Next Generation Dengue Vaccines: a Review of Candidates in Preclinical Developmentଝ

Vaccine 29 (2011) 7276–7284

Contents lists available at ScienceDirect

Vaccine

j ournal homepage: www.elsevier.com/locate/vaccine

Next generation dengue vaccines: A review of candidates in preclinical developmentଝ

a b c a,∗

Julia Schmitz , John Roehrig , Alan Barrett , Joachim Hombach

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 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.

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

various technologies used, while the second part provides a more

Dengue is a mosquito-borne ﬂavivirus disease that has spread detailed description of speciﬁc vaccine projects in preclinical devel-

to most tropical and many subtropical areas and creates a signif- opment.

icant burden of disease and economic costs in endemic countries

[1–4]. As speciﬁc dengue therapeutics are not available and disease

2. Overview of dengue vaccine development

prevention is limited to vector control measures, the development

of a dengue vaccine would represent a major advance in the control

2.1. Dengue immunity and challenges to vaccine development

of the disease (reviewed in [5]). While no licensed dengue vaccine

currently exists, vaccine development has progressed considerably

The disease dengue is caused by four dengue viruses (DENV),

in recent years. Several candidates are currently undergoing either

DENV-1 to DENV-4. Due to their serological and genetic relatedness

clinical evaluation or preclinical development. These have been

they are considered four serotypes of DENV. Multiple DENV can

reviewed recently [6–9].

co-circulate in endemic areas (reviewed in [10]). Infection by one

This review focuses on dengue vaccine candidates in preclin-

serotype confers lasting protection against re-infection by the same

ical development. It is based on published and unpublished data

serotype, but only transient protection against secondary infec-

presented by vaccine developers and researchers at the WHO/IVI

tion by one of the three heterologous serotypes (reviewed in [9]).

Informal Consultation on Next Generation Dengue Vaccines and

Dengue vaccine development efforts therefore aim for a tetrava-

Diagnostics (1–2 November 2010, Atlanta). Additional preclinical

lent vaccine which simultaneously provides long-term protection

candidates from the published literature are included in the review

against all DENV serotypes.

to illustrate the wide range of strategies applied to dengue vaccine

The mechanism of protective immunity against dengue is not

development. The ﬁrst part of the review presents an overview

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

ଝ

Disclaimer: Two of the authors (JH, JS) are staff members of the World Health neutralizing antibody is needed for protective immunity, and it is

Organization. The authors alone are responsible for the views expressed in this pub-

increasingly likely that the contribution of neutralizing antibodies

lication and they do not necessarily represent the decisions, policy or views of the

to protection will not become known until efﬁcacy trials of candi-

World Health Organization.

∗

date vaccines have been completed. In addition, contributions of

Corresponding author. Tel.: +41 22 791 4531; fax: +41 22 791 4860.

E-mail address: [email protected] (J. Hombach). other immune mechanisms such as cytotoxic T cell responses are

J. Schmitz et al. / Vaccine 29 (2011) 7276–7284 7277

less clear (reviewed in [9,11,12]). Further research is needed to bet- tective efﬁcacy of vaccine candidates in NHPs therefore measure

ter deﬁne and validate immune correlates of protection for vaccine prevention or reduction of viremia upon viral challenge [28].

development [11].

Another challenge for vaccine development is the potentially 2.2. Technological approaches to dengue vaccine development

detrimental role of immune enhancement in dengue pathogene-

sis [5]. Severe disease is most commonly observed in secondary, A wide range of vaccine technologies has been applied to

heterologous DENV infections. Antibody-dependent enhancement dengue vaccine development, including live attenuated virus (LAV),

(ADE) of infection has been proposed as the primary mechanism puriﬁed inactivated virus (PIV), recombinant subunits, virus-like

of dengue immunopathogenesis [13,14]. In vitro studies suggest particles (VLPs), and plasmid or viral vectors. Each of these

that the capacity of DENV antibodies to contribute to neutraliza- approaches has its advantages and challenges.

tion or enhancement of infection is determined by multiple factors, LAV vaccines have been shown to produce robust, lasting

including antibody speciﬁcity, antibody afﬁnity, antibody titre and and broad immunity, including humoral and cellular immune

epitope accessibility (reviewed in [15,16]). responses [34]. In addition, their production cost tends be lower

The potential risk of immune enhancement of infection and dis- than for many other vaccine technologies. However, it has often

ease underscores the importance of developing dengue vaccines proven difﬁcult to achieve a level of attenuation which opti-

which produce balanced, long-lasting immunity to all four DENV mally balances low reactogenicity (e.g. avoidance of clinical dengue

serotypes. A tetravalent vaccine eliciting protective, neutralizing symptoms) with sufﬁciently high immunogenicity. In addition, LAV

antibody responses against all serotypes should address theoretical vaccines should replicate well in cell culture in order to facilitate

concerns about vaccine-induced ADE. However, vaccine-induced their production, while their transmissibility by mosquitoes should

ADE might again become problematic as antibody titres wane post- be prevented or strongly reduced. Attenuation strategies for live

vaccination. dengue vaccines include serial cell passage (e.g. in Primary Dog Kid-

DEN virions are composed of a lipid envelope modiﬁed by the ney cells). The ﬁrst tetravalent dengue vaccine was produced in this

insertion of envelope (E) proteins and premembrane/membrane way [35]. Additionally, introduction of selected mutations or cre-

(prM/M) proteins (depending on the maturation state [17]), sur- ation of chimeric viruses by recombinant DNA technology can be

rounding a nucleocapsid composed of capsid (C) proteins and the used. Success of these strategies is however often limited by lack

viral RNA genome (reviewed in [18]). Human antibodies raised of a detailed understanding of the molecular determinants of vir-

against the DEN virion are mostly targeted at the E and prM ulence due to unavailability of animal models, as described above.

proteins [19]. Research to determine the most suitable target epi- There are also concerns about the genetic stability of LAV vaccines,

topes for vaccines is ongoing, and while considerable progress including the possibility of reversion to a more virulent pheno-

has been made in the characterization of the humoral immune type and the theoretical risk of recombination between vaccine

response to DENV, important knowledge gaps still exist. There and wild-type viruses. Furthermore, the need to achieve a bal-

is evidence suggesting that anti-E antibodies have higher type- anced immune response against all four DENV serotypes presents

speciﬁc neutralization capacity and lower ADE potential than a particular challenge for multi-component LAV vaccines. Repli-

anti-prM antibodies. Moreover, most potent DENV neutralizing cation interference between the different serotypes of DENV in

antibodies identiﬁed so far recognize epitopes in domain III of tetravalent formulations has been observed, resulting in a reduc-

the E protein (EDIII) which is involved in binding of DENV to tion of neutralizing antibodies to some serotypes, when compared

cell receptors [19–27]. The conundrum of the human antibody to monovalent formulations. LAV vaccines represent the majority

response to DENV infection is that most antibodies are speciﬁc for of dengue vaccine candidates in clinical evaluation [36–47], and

domain II of the E protein (EDII) [19], which contains a variety of additional candidates are in preclinical development [48–53].

serotype and ﬂavivirus cross-reactive epitopes. The contribution of Nonliving vaccines can have some advantages over LAV

the human antibody bias to EDII to sensitization for ADE has not vaccines, including reduced potential for reactogenicity and bet-

been determined. Because of its antigenic nature, EDIII is consid- ter suitability for immunocompromised individuals. Moreover,

ered an antigenic target of particular relevance for dengue vaccine achievement of a balanced immune response is facilitated by lack of

development. New vaccine strategies may have to be developed replication competition between tetravalent vaccine components.

to enhance the human anti-EDIII response. Antibodies directed However, immune responses to nonliving vaccines tend to be less

against the C protein and against non-structural proteins, such as broad, potent and durable compared to LAV vaccines. Adjuvants

