-

key kidney epithelial LLC-MK2 cells), and reduced virulence for suckling

mice and rhesus monkeys. The DENV2 vaccine strain was tested in six

YFV-immune human volunteers (Bancroft et al., 1981). Five of six had

viremia and seroconverted, including one who had symptoms of mild

DF including fever, headache, and myalgia. A subsequent study tested

the vaccine in 98 volunteers (Bancroft et al., 1984). Seroconversion was

higher in YFV-immune individuals compared with naive volunteers

341Dengue Vaccines

(90% vs. 61%), and peak neutralizing antibody titers were higher in YFV-

immune volunteers as well.

A DENV1 vaccine candidate, 45AZ5, was derived by passaging a clinical

isolate through FRhL cells followed by chemical mutagenesis with

5-azacytidine (McKee et al., 1987). Despite having markers of attenuation

including temperature sensitivity, small plaque size, and reduced virulence in

mice and monkeys, 45AZ5 was genetically unstable and caused DF in two

volunteers. Similarly, a DENV3 vaccine candidate caused DF in recipients

(Innis et al., 1988).

The DENV4 strain H241 was passaged through PDK cells and FRhL

cells to derive the H241, PDK35-TD3 FRhL p3 vaccine strain (Halstead,

Eckels, Putvatana, Larsen, & Marchette, 1984). This strain was attenuated

in vitro and in suckling mice and had low virulence in rhesus monkeys. It

was next tested in five YFV-immune volunteers (Eckels et al., 1984). Only

two subjects seroconverted, and those individuals developed mild clinical

disease. Phenotypically changed virus was isolated from the volunteers with

viremia, indicating the virus was genetically unstable.

A DENV4 vaccine candidate was also developed at WRAIR. The

DENV4 strain 341750 Carib was passaged in PDK cells 20 times and in

FRhL-2 cells 4 times to derive 341750 Carib PDK-20/FRhL-4

(Marchette et al., 1990). The vaccine strain was less virulent than the paren-

tal strain in rhesus monkeys, yet the vaccine strain induced the development

of neutralizing antibodies and hemagglutination inhibition (HAI) antibodies

against DENV4. Monkeys immunized with the vaccine strain were protec-

ted from parental DENV4 challenge. Three doses (103, 104, or 105 plaque-

forming units (PFU)) of the 341750 Carib PDK-20/FRhL-4 vaccine strain

were then tested in human volunteers (Hoke et al., 1990). Five of 8 volun-

teers receiving 105 PFU developed viremia and antibody responses (neutral-

izing, HAI, and IgM) against DENV4. The viremic subjects also developed

rash and slight temperature elevations. The vaccine was deemed safe and rea-

sonably immunogenic and selected for further study as part of a tetravalent

vaccine. The other strains selected were DENV1 45AZ5 PDK-20 FRhL3,

DENV2 S16803 PDK-50 FRhL3, and DENV3 CH53489 PDK-20

FRhL3. These vaccine strains were tested in flavivirus-naive adults as mono-

valent or tetravalent vaccination (Sun et al., 2003). Monovalent recipients

were given one or two doses 1 or 3 months apart, and the tetravalent vaccine

was given in two or three doses at 1–4 month intervals. The doses of

DENV1 and DENV2 were 10-fold higher than DENV3 and DENV4.

The highest reactogenicity was observed with DENV1, and myalgia, rash,


and fever were the most common symptoms. Viremia was detected in some

of the volunteers, most often in DENV3 or tetravalent recipients. Serocon-

version after one monovalent dose was 100% for DENV1, 92% for DENV2,

46% for DENV3, and 58% for DENV4; for tetravalent vaccination, sero-

conversion ranged from 30% to 70%. Seroconversion did not significantly

differ between monovalent and tetravalent recipients, suggesting a lack of

viral interference. The second dose of monovalent vaccination 30 or 90 days

later was less reactogenic than the first dose, but did not boost antibody titers

except to DENV3. Second and third doses of the tetravalent vaccine

increased the number of seroconversions and neutralizing antibody titers.

In collaboration with GSK, a subsequent phase 1 trial in flavivirus-naive

adults tested 16 formulations of the tetravalent vaccine: DENV1 (45AZ5)

PDK-20, DENV2 (S16803) PDK-50, DENV3 (CH53489) PDK-20, and

DENV4 (341750) PDK-20 (Edelman et al., 2003). The formulations were

variably reactogenic, and reactogenicity correlated with immunogenicity.

Viremia was detected in 47% of recipients overall, primarily after the first

dose. Overall, seroconversion to DENV1, 2, 3, and 4 were 69%, 78%,

69%, and 38%, respectively, and the highest neutralizing antibody titers were

against DENV1. There was no consistent effect of a second immunization at

day 28 on neutralizing antibody responses, and no formulation induced a

tetravalent neutralizing antibody response after two doses. The poor

response to the boost was likely due to the presence of heterotypic immu-

nity, which prevented replication of the second dose.

