Vaccine 29 (2011) 72767284

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Next generation dengue vaccines: A review of candidates in preclinical

development
Julia Schmitz a , John Roehrig b , Alan Barrett c , Joachim Hombach a,
a
b
c

Initiative for Vaccine Research, World Health Organization, Geneva, Switzerland

Arboviral Diseases Branch, Centers for Disease Control and Prevention, Fort Collins, CO, USA
Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, TX, USA

a r t i c l e

i n f o

Article history:
Available online 21 July 2011
Keywords:
Dengue vaccines
Dengue
Dengue virus

a b s t r a c t
Dengue represents a major public health problem of growing global importance. In the absence of specic dengue therapeutics, strategies for disease control have increasingly focused on the development
of dengue vaccines. While a licensed dengue vaccine is not yet available, several vaccine candidates
are currently being evaluated in clinical trials and are described in detail in accompanying articles. In
addition, there are a large variety of candidates in preclinical development, which are based on diverse
technologies, ensuring a continued inux of innovation into the development pipeline. Potentially, some
of the current preclinical candidates may become next generation dengue vaccines with superior product proles. This review provides an overview of the various technological approaches to dengue vaccine
development and specically focuses on candidates in preclinical development.
2011 World Health Organization.. Published by Elsevier Ltd. All rights reserved.

1. Introduction
Dengue is a mosquito-borne avivirus disease that has spread
to most tropical and many subtropical areas and creates a significant burden of disease and economic costs in endemic countries
[14]. As specic dengue therapeutics are not available and disease
prevention is limited to vector control measures, the development
of a dengue vaccine would represent a major advance in the control
of the disease (reviewed in [5]). While no licensed dengue vaccine
currently exists, vaccine development has progressed considerably
in recent years. Several candidates are currently undergoing either
clinical evaluation or preclinical development. These have been
reviewed recently [69].
This review focuses on dengue vaccine candidates in preclinical development. It is based on published and unpublished data
presented by vaccine developers and researchers at the WHO/IVI
Informal Consultation on Next Generation Dengue Vaccines and
Diagnostics (12 November 2010, Atlanta). Additional preclinical
candidates from the published literature are included in the review
to illustrate the wide range of strategies applied to dengue vaccine
development. The rst part of the review presents an overview

Disclaimer: Two of the authors (JH, JS) are staff members of the World Health
Organization. The authors alone are responsible for the views expressed in this publication and they do not necessarily represent the decisions, policy or views of the
World Health Organization.
Corresponding author. Tel.: +41 22 791 4531; fax: +41 22 791 4860.
E-mail address: hombachj@who.int (J. Hombach).

of dengue immunity, challenges to vaccine development and the

various technologies used, while the second part provides a more
detailed description of specic vaccine projects in preclinical development.

2. Overview of dengue vaccine development

2.1. Dengue immunity and challenges to vaccine development
The disease dengue is caused by four dengue viruses (DENV),
DENV-1 to DENV-4. Due to their serological and genetic relatedness
they are considered four serotypes of DENV. Multiple DENV can
co-circulate in endemic areas (reviewed in [10]). Infection by one
serotype confers lasting protection against re-infection by the same
serotype, but only transient protection against secondary infection by one of the three heterologous serotypes (reviewed in [9]).
Dengue vaccine development efforts therefore aim for a tetravalent vaccine which simultaneously provides long-term protection
against all DENV serotypes.
The mechanism of protective immunity against dengue is not
fully understood. There is considerable evidence for a major role
of antibody-mediated DENV neutralization in protection against
infection and disease [9]. However, it is unclear what quantity of
neutralizing antibody is needed for protective immunity, and it is
increasingly likely that the contribution of neutralizing antibodies
to protection will not become known until efcacy trials of candidate vaccines have been completed. In addition, contributions of
other immune mechanisms such as cytotoxic T cell responses are

0264-410X/$ see front matter 2011 World Health Organization.. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2011.07.017

J. Schmitz et al. / Vaccine 29 (2011) 72767284

less clear (reviewed in [9,11,12]). Further research is needed to better dene and validate immune correlates of protection for vaccine
development [11].
Another challenge for vaccine development is the potentially
detrimental role of immune enhancement in dengue pathogenesis [5]. Severe disease is most commonly observed in secondary,
heterologous DENV infections. Antibody-dependent enhancement
(ADE) of infection has been proposed as the primary mechanism
of dengue immunopathogenesis [13,14]. In vitro studies suggest
that the capacity of DENV antibodies to contribute to neutralization or enhancement of infection is determined by multiple factors,
including antibody specicity, antibody afnity, antibody titre and
epitope accessibility (reviewed in [15,16]).
The potential risk of immune enhancement of infection and disease underscores the importance of developing dengue vaccines
which produce balanced, long-lasting immunity to all four DENV
serotypes. A tetravalent vaccine eliciting protective, neutralizing
antibody responses against all serotypes should address theoretical
concerns about vaccine-induced ADE. However, vaccine-induced
ADE might again become problematic as antibody titres wane postvaccination.
DEN virions are composed of a lipid envelope modied by the
insertion of envelope (E) proteins and premembrane/membrane
(prM/M) proteins (depending on the maturation state [17]), surrounding a nucleocapsid composed of capsid (C) proteins and the
viral RNA genome (reviewed in [18]). Human antibodies raised
against the DEN virion are mostly targeted at the E and prM
proteins [19]. Research to determine the most suitable target epitopes for vaccines is ongoing, and while considerable progress
has been made in the characterization of the humoral immune
response to DENV, important knowledge gaps still exist. There
is evidence suggesting that anti-E antibodies have higher typespecic neutralization capacity and lower ADE potential than
anti-prM antibodies. Moreover, most potent DENV neutralizing
antibodies identied so far recognize epitopes in domain III of
the E protein (EDIII) which is involved in binding of DENV to
cell receptors [1927]. The conundrum of the human antibody
response to DENV infection is that most antibodies are specic for
domain II of the E protein (EDII) [19], which contains a variety of
serotype and avivirus cross-reactive epitopes. The contribution of
the human antibody bias to EDII to sensitization for ADE has not
been determined. Because of its antigenic nature, EDIII is considered an antigenic target of particular relevance for dengue vaccine
development. New vaccine strategies may have to be developed
to enhance the human anti-EDIII response. Antibodies directed
against the C protein and against non-structural proteins, such as
NS1, have also been detected in DENV-infected individuals, but
their role in dengue immunity or immunopathogenesis remains
unclear [9].
Dengue vaccine development has been hampered by the lack
of an adequate animal model for the disease [28]. Normal mice
do not display signicant viremia or disease when infected with
human DENV isolates. Intracerebral challenge of mice with certain mouse-adapted DENV strains produces a paralysis phenotype,
and this intracerebral challenge model has been used to evaluate protective efcacy of vaccine candidates in mice. Furthermore,
immunocompromised mouse models have been developed which
show some of the characteristics of human disease and can also be
used as an in vivo system for studying ADE [2931]. However, concerns remain about whether the full spectrum of dengue immunity
and disease can be modeled in this immunocompromised setting.
Humanized mice that lack a murine antibody response but demonstrate some symptoms that re-capitulate human infection are also
being investigated [32,33]. Non-human primates (NHPs) are susceptible to infection by DENV and develop viremia, but do not
show signicant clinical signs of infection. Studies to address pro-

