Towards a dengue vaccine: Progress to date and remaining challenges

ARTICLE IN PRESS

Comparative Immunology, Microbiology

& Infectious Diseases 31 (2008) 239–252

0147-9571/$ -

doi:10.1016/j

�CorrespoE-mail ad

www.elsevier.com/locate/cimid

Towards a dengue vaccine: Progress to date andremaining challenges

Bruno Guy, Jeffrey W. Almond�

Sanofi pasteur, 1541 Avenue Marcel Merieux, 69280 Marcy L’Etoile, France

Accepted 12 July 2007

Abstract

The increased incidence and extended geographical reach of Dengue virus over the past two

decades have made the development of an effective vaccine an international urgency. Various

strategies are being pursued, including live, vectored and killed/recombinant preparations. For

all approaches, the challenge is to induce a broad durable immune response against all four

serotypes of Dengue virus simultaneously whilst avoiding the possible exacerbation of risk of

developing the severe forms of disease through incomplete or modified responses. This review

presents the current state of knowledge and discusses the challenges of further clinical

development.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Dengue; Vaccine; Clinical trials; Antibodies; Cellular immunity; Vaccine safety

Resume

La frequence croissante ainsi que l’extension geographique des virus de la dengue lors de ces

deux dernieres decennies ont fait du developpement d’un vaccin efficace une urgence

internationale. Differentes strategies sont actuellement poursuivies, incluant notamment des

vaccins vivants attenues ou vectorises, des vaccins ADN et des formulations sous unitaires

inactivees. Pour toutes ces approches, le defi est d’induire simultanement une reponse

immunitaire large et durable contre les quatre serotypes du virus de la dengue, tout en excluant

see front matter r 2007 Elsevier Ltd. All rights reserved.

.cimid.2007.07.011

nding author. Tel.: +33437379453.

dress: [email protected] (J.W. Almond).

www.elsevier.com/locate/cimid

dx.doi.org/10.1016/j.cimid.2007.07.011

mailto:[email protected]

ARTICLE IN PRESS

B. Guy, J.W. Almond / Comp. Immun. Microbiol. Infect. Dis. 31 (2008) 239–252240

un risque potentiel d’immunopathologie qui serait lie a des reponses incompletes ou

inadaptees. Cette etude presente l’etat actuel de connaissances et examine les defis a relever

lors de futurs developpements cliniques.

r 2007 Elsevier Ltd. All rights reserved.

Mots cles: Dengue; Vaccins; Essais cliniques; Anticorps; Immunite cellulaire; Securite de vaccin

1. Introduction

Dengue virus exists as four closely related but antigenically distinct serotypes(DEN1–4) and is a member of the genus Flavivirus. The virus infects humans viablood feeding by infected Aedes mosquitos (Aedes aegypti and Aedes albopictus).Dengue infection thus occurs only in tropical and subtropical areas where insectvectors are present. Over the past two decades the number of dengue infections hascontinued to grow in the endemic areas of South-east Asia, Central and SouthAmerica and the South Pacific regions. Up to 80 million dengue infections and24,000 deaths are now estimated annually with children bearing the bulk of thedisease burden. Over 100 countries are affected with 43 billion people at risk(Fig. 1).

Infection with any of the four serotypes of Dengue virus is often asymptomatic butcan produce clinical manifestations ranging from a self-limiting dengue fever (DF) to

Fig. 1. Worldwide distribution of Aedes aegypti and dengue fever as reported by WHO in 2001. A similar

distribution has been observed through 2006. Dengue fever has the potential to spread throughout the

zones where Aedes aegypti is present.