A new formulation, containing a higher passage DENV1 and lower pas-

sage DENV4 than the previous formulations (DENV1 (45AZ5) PDK-27,

DENV2 (S16803) PDK-50, DENV3 (CH53489) PDK-20, and DENV4

(341750) PDK-6), was tested in cynomolgus macaques and found to induce

a balanced tetravalent neutralizing antibody response (Koraka, Benton, van

Amerongen, Stittelaar, & Osterhaus, 2007). It was then studied in seven

DENV- and JEV-naive Thai children who were given two doses 6 months

apart (Simasathien et al., 2008). The vaccine was safe, with no severe adverse

events (SAE) observed. Symptoms were more frequently reported after the

first vaccination and included fever, fatigue, headache, myalgia, and arthral-

gia. DENV4 viremia was detected in three volunteers. The vaccine was also

immunogenic: 50% of the children seroconverted to DENV2 and DENV4

after the first dose, and after the second dose, six of seven recipients

seroconverted to all four serotypes.

The new tetravalent formulation was tested side by side with two

older formulations in a double-blind, randomized phase 2 trial in

343Dengue Vaccines

71 flavivirus-naive adults (Sun et al., 2009). Volunteers were given two

doses at 0 and 6 months. The new formulation was immunogenic; 63%

of recipients developed a tetravalent neutralizing antibody response after

two doses. Compared with the older formulations, the new formulation

was less reactogenic and more immunogenic, and was therefore selected

for future studies, including a phase 1/2 trial in infants (Watanaveeradej

et al., 2011). Thirty-four infants (12–15 months of age) received two doses

of the tetravalent DENV vaccine 6 months apart (PDK 27/50/20/6), and

17 infants received a control vaccine. The vaccine was safe; no vaccine-

related SAE were observed, although one subject had transiently elevated

AST/ALT levels. The vaccine was also moderately immunogenic: after

the second dose, 85.7% of recipients had trivalent neutralizing antibody

responses and 53.6% had tetravalent responses.

Two formulations of a new vaccine (TDEN) were produced using

rederived master seeds from the PDK 27/50/20/6 precursor vaccine and

were studied in a placebo-controlled phase 2 trial in 86 adults (Thomas

et al., 2013). The two new formulations (F17 and F19) were compared with

the precursor vaccine (F17/Pre: PDK 27/50/20/6). F19 had fourfold less

DENV4 than F17 and F17/pre). No vaccine-related SAE were observed

in the vaccinees, and symptoms were transient and mild to moderate in

severity. Rash was the only symptom observed more often in DENV vac-

cine recipients versus placebo. DENV4 viremia was detected in some of the

F17/Pre vaccinees and one F17 vaccinated subject; no viremia was detected

for the other serotypes or in the F19 recipients. A second dose at 6 months

increased antibody titers and broadened the response. Tetravalent serocon-

version rates in DENV-unprimed subjects were 60% for F17 and 66.7% for

F19 one month after the second dose. A third dose given 5–12 months later

was ineffective at boosting neutralizing antibody titers. Altogether, the new

formulations were safe andmoderately effective, and the authors state studies

in a larger number of adults and then children are warranted.

9.2. Mahidol UniversityThe DENV2 strain, 16681, was serially passaged through PDK cells 53 times

to obtain 16681-PDK-53, which was tested in a phase 1 trial in Thailand

(Bhamarapravati, Yoksan, Chayaniyayothin, Angsubphakorn, &

Bunyaratvej, 1987). Five JEV- and DENV-naive volunteers and five

JEV-immune volunteers were vaccinated. One patient became viremic,

and all developed neutralizing antibodies that lasted for 1.5 years.


DENV2-specific CD4þ and CD8þ T-cell responses were detected in all

vaccinees (Dharakul et al., 1994).When given in a bivalent formulation with

a DENV4 vaccine strain, 1036 PDK 48, all subjects developed neutralizing

antibodies against DENV2 and DENV4 (Bhamarapravati & Yoksan, 1989).

The 16681-PDK-53 vaccine was also found to be safe and immunogenic in

10 flavivirus-naive American volunteers, who developed a DENV2 neutral-

izing antibody response that lasted for 2 years (Vaughn et al., 1996).

Vaccine strains from each serotype obtained by passage through PDK

cells or primary GMK cells were selected and tested in monovalent, bivalent,

trivalent, and tetravalent vaccinations in Thai adults (Bhamarapravati &

Sutee, 2000). The strains used were DENV1 PDK-13, DENV2 PDK-53,

DENV3 PGMK-30/F3, and DENV4 PDK-48. The vaccine was safe and

did not induce clinically significant symptoms. Of the volunteers that

seroconverted, most had neutralizing antibodies 2 years after monovalent

vaccination. All bivalent and trivalent vaccine recipients seroconverted to

all serotypes in the vaccine, and of the tetravalent recipients, four of six

developed neutralizing antibodies to all four serotypes, whereas two

seroconverted to DENV1, 2, and 3 but not DENV4.

The vaccine strains were produced by Aventis Pasteur and tested in a

phase 1 trial in the United States in 40 flavivirus-naive adults (Kanesa-

thasan et al., 2001). Subjects received a single dose of a monovalent vaccine

or the tetravalent vaccine (containing 3.47–3.9 log10 PFU of each serotype).

Mild symptoms including fever, headache, malaise, rash, and transient neu-

tropenia were observed in the monovalent recipients. Tetravalent vaccina-

tion was more reactogenic than monovalent vaccination, and one volunteer

developed a dengue-like syndrome. Viremia was detected in DENV3 and

DENV4monovalent recipients, and DENV3was detected in the tetravalent

vaccine recipients. All of the of DENV2, 3, and 4 monovalent recipients but

only 60% of the DENV1 recipients seroconverted. Of the tetravalent recip-

ients, only one of ten seroconverted to all four serotypes, and neutralizing

antibody responses were directed primarily to DENV3. The vaccine

induced DENV-specific T-cell responses (as measured by in vitro prolifera-

tion, IFN-g production, and cytotoxicity) in the tetravalent vaccine recip-

ients; however, the responses to the four serotypes were not equivalent

(Rothman et al., 2001).