7277

tective efcacy of vaccine candidates in NHPs therefore measure

prevention or reduction of viremia upon viral challenge [28].
2.2. Technological approaches to dengue vaccine development
A wide range of vaccine technologies has been applied to
dengue vaccine development, including live attenuated virus (LAV),
puried inactivated virus (PIV), recombinant subunits, virus-like
particles (VLPs), and plasmid or viral vectors. Each of these
approaches has its advantages and challenges.
LAV vaccines have been shown to produce robust, lasting
and broad immunity, including humoral and cellular immune
responses [34]. In addition, their production cost tends be lower
than for many other vaccine technologies. However, it has often
proven difcult to achieve a level of attenuation which optimally balances low reactogenicity (e.g. avoidance of clinical dengue
symptoms) with sufciently high immunogenicity. In addition, LAV
vaccines should replicate well in cell culture in order to facilitate
their production, while their transmissibility by mosquitoes should
be prevented or strongly reduced. Attenuation strategies for live
dengue vaccines include serial cell passage (e.g. in Primary Dog Kidney cells). The rst tetravalent dengue vaccine was produced in this
way [35]. Additionally, introduction of selected mutations or creation of chimeric viruses by recombinant DNA technology can be
used. Success of these strategies is however often limited by lack
of a detailed understanding of the molecular determinants of virulence due to unavailability of animal models, as described above.
There are also concerns about the genetic stability of LAV vaccines,
including the possibility of reversion to a more virulent phenotype and the theoretical risk of recombination between vaccine
and wild-type viruses. Furthermore, the need to achieve a balanced immune response against all four DENV serotypes presents
a particular challenge for multi-component LAV vaccines. Replication interference between the different serotypes of DENV in
tetravalent formulations has been observed, resulting in a reduction of neutralizing antibodies to some serotypes, when compared
to monovalent formulations. LAV vaccines represent the majority
of dengue vaccine candidates in clinical evaluation [3647], and
additional candidates are in preclinical development [4853].
Nonliving vaccines can have some advantages over LAV
vaccines, including reduced potential for reactogenicity and better suitability for immunocompromised individuals. Moreover,
achievement of a balanced immune response is facilitated by lack of
replication competition between tetravalent vaccine components.
However, immune responses to nonliving vaccines tend to be less
broad, potent and durable compared to LAV vaccines. Adjuvants
have been shown to improve immunogenicity of nonliving vaccines, but safety concerns raised in relation to some adjuvants need
to be addressed [54]. Also, access to adjuvants has proven difcult
for many vaccine developers.
PIV dengue vaccines are killed wild type virions, containing all
DENV structural proteins and viral RNA. This permits induction of
an immune response to the three DENV structural proteins. PIV
dengue vaccines have not yet reached clinical evaluation, but several candidates are in preclinical development [55,56].
Other nonliving vaccines express particular DENV antigens,
which allows for targeting of the immune response to antigens
with particular desirable properties (e.g. induction of potent neutralizing antibodies). Recombinant subunit dengue vaccines have
been produced in a variety of protein expression systems, including
bacterial, yeast, insect and mammalian cells. Most dengue subunit vaccines express truncated versions of the E protein that have
deleted transmembrane domains, or simply express EDIII alone.
While subunit vaccines often show low reactogenicity, achievement of sufcient immunogenicity usually requires the use of
adjuvants. Various recombinant dengue subunit vaccines are in

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J. Schmitz et al. / Vaccine 29 (2011) 72767284

preclinical development [5764], and the most advanced candidate

[65] is being evaluated in phase 1 clinical trials.
VLP vaccines are virus-like particles that do not contain replicative genetic material, but permit presentation of antigens in a
repetitive, ordered array similar to the virion structure, which is
thought to increase immunogenicity [66]. Several dengue VLP vaccine candidates are currently in preclinical development [6770].
DNA vaccines and virus-vectored vaccines allow for in vivo
expression of antigens, often in the form of DENV VLPs. One of
the advantages of DNA vaccines is their thermostability, which
avoids cold-chain requirements for vaccine storage and transport.
However, DNA vaccines often face challenges in terms of adequate
cellular uptake and expression. Improved DNA vaccine delivery systems have shown some success in addressing these issues [71].
Various DNA dengue vaccines are currently in preclinical development [7281]. The most advanced DNA dengue vaccine candidate
has progressed to phase 1 clinical trials [82].
Virus-vectored dengue vaccines express DENV antigens from a
viral vaccine vector. Viral vectors allow for efcient infection of
target cells. Moreover, several well-characterized vaccine vector
platform technologies exist, some of which are used for licensed
vaccines with a well-known safety record [83]. However, there
are concerns that pre-existing immunity to viral vaccine vectors
could reduce the effectiveness of some virus-vectored dengue
vaccines. Several virus-vectored dengue vaccine candidates are
currently in preclinical development, including candidates based
on non-replicating vectors [84,85], single-cycle vectors [86,87] and
replication-competent vectors [88].
Heterologous prime-boost approaches, which use different vaccine types for the priming and boosting steps of an immunization
schedule, may allow for optimization of immune responses by combining advantages of different vaccine technologies in a unique
order of vaccination. However, the increased complexity of vaccination schedules could make heterologous prime-boost approaches
more difcult to implement in the context of immunization
programmes. Several heterologous prime-boost approaches are
currently evaluated in preclinical studies [89,90].

3. Dengue vaccine candidates in preclinical

development
3.1. Recombinant subunit vaccines
Several recombinant subunit vaccine candidates have been
developed by the Pedro Kour Tropical Medicine Institute (IPK) in
collaboration with the Center for Genetic Engineering and Biotechnology (CIGB) in Cuba (Table 1). One approach is based on fusion
of DENV EDIII to the carrier protein p64k of Neisseria meningitidis.
The EDIII-p64k fusion protein is expressed in E. coli. Monovalent
vaccine candidates for all DENV serotypes were shown to induce
neutralizing antibodies and protect against viral challenge in mice.
DENV-1 and DENV-2 monovalent candidates have also been evaluated in NHPs. Monkeys were immunized subcutaneously with
four doses of the monovalent vaccine (50100 g protein per dose,
formulated in Freunds adjuvant). The monovalent vaccine candidates were found to be immunogenic and provided protection
against viral challenge [58,91]. Adjuvants suitable for human use
are under evaluation, including N. meningitidis serogroup A capsular polysaccharide (CPS-A) adsorbed on aluminium hydroxide
[63]. In another approach, a DENV EDIII-capsid fusion protein was
expressed in E. coli and mixed in vitro with oligodeoxynucleotides
to obtain particulated aggregates. The aggregated DENV-2 EDIIIcapsid fusion protein induced both humoral and cellular immune
responses in NHPs and provided partial protection against viral
challenge [62]. The different subunit vaccine candidates are also

being studied in the context of heterologous prime-boost strategies

[64,92].
A subunit vaccine candidate developed by VaxInnate is based
on fusion of DENV EDIII to a proprietary form of agellin (STF2D),
which serves as an adjuvant. STF2D contains the toll-like receptor
5 (TLR5) activation domain from Salmonella typhimurium agellin
[93]. This approach links adaptive and innate immunity and is
believed to mimic natural infection where antigen and TLR ligand are contained in a single pathogen. Physical linkage of TLR
ligand to antigen has been shown to be more specic and efcient than co-administration, requiring lower doses of adjuvant.
STF2D fusion proteins can be expressed and puried in E. coli. In
immunogenicity studies in mice, monovalent EDIII-STF2D fusion
proteins were shown to elicit neutralizing DENV antibodies and
provide partial protection against viral challenge. Bivalent DENV1/3 and DENV-2/4 constructs were then generated by fusing the
EDIII domains of two different serotypes to separate attachment
points on STF2D. Subcutaneous immunization of mice with three
doses (3 g each) of a DENV-1/3 EDIII-STF2D fusion protein resulted
in robust and balanced titres of neutralizing antibodies against
DENV-1 and DENV-3. It was found that the choice of antigen attachment points (C-terminal fusion versus insertion via replacement
of the agellin D3 domain) affects immunogenicity. Longevity of
the immune response is yet to be studied. Additional bivalent constructs are being generated and tested, with the aim of combining
two suitable bivalent vaccine candidates to a tetravalent vaccine.
In a development approach by the International Centre for
Genetic Engineering and Biotechnology (ICGEB) in India, a tetravalent chimeric EDIII fusion protein was generated, which consists of
the EDIII domains of DENV-1,-2,-3,-4 joined by exible peptide linkers [59]. This single tetravalent approach avoids the complexities
related to producing tetravalent mixtures of monovalent vaccine
components. The tetravalent chimeric EDIII protein was expressed
in Pichia pastoris. Immunization of mice with the tetravalent vaccine candidate adjuvanted with montanide resulted in neutralizing
antibody responses against all DENV serotypes.
A different approach to the development of a single tetravalent subunit vaccine was taken at the Taiwanese National Health
Research Institutes (NHRI). A consensus EDIII amino acid sequence
was derived by sequence analysis of different DENV-1-4 strains,
and the consensus EDIII protein was expressed in E. coli. The single
tetravalent vaccine candidate adjuvanted with alum induced neutralizing antibody responses against all DENV serotypes in mice
[61]. This vaccine candidate was developed further by fusion of
the consensus EDIII with a fragment of the Ag473 lipoprotein of N.
meningitidis. The consensus EDIII-Ag473 fusion protein was found
to induce higher levels of neutralizing antibodies in mice than consensus EDIII formulated in alum [60]. The intrinsic adjuvant activity
of the EDIII-Ag473 lipo-immunogen appears to be due to stimulation of innate immunity through the TLR2 signaling pathway
[94].
3.2. DNA vaccines
A tetravalent DNA vaccine candidate developed by Inovio Pharmaceuticals consists of a DNA plasmid vector that expresses a single
ORF comprising the EDIII domains of all four DENV serotypes separated by proteolytic cleavage sites [80]. In vivo expression of a
single tetravalent EDIII fusion protein, which is subsequently processed into monovalent EDIII proteins by cellular proteases, aims
to ensure that the four EDIII domains are presented in equal ratios
in order to facilitate a balanced immune response. A synthetic consensus sequence for the EDIII domain of each DENV serotype was
derived by sequence comparison of approximately 15 different
strains. Sequences were also human codon optimized to improve
expression levels. Administration of the vaccine is via an improved