NS1, have also been detected in DENV-infected individuals, but have been shown to improve immunogenicity of nonliving vac-

their role in dengue immunity or immunopathogenesis remains cines, but safety concerns raised in relation to some adjuvants need

unclear [9]. to be addressed [54]. Also, access to adjuvants has proven difﬁcult

Dengue vaccine development has been hampered by the lack for many vaccine developers.

of an adequate animal model for the disease [28]. Normal mice PIV dengue vaccines are killed wild type virions, containing all

do not display signiﬁcant viremia or disease when infected with DENV structural proteins and viral RNA. This permits induction of

human DENV isolates. Intracerebral challenge of mice with cer- an immune response to the three DENV structural proteins. PIV

tain mouse-adapted DENV strains produces a paralysis phenotype, dengue vaccines have not yet reached clinical evaluation, but sev-

and this intracerebral challenge model has been used to evalu- eral candidates are in preclinical development [55,56].

ate protective efﬁcacy of vaccine candidates in mice. Furthermore, Other nonliving vaccines express particular DENV antigens,

immunocompromised mouse models have been developed which which allows for targeting of the immune response to antigens

show some of the characteristics of human disease and can also be with particular desirable properties (e.g. induction of potent neu-

used as an in vivo system for studying ADE [29–31]. However, con- tralizing antibodies). Recombinant subunit dengue vaccines have

cerns remain about whether the full spectrum of dengue immunity been produced in a variety of protein expression systems, including

and disease can be modeled in this immunocompromised setting. bacterial, yeast, insect and mammalian cells. Most dengue sub-

Humanized mice that lack a murine antibody response but demon- unit vaccines express truncated versions of the E protein that have

strate some symptoms that re-capitulate human infection are also deleted transmembrane domains, or simply express EDIII alone.

being investigated [32,33]. Non-human primates (NHPs) are sus- While subunit vaccines often show low reactogenicity, achieve-

ceptible to infection by DENV and develop viremia, but do not ment of sufﬁcient immunogenicity usually requires the use of

show signiﬁcant clinical signs of infection. Studies to address pro- 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 being studied in the context of heterologous prime-boost strategies

[65] is being evaluated in phase 1 clinical trials. [64,92].

VLP vaccines are virus-like particles that do not contain replica- A subunit vaccine candidate developed by VaxInnate is based

tive genetic material, but permit presentation of antigens in a on fusion of DENV EDIII to a proprietary form of ﬂagellin (STF2D),

repetitive, ordered array similar to the virion structure, which is which serves as an adjuvant. STF2D contains the toll-like receptor

thought to increase immunogenicity [66]. Several dengue VLP vac- 5 (TLR5) activation domain from Salmonella typhimurium ﬂagellin

cine candidates are currently in preclinical development [67–70]. [93]. This approach links adaptive and innate immunity and is

DNA vaccines and virus-vectored vaccines allow for in vivo believed to mimic natural infection where antigen and TLR lig-

expression of antigens, often in the form of DENV VLPs. One of and are contained in a single pathogen. Physical linkage of TLR

the advantages of DNA vaccines is their thermostability, which ligand to antigen has been shown to be more speciﬁc and efﬁ-

avoids cold-chain requirements for vaccine storage and transport. cient than co-administration, requiring lower doses of adjuvant.

However, DNA vaccines often face challenges in terms of adequate STF2D fusion proteins can be expressed and puriﬁed in E. coli. In

cellular uptake and expression. Improved DNA vaccine delivery sys- immunogenicity studies in mice, monovalent EDIII-STF2D fusion

tems have shown some success in addressing these issues [71]. proteins were shown to elicit neutralizing DENV antibodies and

Various DNA dengue vaccines are currently in preclinical develop- provide partial protection against viral challenge. Bivalent DENV-

ment [72–81]. The most advanced DNA dengue vaccine candidate 1/3 and DENV-2/4 constructs were then generated by fusing the

has progressed to phase 1 clinical trials [82]. EDIII domains of two different serotypes to separate attachment

Virus-vectored dengue vaccines express DENV antigens from a points on STF2D. Subcutaneous immunization of mice with three

viral vaccine vector. Viral vectors allow for efﬁcient infection of doses (3 ␮g each) of a DENV-1/3 EDIII-STF2D fusion protein resulted

target cells. Moreover, several well-characterized vaccine vector in robust and balanced titres of neutralizing antibodies against

platform technologies exist, some of which are used for licensed DENV-1 and DENV-3. It was found that the choice of antigen attach-

vaccines with a well-known safety record [83]. However, there ment points (C-terminal fusion versus insertion via replacement

are concerns that pre-existing immunity to viral vaccine vectors of the ﬂagellin D3 domain) affects immunogenicity. Longevity of

could reduce the effectiveness of some virus-vectored dengue the immune response is yet to be studied. Additional bivalent con-

vaccines. Several virus-vectored dengue vaccine candidates are structs are being generated and tested, with the aim of combining

currently in preclinical development, including candidates based two suitable bivalent vaccine candidates to a tetravalent vaccine.

on non-replicating vectors [84,85], single-cycle vectors [86,87] and In a development approach by the International Centre for

replication-competent vectors [88]. Genetic Engineering and Biotechnology (ICGEB) in India, a tetrava-

Heterologous prime-boost approaches, which use different vac- lent chimeric EDIII fusion protein was generated, which consists of

cine types for the priming and boosting steps of an immunization the EDIII domains of DENV-1,-2,-3,-4 joined by ﬂexible peptide link-

schedule, may allow for optimization of immune responses by com- ers [59]. This single tetravalent approach avoids the complexities

bining advantages of different vaccine technologies in a unique related to producing tetravalent mixtures of monovalent vaccine

order of vaccination. However, the increased complexity of vaccina- components. The tetravalent chimeric EDIII protein was expressed

tion schedules could make heterologous prime-boost approaches in Pichia pastoris. Immunization of mice with the tetravalent vac-

more difﬁcult to implement in the context of immunization cine candidate adjuvanted with montanide resulted in neutralizing

programmes. Several heterologous prime-boost approaches are antibody responses against all DENV serotypes.

currently evaluated in preclinical studies [89,90]. 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

3. Dengue vaccine candidates in preclinical was derived by sequence analysis of different DENV-1-4 strains,

development and the consensus EDIII protein was expressed in E. coli. The single

tetravalent vaccine candidate adjuvanted with alum induced neu-

3.1. Recombinant subunit vaccines tralizing antibody responses against all DENV serotypes in mice

[61]. This vaccine candidate was developed further by fusion of

Several recombinant subunit vaccine candidates have been the consensus EDIII with a fragment of the Ag473 lipoprotein of N.

developed by the Pedro Kourí Tropical Medicine Institute (IPK) in meningitidis. The consensus EDIII-Ag473 fusion protein was found

collaboration with the Center for Genetic Engineering and Biotech- to induce higher levels of neutralizing antibodies in mice than con-

nology (CIGB) in Cuba (Table 1). One approach is based on fusion sensus EDIII formulated in alum [60]. The intrinsic adjuvant activity

of DENV EDIII to the carrier protein p64k of Neisseria meningitidis. of the EDIII-Ag473 lipo-immunogen appears to be due to stim-

The EDIII-p64k fusion protein is expressed in E. coli. Monovalent ulation of innate immunity through the TLR2 signaling pathway

vaccine candidates for all DENV serotypes were shown to induce [94].

neutralizing antibodies and protect against viral challenge in mice.