ARTICLE IN PRESS

B. Guy, J.W. Almond / Comp. Immun. Microbiol. Infect. Dis. 31 (2008) 239–252 241

severe dengue hemorrhagic fever (DHF) and fatal dengue shock syndrome (DSS).DF is characterized by biphasic fever, headache, myalgia, eye pain and rash.Recovery is usually completed in 7–10 days but prolonged asthenia is common.Leukocytes and platelet count decreases are frequently observed. The managementof DF is supportive with bed rest, control of fever and pain with antipyretics/analgesics, and adequate fluid intake. Immune responses provide complete andprobably lifelong protection against homotypic strains, but cross-protection betweenDengue virus serotypes is limited [1]. Consequently, an individual can experiencesecond, third and fourth infections with the different serotypes. Secondary infectionshave been associated with a higher risk of developing the more severe forms ofdisease, DHF and DSS. The pathogenesis of these severe forms is multifactorial andis influenced by the viral strain, age genetic predisposition of the patient and priorimmune status [2]. Rapid viral replication with high viremia seems to play a majorrole in disease severity. However, inappropriate pre-existing heterologous immuneresponses, both humoral and cellular, have been implicated. On the cellular side,low-affinity heterologous T-cell responses, excessive production of soluble pro-inflammatory mediators and complement activation have been linked to diseaseseverity (for a review, see Refs. [3] and [4]). On the humoral side, it has beenhypothesized that one of the mechanisms explaining DHF or DSS is theenhancement of virus replication through the presence of heterotypic, probablynon-neutralizing antibodies from a prior infection (via the Fc receptor onmononuclear leukocytes—the phenomenon of antibody dependent enhancement(ADE) [5,6].

The patho-physiological features of DHF and DSS include leakage of plasmafrom capillaries into the extra-vascular space, tissue effusion, hemorrhagic diathesis,and often accompanying hepatic involvement (for a review, see Refs. [7] and [8]).Treatment involves correction of fluid loss, replacement of coagulation factors, andinfusion of heparin. With appropriate intensive supportive therapy, mortality maybe reduced to less than 1%. The WHO case definition of DHF includes elevatedhematocrit (420% above normal) and thrombocytopenia (o100,000/mm3).However, it has been proposed that these criteria should be re-evaluated [9], as inendemic areas patients often receive supportive treatment in early stages of thedisease.

Since there is no specific treatment for dengue disease, preventive measurespresently rely on vector control and personal protection measures, which are difficultto enforce and maintain, and are expensive. The best method of prevention wouldtherefore be the development of a safe and effective vaccine directed against all fourserotypes of Dengue virus. Such a development is an urgent need, in particular forchildren living in endemic areas [10].

2. Humoral and cellular immunity against Dengue virus

The primary cells infected after inoculation via mosquito saliva are the dendriticcells in the skin [11], which subsequently migrate to the draining lymph nodes. After

ARTICLE IN PRESS


initial replication at the sites of entry, virus appears in blood during the acute febrilephase, generally for 3–5 days, and may be recovered from serum and from peripheralblood mononuclear cells (PBMCs).

In this scenario, pre-existing neutralizing antibodies probably reduces the initialinfection of skin, lymph node and circulating cells, while pre-existing cellularresponses (T helper and cytotoxic T cells) may limit the expansion of infection bykilling infected cells and secreting inflammatory cytokines. Both humoral andcellular arms can thus play a role in protection against dengue disease, but asmentioned above, both arms have also been linked to immunopathology. In thisregard, it is important to monitor antibodies and cellular responses triggered byvaccination, in order to establish which profile(s) of response is (are) induced and ifsuch responses are likely to be protective.

2.1. Protective role of antibodies; antibody-dependant enhancement (ADE)

It is widely accepted that the main markers of protection are seroneutralizingantibodies, as measured for instance by the plaque reduction neutralization test(PRNT), although the titers that need to be obtained for protection is still a matterof debate [12]. Cross-protection between Dengue virus serotypes is limited and ADEhas been proposed to constitute one mechanism leading to severe dengue disease[5,6]. However, as discussed above, the aetiology of DHF appears to be multi-factorial. Different reviewers have addressed this question, proposing more studiesto explain the role of antibodies in DHF [7,8]. A recent study showed that levels ofpre-illness plasma ADE activity in K562 cells did not correlate with the clinicalseverity or viral burden of secondary dengue infection, thus suggesting that in anycase the risk linked to ADE is relative, and that other parameters are involved indengue severity [13]. The results from our own study carried out on sera from Thaichildren vaccinated with live-attenuated virus (LAV) candidates were also in linewith a low/absent risk linked to ADE activity in vitro [14] (see below). All in all,whatever the role of ADE in DHF/DSS aetiology, a vaccine inducing a neutralizingresponse against all four serotypes simultaneously should not induce any risk in thisrespect. This is the goal of current dengue vaccine developments. In this regard, livevaccines inducing long lasting antibodies appear to be among the most promisingcandidates [15–17].