J. Schmitz et al. / Vaccine 29 (2011) 72767284

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Table 1
Dengue vaccine candidates in preclinical development.
Technological
approach

Vaccine developer

Details

Development stage

Recombinant subunit
vaccines

IPK/CIGB

EDIII-p64k fusion proteins and EDIII-capsid fusion

proteins expressed in E. coli [58,6264]
Bivalent EDIII-STF2D fusion proteins expressed in
E. coli
Tetravalent chimeric EDIII expressed in P. pastoris
[59]
Tetravalent consensus EDIII-Ag473 fusion protein
expressed in E. coli [60,61,94]

Monovalent candidates evaluated

in NHPs
Bivalent candidate evaluated in
mice
Tetravalent candidate evaluated in
mice
Tetravalent candidate evaluated in
mice

Kobe University

Tetravalent chimeric EDIII with inbuilt cleavage

sites expressed from plasmid vector [80]
prM/E expressed from plasmid vector [78]

CDC

prM/E expressed from plasmid vector [72,75]

NMRC

Tetravalent shufed prM/E expressed from

plasmid vector [76]

Tetravalent candidate evaluated in

NHPs
Tetravalent candidate evaluated in
mice
Tetravalent candidate evaluated in
NHPs
Tetravalent candidates evaluated
in NHPs

Cytos Biotechnology

EDIII chemically coupled to Q VLPs expressed in

E. coli
EDIII-HBsAg VLPs or ectoE-based VLPs expressed
in P. pastoris
prM/E VLPs expressed in insect cells (Sf9) [69]

VaxInnate
ICGEB
NHRI

DNA vaccines

VLP vaccines

Inovio Pharmaceuticals

ICGEB
Kobe University

Virus-vectored vaccines

Tetravalent candidate evaluated in

mice
Tetravalent candidate evaluated in
NHPs
Monovalent candidates evaluated
in NHPs
Monovalent candidate evaluated in
mice
Tetravalent candidate evaluated in
NHPs

NMRC

Psoralen-inactivated DENV [56]

GSK/WRAIR/FIOCRUZ

Puried inactivated DENV [55]

Monovalent candidate evaluated in

NHPs
Tetravalent candidate evaluated in
NHPs

FIOCRUZ

DENV prM/E expressed from live attenuated

chimeric YF 17D/DEN virus [4850]
DEN/DEN chimeric viruses [51,52]

Monovalent candidates evaluated

in NHPs
Monovalent candidates evaluated
in mice

prM/E expressed from plasmid vector (prime) and

from single cycle VEE virus vector (boost) [89]
Puried inactivated DENV or plasmid vector
expressing prM/E (prime) and live attenuated
DENV (boost) [90]

Monovalent candidates evaluated

in NHPs
Tetravalent candidates evaluated
in NHPs

UNC
UTMB
Themis Bioscience/Institut Pasteur

Live attenuated virus

vaccines

Chiang Mai University/Mahidol

University/NSTDA/BioNet-Asia
Heterologous prime-boost
approaches

Monovalent candidate evaluated in

mice

Tetravalent chimeric EDIII expressed from

replication-decient adenovirus vector [85]
Bivalent prM/E expressed from
replication-decient adenovirus vector [84]
EDIII, E85 or prM/E expressed from single-cycle
VEE virus vector [86]
prM/E expressed from single-cycle West Nile virus
vector [87]
Tetravalent EDIII and DENV-1 ectoM expressed
from live attenuated measles virus vector [88]

ICGEB
GenPhar/NMRC

Puried inactivated virus

vaccines

Tetravalent candidate evaluated in

mice
Monovalent candidates generated

NMRC
NMRC/WRAIR

Abbreviations: CDC: Centers for Disease Control and Prevention, USA; CIGB: Center for Genetic Engineering and Biotechnology, Cuba; FIOCRUZ: Oswaldo Cruz Foundation,
Brazil; GSK: GlaxoSmithKline Biologicals; ICGEB: International Centre for Genetic Engineering and Biotechnology, India; IPK: Pedro Kour Tropical Medicine Institute, Cuba;
NHRI: National Health Research Institutes, Taiwan; NMRC: Naval Medical Research Center, USA; NSTDA: National Science and Technology Development Agency, Thailand;
UNC: University of North Carolina at Chapel Hill, USA; UTMB: University of Texas Medical Branch, USA; WRAIR: Walter Reed Army Institute of Research, USA.

DNA delivery system based on electroporation-enhanced transfection, suitable for both intradermal and intramuscular routes.
Electroporation-based DNA immunization is currently being tested
in phase 1 clinical trials for HIV and inuenza prevention and in
phase 2 clinical trials for hepatitis C, HPV 16/18 and AML/CML cancer therapeutic studies. Immunization of mice with the tetravalent
dengue vaccine candidate, using intramuscular electroporation of
three doses (10 g DNA) at biweekly intervals, resulted in neutralizing antibody titres against all four DENV serotypes. Immunization
of NHPs was also shown to elicit robust tetravalent antibody
responses. Further studies to assess DENV cross-reactive versus
serotype-specic antibody responses are in progress.
Another tetravalent DNA vaccine candidate developed at Kobe
University is composed of a mixture of four plasmid vectors, each
of which expresses the prM and E proteins (prM/E) of one DENV
serotype. Two doses of the tetravalent vaccine (100 g per dose, at
three week intervals) were administered to mice using a needle-

free jet injector. Mice were protected against viral challenge 3

weeks after the last dose, and persistence of neutralizing antibodies
was observed during a 30-week follow-up [95]. Immunogenicity
of the tetravalent DNA vaccine in mice was further increased by
co-immunization with DENV-2 VLPs generated by co-expression
of prM/E in cell culture. Immunogenicity of the DNA vaccine was
also found to be improved by co-immunization with an inactivated
Japanese encephalitis vaccine [78].
A similar tetravalent DNA vaccine candidate based on prM/E
expression has been developed by the U.S. Centers for Disease Control and Prevention (CDC). Transfection of the recombinant plasmid
vectors into cultured cells has been shown to result in secretion of
prM/E containing VLPs, which have an antigenic structure similar
to DEN virions [72,75]. Immunogenicity of a tetravalent mixture
of four monovalent DNA vaccines was evaluated in NHPs (two
dose schedule one month apart; 500 g per monovalent component; administration by intramuscular injection). The tetravalent