In an attempt to achieve a more balanced antibody response, seven tet-

ravalent vaccine formulations were tested that differed in overall viral dose

and the dose of each serotype (Sabchareon et al., 2002). Fifty-nine flavivirus-

naive Thai adults received two vaccine doses 6 months apart. Five volunteers

345Dengue Vaccines

developed a DF-like illness, with headache, fever, and myalgia the most

common symptoms. Some hematologic abnormalities were also observed

including decreases in platelets, neutrophils, and lymphocytes, and some

subjects had increased AST and ALT levels. The second dose was less

reactogenic, but viremia was detected after both doses. After the second

dose, 76% of subjects seroconverted to three serotypes and 71%

seroconverted to all four. The DENV3 component was dominant; viremia

detected after the first dose was mainly DENV3, all subjects seroconverted to

DENV3 after one dose, and neutralizing antibody titers were highest

against DENV3.

Two formulations of a tetravalent vaccine that contained less DENV3

than previous formulations were then tested in Thai children (Sabchareon

et al., 2004). Children 5–12 years of age received three immunizations—

the second was given 3–5 months after first, and the third was given 8–12

months after the second. The vaccines were moderately reactogenic and

induced symptoms including fever, myalgia, and rash. There were five

severe reactions including one DF-like illness. After three doses, 89% and

100% of the recipients seroconverted to all four serotypes. DENV3 was still

dominant, as indicated by a high prevalence of DENV3 viremia and high

neutralizing antibody titers against DENV3.

A planned phase 1b trial to test two formulations of the vaccine in adult

Caucasians in Australia was halted after 10 recipients received one dose and

developed a mild DF-like syndrome due to the DENV3 component

(Kitchener et al., 2006). In an attempt to attenuate DENV3, the vaccine

strain was plaque-purified and adapted to Vero cells (Sanchez et al.,

2006). The Vero-adapted dengue serotype 3 vaccine, VDV3, was attenuated

in vitro and in monkeys and was next tested in 15 volunteers in Hong Kong.

All subjects had adverse reactions and the trial was halted. As a balanced

immune response was not achieved with these vaccine candidates, they were

not pursued further.

9.3. CDC/InviragenAnother live attenuated candidate was developed at the CDC and has been

licensed by Inviragen. Chimeric viruses were cloned with the DENV2

PDK-53 vaccine strain developed at Mahidol University as a backbone,

and the DENV2 structural proteins were replaced with the structural pro-

teins from DENV1, 3, or 4 to create the tetravalent vaccine, DENVax.

Attenuating mutations in PDK-53 are outside of the structural genes


(Butrapet et al., 2000); therefore, all four chimeric strains should retain the

DENV2 PDK-53 attenuation markers. DENV2/DENV1 chimeras were

created using the C, M, and E proteins of the Mahidol DENV1 PDK-13

vaccine virus or wild-type DENV1 16007 and were found to be attenuated

in vitro and in mice (Huang et al., 2000).

DENV2/1, DENV2/3, and DENV2/4 chimeras were created by clon-

ing prM and E from wild-type DENV1 (strain 16007), DENV3 (strain

16562), and DENV4 (strain 1036) into two genetic variants of the DENV2

PDK-53 vaccine virus, or the parental strain, 16681 (Huang et al., 2003).

The chimeras retained the DENV2 PDK-53 attenuation markers, including

temperature sensitivity, small plaque size in LLC-MK2 cells, lack of neu-

rovirulence in newborn mice, and reduced replication in C6/36 mosquito

cells. Monovalent and tetravalent chimeric vaccine (DENVax) formulations

were tested in AG129 mice (Brewoo et al., 2012; Huang et al., 2003).

Monovalent DENVax-1, 2, or 3 significantly protected against lethal

DENV1 or DENV2 challenge. Tetravalent vaccination induced neutraliz-

ing antibody responses against all four serotypes and protected against chal-

lenge with DENV1 or DENV2.

Three different formulations, differing in the dose of each serotype, of

the tetravalent chimeric DENVax vaccine were tested in cynomolgus

macaques (Osorio, Brewoo, et al., 2011). Monkeys were given two vacci-

nations 60 days apart. Low-level DENV2 viremia was detected, yet all mon-

keys developed neutralizing antibodies against all four serotypes after one or

two doses. Monkeys also developed a DENV2-specific T-cell response. The

most balanced antibody response was observed with the formulation con-

taining 103 PFU of DENV1 and DENV2 and 105 PFU of DENV3 and

DENV4. All monkeys were completely protected against challenge with

DENV3 or DENV4 30 days after the second immunization, and the

high-dose formulation (105 PFU of each serotype) completely protected

against DENV1 and DENV2 as well. Based on these results, tetravalent

DENVax is being tested in phase 1 clinical trials (Osorio, Huang,

Kinney, & Stinchcomb, 2011), and a phase 2 study in healthy volunteers

between 1.5 and 45 years of age began in 2011 (Clinicaltrials.gov

NCT01511250).