2.2. Role of cell mediated immunity (CMI)

Cellular immunity against dengue disease has often been compared to a doubleedge sword (for a review, see Refs. [3] and [4]). The important parameters withregard to protection or DHF would seem to be: (i) the balance of the Th response(Th1/Th2); (ii) kinetics of the response; (iii) magnitude, and (iv) the specificity andavidity of the CTL response. To (over) simplify, one can say that a homologousresponse is better than an heterologous one, high avidity is better than low avidity,IFNg is better than TNFa, and more generally Th1 (at a reasonable level) better thanTh2. In particular, it has been shown that heterologous cross-reactive responses tend

ARTICLE IN PRESS


to trigger TNFa while homologous ones trigger IFNg. It has also been shownrecently that regulatory T cells (Tregs) were induced during dengue infection,although their relative frequencies were insufficient to control the immunopathologyof severe disease [18]. Of course, CMI cannot be isolated from antibody response,and one has to take these two arms in consideration to propose a model of eventsleading to DHF or protection [19].

3. Vaccine candidates: stages of development

3.1. Vaccine candidates

Although no licensed vaccine is yet available, several promising candidates areunder development. The ideal dengue vaccine should provide life-long immunityagainst infection by any of the four serotypes and be free from any reactogenicity.It should be suitable for use in infants and provide immune responses that do not atany point in the vaccination process increase the risk of DHF from concomitant orsubsequent exposure to wild-type virus. The current candidates include live-attenuated vaccines live chimeras based on attenuated Dengue virus or YFbackbones, DNA vaccines, adenoviruses, adjuvanted sub-unit proteins, or combina-tions of several technologies [20,21]. These are discussed in greater detail below.

As discussed above, ideally a vaccine should induce both humoral (neutralizingantibodies) and cellular (Th1/CTL) immunity. Live-attenuated vaccines would thusbe optimal in this respect. Attenuated strains must be able to replicate sufficientlywell in vivo to provoke an immune response (ideally against all four serotypes at thesame time), but be restricted in systemic replication sufficiently to avoid inducing anyof the dengue-associated symptoms of fever, headache and arthralgia. Ideally, thestrains should not cause viremia, although a low level viremia comparable to thatcaused by Yellow fever virus (YF) vaccine may be acceptable. Live-attenuatedvaccine strains also need to be genetically stable since any reversion, either duringvaccine batch manufacture or following administration, may adversely affect safety.Moreover, the strains must be incapable of transmission by mosquitoes since thismay facilitate evolutionary change towards virulence. Transmission to mosquitoeswill be unlikely if viremia is low, but mutations restricting replications in themosquito host are also desirable.

The best studied classical live-attenuated vaccine strains have been developed atMahidol University, Bangkok [15,16], and the Walter Reed Institute (WRAIR) andwere derived by the usual empirical method of multiple passages in cell culture,principally primary canine kidney cells and fetal rhesus lung cells [22,23]. Derivativesof the Mahidol strains have also been adapted for growth in Vero cells (Vero denguevaccine (VDV) ) [24]. All these strains generally show good attenuation properties inpre-clinical testing and most have progressed to clinical evaluation as discussedbelow.

A different approach has been adopted by the Laboratory of Infectious Diseases(Bethesda), based on reverse genetics. Here, attenuation has been achieved by

ARTICLE IN PRESS


creating a 30-base deletion in the 30 non-translated region of the genomes of the fourserotypes of Dengue virus [25,26]. This strategy seems to have worked well forDengue virus serotypes 1 and 4, but did not generate suitable candidates for serotypes2 and 3. Thus, Dengue viruses 2 and 3 candidates were generated by constructingchimeras based on the attenuated serotype 4 strain as backbone, but carrying thepremembrane (pr-M) and envelope (E) genes of serotypes 2 or 3 [27]. Although thetetravalent formulation of these strains seems to be attenuated and immunogenic inmonkeys, it has yet to be tested in humans.

Chimeric vaccines, a further series of vaccine candidates, are being developed bySanofi pasteur, in collaboration with Acambis [27,28]. This approach exploits the17-D YF vaccine as a vector to carry the dengue pr-M and E genes, replacing thecorresponding genes from YF. Using infectious cDNAs from wild type strains ofDengue virus and 17D, chimeras have been constructed for each of the four serotypesof Dengue virus (ChimeriVax

TM

CYD1–4). These are currently undergoing phase Iclinical trials [17,28]. The objective of this strategy was to retain the wellcharacterized attenuation phenotype of the YF-17D backbone but incorporatedengue antigenicity. These chimerivax strains grow well in Vero cell culture andappear sufficiently attenuated and immunogenic, both in pre-clinical and clinicalstudies (discussed below). Indeed, preclinical data suggests that for this and otherYF-Flavivirus chimeras, the chimerization process itself is attenuating, thus addingto, and potentially providing a greater stability to, the attenuation phenotype ofYF-17D.

