Vaccine 29 (2011) 7276–7284

Contents lists available at ScienceDirect

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

Next generation dengue vaccines: A review of candidates in preclinical

development夽
Julia Schmitz a , John Roehrig b , Alan Barrett c , Joachim Hombach a,∗
a
Initiative for Vaccine Research, World Health Organization, Geneva, Switzerland
b
Arboviral Diseases Branch, Centers for Disease Control and Prevention, Fort Collins, CO, USA
c
Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, TX, USA

a r t i c l e i n f o a b s t r a c t

Article history: Dengue represents a major public health problem of growing global importance. In the absence of spe-
Available online 21 July 2011 cific 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
Keywords: are currently being evaluated in clinical trials and are described in detail in accompanying articles. In
Dengue vaccines addition, there are a large variety of candidates in preclinical development, which are based on diverse
Dengue
technologies, ensuring a continued influx of innovation into the development pipeline. Potentially, some
Dengue virus
of the current preclinical candidates may become next generation dengue vaccines with superior prod-
uct profiles. This review provides an overview of the various technological approaches to dengue vaccine
development and specifically focuses on candidates in preclinical development.
© 2011 World Health Organization.. Published by Elsevier Ltd. All rights reserved.

1. Introduction of dengue immunity, challenges to vaccine development and the

Dengue is a mosquito-borne flavivirus disease that has spread
to most tropical and many subtropical areas and creates a signif-
icant burden of disease and economic costs in endemic countries
[1–4]. As specific 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 [6–9].
This review focuses on dengue vaccine candidates in preclin-
ical 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 (1–2 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 first part of the review presents an overview
夽 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 pub-
lication 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).
various technologies used, while the second part provides a more
detailed description of specific vaccine projects in preclinical devel-
opment.
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 infec-
tion by one of the three heterologous serotypes (reviewed in [9]).
Dengue vaccine development efforts therefore aim for a tetrava-
lent 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 efficacy trials of candi-
date 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) 7276–7284
less clear (reviewed in [9,11,12]). Further research is needed to bet-
ter define 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 pathogene-
sis [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 neutraliza-
tion or enhancement of infection is determined by multiple factors,
including antibody specificity, antibody affinity, antibody titre and
epitope accessibility (reviewed in [15,16]).
The potential risk of immune enhancement of infection and dis-
ease 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 post-
vaccination.
DEN virions are composed of a lipid envelope modified by the
insertion of envelope (E) proteins and premembrane/membrane
(prM/M) proteins (depending on the maturation state [17]), sur-
rounding 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 epi-
topes 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 type-
specific neutralization capacity and lower ADE potential than
anti-prM antibodies. Moreover, most potent DENV neutralizing
antibodies identified so far recognize epitopes in domain III of
the E protein (EDIII) which is involved in binding of DENV to
cell receptors [19–27]. The conundrum of the human antibody
response to DENV infection is that most antibodies are specific for
domain II of the E protein (EDII) [19], which contains a variety of
serotype and flavivirus 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 consid-
ered 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 significant viremia or disease when infected with
human DENV isolates. Intracerebral challenge of mice with cer-
tain mouse-adapted DENV strains produces a paralysis phenotype,
and this intracerebral challenge model has been used to evalu-
ate protective efficacy 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 [29–31]. However, con-
cerns 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 demon-
strate some symptoms that re-capitulate human infection are also
being investigated [32,33]. Non-human primates (NHPs) are sus-
ceptible to infection by DENV and develop viremia, but do not
show significant clinical signs of infection. Studies to address pro-
7277
tective efficacy 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),
purified 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 difficult to achieve a level of attenuation which opti-
mally balances low reactogenicity (e.g. avoidance of clinical dengue
symptoms) with sufficiently 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 Kid-
ney cells). The first tetravalent dengue vaccine was produced in this
way [35]. Additionally, introduction of selected mutations or cre-
ation 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 vir-