9.4. NIAID/NIHA genetics approach was undertaken by researchers in the Laboratory of

Infectious Diseases at the National Institute of Allergy and Infectious

347Dengue Vaccines

Diseases (NIAID) with the goal of attenuating the virus without significantly

reducing immunogenicity. Reverse genetics was used to introduce dele-

tions, from 30 to 262 nucleotides (nt), into the 30UTR of DENV4 cDNA

(Men, Bray, Clark, Chanock, & Lai, 1996). Mutants that were attenuated in

LLC-MK2 cells were selected and tested in rhesus monkeys. Some mutants

were attenuated in vivo, in terms of reduced viremia and neutralizing anti-

body titers, compared with the parental wild-type DENV4 virus. ADENV4

30 nt 30UTR deletion mutant (rDENV4D30) that was attenuated in mon-

keys was selected and tested in 20 healthy adults in a phase 1 trial (Durbin

et al., 2001). Volunteers received 105 PFU s.c. Low titer viremia was

detected in 14 volunteers, and 100% developed neutralizing antibody

responses against DENV4. The vaccine was well tolerated: Asymptomatic

rash was observed in subjects with viremia, and 5 volunteers had a transient

increase in serum ALT levels. The vaccine was attenuated for mosquitoes as

well. Compared with the wild-type parental virus, the vaccine strain was

restricted in infecting A. aegypti midgut and in disseminating from the mid-

gut to the salivary gland. In addition, vaccine recipients did not transmit the

virus to A. albopictus mosquitoes (Troyer et al., 2001).

The rDENV4D30 vaccine was further evaluated in phase 2 placebo-

controlled trial (Durbin et al., 2005). A dose deescalation was done, and vac-

cinees (20 per group) received 103, 102, or 101 PFU. All doses were well

tolerated and immunogenic. Some recipients developed a mild rash and

neutropenia, but only 1/60 had an elevated serum ALT level. Almost all

recipients (97%) seroconverted (defined as a � fourfold increase in neutral-

izing antibody titers) to DENV4 after a single inoculation. These results

supported the inclusion of this vaccine strain in a tetravalent formulation.

In parallel, DENV4 mutants were generated in an attempt to derive a

vaccine candidate that would not induce the hepatotoxicity observed in vol-

unteers receiving 105 PFU of the rDENV4D30 vaccine (Hanley, Lee,

Blaney, Murphy, & Whitehead, 2002). Five attenuating mutations were

introduced into rDENV4D30 and were tested in SCID-HuH-7 mice and

rhesus monkeys (Hanley et al., 2004). One mutant (rDENV4D30-200,201) that was significantly attenuated in rhesus monkeys compared with

wild-type DENV4 and rDENV4D30 was selected and tested in a phase 1

trial (McArthur et al., 2008). Volunteers received 105 PFU of

rDENV4D30-200,201, which was well tolerated; no ALT elevations or

viremia were detected, and all 20 volunteers seroconverted after one dose.

Toward the goal of creating a tetravalent vaccine, the group introduced

the 30 nt 30UTR deletion into a full-length DENV1 cDNA clone to create


rDENV1D30 (Whitehead, Falgout, et al., 2003). This virus was attenuated

similarly to rDENV4D30 in rhesus monkeys and completely protected

against DENV1 challenge, with no viremia detected in vaccinated monkeys.

A phase 1 study of the rDENV1D30 DENV1 vaccine was conducted in

adult volunteers (Durbin et al., 2006a). Twenty vaccinees received 103

PFU, which was well tolerated. The most common adverse events were

an asymptomatic rash and neutropenia, which were observed in 40% and

45% of the recipients, respectively. Viremia was detected in 9/20 subjects

and was slightly higher titer than rDENV4D30-induced viremia. The vac-

cine was highly immunogenic, as 95% of the recipients seroconverted and

had neutralizing antibodies against DENV1 that lasted for the 6 months of

the study. A subsequent study found a second immunization with

rDENV1D30 4 or 6 months after the first dose was safe; however, it was

not infectious and it did not boost antibody titers, indicating the first vacci-

nation induced sterilizing immunity that lasted for at least 6 months (Durbin,

Whitehead, et al., 2011).

For DENV3, unlike DENV1 and DENV4, the D30 mutation was not

sufficiently attenuating. rDENV3D30 was not attenuated in mosquitoes,

SCID-HuH-7 mice, or monkeys (Blaney, Hanson, Firestone, et al.,

2004). As an alternate attenuating strategy, the DENV3 M and E proteins

were cloned into the rDENV4 backbone to create rDENV3/4(ME) and

rDENV3/4D30(ME) chimeras, which were attenuated in mice, mosqui-

toes, and rhesus monkeys. The two chimeras were comparably attenuated,

indicating the D30 mutation did not confer additional attenuation. No vire-

mia was detected in immunized monkeys yet all seroconverted, and they

were protected against challenge with the parental DENV3.

Additional DENV3 vaccine candidates were created, including

rDENV3D30/31, which contains an additional 31 nt deletion in the

30UTR, and rDENV3-30D4D30, which was created by replacing the entire30UTR of rDENV3 with the 30UTR of rDENV4D30 (Blaney et al., 2008).Both viruses were attenuated in SCID-HuH-7 mice and rhesus monkeys;

immunization of monkeys resulted in neutralizing antibody responses and

protection from wild-type DENV3 challenge. rDENV3D30/31 was also

attenuated for mosquitoes.