The inherent difficulties of designing strains of a sufficient level of attenuationwhich are immunogenic and non-interfering when administered as a tetravalentmixture, has led other groups to concentrate on inactivated vaccine approaches.Although less likely to induce a cellular response comparable to natural infection,killed vaccines offer in principle a more reliable way of achieving a balanced immuneresponse against each of the four serotypes at the same time, thus potentiallyavoiding the possible elevation of the risk of DHF. A purified inactivated dengue 2vaccine candidate has been developed and induced a good level of neutralizingantibody and protection against viremia in primates [29]. So far the testing of thiscandidate in humans has not been reported. Recombinant envelope proteinsprepared in a variety of expression systems have also been described. Perhaps themost advanced and promising of these are the truncated E proteins described byGuzman and colleagues which at high doses and with adjuvants, were able to protectmonkeys against infection [29,30]. Finally, DNA vaccines [31] and dengue genesvectored by adenoviruses [32] have been constructed, although these are still in earlystage development.

3.2. Interferences

As discussed above, the goal of dengue vaccine development is to induce asignificant and balanced immune response against all four serotypes to avoid anyrisk of immunopotentiation. However, one of the main difficulties linked to suchdevelopment is the existence of interferences between serotypes, i.e. after vaccination

ARTICLE IN PRESS


responses against only one or two serotypes will dominate while the responsesagainst the other serotypes will be low or even absent as has been observed in someclinical trials [15,33]. Extensive preclinical studies in monkeys have therefore beenconducted, and different formulations and schedules identified that induce suchbalanced responses (Guy et al., unpublished observations). These conditions willsoon be tested in clinical trials.

An interesting alternative approach consists of combining protective epitopes ofall four serotypes in a chimeric envelope by DNA shuffling, which inducedseroneutralizing antibodies in animal models [34,35].

4. Preclinical studies

Before entering clinical trials, candidate vaccines must be tested for safety andimmunogenicity in preclinical studies. For dengue, such evaluations can beconducted both in vitro on human cells and in vivo in animals, mostly in non-human primates (NHP) although some murine models have also been developed.

4.1. Animal models

4.1.1. Monkeys

Some NHP including Rhesus (Macaca mulatta) and Cynomolgus monkeys(Macaca fascicularis) are sensitive to infection by Dengue virus and the YF. WHOrecognized this species as a good model to assess the neurotropism and theviscerotropism of attenuated YF vaccines [36]. NHP studies can also provide usefulinformation on vaccine candidates with respect to immunogenicity (seroneutralizingantibodies) and viremia [23,29,37]. Viremia is measured as an indirect indicator ofsafety, as monkeys do not show clinical signs, but only a transient viremia isobserved.

Although studies on monkeys indicate that responses can be monitored in thisspecies, extrapolation to the human situation should be done only on a case-by-casebasis. The most solid and numerous data concern immunogenicity and viremia,which has been identified in particular as one of the factors associated with virulenceand disease severity in humans [2]. However, the absolute threshold values forprotective immunogenicity and acceptable viremia are difficult to establish, and ingeneral are ranked as compared to those induced by wild-type viruses. Nevertheless,the relative efficacy of different formulations or schedules can be compared withinthe same experiment, allowing useful parameters, for instance interferences, to bemonitored [37].

4.1.2. Mice

In addition to monkey models, a mouse model of neurovirulence has been used todiscriminate neurotropism of the different vaccine candidates. Some normal versusknockout mice have also been used to monitor the role of some immune factors inprotection against dengue disease. For instance, it has been shown in such models

ARTICLE IN PRESS


that interferon (IFN) a/b were critical for early immune responses to DEN infectionwhile IFNg-mediated immune responses were involved in both early and late viralclearance [38]. The IFN system in this murine model was shown to play a moreimportant role than T- and B-cell-dependent immunity in resistance to primaryDEN. AG129 mice lacking IFN receptors have also been used to set up a model ofsevere dengue disease showing increased vascular permeability by using a novelDEN strain, D2S10, which was generated by alternately passaging a non-mouse-adapted DEN strain between mosquito cells and mice [39].