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vaccine was found to stimulate a balanced immunity that lasted

for 10 months and protected from viral challenge. Pre-existing
antibodies against DENV or other aviviruses were shown to
improve immunogenicity of the vaccine. Moreover, there is evidence suggesting that the safety and immunogenicity of the vaccine
candidates could be further improved by manipulating antigenic
determinants of the E protein. In order to reduce the potential for
ADE by cross-reactive antibodies, avivirus group cross-reactive
epitopes and DENV serocomplex cross-reactive epitopes in the
E protein were mapped with monoclonal antibody panels and
mutated to reduce cross-reactivity, resulting in the formation of
cross-reactivity reduced VLPs in cell culture [96]. When evaluated
in mice, cross-reactivity reduced DENV-1 and DENV-2 VLP vaccines continued to elicit high levels of neutralizing antibodies, but
showed reduced ADE potential compared to wild type vaccines
both in vitro (K562 cell-based assay) and in vivo (AG129 mouse
model [29,97]). Immunogenicity of the cross-reactivity reduced
vaccines was further improved by incorporating a CD4 epitope from
the transmembrane domain of West Nile virus E protein into the
expression construct.
In a single tetravalent approach developed at the U.S. Naval
Medical Research Center (NMRC), a prM/E construct containing a
chimeric shufed E protein is expressed from a plasmid vector
[76]. Various chimeric E proteins containing sequence portions of
all DENV serotypes were derived by DNA shufing technology. The
shufed constructs were screened for antigen expression in vitro
and for immunogenicity in mice. Three selected constructs were
evaluated as single tetravalent DNA vaccine candidates in NHPs.
Two of these candidates were found to induce tetravalent neutralizing antibody responses in the majority of animals and to provide
partial protection against viral challenge.
3.3. VLP vaccines
A VLP dengue vaccine candidate developed by Cytos Biotechnology is based on chemical coupling of recombinant EDIII to carrier
VLPs derived from bacteriophage Q [98]. A similar approach has
recently been applied to the development of a West Nile virus vaccine [99]. About 6090 EDIII molecules can be coupled to one VLP
carrier, resulting in the formation of a highly repetitive, ordered
array of antigen, which is expected to increase immunogenicity.
Q VLPs also contain E. coli-derived ssRNA, which has been shown
to be required for IgG isotype switching in mice immunized with
Q VLPs [100,101] and is used as a built-in adjuvant to improve the
antibody response to Q VLP-based vaccines. Q VLPs can be economically produced in E. coli, resulting in 2 g/l culture of GMP grade
material. Q VLP based vaccine candidates are in advanced clinical
evaluation for other target antigens. Four monovalent dengue vaccine candidates were generated by coupling the EDIII domain of
the different DENV serotypes to the carrier VLP. Immunogenicity
studies in mice were performed to compare monovalent vaccines
individually and in a tetravalent formulation (three doses at 10
day intervals; 50 g per dose of monovalent vaccine and 200 g
per dose of tetravalent vaccine; subcutaneous administration with
alum). The neutralizing antibody response observed to DENV-4
was weaker than for the other serotypes. However, an improved
formulation with increased representation of the DENV-4 monovalent component induced a potent neutralizing antibody response
against all four serotypes.
VLP vaccine candidates have also been developed by the ICGEB.
In a rst approach, DENV E was expressed as a fusion protein with
Hepatitis B virus surface antigen (HBsAg) in P. pastoris [67]. HBsAg
expressed in P. pastoris has been shown to assemble into highly
immunogenic VLPs. Furthermore, high protein yields (up to 7 gram
HBsAg per liter culture [102]) make P. pastoris an attractive system for affordable production of protein-based vaccines. While

expression of EDIII alone does not result in VLP formation, mosaic

VLPs composed of varying ratios of EDIII-HBsAg and HBsAg could
be generated by co-expression of the EDIII-HBsAg fusion protein
with 14 copies of HBsAg. In a second approach, a fusion protein consisting of the E protein ectodomain (ectoE) and 30 amino
residues of the prM protein was expressed in P. pastoris, which
also resulted in VLP formation. Structural characterization of the
VLPs, their functional evaluation for immunogenicity, and evaluation of prime-boost protocols combining VLP vaccine candidates
with rAd5 vectored vaccine candidates are planned.
In a VLP vaccine development approach employed at Kobe
University, prM/E is expressed from a plasmid vector transfected
into cell culture-based expression systems and secreted VLPs are
puried. A DENV-2 VLP vaccine candidate was found to be immunogenic and protective in mice [103]. Expression of VLPs by transient
transfection of insect cells (Sf9 cell line) resulted in 10100-fold
improved yields compared to mammalian expression systems
[69]. Immunogenicity of the DENV-2 VLP vaccine candidate in
mice could be improved by co-immunization with a DNA vaccine
expressing prM/E, which also functions as a CpG adjuvant.
3.4. Virus-vectored vaccines
A virus-vectored dengue vaccine developed by ICGEB is based
on expression of a tetravalent chimeric EDIII fusion protein from
a replication-decient adenovirus vaccine vector [85]. Advantages
of adenovirus vaccine vectors include a large insert capacity, efcient delivery and high expression levels of antigens in a variety of
cells, and a well-documented human safety record. Immunization
of mice with two doses of the single component tetravalent dengue
vaccine candidate (administered 30 days apart by the intraperitoneal route) resulted in balanced neutralizing antibody responses
against all four DENV serotypes. Of note, prior immunity to the adenovirus vaccine vector did not impair the potency of the dengue
vaccine candidate. Presence of antibodies against the vector in the
sera of immunized mice enabled uptake of the vector into U937
human monocytic cells, which are not susceptible to infection in the
absence of antibodies against the vector. This effect is presumably
mediated by the Fc receptor pathway [104]. The results suggest that
low levels of pre-existing antibodies against the adenovirus vector may in fact even facilitate uptake of the virus-vectored dengue
vaccine into antigen-presenting cells.
Another virus-vectored vaccine approach developed by GenPhar in collaboration with the NMRC is based on expression
of DENV prM/E from a replication-decient complex adenovirus
vaccine vector capable of accommodating multiple large antigen
inserts [84]. The tetravalent vaccine is composed of two bivalent
vaccines, each of which expresses prM and E proteins of two different DENV serotypes. NHPs were immunized with two doses of
tetravalent vaccine 8 weeks apart, followed by viral challenge at
36 months. Neutralizing antibody responses were induced against
all serotypes. Viremia from challenge with DENV-1 and DENV-3
was blocked, while viremia from challenge with DENV-2 and DENV4 was signicantly reduced. Further development of the vaccine
constructs, including optimization of all prM and E gene sequences
for human codon usage, resulted in an improved tetravalent formulation, which induced a more balanced immune response to all
four serotypes. The candidate vaccine is scheduled to enter clinical
evaluation shortly.
A virus-vectored vaccine candidate developed at the University
of North Carolina at Chapel Hill (UNC) is based on expression of
DENV antigens from a single-cycle Venezuelan equine encephalitis
(VEE) virus vaccine vector [86]. Packaging technologies based on
helper RNAs allow for the production of infectious single-cycle VEE
virus particles also referred to as virus replicon particles (VRPs). VEE
VRPs have been shown to infect human dendritic cells and express

J. Schmitz et al. / Vaccine 29 (2011) 72767284

high levels of recombinant antigens, inducing both innate and

adaptive immune responses. Different monovalent vaccine candidates expressing DENV prM/E proteins, C-terminally truncated E
proteins (E81, E85 [81% or 85% of E]) or EDIII were evaluated for
immunogenicity in mice. Vaccine candidates expressing truncated
E proteins were found to elicit higher neutralizing antibody titres
than candidates expressing prM/E. Immunogenicity and protective efcacy of the monovalent candidates were evaluated further
in NHP studies. Three doses (108 IU each) of a monovalent vaccine candidate expressing either DENV-3 E85 or DENV-3 prM/E
were administered subcutaneously at weeks 0, 7, and 25, and viral
challenge was performed at week 41. Higher neutralizing antibody titres were observed with the DENV-3 E85 based vaccine,
which also completely protected all monkeys against viral challenge, while immunization with the DENV-3 prM/E based vaccine
resulted in only partial protection. Neutralizing antibodies against
DENV-3 E85 were serotype-specic and largely directed against
EDIII epitopes. Immunogenicity and protective efcacy studies of a
tetravalent E85 based vaccine candidate in NHPs are in progress.
Another approach taken at the University of Texas Medical
Branch (UTMB) is based on a single-cycle, capsid-gene deleted West
Nile virus vaccine vector [105]. Infectious single-cycle WNV particles are produced by packaging technologies, which supply the
capsid protein in trans. Infected target cells are unable to produce
viral progeny, but the single replication cycle results in efcient
production of antigen including secretion of VLPs from infected
cells. A DENV-2 vaccine candidate was generated by replacing the
prM/E genes of the WNV vaccine vector with the prM/E genes of
DENV-2 [87]. The vaccine was tested for potency and efcacy in a
mouse model of dengue pathogenesis (AG129 mice [97]). A single
intraperitoneal immunization of mice produced a dose-dependent
DENV-2 neutralizing antibody response and resulted in a protective
effect against viral challenge.
A virus-vectored dengue vaccine candidate developed by
Themis Bioscience and Institut Pasteur is based on expression of
a single tetravalent DENV antigen construct from a live attenuated
measles virus vaccine vector [88]. This vaccine vector technology
allows for integration of large antigen inserts and has been shown
to induce strong neutralizing antibodies and cellular immune
responses even in the presence of pre-existing immunity to measles
virus. The dengue vaccine candidate expresses a construct containing the EDIII domains of DENV-1-4 as well as the DENV-1 M protein
ectodomain (ectoM). The single component tetravalent vaccineinduced neutralizing antibodies against all DENV serotypes in mice.
Inclusion of ectoM in the vaccine construct was found to provide an
adjuvant activity. Following further improvements of the vaccine
construct, immunogenicity and efcacy studies have been initiated
in NHPs. Establishment of a GMP process and initiation of clinical
trials are planned in 2011 and 2012, respectively.
3.5. Puried inactivated virus vaccines
A puried psoralen-inactivated DENV-1 vaccine candidate has
been developed by the NMRC [56]. Following immunogenicity
studies in mice, the monovalent candidate adjuvanted in alum was
evaluated in NHPs using a three-dose schedule (intradermal injection, biweekly intervals). Immunized monkeys showed a DENV-1
neutralizing antibody response and reduced duration of viremia
upon viral challenge on day 132, suggesting that the vaccine candidate provides partial protection in NHPs.
PIV vaccine candidates are also being developed by GlaxoSmithKline (GSK) in collaboration with the Walter Reed Army
Institute of Research (WRAIR) and the Oswaldo Cruz Foundation
(FIOCRUZ) [55]. Different approaches for virus inactivation and
purication and various adjuvant systems are currently under
evaluation. A pilot study was carried out in NHPs to test a tetrava-