Similarly, the D30 mutation in DENV2 did not sufficiently attenuate the

virus to be considered for a human vaccine. rDENV2D30 was attenuated inSCID-HuH-7 mice and not infectious for A. aegypti mosquitoes, but was

only slightly attenuated in rhesus monkeys compared with rDENV2 and

wild-type DENV2 (Blaney, Hanson, Hanley, et al., 2004). To further

349Dengue Vaccines

attenuate rDENV2D30, a point mutation in NS3 that had been previously

demonstrated to attenuate rDENV4D30 (Hanley et al., 2004) was made.

rDENV2D30-4995 was found to be further attenuated in SCID-HuH-7

mice compared with rDENV2D30. In other approaches to create DENV2

vaccine candidates, the structural genes (CME or ME) of DENV2 were

cloned into rDENV4 or rDENV4D30 (Whitehead, Hanley, et al., 2003).

Chimeras (without the D30 deletion) were attenuated in SCID-HuH-7

mice, mosquitoes, and rhesus monkeys. rDENV2/4D30(CME) was more

attenuated than rDENV2/4(CME) and did not replicate in monkeys;

rDENV2/4(ME) was similarly attenuated when cloned with or without

the D30 deletion.

Due to its attenuation and immunogenicity, rDENV2/4D30(ME) was

deemed a promising vaccine candidate and was tested in 20 DENV-naive

adults (Durbin et al., 2006b). The volunteers received 103 PFU, which

was safe and immunogenic. A mild asymptomatic rash and mild neutropenia

were observed in some subjects. All volunteers seroconverted to DENV2

and neutralizing antibodies were maintained for the 6 months of the study.

Low magnitude viremia was detected in 11 volunteers, and the D30 muta-

tion was unchanged in the viremic volunteers, confirming that the mutation

was stable.

Three tetravalent vaccine formulations were tested in animals (Blaney

et al., 2005). TV-1 was composed of 105 PFU of the four D30 viruses;

TV-2 contained 105 PFU of rDENV1D30, rDENV4D30, rDENV2/

4D30, and rDENV3/4D30; and TV-3 contained 105 PFU of rDENV1D30,rDENV2D30, and rDENV4D30, and 106 PFU of rDENV3/4D30. TV-1and TV-2 were attenuated in SCID-HuH-7mice, and all three formulations

were attenuated in rhesus monkeys. TV-1- and TV-3-immunized monkeys

all seroconverted after one dose, whereas TV-2 required a booster immu-

nization to achieve high titers against DENV2 and DENV3. Boosting at

4 months, but not 1 month, increased neutralizing antibody titers.

A single dose of TV-2 protected against challenge with DENV1, 3, and

4, and two doses protected from challenge with DENV2. Two doses of

TV-3 also completely protected against DENV2 challenge. These results

supported testing TV-2 and TV-3 in clinical trials.

A phase 1 trial investigated a single dose of four different formulations of

a live tetravalent vaccine in 113 flavivirus-naive volunteers (Durbin et al.,

2013). The vaccines were well tolerated, with no SAE or fever induced

in any subject. The only side effect that occurred with a significantly higher

incidence in vaccinees compared with placebo recipients was an


asymptomatic rash observed in 64.2% of vaccinees. Low-level viremia was

detected in most (73%) recipients, and in the majority (64%) of viremic sub-

jects, one serotype of virus was detected. One dose of each formulation

induced a trivalent or better neutralizing antibody response in 75–90% of

the volunteers. Black race correlated with lower seropositivity and a reduced

incidence of viremia, which was interesting as the black race is associated

with resistance to DENV infection (Blanton et al., 2008; Halstead et al.,

2001). Formulation TV003, containing 103 PFU each of rDENV1D30,rDENV2/4D30, rDENV3D30/31, and rDENV4D30, induced the most

balanced neutralizing antibody response and a trivalent or better response

in 90% of recipients after a single dose. However, only 50% of recipients

seroconverted to DENV2. Phase 1 trials testing two different formulations

(TV003 and TV005, which contains a higher dose of rDENV2/4D30 than

TV003) of the tetravalent vaccine (TetraVax-DV) began in 2011 in

flavivirus-naive adults (Clinicaltrials.gov NCT01436422) and flavivirus-

immune adults (NCT01506570). A phase 2 trial in Brazil is planned.

The safety and immunogenicity of vaccination of DENV-immune indi-

viduals was investigated (Durbin, Schmidt, et al., 2011). Individuals who

had received a monovalent DENV vaccine were given a second immuniza-

tion with a heterotypic monovalent attenuated vaccine 0.6–7.4 years later.

Replication and safety were comparable in immunized and naive volunteers.

In contrast to naive individuals, most volunteers who received a second

DENV vaccination developed a broad, heterotypic neutralizing antibody

response. However, in one cohort, preexisting DENV2 immunity impaired

seroconversion to a DENV1 vaccine.

The D30 vaccines have a number of advantages. Attenuation is due to

deletions in 30UTR, so both T-cell and antibody responses can be induced

against wild-type DENV structural and nonstructural proteins. Deletion

mutants are more stable than point mutations and therefore these strains

are unlikely to revert to wild-type viruses. In addition, as the four vaccine

strains contain the same deletion, potential recombination between the four

viruses will not lead to reversion of wild-type virus.