4.2. In vitro CMI

The first cells encountering virus after infection are skin dendritic cells, the mostefficient antigen-presenting cells (APC). Virus–dendritic cells interactions have beenstudied over several years for wild type Dengue virus [40,41], but are also of interestto investigate the immune consequences of infection with the vaccine candidatesversus their wild type parents. For example, flow cytometry has been used tocompare the consequences of infection by Dengue virus serotype 2 live attenuatedvaccine (LAV2) or its parental strain DEN2 16681 [42]. Both parental andattenuated strains increased the expression of some phenotypic markers, demon-strating cellular activation. Stimulated DCs also produced TNFa, and to a lowerextent, IL6. Of importance, while parental virus induced cytokine production both inthe infected and non-infected populations, LAV2-induced cytokine production wasrestricted to the infected population. This limited activation triggered by LAV2 is inagreement with its level of attenuation. Similar studies were also conducted with theChimeriVax

TM

dengue vaccines (CYD) and the consequences of CYD1–4 infection ofDCs on activation/maturation and the secretion of pro- and anti-inflammatorycytokines, chemokines and type I interferons were investigated [43]. In CYD-infectedDCs, an up-regulation of activation markers, and secretion of type I interferons,MCP-1/CCL2, IL-6, and low amounts of TNF-a were observed. Parental Dengue

viruses induced a similar array of cytokines, but more TNF-a, less IL-6 and lessMCP-1/CCL-2 than induced by CYD. Chimeras thus induced DCs maturation anda controlled response, accompanied by limited inflammatory cytokine productionand consistent expression of anti-viral interferons, in agreement with clinicalobservations of safety and immunogenicity. These in vitro studies using primaryhuman dendritic cells may thus provide useful information on the immunogenicityand indirectly on safety of dengue vaccine candidates.

4.3. Transmission by mosquitoes

When developing live-attenuated vaccines, and in particular when they aregenetically modified organisms (GMOs), it is important to monitor their potentialfor transmissibility in the field. There is a theoretical possibility that mosquitovectors could become infected when feeding on the blood of a vaccinated host. Theability of ChimeriVax

TM

dengue vaccines to replicate in the principal mosquitovectors of YF and Dengue virus, was evaluated using A. albopictus mosquito cell

ARTICLE IN PRESS


culture (C6/36) and in A. aegypti mosquitos [44]. The growth kinetics of theindividual ChimeriVax

TM

Dengue viruses and the tetravalent formulation werecompared to the YF 17D virus and corresponding parent WT Dengue viruses. It wasshown that the chimeric viruses were not able to infect and replicate in A. aegypti

midgut tissue and were even more attenuated than YF 17D virus in mosquitoes.Thus the risk of secondary infection after vaccination is considered to be minimal forthese vaccine candidates.

5. Clinical studies

5.1. Protective antibodies

As discussed, the main markers of protection are considered to be seroneutralizingantibodies, as measured for instance by the PRNT. End points measured in clinicalstudies are usually the number of volunteers seroconverting against each serotype(PRNT50 titer X1/10), as well as the magnitude of this response (GMT). Such aresponse is usually measured against the vaccine itself, and against its homologousparental strain. In addition, it is important to measure cross-reactive responsesagainst other isolates within the same serotype, and also against other serotypes.These responses are measured shortly after each immunization (usually 1 monthafter), but long-term persistence is also an important parameter to monitor, inparticular with regard to the potential risk of immunopotentiation linked to awaning immune response against one or several serotypes.

Such parameters have been measured after immunization with tetravalent LAVs,which allowed studying serotype immunodominance and the importance of boost(s)[15]. It was shown in this study that after one dose, 58% of dengue vaccine recipientsseroconverted against at least three serotypes while 35% seroconverted against allfour; after the second dose, seroconversion was 76% and 71%, respectively. Allsubjects seroconverted to serotype 3 after one dose; serotype 4 elicited the lowestprimary response but the highest increase in seroconversion after the second dose.A third dose was shown in another study to induce seroconversion against all fourserotypes in almost all vaccine recipients [16]. More recently, a study performed withmonovalent ChimeriVax

TM

dengue 2 (CYD2) demonstrated its safety andimmunogenicity, and the fact that preimmunity to YF virus allowed CYD2 toinduce a long lasting and cross neutralizing antibody response to all four DENserotypes [17]. In fact, the influence of prior homologous (dengue) or heterologousYF, Japanese encephalitis virus (JEV)) antiflavivirus immunity is an importantparameter to consider in clinical studies, in particular in flavivirus-endemic areas inwhich a significant proportion of volunteers presents such pre-immunity.