7281

lent inactivated virus vaccine candidate together with different

proprietary adjuvant systems (AS01, AS03, AS04). Two doses of
adjuvanted vaccine were given 30 days apart, followed by viral
challenge 4 months later. All immunized animals were found to
be protected against viral challenge. The highest neutralizing antibody responses were obtained with the AS03 adjuvant system.
Clinical trials of the adjuvanted PIV vaccine are planned to start
in 2012, with a rst assessment of protective efcacy envisaged for
201415.
3.6. Live attenuated virus vaccines
A live attenuated dengue vaccine candidate developed by
FIOCRUZ uses the live attenuated yellow fever vaccine 17DD substrain as a genetic backbone. Chimeric YF 17D/DEN viruses were
constructed by replacing the YFV prM/E genes with those of DENV
[106]. Monovalent dengue vaccine candidates for different DENV
serotypes were generated and evaluated in NHPs. In attenuation studies, the monovalent vaccines produced only low levels
of viremia and showed reduced neurotropism compared to the
YF 17D vaccine. Neutralizing antibody responses and protection
against viral challenge were also shown [4850]. DEN viruses used
for the construction of chimeric YF 17D/DEN viruses in this vaccine project include strains isolated from Latin American patients,
while the DENV strains that provided the basis for a tetravalent
dengue vaccine candidate developed by Sano Pasteur, which is
currently in advanced clinical evaluation [4447,107], were isolated from Asian patients. Different sets of chimeric YF 17D/DEN
viruses are thus available for the development of a tetravalent live
attenuated dengue vaccine.
Another live attenuated vaccine candidate has been developed in collaboration between Chiang Mai University, Mahidol
University, the Thai National Science and Technology Development Agency (NSTDA) and BioNet-Asia [51,52]. DEN/DEN chimeric
viruses were constructed which contain the prM/E coding region of
recent dengue clinical isolates in the genetic background of attenuated DENV. Monovalent vaccine candidates have been evaluated
for neurovirulence and immunogenicity in mice. Safety and efcacy
studies in NHPs are planned.
3.7. Heterologous prime-boost approaches
A heterologous prime-boost approach developed by the NMRC
uses a DNA vaccine candidate and a virus-vectored vaccine candidate [89]. The monovalent DNA vaccine, which has been evaluated
in a phase 1 clinical trial, consists of a plasmid vector expressing
DENV-1 prM/E and is administered intramuscularly with a needlefree Biojector system [82]. The virus-vectored vaccine expresses
the same DENV-1 prM/E sequence from a single-cycle VEE virus
vector. A three-dose prime-boost regimen was evaluated in NHPs,
which consists of two doses of the DNA vaccine at days 0 and 28
followed by intramuscular injection of the virus-vectored vaccine
at day 117. When compared to homologous three dose schedules
of either the DNA or the virus-vectored vaccines, the heterologous
prime-boost approach was found to improve neutralizing antibody
responses and protection against viral challenge [89].
Another heterologous prime-boost approach developed by the
NMRC and the WRAIR uses non-replicating vaccines for priming,
followed by boosting with a LAV vaccine candidate [90]. Two nonreplicating vaccines were evaluated: a tetravalent DNA vaccine
composed of plasmid vectors expressing DENV prM/E [82] and a
tetravalent PIV vaccine consisting of formalin-inactivated DENV
adjuvanted with alum [55]. The tetravalent LAV vaccine candidate,
which was developed by GSK and WRAIR using serial passage of
clinical DENV isolates in PDK cells, has reached phase 2 clinical trials
[42]. To determine which of the two non-replicating vaccine candi-

7282

J. Schmitz et al. / Vaccine 29 (2011) 72767284

dates was more effective for priming, a DNA-DNA-LAV vaccination

schedule (at -30, 0 and 60 days) was compared to a PIV-LAV vaccination schedule (at 0 and 60 days) in NHPs. Both regimens were
found to elicit neutralizing antibody responses. However, priming
with the DNA vaccine provided only partial protection against viral
challenge 6 months after the last dose, as evidenced by the observation of breakthrough viremia. By contrast, priming with one dose
of PIV vaccine followed by a LAV vaccine booster dose two months
later resulted in complete protection against challenge with any of
the four DENV serotypes [90].
While heterologous prime-boost strategies may offer advantages over other vaccination approaches, recent investigations with
West Nile virus vaccine candidates suggest that the order of antigens used in a heterologous prime-boost approach was important
in terms of the specicity and magnitude of the immune response
[108]. Similar results can be expected for dengue vaccines.
4. Conclusions
Efforts for dengue vaccine development have faced multiple
challenges, including the need to induce a balanced and lasting immunity against four DENV serotypes, uncertainties around
immune correlates of protection, potential immune enhancement
of disease, and lack of suitable animal model. However, considerable progress has been made in recent years, which has resulted in
an advanced dengue vaccine pipeline, with a lead candidate entering phase 3 clinical trials [4447] and several other candidates in
earlier stages of clinical evaluation [3643,65,82]. The majority of
dengue vaccine candidates currently in clinical development are
based on the same immunization regimen (namely a tetravalent
LAV) derived using different technologies. However, some uncertainties around the utilization of LAVs remain, including potential
imbalances in immunity due to immune interference.
A large number of diverse dengue vaccine candidates are in
preclinical development. These preclinical candidates ensure a continued inux of innovation into the vaccine pipeline, which is
critical for maximizing the chances of success for dengue vaccine development. One possible scenario is that a rst generation
of licensed dengue vaccines will arise from candidates currently
in clinical development, while some of the current preclinical
stage candidates could become next generation dengue vaccines
licensed at a later stage. Next generation dengue vaccines could
serve to complement existing vaccines. For example, prime-boost
approaches combining live and non-replicating vaccines may allow
for balancing the relative advantages and shortcomings of these
technologies. Furthermore, next generation dengue vaccines could
have a superior product prole compared to existing vaccines,
including efcacy, dose-scheduling, and stability. There are indications that candidates which are currently in advanced clinical
development may require long, multi-dose immunization schedules (e.g. three doses over a 12 month period). Next generation
dengue vaccines could have more compressed schedules requiring fewer doses, or characteristics that make them more suitable
for certain target groups (e.g. particular age groups, immunocompromised individuals, travelers). Finally, next generation dengue
vaccines could generate much-welcomed competition, leading to
more affordable and cost-effective vaccines, which would facilitate
their use particularly in endemic developing countries.
Acknowledgements
Participants of the WHO/IVI Informal Consultation on Next Generation Dengue Vaccines and Diagnostics (12 November 2010,
Atlanta) are thanked for sharing unpublished data. Preclinical stage
dengue vaccine candidates were presented at the consultation by

Drs. Martin Bachmann (Cytos Biotechnology), Gwong-Jen Chang

(CDC), John Dong (GenPhar), Bruce Innis (GSK), Niranjan Y. Sardesai
(Inovio Pharmaceuticals) and David B. Weiner (University of Pennsylvania), Alan Shaw (VaxInnate), Sathyamangalam Swaminathan
(ICGEB), Erich Tauber (Themis Bioscience) and Laura White (UNC).
Financial support of the consultation from the Bill and Melinda
Gates Foundation is kindly acknowledged.