DENV-1 and DENV-2 monovalent candidates have also been eval- 3.2. DNA vaccines

uated in NHPs. Monkeys were immunized subcutaneously with

four doses of the monovalent vaccine (50–100 ␮g protein per dose, A tetravalent DNA vaccine candidate developed by Inovio Phar-

formulated in Freund’s adjuvant). The monovalent vaccine can- maceuticals consists of a DNA plasmid vector that expresses a single

didates were found to be immunogenic and provided protection ORF comprising the EDIII domains of all four DENV serotypes sep-

against viral challenge [58,91]. Adjuvants suitable for human use arated by proteolytic cleavage sites [80]. In vivo expression of a

are under evaluation, including N. meningitidis serogroup A cap- single tetravalent EDIII fusion protein, which is subsequently pro-

sular polysaccharide (CPS-A) adsorbed on aluminium hydroxide cessed into monovalent EDIII proteins by cellular proteases, aims

[63]. In another approach, a DENV EDIII-capsid fusion protein was to ensure that the four EDIII domains are presented in equal ratios

expressed in E. coli and mixed in vitro with oligodeoxynucleotides in order to facilitate a balanced immune response. A synthetic con-

to obtain particulated aggregates. The aggregated DENV-2 EDIII- sensus sequence for the EDIII domain of each DENV serotype was

capsid fusion protein induced both humoral and cellular immune derived by sequence comparison of approximately 15 different

responses in NHPs and provided partial protection against viral strains. Sequences were also human codon optimized to improve

challenge [62]. The different subunit vaccine candidates are also 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 NMRC prM/E expressed from plasmid vector (prime) and Monovalent candidates evaluated

approaches 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- free jet injector. Mice were protected against viral challenge 3

fection, suitable for both intradermal and intramuscular routes. weeks after the last dose, and persistence of neutralizing antibodies

Electroporation-based DNA immunization is currently being tested was observed during a 30-week follow-up [95]. Immunogenicity

in phase 1 clinical trials for HIV and inﬂuenza prevention and in of the tetravalent DNA vaccine in mice was further increased by

phase 2 clinical trials for hepatitis C, HPV 16/18 and AML/CML can- co-immunization with DENV-2 VLPs generated by co-expression

cer therapeutic studies. Immunization of mice with the tetravalent of prM/E in cell culture. Immunogenicity of the DNA vaccine was

dengue vaccine candidate, using intramuscular electroporation of also found to be improved by co-immunization with an inactivated

three doses (10 ␮g DNA) at biweekly intervals, resulted in neutral- Japanese encephalitis vaccine [78].

izing antibody titres against all four DENV serotypes. Immunization A similar tetravalent DNA vaccine candidate based on prM/E

of NHPs was also shown to elicit robust tetravalent antibody expression has been developed by the U.S. Centers for Disease Con-

responses. Further studies to assess DENV cross-reactive versus trol and Prevention (CDC). Transfection of the recombinant plasmid

serotype-speciﬁc antibody responses are in progress. vectors into cultured cells has been shown to result in secretion of

Another tetravalent DNA vaccine candidate developed at Kobe prM/E containing VLPs, which have an antigenic structure similar

University is composed of a mixture of four plasmid vectors, each to DEN virions [72,75]. Immunogenicity of a tetravalent mixture

of which expresses the prM and E proteins (prM/E) of one DENV of four monovalent DNA vaccines was evaluated in NHPs (two

␮ ␮

serotype. Two doses of the tetravalent vaccine (100 g per dose, at dose schedule one month apart; 500 g per monovalent compo-

three week intervals) were administered to mice using a needle- 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 expression of EDIII alone does not result in VLP formation, mosaic

for 10 months and protected from viral challenge. Pre-existing VLPs composed of varying ratios of EDIII-HBsAg and HBsAg could

antibodies against DENV or other ﬂaviviruses were shown to be generated by co-expression of the EDIII-HBsAg fusion protein

improve immunogenicity of the vaccine. Moreover, there is evi- with 1–4 copies of HBsAg. In a second approach, a fusion pro-

dence suggesting that the safety and immunogenicity of the vaccine tein consisting of the E protein ectodomain (ectoE) and 30 amino

candidates could be further improved by manipulating antigenic residues of the prM protein was expressed in P. pastoris, which

determinants of the E protein. In order to reduce the potential for also resulted in VLP formation. Structural characterization of the

ADE by cross-reactive antibodies, ﬂavivirus group cross-reactive VLPs, their functional evaluation for immunogenicity, and evalu-

epitopes and DENV serocomplex cross-reactive epitopes in the ation of prime-boost protocols combining VLP vaccine candidates

E protein were mapped with monoclonal antibody panels and with rAd5 vectored vaccine candidates are planned.

mutated to reduce cross-reactivity, resulting in the formation of In a VLP vaccine development approach employed at Kobe

cross-reactivity reduced VLPs in cell culture [96]. When evaluated University, prM/E is expressed from a plasmid vector transfected

in mice, cross-reactivity reduced DENV-1 and DENV-2 VLP vac- into cell culture-based expression systems and secreted VLPs are

cines continued to elicit high levels of neutralizing antibodies, but puriﬁed. A DENV-2 VLP vaccine candidate was found to be immuno-

showed reduced ADE potential compared to wild type vaccines genic and protective in mice [103]. Expression of VLPs by transient

both in vitro (K562 cell-based assay) and in vivo (AG129 mouse transfection of insect cells (Sf9 cell line) resulted in 10–100-fold

model [29,97]). Immunogenicity of the cross-reactivity reduced improved yields compared to mammalian expression systems

vaccines was further improved by incorporating a CD4 epitope from [69]. Immunogenicity of the DENV-2 VLP vaccine candidate in

the transmembrane domain of West Nile virus E protein into the mice could be improved by co-immunization with a DNA vaccine

expression construct. expressing prM/E, which also functions as a CpG adjuvant.

In a single tetravalent approach developed at the U.S. Naval

Medical Research Center (NMRC), a prM/E construct containing a 3.4. Virus-vectored vaccines

chimeric “shufﬂed” E protein is expressed from a plasmid vector

[76]. Various chimeric E proteins containing sequence portions of A virus-vectored dengue vaccine developed by ICGEB is based

all DENV serotypes were derived by DNA shufﬂing technology. The on expression of a tetravalent chimeric EDIII fusion protein from

shufﬂed constructs were screened for antigen expression in vitro a replication-deﬁcient adenovirus vaccine vector [85]. Advantages

and for immunogenicity in mice. Three selected constructs were of adenovirus vaccine vectors include a large insert capacity, efﬁ-

evaluated as single tetravalent DNA vaccine candidates in NHPs. cient delivery and high expression levels of antigens in a variety of

Two of these candidates were found to induce tetravalent neutral- cells, and a well-documented human safety record. Immunization

izing antibody responses in the majority of animals and to provide of mice with two doses of the single component tetravalent dengue

partial protection against viral challenge. vaccine candidate (administered 30 days apart by the intraperi-

toneal route) resulted in balanced neutralizing antibody responses

3.3. VLP vaccines against all four DENV serotypes. Of note, prior immunity to the ade-

novirus vaccine vector did not impair the potency of the dengue

A VLP dengue vaccine candidate developed by Cytos Biotechnol- vaccine candidate. Presence of antibodies against the vector in the

ogy is based on chemical coupling of recombinant EDIII to carrier sera of immunized mice enabled uptake of the vector into U937

VLPs derived from bacteriophage Q␤ [98]. A similar approach has human monocytic cells, which are not susceptible to infection in the

recently been applied to the development of a West Nile virus vac- absence of antibodies against the vector. This effect is presumably

cine [99]. About 60–90 EDIII molecules can be coupled to one VLP mediated by the Fc receptor pathway [104]. The results suggest that

carrier, resulting in the formation of a highly repetitive, ordered low levels of pre-existing antibodies against the adenovirus vec-

array of antigen, which is expected to increase immunogenicity. tor may in fact even facilitate uptake of the virus-vectored dengue

␤

Q VLPs also contain E. coli-derived ssRNA, which has been shown vaccine into antigen-presenting cells.

to be required for IgG isotype switching in mice immunized with Another virus-vectored vaccine approach developed by Gen-