5.2. Potential sensitization

As discussed in a previous section, the potential risk of ADE induced aftervaccination with first-generation vaccine candidates has been explored. To document

ARTICLE IN PRESS


this theoretical risk, an epidemiological study (EPI10) was conducted in 2001 toevaluate the occurrence of dengue disease, the neutralizing antibody response againsteach serotype and in vitro ADE in 113 children (4–15 years old) vaccinated with asingle dose of tetravalent vaccine between 1992 and 1997 in comparison to a controlpopulation of 226 children [14]. Sera were examined for immune enhancementcapacity by a highly reproducible flow cytometric assay in Fc receptor-bearing K562human cells. In parallel assays employing each serotype of Dengue virus, no or onlyminimal ADE was observed when sera from vaccinated or control subjects were usedat a low serum dilution that approximated the in vivo condition. These findingsshowed in particular that as soon as a broad response against all four serotypes wasinduced, the potential ADE risk was absent or minimal. They also showed thatclinically relevant immune enhancement may not be likely if this is not uniformlyachieved after the first immunization anyway. However, the study was not designedto investigate potential links between in vitro ADE activity and DHF. This has beeninvestigated by others [6], whose findings suggested a positive correlation betweenDHF and ADE using undiluted sera. The results should nevertheless be viewed inthe context of the epidemiological study from which the sera were selected.Collectively, there were no apparent differences in the frequency of DHF amongvaccinated or site-control children [45], and although a relatively small cohort hasbeen followed, it would appear that the unidose tetravalent live-attenuated vaccinehas not increased the number of DHF cases, in either children who developed anantibody response against only one serotype or several serotypes after vaccination.Since sera from DHF subjects were not found among the randomly chosen samplesfor the ADE study, is was concluded that the ADE activities monitored in vitro, evenwhen detectable, may reasonably be considered to reflect uncomplicated infection byDengue virus acquired naturally or by vaccination, or both.

5.3. Innate and specific cellular immunity

In view of the different hypotheses and experimental findings implicating T cellactivities in the pathogenesis of DHF [4], it is important to monitor T cellresponses in parallel to antibodies in the course of dengue vaccine trials. This wasdone in initial studies with first generation LAVs [46], and more recently withVDV3, a clonal derivative of the Mahidol live-attenuated dengue 3 vaccine preparedin Vero cells. This allowed the linkage of some cellular immune parameters toreactogenicity and safety [24]. In fact, despite satisfactory preclinical evaluation,VDV3 was found to be reactogenic in humans. Early innate and specific pro/anti-inflammatory cytokine serum responses as well as adaptive CD4/CD8 responseswere monitored 1 month after immunization, which led to the proposition that,among other factors, a too high adaptive immunity may have contributed to VDV3reactogenicity, even after primary vaccination. In addition, the unexpecteddiscordance observed between preclinical results and clinical outcome in humansled to a reconsideration of some preclinical acceptance criteria, and lessons learnedfrom these results have helped to pursue the development of alternative safe andimmunogenic vaccines.

ARTICLE IN PRESS


Similar endpoints are presently being measured in ongoing trials withChimeriVax

TM

vaccines, in which cellular responses induced both against the YFbackbone (NS proteins) and dengue E proteins are being measured. The excellentsafety and immunogenicity of CYDs are indeed reflected by the level and quality ofcellular responses that have been measured so far (Forrat et al., unpublishedobservations).

5.4. Medical development

Medical development will have to take into consideration the several parameterspresented in the preceding sections. In particular, in addition to efficacy againstdengue disease, long-term safety and immunogenicity follow up will have to beperformed in Phase III efficacy trials. In this respect, the risk of sensitization will beassessed by the incidence of severe wild type dengue cases in vaccinated subjectscompared to the controls. Sites where such efficacy trials will be performed arecurrently under investigation and several cohorts are already being followed long-term to evaluate the prevalence and incidence of dengue in these areas (for instancein Thailand, Vietnam, Central and South America). The number of volunteers to beincorporated in Phase III trials will depend on such evaluations.

6. Conclusion

Although dengue vaccine development has been slow over previous decades,progress is now being made at an unprecedented rate. The new LAV andChimeriVax

TM

candidates seem to have near optimal characteristics in pre-clinicaltests and so far, the clinical data generated in humans is very promising. Killedapproaches too have begun to generate encouraging data. Nevertheless thechallenges ahead are significant. Clinical trials will need to address safety fromDHF/DSS and will be large and lengthy. Further development will require diligentclinical site preparation and international determination. The increased incidenceand global reach of DF over the past two decades have made this one of the moretroublesome pathogens, especially of children. An effective vaccine is now urgent.The present vaccine candidates give hope that protection is now within our reach;hopefully within the next decade.