References
[1] Gubler DJ. Epidemic dengue/dengue hemorrhagic fever as a public health,
social and economic problem in the 21st century. Trends Microbiol
2002;10:1003.
[2] Suaya JA, Shepard DS, Siqueira JB, Martelli CT, Lum LC, Tan LH, et al. Cost of
dengue cases in eight countries in the Americas and Asia: a prospective study.
Am J Trop Med Hyg 2009;80:84655.
[3] World Health Organization. Dengue guidelines for diagnosis, treatment,
prevention and control. Geneva: World Health Organization; 2009. Available
http://whqlibdoc.who.int/publications/2009/9789241547871 eng.pdf
at:
[accessed 21 June 2011].
[4] Shepard DS, Coudeville L, Halasa YA, Zambrano B, Dayan GH. Economic impact
of dengue illness in the Americas. Am J Trop Med Hyg 2011;84:2007.
[5] Whitehorn J, Farrar J. Dengue. Br Med Bull 2010;95:16173.
[6] Durbin AP, Whitehead SS. Dengue vaccine candidates in development. Curr
Top Microbiol Immunol 2010;338:12943.
[7] Swaminathan S, Batra G, Khanna N. Dengue vaccines: state of the art. Expert
Opin Ther Pat 2010;20:81935.
[8] Guzman MG. Dengue vaccines: new developments. Drugs Future
2011;36:4562.
[9] Murphy BR, Whitehead SS. Immune response to dengue virus and prospects
for a vaccine. Annu Rev Immunol 2011;29:587619.
[10] Halstead SB. Dengue. Lancet 2007;370:164452.
[11] Hombach J, Cardosa MJ, Sabchareon A, Vaughn DW, Barrett AD. Scientic
consultation on immunological correlates of protection induced by dengue
vaccines. Vaccine 2007;25:41309.
[12] Thomas SJ, Hombach J, Barrett A. Scientic consultation on cell mediated immunity (CMI) in dengue and dengue vaccine development. Vaccine
2009;27:35568.
[13] Halstead SB. Neutralization and antibody-dependent enhancement of dengue
viruses. Adv Virus Res 2003;60:42167.
[14] Halstead SB. Antibodies determine virulence in dengue. Ann N Y Acad Sci
2009;1171(Suppl. 1):E4856.
[15] Pierson TC, Fremont DH, Kuhn RJ, Diamond MS. Structural insights into
the mechanisms of antibody-mediated neutralization of avivirus infection:
implications for vaccine development. Cell Host Microbe 2008;4:22938.
[16] Dowd KA, Pierson TC. Antibody-mediated neutralization of aviviruses: a
reductionist view. Virology 2011;411:30615.
[17] Junjhon J, Edwards TJ, Utaipat U, Bowman VD, Holdaway HA, Zhang W, et al.
Inuence of pr-M cleavage on the heterogeneity of extracellular dengue virus
particles. J Virol 2010;84:83538.
[18] Perera R, Kuhn RJ. Structural proteomics of dengue virus. Curr Opin Microbiol
2008;11:36977.
[19] Beltramello M, Williams KL, Simmons CP, Macagno A, Simonelli L, Quyen NT,
et al. The human immune response to dengue virus is dominated by highly
cross-reactive antibodies endowed with neutralizing and enhancing activity.
Cell Host Microbe 2010;8:27183.
[20] Crill WD, Roehrig JT. Monoclonal antibodies that bind to domain III of dengue
virus E glycoprotein are the most efcient blockers of virus adsorption to Vero
cells. J Virol 2001;75:776973.
[21] Roehrig JT. Antigenic structure of avivirus proteins. Adv Virus Res
2003;59:14175.
[22] Brien JD, Austin SK, Sukupolvi-Petty S, OBrien KM, Johnson S, Fremont DH,
et al. Genotype-specic neutralization and protection by antibodies against
dengue virus type 3. J Virol 2010;84:1063043.
[23] Dejnirattisai W, Jumnainsong A, Onsirisakul N, Fitton P, Vasanawathana S,
Limpitikul W, et al. Cross-reacting antibodies enhance dengue virus infection
in humans. Science 2010;328:7458.
[24] Rodenhuis-Zybert IA, van der Schaar HM, da Silva Voorham JM, van der
Ende-Metselaar H, Lei HY, Wilschut J, et al. Immature dengue virus: a veiled
pathogen? PLoS Pathog 2010;6:e1000718.
[25] Shrestha B, Brien JD, Sukupolvi-Petty S, Austin SK, Edeling MA, Kim T, et al. The
development of therapeutic antibodies that neutralize homologous and heterologous genotypes of dengue virus type 1. PLoS Pathog 2010;6:e1000823.
[26] Sukupolvi-Petty S, Austin SK, Engle M, Brien JD, Dowd KA, Williams KL, et al.
Structure and function analysis of therapeutic monoclonal antibodies against
dengue virus type 2. J Virol 2010;84:922739.
[27] Wahala WM, Donaldson EF, de Alwis R, Accavitti-Loper MA, Baric RS, de Silva
AM. Natural strain variation and antibody neutralization of dengue serotype
3 viruses. PLoS Pathog 2010;6:e1000821.
[28] Cassetti MC, Durbin A, Harris E, Rico-Hesse R, Roehrig J, Rothman A,
et al. Report of an NIAID workshop on dengue animal models. Vaccine
2010;28:422934.