␤

Q VLPs [100,101] and is used as a built-in adjuvant to improve the Phar in collaboration with the NMRC is based on expression

antibody response to Q␤ VLP-based vaccines. Q␤ VLPs can be eco- of DENV prM/E from a replication-deﬁcient complex adenovirus

nomically produced in E. coli, resulting in 2 g/l culture of GMP grade vaccine vector capable of accommodating multiple large antigen

material. Q␤ VLP based vaccine candidates are in advanced clinical inserts [84]. The tetravalent vaccine is composed of two bivalent

evaluation for other target antigens. Four monovalent dengue vac- vaccines, each of which expresses prM and E proteins of two dif-

cine candidates were generated by coupling the EDIII domain of ferent DENV serotypes. NHPs were immunized with two doses of

the different DENV serotypes to the carrier VLP. Immunogenicity tetravalent vaccine 8 weeks apart, followed by viral challenge at

studies in mice were performed to compare monovalent vaccines 3–6 months. Neutralizing antibody responses were induced against

individually and in a tetravalent formulation (three doses at 10 all serotypes. Viremia from challenge with DENV-1 and DENV-3

day intervals; 50 ␮g per dose of monovalent vaccine and 200 ␮g was blocked, while viremia from challenge with DENV-2 and DENV-

per dose of tetravalent vaccine; subcutaneous administration with 4 was signiﬁcantly reduced. Further development of the vaccine

alum). The neutralizing antibody response observed to DENV-4 constructs, including optimization of all prM and E gene sequences

was weaker than for the other serotypes. However, an improved for human codon usage, resulted in an improved tetravalent for-

formulation with increased representation of the DENV-4 monova- mulation, which induced a more balanced immune response to all

lent component induced a potent neutralizing antibody response four serotypes. The candidate vaccine is scheduled to enter clinical

against all four serotypes. evaluation shortly.

VLP vaccine candidates have also been developed by the ICGEB. A virus-vectored vaccine candidate developed at the University

In a ﬁrst approach, DENV E was expressed as a fusion protein with of North Carolina at Chapel Hill (UNC) is based on expression of

Hepatitis B virus surface antigen (HBsAg) in P. pastoris [67]. HBsAg DENV antigens from a single-cycle Venezuelan equine encephalitis

expressed in P. pastoris has been shown to assemble into highly (VEE) virus vaccine vector [86]. Packaging technologies based on

immunogenic VLPs. Furthermore, high protein yields (up to 7 gram helper RNAs allow for the production of infectious single-cycle VEE

HBsAg per liter culture [102]) make P. pastoris an attractive sys- virus particles also referred to as virus replicon particles (VRPs). VEE

tem for affordable production of protein-based vaccines. While VRPs have been shown to infect human dendritic cells and express

J. Schmitz et al. / Vaccine 29 (2011) 7276–7284 7281

high levels of recombinant antigens, inducing both innate and lent inactivated virus vaccine candidate together with different

adaptive immune responses. Different monovalent vaccine candi- proprietary adjuvant systems (AS01, AS03, AS04). Two doses of

dates expressing DENV prM/E proteins, C-terminally truncated E adjuvanted vaccine were given 30 days apart, followed by viral

proteins (E81, E85 [81% or 85% of E]) or EDIII were evaluated for challenge 4 months later. All immunized animals were found to

immunogenicity in mice. Vaccine candidates expressing truncated be protected against viral challenge. The highest neutralizing anti-

E proteins were found to elicit higher neutralizing antibody titres body responses were obtained with the AS03 adjuvant system.

than candidates expressing prM/E. Immunogenicity and protec- Clinical trials of the adjuvanted PIV vaccine are planned to start

tive efficacy of the monovalent candidates were evaluated further in 2012, with a first assessment of protective efficacy envisaged for

in NHP studies. Three doses (10 IU each) of a monovalent vac- 2014–15.

cine candidate expressing either DENV-3 E85 or DENV-3 prM/E

were administered subcutaneously at weeks 0, 7, and 25, and viral 3.6. Live attenuated virus vaccines

challenge was performed at week 41. Higher neutralizing anti-

body titres were observed with the DENV-3 E85 based vaccine, A live attenuated dengue vaccine candidate developed by

which also completely protected all monkeys against viral chal- FIOCRUZ uses the live attenuated yellow fever vaccine 17DD sub-

lenge, while immunization with the DENV-3 prM/E based vaccine strain as a genetic backbone. Chimeric YF 17D/DEN viruses were

resulted in only partial protection. Neutralizing antibodies against constructed by replacing the YFV prM/E genes with those of DENV

DENV-3 E85 were serotype-speciﬁc and largely directed against [106]. Monovalent dengue vaccine candidates for different DENV

EDIII epitopes. Immunogenicity and protective efﬁcacy studies of a serotypes were generated and evaluated in NHPs. In attenua-

tetravalent E85 based vaccine candidate in NHPs are in progress. tion studies, the monovalent vaccines produced only low levels

Another approach taken at the University of Texas Medical of viremia and showed reduced neurotropism compared to the

Branch (UTMB) is based on a single-cycle, capsid-gene deleted West YF 17D vaccine. Neutralizing antibody responses and protection

Nile virus vaccine vector [105]. Infectious single-cycle WNV par- against viral challenge were also shown [48–50]. DEN viruses used

ticles are produced by packaging technologies, which supply the for the construction of chimeric YF 17D/DEN viruses in this vac-

capsid protein in trans. Infected target cells are unable to produce cine project include strains isolated from Latin American patients,

viral progeny, but the single replication cycle results in efﬁcient while the DENV strains that provided the basis for a tetravalent

production of antigen including secretion of VLPs from infected dengue vaccine candidate developed by Sanoﬁ Pasteur, which is

cells. A DENV-2 vaccine candidate was generated by replacing the currently in advanced clinical evaluation [44–47,107], were iso-

prM/E genes of the WNV vaccine vector with the prM/E genes of lated from Asian patients. Different sets of chimeric YF 17D/DEN

DENV-2 [87]. The vaccine was tested for potency and efﬁcacy in a viruses are thus available for the development of a tetravalent live

mouse model of dengue pathogenesis (AG129 mice [97]). A single attenuated dengue vaccine.

intraperitoneal immunization of mice produced a dose-dependent Another live attenuated vaccine candidate has been devel-

DENV-2 neutralizing antibody response and resulted in a protective oped in collaboration between Chiang Mai University, Mahidol

effect against viral challenge. University, the Thai National Science and Technology Develop-

A virus-vectored dengue vaccine candidate developed by ment Agency (NSTDA) and BioNet-Asia [51,52]. DEN/DEN chimeric

Themis Bioscience and Institut Pasteur is based on expression of viruses were constructed which contain the prM/E coding region of

a single tetravalent DENV antigen construct from a live attenuated recent dengue clinical isolates in the genetic background of atten-

measles virus vaccine vector [88]. This vaccine vector technology uated DENV. Monovalent vaccine candidates have been evaluated

allows for integration of large antigen inserts and has been shown for neurovirulence and immunogenicity in mice. Safety and efﬁcacy

to induce strong neutralizing antibodies and cellular immune studies in NHPs are planned.

responses even in the presence of pre-existing immunity to measles

virus. The dengue vaccine candidate expresses a construct contain- 3.7. Heterologous prime-boost approaches

ing the EDIII domains of DENV-1-4 as well as the DENV-1 M protein

ectodomain (ectoM). The single component tetravalent vaccine- A heterologous prime-boost approach developed by the NMRC

induced neutralizing antibodies against all DENV serotypes in mice. uses a DNA vaccine candidate and a virus-vectored vaccine candi-