References

[1] Adams B, Holmes EC, Zhang C, Mammen Jr. MP, Nimmannitya S, Kalayanarooj S, et al. Cross-

protective immunity can account for the alternating epidemic pattern of dengue virus serotypes

circulating in Bangkok. Proc Natl Acad Sci USA 2006;103:14234–9.

[2] Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, Suntayakorn S, et al. Dengue

viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect

Dis 2000;181:2–9.

[3] Rothman AL. Dengue: defining protective versus pathologic immunity. J Clin Invest 2004;113(7):946–51.

[4] Green S, Rothman A. Immunopathological mechanisms in dengue and dengue hemorrhagic fever.

Curr Opin Infect Dis 2006;19:429–36.

ARTICLE IN PRESS


[5] Halstead SB, O’Rourke EJ. Antibody-enhanced dengue virus infection in primate leukocytes. Nature

1977;265:739–41.

[6] Kliks SC, Nisalak A, Brandt WE, Wahl L, Burke D. Antibody-dependent enhancement of dengue

virus growth in human monocytes as a risk factor for dengue hemorrhagic fever. Am J TropMed Hyg

1989;40:444–51.

[7] Rigau-Perez JG, Clark GG, Gubler DJ, Reiter P, Sanders EJ, Vorndam AV. Dengue and dengue

haemorrhagic fever. Lancet 1998;352:971–7.

[8] Guzman MG, Kouri G. Dengue: an update. Lancet Infect Dis 2002;2:33–42.

[9] Deen JL, Harris E, Wills B, Balmaseda A, Hammond SN, Rocha C, et al. The WHO dengue

classification and case definitions: time for a reassessment. Lancet 2006;368:170–3.

[10] Almond J, Clemens J, Engers H, Halstead S, Khiem HB, Pablos-Mendez A, et al. Accelerating the

development and introduction of a dengue vaccine for poor children, 5–8 December 2001, Ho Chi

Minh City, VietNam. Vaccine 2002;20:3043–6.

[11] Marovich M, Grouard-Vogel G, Louder M, Eller M, Sun W, Wu SJ, et al. Human dendritic cells as

targets of dengue virus infection. Inves Dermatol Symp Proc 2001;6(3):219–24.

[12] Endy TP, Nisalak A, Chunsuttitwat S, Vaughn DW, Green S, Ennis FA, et al. Relationship of

preexisting dengue virus (DV) neutralizing antibody levels to viremia and severity of disease in a

prospective cohort study of DV infection in Thailand. J Infect Dis 2004;189:990–1000.

[13] Laoprasopwattana K, Libraty DH, Endy TP, Nisalak A, Chunsuttiwat S, Vaughn DW, et al. Dengue

virus (DV) enhancing antibody activity in preillness plasma does not predict subsequent disease

severity or viremia in secondary DV infection. J Infect Dis 2005;192:510–9.

[14] Guy B, Chanthavanich P, Gimenez S, Sirivichayakul C, Sabchareon A, Begue S, et al. Evaluation

by flow cytometry of antibody-dependent enhancement (ADE) of dengue infection by sera from

Thai children immunized with a live-attenuated tetravalent dengue vaccine. Vaccine 2004;22:

3563–74.

[15] Sabchareon A, Lang J, Chanthavanich P, Yoksan S, Forrat R, Attanath P, et al. Safety and

immunogenicity of tetravalent live-attenuated dengue vaccines in Thai adult volunteers: role of

serotype concentration, ratio, and multiple doses. Am J Trop Med Hyg 2002;66:264–72.

[16] Sabchareon A, Lang J, Chanthavanich P, Yoksan S, Forrat R, Attanath P, et al. Safety and

immunogenicity of a three dose regimen of two tetravalent live-attenuated dengue vaccines in five- to

twelve-year-old Thai children. Pediatr Infect Dis J 2004;23:99–109.

[17] Guirakhoo F, Kitchener S, Morrison D, Forrat R, McCarthy K, Nichols R, et al. Live attenuated

chimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vaccine: Phase I clinical trial for safety and

immunogenicity: effect of yellow fever pre-immunity in induction of cross neutralizing antibody

responses to all 4 dengue serotypes. Hum Vaccine 2006;2:60–7.