J. Schmitz et al. / Vaccine 29 (2011) 72767284

[29] Williams KL, Zompi S, Beatty PR, Harris E. A mouse model for studying dengue
virus pathogenesis and immune response. Ann N Y Acad Sci 2009;1171(Suppl.
1):E1223.
[30] Balsitis SJ, Williams KL, Lachica R, Flores D, Kyle JL, Mehlhop E, et al. Lethal
antibody enhancement of dengue disease in mice is prevented by Fc modication. PLoS Pathog 2010;6:e1000790.
[31] Zellweger RM, Prestwood TR, Shresta S. Enhanced infection of liver sinusoidal endothelial cells in a mouse model of antibody-induced severe dengue
disease. Cell Host Microbe 2010;7:12839.
[32] Kuruvilla JG, Troyer RM, Devi S, Akkina R. Dengue virus infection and immune
response in humanized RAG2(/)gamma(c)(/) (RAG-hu) mice. Virology
2007;369:14352.
[33] Mota J, Rico-Hesse R. Humanized mice show clinical signs of dengue fever
according to infecting virus genotype. J Virol 2009;83:863845.
[34] Lauring AS, Jones JO, Andino R. Rationalizing the development of live attenuated virus vaccines. Nat Biotechnol 2010;28:5739.
[35] Bhamarapravati N, Sutee Y. Live attenuated tetravalent dengue vaccine. Vaccine 2000;18(Suppl. 2):447.
[36] Huang CY, Butrapet S, Tsuchiya KR, Bhamarapravati N, Gubler DJ, Kinney RM.
Dengue 2 PDK-53 virus as a chimeric carrier for tetravalent dengue vaccine
development. J Virol 2003;77:1143647.
[37] Durbin AP, Whitehead SS, McArthur J, Perreault JR, Blaney Jr JE, Thumar B, et al.
rDEN4delta30, a live attenuated dengue virus type 4 vaccine candidate, is safe,
immunogenic, and highly infectious in healthy adult volunteers. J Infect Dis
2005;191:7108.
[38] Durbin AP, McArthur J, Marron JA, Blaney Jr JE, Thumar B, Wanionek K,
et al. The live attenuated dengue serotype 1 vaccine rDEN1Delta30 is
safe and highly immunogenic in healthy adult volunteers. Hum Vaccin
2006;2:16773.
[39] Durbin AP, McArthur JH, Marron JA, Blaney JE, Thumar B, Wanionek K, et al.
rDEN2/4Delta30(ME), a live attenuated chimeric dengue serotype 2 vaccine
is safe and highly immunogenic in healthy dengue-naive adults. Hum Vaccin
2006;2:25560.
[40] McArthur JH, Durbin AP, Marron JA, Wanionek KA, Thumar B, Pierro DJ,
et al. Phase I clinical evaluation of rDEN4Delta30-200,201: a live attenuated
dengue 4 vaccine candidate designed for decreased hepatotoxicity. Am J Trop
Med Hyg 2008;79:67884.
[41] Simasathien S, Thomas SJ, Watanaveeradej V, Nisalak A, Barberousse C, Innis
BL, et al. Safety and immunogenicity of a tetravalent live-attenuated dengue
vaccine in avivirus naive children. Am J Trop Med Hyg 2008;78:42633.
[42] Sun W, Cunningham D, Wasserman SS, Perry J, Putnak JR, Eckels KH, et al.
Phase 2 clinical trial of three formulations of tetravalent live-attenuated
dengue vaccine in avivirus-naive adults. Hum Vaccin 2009;5:3340.
[43] Wright PF, Durbin AP, Whitehead SS, Ikizler MR, Henderson S, Blaney JE, et al.
Phase 1 trial of the dengue virus type 4 vaccine candidate rDEN4Delta30-4995
in healthy adult volunteers. Am J Trop Med Hyg 2009;81:83441.
[44] Guy B, Guirakhoo F, Barban V, Higgs S, Monath TP, Lang J. Preclinical and
clinical development of YFV 17D-based chimeric vaccines against dengue,
West Nile and Japanese encephalitis viruses. Vaccine 2010;28:63249.
[45] Morrison D, Legg TJ, Billings CW, Forrat R, Yoksan S, Lang J. A novel tetravalent
dengue vaccine is well tolerated and immunogenic against all 4 serotypes in
avivirus-naive adults. J Infect Dis 2010;201:3707.
[46] Guy B, Almond J, Lang J. Dengue vaccine prospects: a step forward. Lancet
2011;377:3812.
[47] Poo J, Galan F, Forrat R, Zambrano B, Lang J, Dayan GH. Live-attenuated tetravalent dengue vaccine in dengue-naive children, adolescents, and adults in
Mexico City: randomized controlled phase 1 trial of safety and immunogenicity. Pediatr Infect Dis J 2011;30:E917.
[48] Galler R, Marchevsky RS, Caride E, Almeida LF, Yamamura AM, Jabor AV, et al.
Attenuation and immunogenicity of recombinant yellow fever 17D-dengue
type 2 virus for rhesus monkeys. Braz J Med Biol Res 2005;38:183546.
[49] Mateu GP, Marchevsky RS, Liprandi F, Bonaldo MC, Coutinho ES, Dieudonne
M, et al. Construction and biological properties of yellow fever 17D/dengue
type 1 recombinant virus. Trans R Soc Trop Med Hyg 2007;101:28998.
[50] Trindade GF, Marchevsky RS, Fillipis AM, Nogueira RM, Bonaldo MC, Acero PC,
et al. Limited replication of yellow fever 17DD and 17D-Dengue recombinant
viruses in rhesus monkeys. An Acad Bras Cienc 2008;80:31121.
[51] BioNet-Asia. BioNet-Asia to enter development of a dengue vaccine.
Press release 2011. Available at: http://www.bionet-asia.com/feb2111.html
[accessed 21 June 2011].
[52] National Science and Technology Development Agency. Chimeric dengue vaccine licensing agreement. Press release 2011. Available at: http://www.nstda.
or.th/eng/index.php/research-themes/health-and-medicine/item/177-chim
eric-dengue-vaccine-licensing-agreement [accessed 21 June 2011].
[53] Smith KM, Nanda K, Spears CJ, Ribeiro M, Vancini R, Piper A, et al. Structural mutants of dengue virus 2 transmembrane domains exhibit host-range
phenotype. Virol J 2011;8:289.
[54] Leroux-Roels G. Unmet needs in modern vaccinology: adjuvants to improve
the immune response. Vaccine 2010;28(Suppl. 3):C2536.
[55] Putnak JR, Coller BA, Voss G, Vaughn DW, Clements D, Peters I, et al. An evaluation of dengue type-2 inactivated, recombinant subunit, and live-attenuated
vaccine candidates in the rhesus macaque model. Vaccine 2005;23:
444252.
[56] Maves RC, Ore RM, Porter KR, Kochel TJ. Immunogenicity and protective efcacy of a psoralen-inactivated dengue-1 virus vaccine candidate in Aotus
nancymaae monkeys. Vaccine 2011;29:26916.

7283

[57] Simmons M, Murphy GS, Hayes CG. Short report: antibody responses of mice
immunized with a tetravalent dengue recombinant protein subunit vaccine.
Am J Trop Med Hyg 2001;65:15961.
[58] Bernardo L, Izquierdo A, Alvarez M, Rosario D, Prado I, Lopez C, et al. Immunogenicity and protective efcacy of a recombinant fusion protein containing
the domain III of the dengue 1 envelope protein in non-human primates.
Antiviral Res 2008;80:1949.
[59] Etemad B, Batra G, Raut R, Dahiya S, Khanam S, Swaminathan S, et al. An
envelope domain III-based chimeric antigen produced in Pichia pastoris elicits
neutralizing antibodies against all four dengue virus serotypes. Am J Trop Med
Hyg 2008;79:35363.
[60] Chen HW, Liu SJ, Liu HH, Kwok Y, Lin CL, Lin LH, et al. A novel technology for
the production of a heterologous lipoprotein immunogen in high yield has
implications for the eld of vaccine design. Vaccine 2009;27:14009.
[61] Leng CH, Liu SJ, Tsai JP, Li YS, Chen MY, Liu HH, et al. A novel dengue vaccine
candidate that induces cross-neutralizing antibodies and memory immunity.
Microbes Infect 2009;11:28895.
[62] Valdes I, Bernardo L, Gil L, Pavon A, Lazo L, Lopez C, et al. A novel fusion protein
domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice. Virology 2009;394:24958.
[63] Valdes I, Hermida L, Martin J, Menendez T, Gil L, Lazo L, et al. Immunological evaluation in nonhuman primates of formulations based on the chimeric
protein P64k-domain III of dengue 2 and two components of Neisseria meningitidis. Vaccine 2009;27:9951001.
[64] Valdes I, Gil L, Romero Y, Castro J, Puente P, Lazo L, et al. The chimeric protein
domain III-capsid of dengue virus serotype 2 (DEN-2) successfully boosts neutralizing antibodies generated in monkeys upon infection with DEN-2. Clin
Vaccine Immunol 2011;18:4559.
[65] Clements DE, Coller BA, Lieberman MM, Ogata S, Wang G, Harada KE, et al.
Development of a recombinant tetravalent dengue virus vaccine: immunogenicity and efcacy studies in mice and monkeys. Vaccine 2010;28:270515.
[66] Jennings GT, Bachmann MF. The coming of age of virus-like particle vaccines.
Biol Chem 2008;389:52136.
[67] Bisht H, Chugh DA, Raje M, Swaminathan SS, Khanna N. Recombinant
dengue virus type 2 envelope/hepatitis B surface antigen hybrid protein
expressed in Pichia pastoris can function as a bivalent immunogen. J Biotechnol 2002;99:97110.
[68] Amexis G, Young NS. Parvovirus B19 empty capsids as antigen carriers
for presentation of antigenic determinants of dengue 2 virus. J Infect Dis
2006;194:7904.
[69] Kuwahara M, Konishi E. Evaluation of extracellular subviral particles of
dengue virus type 2 and Japanese encephalitis virus produced by Spodoptera
frugiperda cells for use as vaccine and diagnostic antigens. Clin Vaccine
Immunol 2010;17:15606.
[70] Liu W, Jiang H, Zhou J, Yang X, Tang Y, Fang D, et al. Recombinant dengue viruslike particles from Pichia pastoris: efcient production and immunological
properties. Virus Genes 2010;40:539.
[71] Kutzler MA, Weiner DB. DNA vaccines: ready for prime time? Nat Rev Genet
2008;9:77688.
[72] Chang GJ, Hunt AR, Holmes DA, Springeld T, Chiueh TS, Roehrig JT, et al.
Enhancing biosynthesis and secretion of premembrane and envelope proteins
by the chimeric plasmid of dengue virus type 2 and Japanese encephalitis
virus. Virology 2003;306:17080.
[73] Wu SF, Liao CL, Lin YL, Yeh CT, Chen LK, Huang YF, et al. Evaluation of protective
efcacy and immune mechanisms of using a non-structural protein NS1 in
DNA vaccine against dengue 2 virus in mice. Vaccine 2003;21:391929.
[74] Mota J, Acosta M, Argotte R, Figueroa R, Mendez A, Ramos C. Induction of
protective antibodies against dengue virus by tetravalent DNA immunization
of mice with domain III of the envelope protein. Vaccine 2005;23:346976.
[75] Purdy DE, Chang GJ. Secretion of noninfectious dengue virus-like particles
and identication of amino acids in the stem region involved in intracellular
retention of envelope protein. Virology 2005;333:23950.
[76] Raviprakash K, Apt D, Brinkman A, Skinner C, Yang S, Dawes G, et al. A chimeric
tetravalent dengue DNA vaccine elicits neutralizing antibody to all four virus
serotypes in rhesus macaques. Virology 2006;353:16673.
[77] Costa SM, Azevedo AS, Paes MV, Sarges FS, Freire MS, Alves AM. DNA vaccines
against dengue virus based on the ns1 gene: the inuence of different signal sequences on the protein expression and its correlation to the immune
response elicited in mice. Virology 2007;358:41323.
[78] Imoto J, Konishi E. Dengue tetravalent DNA vaccine increases its immunogenicity in mice when mixed with a dengue type 2 subunit vaccine or an
inactivated Japanese encephalitis vaccine. Vaccine 2007;25:107684.
[79] De Paula SO, Lima DM, de Oliveira Franca RF, Gomes-Ruiz AC, da Fonseca BA. A
DNA vaccine candidate expressing dengue-3 virus prM and E proteins elicits
neutralizing antibodies and protects mice against lethal challenge. Arch Virol
2008;153:221523.
[80] Ramanathan MP, Kuo YC, Selling BH, Li Q, Sardesai NY, Kim JJ, et al. Development of a novel DNA SynCon tetravalent dengue vaccine that elicits immune
responses against four serotypes. Vaccine 2009;27:644453.
[81] Lima DM, de Paula SO, Franca RF, Palma PV, Morais FR, Gomes-Ruiz AC, et al. A
DNA vaccine candidate encoding the structural prM/E proteins elicits a strong
immune response and protects mice against dengue-4 virus infection. Vaccine
2011;29:8318.
[82] Beckett CG, Tjaden J, Burgess T, Danko JR, Tamminga C, Simmons M, et al.
Evaluation of a prototype dengue-1 DNA vaccine in a Phase 1 clinical trial.
Vaccine 2011;29:9608.