Inclusion of ectoM in the vaccine construct was found to provide an date [89]. The monovalent DNA vaccine, which has been evaluated

adjuvant activity. Following further improvements of the vaccine in a phase 1 clinical trial, consists of a plasmid vector expressing

construct, immunogenicity and efﬁcacy studies have been initiated DENV-1 prM/E and is administered intramuscularly with a needle-

in NHPs. Establishment of a GMP process and initiation of clinical free Biojector system [82]. The virus-vectored vaccine expresses

trials are planned in 2011 and 2012, respectively. the same DENV-1 prM/E sequence from a single-cycle VEE virus

vector. A three-dose prime-boost regimen was evaluated in NHPs,

3.5. Puriﬁed inactivated virus vaccines which consists of two doses of the DNA vaccine at days 0 and 28

followed by intramuscular injection of the virus-vectored vaccine

A puriﬁed psoralen-inactivated DENV-1 vaccine candidate has at day 117. When compared to homologous three dose schedules

been developed by the NMRC [56]. Following immunogenicity of either the DNA or the virus-vectored vaccines, the heterologous

studies in mice, the monovalent candidate adjuvanted in alum was prime-boost approach was found to improve neutralizing antibody

evaluated in NHPs using a three-dose schedule (intradermal injec- responses and protection against viral challenge [89].

tion, biweekly intervals). Immunized monkeys showed a DENV-1 Another heterologous prime-boost approach developed by the

neutralizing antibody response and reduced duration of viremia NMRC and the WRAIR uses non-replicating vaccines for priming,

upon viral challenge on day 132, suggesting that the vaccine can- followed by boosting with a LAV vaccine candidate [90]. Two non-

didate provides partial protection in NHPs. replicating vaccines were evaluated: a tetravalent DNA vaccine

PIV vaccine candidates are also being developed by Glaxo- composed of plasmid vectors expressing DENV prM/E [82] and a

SmithKline (GSK) in collaboration with the Walter Reed Army tetravalent PIV vaccine consisting of formalin-inactivated DENV

Institute of Research (WRAIR) and the Oswaldo Cruz Foundation adjuvanted with alum [55]. The tetravalent LAV vaccine candidate,

(FIOCRUZ) [55]. Different approaches for virus inactivation and which was developed by GSK and WRAIR using serial passage of

puriﬁcation and various adjuvant systems are currently under clinical DENV isolates in PDK cells, has reached phase 2 clinical trials

evaluation. A pilot study was carried out in NHPs to test a tetrava- [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 Drs. Martin Bachmann (Cytos Biotechnology), Gwong-Jen Chang

schedule (at -30, 0 and 60 days) was compared to a PIV-LAV vac- (CDC), John Dong (GenPhar), Bruce Innis (GSK), Niranjan Y. Sardesai

cination schedule (at 0 and 60 days) in NHPs. Both regimens were (Inovio Pharmaceuticals) and David B. Weiner (University of Penn-

found to elicit neutralizing antibody responses. However, priming sylvania), Alan Shaw (VaxInnate), Sathyamangalam Swaminathan

with the DNA vaccine provided only partial protection against viral (ICGEB), Erich Tauber (Themis Bioscience) and Laura White (UNC).

challenge 6 months after the last dose, as evidenced by the obser- Financial support of the consultation from the Bill and Melinda

vation of breakthrough viremia. By contrast, priming with one dose Gates Foundation is kindly acknowledged.

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]. References

While heterologous prime-boost strategies may offer advan-

tages over other vaccination approaches, recent investigations with [1] Gubler DJ. Epidemic dengue/dengue hemorrhagic fever as a public health,

social and economic problem in the 21st century. Trends Microbiol

West Nile virus vaccine candidates suggest that the order of anti-

2002;10:100–3.

gens used in a heterologous prime-boost approach was important

[2] Suaya JA, Shepard DS, Siqueira JB, Martelli CT, Lum LC, Tan LH, et al. Cost of

in terms of the speciﬁcity and magnitude of the immune response dengue cases in eight countries in the Americas and Asia: a prospective study.

Am J Trop Med Hyg 2009;80:846–55.

[108]. Similar results can be expected for dengue vaccines.

[3] World Health Organization. Dengue guidelines for diagnosis, treatment,

prevention and control. Geneva: World Health Organization; 2009. Available

at: http://whqlibdoc.who.int/publications/2009/9789241547871 eng.pdf

4. Conclusions

[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;84:200–7.

Efforts for dengue vaccine development have faced multiple

[5] Whitehorn J, Farrar J. Dengue. Br Med Bull 2010;95:161–73.

challenges, including the need to induce a balanced and last-

[6] Durbin AP, Whitehead SS. Dengue vaccine candidates in development. Curr

ing immunity against four DENV serotypes, uncertainties around Top Microbiol Immunol 2010;338:129–43.

[7] Swaminathan S, Batra G, Khanna N. Dengue vaccines: state of the art. Expert

immune correlates of protection, potential immune enhancement

Opin Ther Pat 2010;20:819–35.

of disease, and lack of suitable animal model. However, consider-

[8] Guzman MG. Dengue vaccines: new developments. Drugs Future

able progress has been made in recent years, which has resulted in 2011;36:45–62.

an advanced dengue vaccine pipeline, with a lead candidate enter- [9] Murphy BR, Whitehead SS. Immune response to dengue virus and prospects

for a vaccine. Annu Rev Immunol 2011;29:587–619.

ing phase 3 clinical trials [44–47] and several other candidates in

[10] Halstead SB. Dengue. Lancet 2007;370:1644–52.

earlier stages of clinical evaluation [36–43,65,82]. The majority of

[11] Hombach J, Cardosa MJ, Sabchareon A, Vaughn DW, Barrett AD. Scientiﬁc

dengue vaccine candidates currently in clinical development are consultation on immunological correlates of protection induced by dengue

vaccines. Vaccine 2007;25:4130–9.

based on the same immunization regimen (namely a tetravalent

[12] Thomas SJ, Hombach J, Barrett A. Scientiﬁc consultation on cell medi-

LAV) derived using different technologies. However, some uncer-

ated immunity (CMI) in dengue and dengue vaccine development. Vaccine

tainties around the utilization of LAVs remain, including potential 2009;27:355–68.

[13] Halstead SB. Neutralization and antibody-dependent enhancement of dengue

imbalances in immunity due to immune interference.

viruses. Adv Virus Res 2003;60:421–67.

A large number of diverse dengue vaccine candidates are in

[14] Halstead SB. Antibodies determine virulence in dengue. Ann N Y Acad Sci

preclinical development. These preclinical candidates ensure a con- 2009;1171(Suppl. 1):E48–56.

[15] Pierson TC, Fremont DH, Kuhn RJ, Diamond MS. Structural insights into

tinued inﬂux of innovation into the vaccine pipeline, which is

the mechanisms of antibody-mediated neutralization of ﬂavivirus infection:

critical for maximizing the chances of success for dengue vac-

implications for vaccine development. Cell Host Microbe 2008;4:229–38.

cine development. One possible scenario is that a ﬁrst generation [16] Dowd KA, Pierson TC. Antibody-mediated neutralization of ﬂaviviruses: a

reductionist view. Virology 2011;411:306–15.

of licensed dengue vaccines will arise from candidates currently

[17] Junjhon J, Edwards TJ, Utaipat U, Bowman VD, Holdaway HA, Zhang W, et al.

in clinical development, while some of the current preclinical

Inﬂuence of pr-M cleavage on the heterogeneity of extracellular dengue virus

stage candidates could become next generation dengue vaccines particles. J Virol 2010;84:8353–8.

licensed at a later stage. Next generation dengue vaccines could [18] Perera R, Kuhn RJ. Structural proteomics of dengue virus. Curr Opin Microbiol

2008;11:369–77.

serve to complement existing vaccines. For example, prime-boost

[19] Beltramello M, Williams KL, Simmons CP, Macagno A, Simonelli L, Quyen NT,

approaches combining live and non-replicating vaccines may allow

et al. The human immune response to dengue virus is dominated by highly

for balancing the relative advantages and shortcomings of these cross-reactive antibodies endowed with neutralizing and enhancing activity.