ulence 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 pheno-
type and the theoretical risk of recombination between vaccine
and wild-type viruses. Furthermore, the need to achieve a bal-
anced immune response against all four DENV serotypes presents
a particular challenge for multi-component LAV vaccines. Repli-
cation interference between the different serotypes of DENV in
tetravalent formulations has been observed, resulting in a reduc-
tion of neutralizing antibodies to some serotypes, when compared
to monovalent formulations. LAV vaccines represent the majority
of dengue vaccine candidates in clinical evaluation [36–47], and
additional candidates are in preclinical development [48–53].
Nonliving vaccines can have some advantages over LAV
vaccines, including reduced potential for reactogenicity and bet-
ter 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 vac-
cines, but safety concerns raised in relation to some adjuvants need
to be addressed [54]. Also, access to adjuvants has proven difficult
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 sev-
eral 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 neu-
tralizing antibodies). Recombinant subunit dengue vaccines have
been produced in a variety of protein expression systems, including
bacterial, yeast, insect and mammalian cells. Most dengue sub-
unit 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, achieve-
ment of sufficient immunogenicity usually requires the use of
adjuvants. Various recombinant dengue subunit vaccines are in
7278 J. Schmitz et al. / Vaccine 29 (2011) 7276–7284
preclinical development [57–64], and the most advanced candidate
[65] is being evaluated in phase 1 clinical trials.
VLP vaccines are virus-like particles that do not contain replica-
tive 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 vac-
cine candidates are currently in preclinical development [67–70].
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 sys-
tems have shown some success in addressing these issues [71].
Various DNA dengue vaccines are currently in preclinical develop-
ment [72–81]. 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 efficient 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 vac-
cine types for the priming and boosting steps of an immunization
schedule, may allow for optimization of immune responses by com-
bining advantages of different vaccine technologies in a unique
order of vaccination. However, the increased complexity of vaccina-
tion schedules could make heterologous prime-boost approaches
more difficult 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 Kourí Tropical Medicine Institute (IPK) in
collaboration with the Center for Genetic Engineering and Biotech-
nology (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 eval-
uated in NHPs. Monkeys were immunized subcutaneously with
four doses of the monovalent vaccine (50–100 ␮g protein per dose,
formulated in Freund’s adjuvant). The monovalent vaccine can-
didates 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 cap-
sular 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 EDIII-
capsid 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 flagellin (STF2D),
which serves as an adjuvant. STF2D contains the toll-like receptor
5 (TLR5) activation domain from Salmonella typhimurium flagellin
[93]. This approach links adaptive and innate immunity and is
believed to mimic natural infection where antigen and TLR lig-
and are contained in a single pathogen. Physical linkage of TLR
ligand to antigen has been shown to be more specific and effi-
cient than co-administration, requiring lower doses of adjuvant.
STF2D fusion proteins can be expressed and purified 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 DENV-
1/3 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 attach-
ment points (C-terminal fusion versus insertion via replacement
of the flagellin D3 domain) affects immunogenicity. Longevity of
the immune response is yet to be studied. Additional bivalent con-
structs 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 tetrava-
lent chimeric EDIII fusion protein was generated, which consists of
the EDIII domains of DENV-1,-2,-3,-4 joined by flexible peptide link-
ers [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 vac-
cine candidate adjuvanted with montanide resulted in neutralizing
antibody responses against all DENV serotypes.
A different approach to the development of a single tetrava-
lent 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 neu-
tralizing 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 con-
sensus EDIII formulated in alum [60]. The intrinsic adjuvant activity
of the EDIII-Ag473 lipo-immunogen appears to be due to stim-
ulation of innate immunity through the TLR2 signaling pathway
[94].
3.2. DNA vaccines
A tetravalent DNA vaccine candidate developed by Inovio Phar-
maceuticals consists of a DNA plasmid vector that expresses a single
ORF comprising the EDIII domains of all four DENV serotypes sep-
arated by proteolytic cleavage sites [80]. In vivo expression of a
single tetravalent EDIII fusion protein, which is subsequently pro-
cessed 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 con-
sensus 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) 7276–7284 7279

Table 1
Dengue vaccine candidates in preclinical development.