7284

J. Schmitz et al. / Vaccine 29 (2011) 72767284

[83] Liniger M, Zuniga A, Naim HY. Use of viral vectors for the development of
vaccines. Expert Rev Vaccines 2007;6:25566.
[84] Raviprakash K, Wang D, Ewing D, Holman DH, Block K, Woraratanadharm
J, et al. A tetravalent dengue vaccine based on a complex adenovirus vector
provides signicant protection in rhesus monkeys against all four serotypes
of dengue virus. J Virol 2008;82:692734.
[85] Khanam S, Pilankatta R, Khanna N, Swaminathan S. An adenovirus type 5
(AdV5) vector encoding an envelope domain III-based tetravalent antigen
elicits immune responses against all four dengue viruses in the presence of
prior AdV5 immunity. Vaccine 2009;27:601121.
[86] White LJ, Parsons MM, Whitmore AC, Williams BM, de Silva A, Johnston RE.
An immunogenic and protective alphavirus replicon particle-based dengue
vaccine overcomes maternal antibody interference in weanling mice. J Virol
2007;81:1032939.
[87] Suzuki R, Winkelmann ER, Mason PW. Construction and characterization of a
single-cycle chimeric avivirus vaccine candidate that protects mice against
lethal challenge with dengue virus type 2. J Virol 2009;83:187080.
[88] Brandler S, Rufe C, Najburg V, Frenkiel MP, Bedouelle H, Despres P, et al.
Pediatric measles vaccine expressing a dengue tetravalent antigen elicits
neutralizing antibodies against all four dengue viruses. Vaccine 2010;28:
67309.
[89] Chen L, Ewing D, Subramanian H, Block K, Rayner J, Alterson KD, et al. A
heterologous DNA prime-Venezuelan equine encephalitis virus replicon particle boost dengue vaccine regimen affords complete protection from virus
challenge in cynomolgus macaques. J Virol 2007;81:116349.
[90] Simmons M, Burgess T, Lynch J, Putnak R. Protection against dengue virus by
non-replicating and live attenuated vaccines used together in a prime boost
vaccination strategy. Virology 2010;396:2808.
[91] Hermida L, Bernardo L, Martin J, Alvarez M, Prado I, Lopez C, et al. A recombinant fusion protein containing the domain III of the dengue-2 envelope
protein is immunogenic and protective in nonhuman primates. Vaccine
2006;24:316571.
[92] Valdes I, Hermida L, Gil L, Lazo L, Castro J, Martin J, et al. Heterologous primeboost strategy in non-human primates combining the infective dengue virus
and a recombinant protein in a formulation suitable for human use. Int J Infect
Dis 2010;14:e37783.
[93] McDonald WF, Huleatt JW, Foellmer HG, Hewitt D, Tang J, Desai P, et al. A West
Nile virus recombinant protein vaccine that coactivates innate and adaptive
immunity. J Infect Dis 2007;195:160717.
[94] Leng CH, Chen HW, Chang LS, Liu HH, Liu HY, Sher YP, et al. A recombinant
lipoprotein containing an unsaturated fatty acid activates NF-kappaB through
the TLR2 signaling pathway and induces a differential gene prole from a
synthetic lipopeptide. Mol Immunol 2010;47:201521.

[95] Konishi E, Kosugi S, Imoto J. Dengue tetravalent DNA vaccine inducing neutralizing antibody and anamnestic responses to four serotypes in mice. Vaccine
2006;24:22007.
[96] Crill WD, Hughes HR, Delorey MJ, Chang GJ. Humoral immune responses of
dengue fever patients using epitope-specic serotype-2 virus-like particle
antigens. PLoS One 2009;4:e4991.
[97] Johnson AJ, Roehrig JT. New mouse model for dengue virus vaccine testing. J
Virol 1999;73:7836.
[98] Spohn G, Keller I, Beck M, Grest P, Jennings GT, Bachmann MF. Active immunization with IL-1 displayed on virus-like particles protects from autoimmune
arthritis. Eur J Immunol 2008;38:87787.
[99] Spohn G, Jennings GT, Martina BE, Keller I, Beck M, Pumpens P, et al. A VLPbased vaccine targeting domain III of the West Nile virus E protein protects
from lethal infection in mice. Virol J 2010;7:146.
[100] Jegerlehner A, Maurer P, Bessa J, Hinton HJ, Kopf M, Bachmann MF. TLR9 signaling in B cells determines class switch recombination to IgG2a. J Immunol
2007;178:241520.
[101] Bessa J, Jegerlehner A, Hinton HJ, Pumpens P, Saudan P, Schneider P, et al.
Alveolar macrophages and lung dendritic cells sense RNA and drive mucosal
IgA responses. J Immunol 2009;183:378899.
[102] Gurramkonda C, Adnan A, Gabel T, Lunsdorf H, Ross A, Nemani SK, et al.
Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: application to intracellular production
of Hepatitis B surface antigen. Microb Cell Fact 2009;8:13.
[103] Konishi E, Fujii A. Dengue type 2 virus subviral extracellular particles produced by a stably transfected mammalian cell line and their evaluation for a
subunit vaccine. Vaccine 2002;20:105867.
[104] Leopold PL, Wendland RL, Vincent T, Crystal RG. Neutralized adenovirusimmune complexes can mediate effective gene transfer via an Fc
receptor-dependent infection pathway. J Virol 2006;80:1023747.
[105] Widman DG, Frolov I, Mason PW. Third-generation avivirus vaccines
based on single-cycle, encapsidation-defective viruses. Adv Virus Res
2008;72:77126.
[106] Caufour PS, Motta MC, Yamamura AM, Vazquez S, Ferreira II, Jabor AV, et al.
Construction, characterization and immunogenicity of recombinant yellow
fever 17D-dengue type 2 viruses. Virus Res 2001;79:114.
[107] Guirakhoo F, Weltzin R, Chambers TJ, Zhang ZX, Soike K, Ratterree
M, et al. Recombinant chimeric yellow fever-dengue type 2 virus is
immunogenic and protective in nonhuman primates. J Virol 2000;74:
547785.
[108] Zlatkovic J, Stiasny K, Heinz FX. Immunodominance and functional activities
of antibody responses to inactivated West Nile virus and recombinant subunit
vaccines in mice. J Virol 2011;85:19942003.