9.5. DENV ChimerasChimeric viruses were constructed using recombinant DNA technology

(Bray & Lai, 1991). Using the cDNA of DENV4, the C, prM, and

E genes were replaced with structural genes from DENV1 or DENV2.

The DENV2/DENV4 chimera was attenuated, providing a proof of

351Dengue Vaccines

concept for producing attenuated, chimeric dengue vaccine strains. The

chimeras were attenuated in rhesus monkeys (Bray, Men, & Lai, 1996).

Monkeys vaccinated with DENV1/DENV4 or DENV2/DENV4 chimeras

developed neutralizing antibodies against DENV1 and DENV2, respec-

tively, and were protected against challenge with DENV1 or DENV2.

Monkeys immunized with an equal mixture of DENV1/DENV4 and

DENV2/DENV4 chimeras were protected from challenge with DENV1

or DENV2.

9.6. Acambis/Sanofi Pasteur (ChimeriVax)Research begun at the NIH and St. Louis University (Bray & Lai, 1991;

Chambers, Nestorowicz, Mason, & Rice, 1999) and continued at Acambis

(now part of Sanofi Pasteur) resulted in the creation of chimeric viruses con-

taining the DENV structural proteins on the YF 17D backbone. The YF

17D vaccine backbone was selected because of the safety, long duration

of immunity, and rapid onset of immunity induced by the YFV 17D vac-

cine, which has been used for over 60 years. To create a DENV2 chimeric

strain, ChimeriVax-DENV2, the prM and E genes from the DENV2 PUO-

218 strain were cloned into a cDNA infectious clone of 17D (Guirakhoo

et al., 2000). ChimeriVax-DENV2 was nonneurovirulent for 4-week-old

mice and was genetically stable. Inoculation of rhesus monkeys resulted

in brief viremia, a neutralizing antibody response, and complete protection

from challenge with wild-type DENV2. DENV1, DENV3, and DENV4

chimeras were then constructed using the prM/E sequences from DENV

clinical isolates (Guirakhoo et al., 2001). The chimeras replicated to high

titers in Vero cells, were nonneurovirulent in 4-week-old mice, and were

immunogenic in rhesus monkeys. Monkeys immunized with a tetravalent

vaccine (ChimeriVax-DENV1–4) seroconverted to all four viruses after

one dose (except 1 of 6 did not seroconvert to DENV4). Preexisting immu-

nity from YF 17D vaccination (YF-VAX) did not significantly affect the

neutralizing antibody response.

A phase 1 trial found the safety profiles of YF-VAX and ChimeriVax-

DENV2 were similar, and no SEA were observed (Guirakhoo et al.,

2006). All recipients seroconverted to DENV2 after vaccination with 5

log10 PFU of the vaccine, and preexisting immunity to YFV did not inter-

fere with DENV2 seroconversion. In fact, all YFV-immune subjects also

seroconverted to the other DENV serotypes, whereas seroconversion to

the other serotypes was low in YFV-naive subjects.


Vaccine lot viruses of ChimeriVax-DENV1–4 were made using current

good manufacturing practice (cGMP) (Guirakhoo et al., 2004). Neu-

rovirulence was tested in cynomolgus monkeys after i.c. inoculation with

the tetravalent vaccine and was found to be reduced compared with

YF-VAX vaccination. Vaccine induced-protection was also tested in

cynomolgus monkeys. Monkeys received a single immunization s.c. with

a high or low dose (3 or 5 log10 PFU of each vaccine strain) of the tetravalent

vaccine and were challenged with wild-type DENV strains 6 months later.

All monkeys seroconverted to all four serotypes, and 22/24 were protected

from challenge.

Viral interference was studied in cynomolgus monkeys vaccinated with

the chimeric vaccine strains (Guy et al., 2009). Interference was observed in

monkeys given equivalent doses of each chimeric vaccine strain, with

DENV4 dominating, and several approaches were investigated to overcome

the interference. Immunization with bivalent vaccines at separate sites with

different draining lymph nodes, preexisting flavivirus immunity, decreasing

the dose of the dominant serotype, and boosting at 1 year all improved the

development of a balanced antibody response.

The ChimeriVax strains were highly attenuated for A. albopictus and

A. aegypti mosquitoes in terms of infection and dissemination (Higgs

et al., 2006; Johnson et al., 2004). Growth of the vaccine strains was also

studied in human myeloid DC and hepatic cell lines in vitro (Brandler

et al., 2005). The vaccine strains were not attenuated for replication in

DC compared with wild-type DENV or YF 17D but replicated to lower

titers than YF 17D in HepG2 and THLE-3 cells (but not HuH-7 cells),

suggesting the vaccine strains may be less hepatotropic than YF 17D and

therefore have less risk of inducing the hepatic failure that has been occasion-

ally been observed after YF 17D vaccination. Importantly, the chimeric

viruses were found to be genetically and phenotypically stable throughout

the manufacturing process (Mantel et al., 2011; Monath et al., 2005).