Technological Vaccine developer Details Development stage

approach

Recombinant subunit IPK/CIGB EDIII-p64k fusion proteins and EDIII-capsid fusion Monovalent candidates evaluated
vaccines proteins expressed in E. coli [58,62–64] in NHPs
VaxInnate Bivalent EDIII-STF2D fusion proteins expressed in Bivalent candidate evaluated in
E. coli mice
ICGEB Tetravalent chimeric EDIII expressed in P. pastoris Tetravalent candidate evaluated in
[59] mice
NHRI Tetravalent consensus EDIII-Ag473 fusion protein Tetravalent candidate evaluated in
expressed in E. coli [60,61,94] mice

DNA vaccines Inovio Pharmaceuticals Tetravalent chimeric EDIII with inbuilt cleavage Tetravalent candidate evaluated in
sites expressed from plasmid vector [80] NHPs
Kobe University prM/E expressed from plasmid vector [78] Tetravalent candidate evaluated in
mice
CDC prM/E expressed from plasmid vector [72,75] Tetravalent candidate evaluated in
NHPs
NMRC Tetravalent “shuffled” prM/E expressed from Tetravalent candidates evaluated
plasmid vector [76] in NHPs

VLP vaccines Cytos Biotechnology EDIII chemically coupled to Qß VLPs expressed in Tetravalent candidate evaluated in
E. coli mice
ICGEB EDIII-HBsAg VLPs or ectoE-based VLPs expressed Monovalent candidates generated
in P. pastoris
Kobe University prM/E VLPs expressed in insect cells (Sf9) [69] Monovalent candidate evaluated in
mice

Virus-vectored vaccines
ICGEB Tetravalent chimeric EDIII expressed from Tetravalent candidate evaluated in
replication-deficient adenovirus vector [85] mice
GenPhar/NMRC Bivalent prM/E expressed from Tetravalent candidate evaluated in
replication-deficient adenovirus vector [84] NHPs
UNC EDIII, E85 or prM/E expressed from single-cycle Monovalent candidates evaluated
VEE virus vector [86] in NHPs
UTMB prM/E expressed from single-cycle West Nile virus Monovalent candidate evaluated in
vector [87] mice
Themis Bioscience/Institut Pasteur Tetravalent EDIII and DENV-1 ectoM expressed Tetravalent candidate evaluated in
from live attenuated measles virus vector [88] NHPs

Purified inactivated virus NMRC Psoralen-inactivated DENV [56] Monovalent candidate evaluated in
vaccines NHPs
GSK/WRAIR/FIOCRUZ Purified inactivated DENV [55] Tetravalent candidate evaluated in
NHPs

Live attenuated virus FIOCRUZ DENV prM/E expressed from live attenuated Monovalent candidates evaluated
vaccines chimeric YF 17D/DEN virus [48–50] in NHPs
Chiang Mai University/Mahidol DEN/DEN chimeric viruses [51,52] Monovalent candidates evaluated
University/NSTDA/BioNet-Asia in mice

Heterologous prime-boost
approaches
NMRC prM/E expressed from plasmid vector (prime) and Monovalent candidates evaluated
from single cycle VEE virus vector (boost) [89] in NHPs
NMRC/WRAIR Purified inactivated DENV or plasmid vector Tetravalent candidates evaluated
expressing prM/E (prime) and live attenuated in NHPs
DENV (boost) [90]