Cell Host Microbe 2010;8:271–83.

technologies. Furthermore, next generation dengue vaccines could

[20] Crill WD, Roehrig JT. Monoclonal antibodies that bind to domain III of dengue

have a superior product proﬁle compared to existing vaccines,

virus E glycoprotein are the most efﬁcient blockers of virus adsorption to Vero

including efﬁcacy, dose-scheduling, and stability. There are indi- cells. J Virol 2001;75:7769–73.

[21] Roehrig JT. Antigenic structure of ﬂavivirus proteins. Adv Virus Res

cations that candidates which are currently in advanced clinical

2003;59:141–75.

development may require long, multi-dose immunization sched-

[22] Brien JD, Austin SK, Sukupolvi-Petty S, O’Brien KM, Johnson S, Fremont DH,

ules (e.g. three doses over a 12 month period). Next generation et al. Genotype-speciﬁc neutralization and protection by antibodies against

dengue virus type 3. J Virol 2010;84:10630–43.

dengue vaccines could have more compressed schedules requir-

[23] Dejnirattisai W, Jumnainsong A, Onsirisakul N, Fitton P, Vasanawathana S,

ing fewer doses, or characteristics that make them more suitable

Limpitikul W, et al. Cross-reacting antibodies enhance dengue virus infection

for certain target groups (e.g. particular age groups, immunocom- in humans. Science 2010;328:745–8.

[24] Rodenhuis-Zybert IA, van der Schaar HM, da Silva Voorham JM, van der

promised individuals, travelers). Finally, next generation dengue

Ende-Metselaar H, Lei HY, Wilschut J, et al. Immature dengue virus: a veiled

vaccines could generate much-welcomed competition, leading to

pathogen? PLoS Pathog 2010;6:e1000718.

more affordable and cost-effective vaccines, which would facilitate [25] Shrestha B, Brien JD, Sukupolvi-Petty S, Austin SK, Edeling MA, Kim T, et al. The

their use particularly in endemic developing countries. development of therapeutic antibodies that neutralize homologous and het-

erologous 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.

Acknowledgements Structure and function analysis of therapeutic monoclonal antibodies against

dengue virus type 2. J Virol 2010;84:9227–39.

[27] Wahala WM, Donaldson EF, de Alwis R, Accavitti-Loper MA, Baric RS, de Silva

Participants of the WHO/IVI Informal Consultation on Next Gen- AM. Natural strain variation and antibody neutralization of dengue serotype

3 viruses. PLoS Pathog 2010;6:e1000821.

eration Dengue Vaccines and Diagnostics (1–2 November 2010,

[28] Cassetti MC, Durbin A, Harris E, Rico-Hesse R, Roehrig J, Rothman A,

Atlanta) are thanked for sharing unpublished data. Preclinical stage

et al. Report of an NIAID workshop on dengue animal models. Vaccine

dengue vaccine candidates were presented at the consultation by 2010;28:4229–34.

J. Schmitz et al. / Vaccine 29 (2011) 7276–7284 7283

[29] Williams KL, Zompi S, Beatty PR, Harris E. A mouse model for studying dengue [57] Simmons M, Murphy GS, Hayes CG. Short report: antibody responses of mice

virus pathogenesis and immune response. Ann N Y Acad Sci 2009;1171(Suppl. immunized with a tetravalent dengue recombinant protein subunit vaccine.

1):E12–23. Am J Trop Med Hyg 2001;65:159–61.

[30] Balsitis SJ, Williams KL, Lachica R, Flores D, Kyle JL, Mehlhop E, et al. Lethal [58] Bernardo L, Izquierdo A, Alvarez M, Rosario D, Prado I, Lopez C, et al. Immuno-

antibody enhancement of dengue disease in mice is prevented by Fc modiﬁ- genicity and protective efﬁcacy of a recombinant fusion protein containing

cation. PLoS Pathog 2010;6:e1000790. the domain III of the dengue 1 envelope protein in non-human primates.

[31] Zellweger RM, Prestwood TR, Shresta S. Enhanced infection of liver sinu- Antiviral Res 2008;80:194–9.

soidal endothelial cells in a mouse model of antibody-induced severe dengue [59] Etemad B, Batra G, Raut R, Dahiya S, Khanam S, Swaminathan S, et al. An

disease. Cell Host Microbe 2010;7:128–39. envelope domain III-based chimeric antigen produced in Pichia pastoris elicits

[32] Kuruvilla JG, Troyer RM, Devi S, Akkina R. Dengue virus infection and immune neutralizing antibodies against all four dengue virus serotypes. Am J Trop Med

response in humanized RAG2(−/−)gamma(c)(−/−) (RAG-hu) mice. Virology Hyg 2008;79:353–63.

2007;369:143–52. [60] Chen HW, Liu SJ, Liu HH, Kwok Y, Lin CL, Lin LH, et al. A novel technology for

[33] Mota J, Rico-Hesse R. Humanized mice show clinical signs of dengue fever the production of a heterologous lipoprotein immunogen in high yield has

according to infecting virus genotype. J Virol 2009;83:8638–45. implications for the ﬁeld of vaccine design. Vaccine 2009;27:1400–9.

[34] Lauring AS, Jones JO, Andino R. Rationalizing the development of live attenu- [61] Leng CH, Liu SJ, Tsai JP, Li YS, Chen MY, Liu HH, et al. A novel dengue vaccine

ated virus vaccines. Nat Biotechnol 2010;28:573–9. candidate that induces cross-neutralizing antibodies and memory immunity.

[35] Bhamarapravati N, Sutee Y. Live attenuated tetravalent dengue vaccine. Vac- Microbes Infect 2009;11:288–95.

cine 2000;18(Suppl. 2):44–7. [62] Valdes I, Bernardo L, Gil L, Pavon A, Lazo L, Lopez C, et al. A novel fusion protein

[36] Huang CY, Butrapet S, Tsuchiya KR, Bhamarapravati N, Gubler DJ, Kinney RM. domain III-capsid from dengue-2, in a highly aggregated form, induces a func-

Dengue 2 PDK-53 virus as a chimeric carrier for tetravalent dengue vaccine tional immune response and protection in mice. Virology 2009;394:249–58.

development. J Virol 2003;77:11436–47. [63] Valdes I, Hermida L, Martin J, Menendez T, Gil L, Lazo L, et al. Immunologi-

[37] Durbin AP, Whitehead SS, McArthur J, Perreault JR, Blaney Jr JE, Thumar B, et al. cal evaluation in nonhuman primates of formulations based on the chimeric

rDEN4delta30, a live attenuated dengue virus type 4 vaccine candidate, is safe, protein P64k-domain III of dengue 2 and two components of Neisseria menin-

immunogenic, and highly infectious in healthy adult volunteers. J Infect Dis gitidis. Vaccine 2009;27:995–1001.

2005;191:710–8. [64] Valdes I, Gil L, Romero Y, Castro J, Puente P, Lazo L, et al. The chimeric protein

[38] Durbin AP, McArthur J, Marron JA, Blaney Jr JE, Thumar B, Wanionek K, domain III-capsid of dengue virus serotype 2 (DEN-2) successfully boosts neu-

et al. The live attenuated dengue serotype 1 vaccine rDEN1Delta30 is tralizing antibodies generated in monkeys upon infection with DEN-2. Clin

safe and highly immunogenic in healthy adult volunteers. Hum Vaccin Vaccine Immunol 2011;18:455–9.

2006;2:167–73. [65] Clements DE, Coller BA, Lieberman MM, Ogata S, Wang G, Harada KE, et al.