A tetravalent vaccine (TDV), containing�5 log10 tissue culture infective

doses (TCID50) of each recombinant serotype, was tested in flavivirus-naive

adults (Morrison et al., 2010). Two groups of 33 volunteers received the

vaccine at 0, 4, and 12–15 months or saline for first injection followed by

two doses of the TDV. The vaccine was safe, with no vaccine-related

SAE. Low-level viremia was observed primarily after the first dose and

was mainly DENV4. Each dose of the vaccine increased neutralizing anti-

body titers, and all volunteers receiving three doses seroconverted to all four

serotypes. The TDV was tested in children and adolescents (2–5, 6–11, or

353Dengue Vaccines

12–17 years of age) and adults in a nondengue endemic area (Mexico City)

(Poo et al., 2010). Subjects received three doses at 0, 3.5, and 12 months or

YF-VAX followed by two doses of TDV. The vaccine was safe, with no

vaccine-related SAE reported, and immunogenic. Seropositivity against

each serotype after three doses of TDV ranged from 77% to 92% and from

85% to 94% in the YF/TDV recipients. A phase 1 trial was then conducted

in the Philippines, a dengue-endemic country (Capeding et al., 2011). Chil-

dren, adolescents, and adults received three doses of the TDV vaccine at 0,

3.5, and 12 months. Reactogenicity was similar in adults and children, with

headache, injection site pain, fever, and myalgia most frequently reported.

A low level of viremia (primarily DENV4) was detected in some recipients,

most frequently after the first dose. After three doses, 100% of adults

seroconverted to all four serotypes, and seroconversion ranged from 83%

to 100% in children/adolescents. CD8þ T-cell responses against YF 17D

NS3 and DENV-specific CD4þ T-cell responses were detected in volun-

teers vaccinated with the tetravalent chimeric vaccine (Guy et al., 2008).

IFN-g dominated over TNF for both CD4þ and CD8þ T-cell responses.

After one vaccine dose, responses were serotype-specific and dominated by

DENV4 but broadened after a booster immunization.

A phase 2a study was designed to examine the safety and efficacy of TDV

vaccination in flavivirus-immune individuals (Qiao, Shaw, Forrat, Wartel-

Tram, & Lang, 2011). One dose of the TDV was given to persons who had

been vaccinated with monovalent live attenuated DENV1 or DENV2 vac-

cines, or YF-VAX 1 year prior, or flavivirus-naive adult volunteers. Prior fla-

vivirus immunity did not increase reactogenicity or the incidence of viremia,

but it did increase immunogenicity. In flavivirus-naive recipients, the neutral-

izing antibody response after one dose of TDVwas directed predominantly to

DENV3 andDENV4,whereas inDENV1-,DENV2-, andYF-primed recip-

ients a more balanced neutralizing antibody response was observed.

A phase 2 study was conducted in 199 children (2–11 years of age) in

Peru who had varying levels of preexisting flavivirus immunity from YF

vaccination (Lanata et al., 2012). Children received 3 doses of TDV at 0,

6, and 12 months. The reactogenicity observed was similar to previous stud-

ies; injection site pain, headache, malaise, fever were most commonly

reported and decreased with subsequent vaccinations. No vaccine-related

SAE were reported. Viremia was detected in 44% of the 97 individuals

tested and was mainly DENV4. Vaccination was immunogenic as well

and resulted in 94% of recipients seroconverting to all four DENV serotypes

with comparable neutralizing antibody titers to the four serotypes.


Results of a phase 2b study of TDV were reported in 2012. The CYD-

TDV vaccine was given to children 4–11 years of age in dengue-endemic

Thailand (Sabchareon et al., 2012). The primary analysis included data

from 2452 vaccine recipients and 1221 controls. More than 90% of the

children had preexisting antibodies against DENV or JEV, and 70% were

seropositive against at least one DENV serotype. Three injections of the

vaccine were given at 0, 6, and 12 months, and the subjects were followed

for 13 months after the last dose. The vaccine was safe with no vaccine-

related SAE and immunogenic. Neutralizing antibody titers increased after

one dose and increased further after the second and third doses and

then decreased 1 year later. However, the overall protective efficacy in

preventing symptomatic dengue infection was only 30.2%. The efficacy

for the individual serotypes was 55.6% for DENV1, 9.2% for DENV2,

75.3% for DENV3, and 100% for DENV4. DENV2 was the most com-

mon infecting serotype, which skewed the overall efficacy. The antibody

neutralization data did not correlate with protection, as neutralizing anti-

body titers (measured by PRNT50) increased after each dose and were

highest against DENV2 and DENV3, yet the subjects were not protected

against DENV2 infection. The authors suggest in the future performing

neutralization studies on cells that express FcR, which are targets of DENV

in vivo. The PRNT also does not distinguish between balanced neutralizing

antibody responses to the four serotypes, or less protective cross-reactive

responses. In addition, antibodies have other functions besides neutraliza-

tion, including ADCC, which may be important for protection. Another

potential reason for the low efficacy includes an antigenic mismatch

between the DENV2 vaccine strain and the DENV2 strain that resulted

in infections. Finally, the lack of a DENV-specific T-cell response may

have contributed to the poor efficacy, as these chimeric vaccines consist

of YFV, not DENV, nonstructural proteins, which are the dominant tar-

gets of the anti-DENV T-cell response in humans and mouse models

(Weiskopf et al., 2013, 2011; Yauch et al., 2010).

Despite the disappointing protection observed, the study results were

informative andmay spur investigations that lead to the identification of cor-

relates of protection. Importantly, the vaccine was safe, with no vaccine-

related SAE induced, and there was no disease enhancement observed in

the presence of nonprotective immunity during the short duration of the

study. Phase 3 studies involving 30,000 individuals in Latin America and

Asia started in 2011 and will provide more data on the efficacy of this vaccine

(Clinicaltrials.gov NCT01374516 and NCT01373281).