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 Kourí 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 trans-
fection, suitable for both intradermal and intramuscular routes.
Electroporation-based DNA immunization is currently being tested
in phase 1 clinical trials for HIV and influenza prevention and in
phase 2 clinical trials for hepatitis C, HPV 16/18 and AML/CML can-
cer 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 neutral-
izing 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-specific 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 Con-
trol 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 compo-
nent; administration by intramuscular injection). The tetravalent
7280 J. Schmitz et al. / Vaccine 29 (2011) 7276–7284
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 flaviviruses were shown to
improve immunogenicity of the vaccine. Moreover, there is evi-
dence 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, flavivirus 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 vac-
cines 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 “shuffled” E protein is expressed from a plasmid vector
[76]. Various chimeric E proteins containing sequence portions of
all DENV serotypes were derived by DNA shuffling technology. The
shuffled 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 neutral-
izing 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 Biotechnol-
ogy 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 vac-
cine [99]. About 60–90 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 eco-
nomically 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 vac-
cine 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 monova-
lent component induced a potent neutralizing antibody response
against all four serotypes.
VLP vaccine candidates have also been developed by the ICGEB.
In a first 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 sys-
tem 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 1–4 copies of HBsAg. In a second approach, a fusion pro-
tein 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 evalu-
ation 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
purified. A DENV-2 VLP vaccine candidate was found to be immuno-
genic and protective in mice [103]. Expression of VLPs by transient
transfection of insect cells (Sf9 cell line) resulted in 10–100-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-deficient adenovirus vaccine vector [85]. Advantages
of adenovirus vaccine vectors include a large insert capacity, effi-
cient 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 intraperi-
toneal route) resulted in balanced neutralizing antibody responses
against all four DENV serotypes. Of note, prior immunity to the ade-
novirus 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 vec-
tor may in fact even facilitate uptake of the virus-vectored dengue
vaccine into antigen-presenting cells.
Another virus-vectored vaccine approach developed by Gen-
Phar in collaboration with the NMRC is based on expression
of DENV prM/E from a replication-deficient 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 dif-
ferent DENV serotypes. NHPs were immunized with two doses of
tetravalent vaccine 8 weeks apart, followed by viral challenge at
3–6 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 DENV-
4 was significantly 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 for-
mulation, 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) 7276–7284
high levels of recombinant antigens, inducing both innate and
adaptive immune responses. Different monovalent vaccine candi-
dates 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 protec-
tive efficacy of the monovalent candidates were evaluated further
in NHP studies. Three doses (108 IU each) of a monovalent vac-
cine 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 anti-
body titres were observed with the DENV-3 E85 based vaccine,
which also completely protected all monkeys against viral chal-
lenge, while immunization with the DENV-3 prM/E based vaccine
resulted in only partial protection. Neutralizing antibodies against
DENV-3 E85 were serotype-specific and largely directed against
EDIII epitopes. Immunogenicity and protective efficacy 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 par-
ticles 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 efficient
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 efficacy 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 contain-
ing the EDIII domains of DENV-1-4 as well as the DENV-1 M protein
ectodomain (ectoM). The single component tetravalent vaccine-
induced 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 efficacy 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. Purified inactivated virus vaccines
A purified 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 injec-
tion, 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 can-
didate provides partial protection in NHPs.
PIV vaccine candidates are also being developed by Glaxo-
SmithKline (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
purification 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 anti-
body responses were obtained with the AS03 adjuvant system.
Clinical trials of the adjuvanted PIV vaccine are planned to start
in 2012, with a first assessment of protective efficacy envisaged for
2014–15.
3.6. Live attenuated virus vaccines
A live attenuated dengue vaccine candidate developed by
FIOCRUZ uses the live attenuated yellow fever vaccine 17DD sub-
strain 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 attenua-
tion 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 [48–50]. DEN viruses used
for the construction of chimeric YF 17D/DEN viruses in this vac-
cine project include strains isolated from Latin American patients,
while the DENV strains that provided the basis for a tetravalent
dengue vaccine candidate developed by Sanofi Pasteur, which is
currently in advanced clinical evaluation [44–47,107], were iso-
lated 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 devel-
oped in collaboration between Chiang Mai University, Mahidol
University, the Thai National Science and Technology Develop-
ment 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 atten-
uated DENV. Monovalent vaccine candidates have been evaluated
for neurovirulence and immunogenicity in mice. Safety and efficacy
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 candi-
date [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 needle-
free 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 non-
replicating 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) 7276–7284
dates was more effective for priming, a DNA-DNA-LAV vaccination
schedule (at -30, 0 and 60 days) was compared to a PIV-LAV vac-
cination 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 obser-
vation 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 advan-
tages over other vaccination approaches, recent investigations with
West Nile virus vaccine candidates suggest that the order of anti-
gens used in a heterologous prime-boost approach was important
in terms of the specificity 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 last-
ing immunity against four DENV serotypes, uncertainties around
immune correlates of protection, potential immune enhancement
of disease, and lack of suitable animal model. However, consider-
able progress has been made in recent years, which has resulted in
an advanced dengue vaccine pipeline, with a lead candidate enter-
ing phase 3 clinical trials [44–47] and several other candidates in
earlier stages of clinical evaluation [36–43,65,82]. 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 uncer-
tainties 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 con-
tinued influx of innovation into the vaccine pipeline, which is
critical for maximizing the chances of success for dengue vac-
cine development. One possible scenario is that a first 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 profile compared to existing vaccines,
including efficacy, dose-scheduling, and stability. There are indi-
cations that candidates which are currently in advanced clinical
development may require long, multi-dose immunization sched-
ules (e.g. three doses over a 12 month period). Next generation
dengue vaccines could have more compressed schedules requir-
ing fewer doses, or characteristics that make them more suitable
for certain target groups (e.g. particular age groups, immunocom-
promised 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 Gen-
eration Dengue Vaccines and Diagnostics (1–2 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 Penn-
sylvania), 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.
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