[39] Durbin AP, McArthur JH, Marron JA, Blaney JE, Thumar B, Wanionek K, et al. Development of a recombinant tetravalent dengue virus vaccine: immuno-

rDEN2/4Delta30(ME), a live attenuated chimeric dengue serotype 2 vaccine genicity and efﬁcacy studies in mice and monkeys. Vaccine 2010;28:2705–15.

is safe and highly immunogenic in healthy dengue-naive adults. Hum Vaccin [66] Jennings GT, Bachmann MF. The coming of age of virus-like particle vaccines.

2006;2:255–60. Biol Chem 2008;389:521–36.

[40] McArthur JH, Durbin AP, Marron JA, Wanionek KA, Thumar B, Pierro DJ, [67] Bisht H, Chugh DA, Raje M, Swaminathan SS, Khanna N. Recombinant

et al. Phase I clinical evaluation of rDEN4Delta30-200,201: a live attenuated dengue virus type 2 envelope/hepatitis B surface antigen hybrid protein

dengue 4 vaccine candidate designed for decreased hepatotoxicity. Am J Trop expressed in Pichia pastoris can function as a bivalent immunogen. J Biotech-

Med Hyg 2008;79:678–84. nol 2002;99:97–110.

[41] Simasathien S, Thomas SJ, Watanaveeradej V, Nisalak A, Barberousse C, Innis [68] Amexis G, Young NS. Parvovirus B19 empty capsids as antigen carriers

BL, et al. Safety and immunogenicity of a tetravalent live-attenuated dengue for presentation of antigenic determinants of dengue 2 virus. J Infect Dis

vaccine in ﬂavivirus naive children. Am J Trop Med Hyg 2008;78:426–33. 2006;194:790–4.

[42] Sun W, Cunningham D, Wasserman SS, Perry J, Putnak JR, Eckels KH, et al. [69] Kuwahara M, Konishi E. Evaluation of extracellular subviral particles of

Phase 2 clinical trial of three formulations of tetravalent live-attenuated dengue virus type 2 and Japanese encephalitis virus produced by Spodoptera

dengue vaccine in ﬂavivirus-naive adults. Hum Vaccin 2009;5:33–40. frugiperda cells for use as vaccine and diagnostic antigens. Clin Vaccine

[43] Wright PF, Durbin AP, Whitehead SS, Ikizler MR, Henderson S, Blaney JE, et al. Immunol 2010;17:1560–6.

Phase 1 trial of the dengue virus type 4 vaccine candidate rDEN4Delta30-4995 [70] Liu W, Jiang H, Zhou J, Yang X, Tang Y, Fang D, et al. Recombinant dengue virus-

in healthy adult volunteers. Am J Trop Med Hyg 2009;81:834–41. like particles from Pichia pastoris: efﬁcient production and immunological

[44] Guy B, Guirakhoo F, Barban V, Higgs S, Monath TP, Lang J. Preclinical and properties. Virus Genes 2010;40:53–9.

clinical development of YFV 17D-based chimeric vaccines against dengue, [71] Kutzler MA, Weiner DB. DNA vaccines: ready for prime time? Nat Rev Genet

West Nile and Japanese encephalitis viruses. Vaccine 2010;28:632–49. 2008;9:776–88.

[45] Morrison D, Legg TJ, Billings CW, Forrat R, Yoksan S, Lang J. A novel tetravalent [72] Chang GJ, Hunt AR, Holmes DA, Springﬁeld T, Chiueh TS, Roehrig JT, et al.

dengue vaccine is well tolerated and immunogenic against all 4 serotypes in Enhancing biosynthesis and secretion of premembrane and envelope proteins

ﬂavivirus-naive adults. J Infect Dis 2010;201:370–7. by the chimeric plasmid of dengue virus type 2 and Japanese encephalitis

[46] Guy B, Almond J, Lang J. Dengue vaccine prospects: a step forward. Lancet virus. Virology 2003;306:170–80.

2011;377:381–2. [73] Wu SF, Liao CL, Lin YL, Yeh CT, Chen LK, Huang YF, et al. Evaluation of protective

[47] Poo J, Galan F, Forrat R, Zambrano B, Lang J, Dayan GH. Live-attenuated tetrava- efﬁcacy and immune mechanisms of using a non-structural protein NS1 in

lent dengue vaccine in dengue-naive children, adolescents, and adults in DNA vaccine against dengue 2 virus in mice. Vaccine 2003;21:3919–29.

Mexico City: randomized controlled phase 1 trial of safety and immunogenic- [74] Mota J, Acosta M, Argotte R, Figueroa R, Mendez A, Ramos C. Induction of

ity. Pediatr Infect Dis J 2011;30:E9–17. protective antibodies against dengue virus by tetravalent DNA immunization

[48] Galler R, Marchevsky RS, Caride E, Almeida LF, Yamamura AM, Jabor AV, et al. of mice with domain III of the envelope protein. Vaccine 2005;23:3469–76.

Attenuation and immunogenicity of recombinant yellow fever 17D-dengue [75] Purdy DE, Chang GJ. Secretion of noninfectious dengue virus-like particles

type 2 virus for rhesus monkeys. Braz J Med Biol Res 2005;38:1835–46. and identiﬁcation of amino acids in the stem region involved in intracellular

[49] Mateu GP, Marchevsky RS, Liprandi F, Bonaldo MC, Coutinho ES, Dieudonne retention of envelope protein. Virology 2005;333:239–50.

M, et al. Construction and biological properties of yellow fever 17D/dengue [76] Raviprakash K, Apt D, Brinkman A, Skinner C, Yang S, Dawes G, et al. A chimeric

type 1 recombinant virus. Trans R Soc Trop Med Hyg 2007;101:289–98. tetravalent dengue DNA vaccine elicits neutralizing antibody to all four virus

[50] Trindade GF, Marchevsky RS, Fillipis AM, Nogueira RM, Bonaldo MC, Acero PC, serotypes in rhesus macaques. Virology 2006;353:166–73.

et al. Limited replication of yellow fever 17DD and 17D-Dengue recombinant [77] Costa SM, Azevedo AS, Paes MV, Sarges FS, Freire MS, Alves AM. DNA vaccines

viruses in rhesus monkeys. An Acad Bras Cienc 2008;80:311–21. against dengue virus based on the ns1 gene: the inﬂuence of different sig-

[51] BioNet-Asia. BioNet-Asia to enter development of a dengue vaccine. nal sequences on the protein expression and its correlation to the immune

Press release 2011. Available at: http://www.bionet-asia.com/feb2111.html response elicited in mice. Virology 2007;358:413–23.

[accessed 21 June 2011]. [78] Imoto J, Konishi E. Dengue tetravalent DNA vaccine increases its immuno-

[52] National Science and Technology Development Agency. Chimeric dengue vac- genicity in mice when mixed with a dengue type 2 subunit vaccine or an

cine licensing agreement. Press release 2011. Available at: http://www.nstda. inactivated Japanese encephalitis vaccine. Vaccine 2007;25:1076–84.

or.th/eng/index.php/research-themes/health-and-medicine/item/177-chim [79] De Paula SO, Lima DM, de Oliveira Franca RF, Gomes-Ruiz AC, da Fonseca BA. A

eric-dengue-vaccine-licensing-agreement [accessed 21 June 2011]. DNA vaccine candidate expressing dengue-3 virus prM and E proteins elicits

[53] Smith KM, Nanda K, Spears CJ, Ribeiro M, Vancini R, Piper A, et al. Struc- neutralizing antibodies and protects mice against lethal challenge. Arch Virol

tural mutants of dengue virus 2 transmembrane domains exhibit host-range 2008;153:2215–23.

phenotype. Virol J 2011;8:289. [80] Ramanathan MP, Kuo YC, Selling BH, Li Q, Sardesai NY, Kim JJ, et al. Develop-

[54] Leroux-Roels G. Unmet needs in modern vaccinology: adjuvants to improve ment of a novel DNA SynCon tetravalent dengue vaccine that elicits immune

the immune response. Vaccine 2010;28(Suppl. 3):C25–36. responses against four serotypes. Vaccine 2009;27:6444–53.