355Dengue Vaccines

10. MOVING FORWARD

Years of dengue vaccine research have brought us close to the point of

having a licensed vaccine. Although the results of the CYD-TDV phase 2b

trial were disappointing, the findings were important in directing future vac-

cine development and will hopefully lead to the identification of immune

correlates of protection. The trial results highlighted the need to study

pre- and postvaccination immune responses in both flavivirus-naive and

flavivirus-immune individuals in more detail. The lack of efficacy against

DENV2 despite neutralizing antibodies measured by PRNT using Vero

cells suggests neutralization assays on cell types that express FcR may be

more relevant. In addition to examining neutralization, other antibody

functions can be studied as well. The titer, class, subclass, and avidity of anti-

bodies specific for E, prM, and NS1 can be determined. The ability of

vaccine-induced antibodies to mediate ADCC and fix complement can also

be analyzed. The magnitude, breadth, and functionality, including cytokine

production and cytotoxicity, of both CD4þ and CD8þ T-cell responses

should also be investigated. As mentioned earlier, recent studies point to

an important protective role for CD8þ T cells in the immune response

to DENV. Vaccines that induce robust T- and B-cell responses may prove

to be superior to those vaccines that induce robust antibody responses but

weak T-cell responses.

Overall, the vaccines currently in clinical trials are safe, and no disease

enhancement has been observed in vaccinated humans to date. However,

long-term studies, both in NHP and humans, are required to ensure waning

immunity does not predispose vaccinees to severe dengue disease. The

WHO recommends following subjects for approximately 3–5 years after

the last vaccination (WHO, 2011). Although no disease enhancement fol-

lowing DENV vaccination has been reported, recent studies of the human

antibody response to DENV found prM/M-specific antibodies are broadly

cross-reactive and weakly or nonneutralizing (Beltramello et al., 2010; de

Alwis et al., 2011; Dejnirattisai et al., 2010), suggesting it may be prudent

to minimize the anti-prM antibody response to avoid ADE.

Animal models provide the necessary tools for dissecting the mechanisms

of vaccine-mediated protection. As some of the vaccine studies discussed

earlier suggest that vaccine-induced immune responses differ in flavivirus-

naive versus flavivirus-immune individuals, animal models provide the tools

to evaluate vaccine-induced immune responses under well-defined naive


versus immune infection settings. Thus, vaccine-induced immune responses

in animal models of dengue disease should be studied in more detail, includ-

ing analyzing the magnitude and quality of the T-cell responses. The existing

murine and NHP animal models can also be improved, and/or new models

developed. Manipulating the virus or mouse immune system may lead to

more relevant models (Zompi & Harris, 2012). For instance, passaging of

DENV though monkeys may result in the isolation of a strain more virulent

for monkeys. Mice lacking only the type I IFN receptor may prove to be a

more relevant model than AG129 mice. In addition, adoptive transfer stud-

ies may be useful for studying subunit and inactivated vaccines. Wild-type

mice can be immunized with these nonreplicating vaccines, followed by

transfer of immune components from the vaccinated wild-type mice into

IFN receptor-deficient mice. The IFN receptor-deficient mouse models

serve as a stringent challenge assay, and the adoptive transfer system allows

for thorough analysis of vaccine-induced humoral versus cellular response in

normal mice.

The lack of an adequate animal model for evaluating live attenuated den-

gue vaccine-induced immune responses has prompted the development of a

dengue human challenge model (DHCM). In a recent study, subjects pre-

viously vaccinated with the WRAIR/GSK live attenuated tetravalent vac-

cine (TDV) were challenged with underattenuated DENV strains to

evaluate the safety of challenge with the underattenuated strains and to eval-

uate the relationship between vaccine-induced neutralizing antibody titers

and protection (Sun et al., 2013). Subjects who had received the TDV

12–42 months previously, or naive controls, were challenged with under-

attenuated DENV1 or DENV3. All 5 vaccinated subjects challenged with

DENV1 were protected, and 2 of 5 challenged with DENV3 were protec-

ted. The 4 naive control recipients developed DF upon challenge. Neutral-

izing antibody titers correlated with protection in all but 1 subject who was

protected from DENV1 challenge despite no detectable neutralizing anti-

bodies. The DENV3 challenge was associated with significant elevations

in AST/ALT. This study demonstrated the feasibility of human challenge

to evaluate DENV vaccine candidates. A DHCM workshop, sponsored

by the WRAIR and the NIH, was held in 2011, and the consensus was that

a DHCM could be developed safely, if appropriate challenge strains can be

identified and produced under cGMP (Durbin & Whitehead, 2013). Safety

is a major concern for a DHCM, as challenge of vaccine recipients with

underattenuated strains could put the subjects at risk for developing severe

disease. Additionally, there is no approved therapeutic that could be used to

357Dengue Vaccines

treat recipients who develop DF or DHF/DSS. However, a DHCM could

provide valuable information on the immune response to DENV and poten-

tially lead to the identification of immune correlates of protection.

A DHCM could also be useful for selecting vaccine candidates for field

studies.

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Dengue Virus Vaccine Development · 2020-03-13 · Dengue virus (DENV) is a significant cause of morbidity and mortality in tropical and subtropical regions, causing hundreds of millions