[55] Putnak JR, Coller BA, Voss G, Vaughn DW, Clements D, Peters I, et al. An evalu- [81] Lima DM, de Paula SO, Franca RF, Palma PV, Morais FR, Gomes-Ruiz AC, et al. A

ation of dengue type-2 inactivated, recombinant subunit, and live-attenuated DNA vaccine candidate encoding the structural prM/E proteins elicits a strong

vaccine candidates in the rhesus macaque model. Vaccine 2005;23: immune response and protects mice against dengue-4 virus infection. Vaccine

4442–52. 2011;29:831–8.

[56] Maves RC, Ore RM, Porter KR, Kochel TJ. Immunogenicity and protective efﬁ- [82] Beckett CG, Tjaden J, Burgess T, Danko JR, Tamminga C, Simmons M, et al.

cacy of a psoralen-inactivated dengue-1 virus vaccine candidate in Aotus Evaluation of a prototype dengue-1 DNA vaccine in a Phase 1 clinical trial.

nancymaae monkeys. Vaccine 2011;29:2691–6. Vaccine 2011;29:960–8.

7284 J. Schmitz et al. / Vaccine 29 (2011) 7276–7284

[83] Liniger M, Zuniga A, Naim HY. Use of viral vectors for the development of [95] Konishi E, Kosugi S, Imoto J. Dengue tetravalent DNA vaccine inducing neutral-

vaccines. Expert Rev Vaccines 2007;6:255–66. izing antibody and anamnestic responses to four serotypes in mice. Vaccine

[84] Raviprakash K, Wang D, Ewing D, Holman DH, Block K, Woraratanadharm 2006;24:2200–7.

J, et al. A tetravalent dengue vaccine based on a complex adenovirus vector [96] Crill WD, Hughes HR, Delorey MJ, Chang GJ. Humoral immune responses of

provides signiﬁcant protection in rhesus monkeys against all four serotypes dengue fever patients using epitope-speciﬁc serotype-2 virus-like particle

of dengue virus. J Virol 2008;82:6927–34. antigens. PLoS One 2009;4:e4991.

[85] Khanam S, Pilankatta R, Khanna N, Swaminathan S. An adenovirus type 5 [97] Johnson AJ, Roehrig JT. New mouse model for dengue virus vaccine testing. J

(AdV5) vector encoding an envelope domain III-based tetravalent antigen Virol 1999;73:783–6.

elicits immune responses against all four dengue viruses in the presence of [98] Spohn G, Keller I, Beck M, Grest P, Jennings GT, Bachmann MF. Active immu-

prior AdV5 immunity. Vaccine 2009;27:6011–21. nization with IL-1 displayed on virus-like particles protects from autoimmune

[86] White LJ, Parsons MM, Whitmore AC, Williams BM, de Silva A, Johnston RE. arthritis. Eur J Immunol 2008;38:877–87.

An immunogenic and protective alphavirus replicon particle-based dengue [99] Spohn G, Jennings GT, Martina BE, Keller I, Beck M, Pumpens P, et al. A VLP-

vaccine overcomes maternal antibody interference in weanling mice. J Virol based vaccine targeting domain III of the West Nile virus E protein protects

2007;81:10329–39. from lethal infection in mice. Virol J 2010;7:146.

[87] Suzuki R, Winkelmann ER, Mason PW. Construction and characterization of a [100] Jegerlehner A, Maurer P, Bessa J, Hinton HJ, Kopf M, Bachmann MF. TLR9 sig-

single-cycle chimeric ﬂavivirus vaccine candidate that protects mice against naling in B cells determines class switch recombination to IgG2a. J Immunol

lethal challenge with dengue virus type 2. J Virol 2009;83:1870–80. 2007;178:2415–20.

[88] Brandler S, Rufﬁe C, Najburg V, Frenkiel MP, Bedouelle H, Despres P, et al. [101] Bessa J, Jegerlehner A, Hinton HJ, Pumpens P, Saudan P, Schneider P, et al.

Pediatric measles vaccine expressing a dengue tetravalent antigen elicits Alveolar macrophages and lung dendritic cells sense RNA and drive mucosal

neutralizing antibodies against all four dengue viruses. Vaccine 2010;28: IgA responses. J Immunol 2009;183:3788–99.

6730–9. [102] Gurramkonda C, Adnan A, Gabel T, Lunsdorf H, Ross A, Nemani SK, et al.

[89] Chen L, Ewing D, Subramanian H, Block K, Rayner J, Alterson KD, et al. A Simple high-cell density fed-batch technique for high-level recombinant pro-

heterologous DNA prime-Venezuelan equine encephalitis virus replicon par- tein production with Pichia pastoris: application to intracellular production

ticle boost dengue vaccine regimen affords complete protection from virus of Hepatitis B surface antigen. Microb Cell Fact 2009;8:13.

challenge in cynomolgus macaques. J Virol 2007;81:11634–9. [103] Konishi E, Fujii A. Dengue type 2 virus subviral extracellular particles pro-

[90] Simmons M, Burgess T, Lynch J, Putnak R. Protection against dengue virus by duced by a stably transfected mammalian cell line and their evaluation for a

non-replicating and live attenuated vaccines used together in a prime boost subunit vaccine. Vaccine 2002;20:1058–67.

vaccination strategy. Virology 2010;396:280–8. [104] Leopold PL, Wendland RL, Vincent T, Crystal RG. Neutralized adenovirus-

[91] Hermida L, Bernardo L, Martin J, Alvarez M, Prado I, Lopez C, et al. A recom- immune complexes can mediate effective gene transfer via an Fc

binant fusion protein containing the domain III of the dengue-2 envelope receptor-dependent infection pathway. J Virol 2006;80:10237–47.

protein is immunogenic and protective in nonhuman primates. Vaccine [105] Widman DG, Frolov I, Mason PW. Third-generation ﬂavivirus vaccines

2006;24:3165–71. based on single-cycle, encapsidation-defective viruses. Adv Virus Res

[92] Valdes I, Hermida L, Gil L, Lazo L, Castro J, Martin J, et al. Heterologous prime- 2008;72:77–126.

boost strategy in non-human primates combining the infective dengue virus [106] Caufour PS, Motta MC, Yamamura AM, Vazquez S, Ferreira II, Jabor AV, et al.

and a recombinant protein in a formulation suitable for human use. Int J Infect Construction, characterization and immunogenicity of recombinant yellow

Dis 2010;14:e377–83. fever 17D-dengue type 2 viruses. Virus Res 2001;79:1–14.

[93] McDonald WF, Huleatt JW, Foellmer HG, Hewitt D, Tang J, Desai P, et al. A West [107] Guirakhoo F, Weltzin R, Chambers TJ, Zhang ZX, Soike K, Ratterree

Nile virus recombinant protein vaccine that coactivates innate and adaptive M, et al. Recombinant chimeric yellow fever-dengue type 2 virus is

immunity. J Infect Dis 2007;195:1607–17. immunogenic and protective in nonhuman primates. J Virol 2000;74:

[94] Leng CH, Chen HW, Chang LS, Liu HH, Liu HY, Sher YP, et al. A recombinant 5477–85.

lipoprotein containing an unsaturated fatty acid activates NF-kappaB through [108] Zlatkovic J, Stiasny K, Heinz FX. Immunodominance and functional activities

the TLR2 signaling pathway and induces a differential gene proﬁle from a of antibody responses to inactivated West Nile virus and recombinant subunit

synthetic lipopeptide. Mol Immunol 2010;47:2015–21. vaccines in mice. J Virol 2011;85:1994–2003.