[18] Luhn K, Simmons CP, Moran E, Dung NT, Chau TN, Quyen NT, et al. Increased frequencies of

CD4+CD25high regulatory T cells in acute dengue infection. J Exp Med 2007.

[19] Rothman AL, Ennis FA. Immunopathogenesis of dengue hemorrhagic fever. Virology 1999;257:1–6.

[20] Simmons M, Porter KR, Hayes CG, Vaughn DW, Putnak R. Characterization of antibody responses

to combinations of a dengue virus type 2 DNA vaccine and two dengue virus type 2 protein vaccines

in rhesus macaques. J Virol 2006;80:9577–85.

[21] Khanam S, Rajendra P, Khanna N, Swaminathan S. An adenovirus prime/plasmid boost strategy for

induction of equipotent immune responses to two dengue virus serotypes. BMC Biotechnol

2007;7:10–7.

[22] Innis BL, Eckels KH. Progress in development of a live-attenuated, tetravalent dengue virus vaccine

by the United States Army Medical Research and Materiel Command. Am J Trop Med Hyg 2003;

69:1–4.

[23] Eckels KH, Dubois DR, Putnak R, Vaughn DW, Innis BL, Henchal EA, et al. Modification of

dengue virus strains by passage in primary dog kidney cells: preparation of candidate vaccines and

immunization of monkeys. Am J Trop Med Hyg 2003;69(6 Suppl):12–6.

[24] Sanchez V, Gimenez S, Tomlinson B, Chan PK, Thomas GN, Forrat R, et al. Innate and adaptive

cellular immunity in flavivirus-naive human recipients of a live-attenuated dengue serotype 3 vaccine

produced in Vero cells (VDV3). Vaccine 2006;24:4914–26.

ARTICLE IN PRESS


[25] Men R, Bray M, Clark D, Chanock RM, Lai CJ. Dengue type 4 virus mutants containing deletions in

the 30 noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered

viremia pattern and immunogenicity in rhesus monkeys. J Virol 1996;70:3930–7.

[26] Whitehead SS, et al. A live, attenuated Dengue virus type 1 vaccine candidate with a 30-nucleotide

deletion in the 30 untranslated region is highly attenuated and immunogenic in monkeys. J Virol

2003;77:1653–7.

[27] Blaney JE, Durbin AP, Murphy BR, Whitehead SS. Development of a live attenuated dengue virus

vaccine using reverse genetics. Viral Immunol 2006;19:10–32.

[28] Guirakhoo F, Arroyo J, Pugachev KV, Miller C, Zhang ZX, Weltzin R, et al. Construction, safety,

and immunogenicity in nonhuman primates of a chimeric yellow fever-dengue virus tetravalent

vaccine. J Virol 2001;75:7290–304.

[29] Putnak R J, Coller BA, Voss G, Vaughn DW, Clements D, Peters I, et al. An evaluation of dengue

type-2 inactivated, recombinant subunit, and live-attenuated vaccine candidates in the rhesus

macaque model. Vaccine 2005;23:4442–52.

[30] Khanam S, Etemad B, Khanna N, Swaminathan S. Induction of neutralizing antibodies specific to

dengue virus serotypes 2 and 4 by a bivalent antigen composed of linked envelope domains III of

these two serotypes. Am J Trop Med Hyg 2006;74:266–77.

[31] Putnak R, Porter K, Schmaljohn C. DNA vaccines for flaviviruses. Adv Virus Res 2003;61:445–68.

[32] Holman DH, Wang D, Raviprakash K, Raja NU, Luo M, Zhang J, et al. Two complex, adenovirus-

based vaccines that together induce immune responses to all four dengue virus serotypes. Clin

Vaccine Immunol 2007;14:182–9.

[33] Kitchener S, Nissen M, Nasveld P, Forrat R, Yoksan S, Lang J, et al. Immunogenicity and safety of

two live-attenuated tetravalent dengue vaccine formulations in healthy Australian adults. Vaccine

2006;24:1238–41.

[34] Raviprakash K, Apt D, Brinkman A, Skinner C, Yang S, Dawes G, et al. A chimeric tetravalent

dengue DNA vaccine elicits neutralizing antibody to all four virus serotypes in rhesus macaques.

Virology 2006;353:166–73.

[35] Apt D, Raviprakash K, Brinkman A, Semyonov A, Yang S, Skinner C, et al. Tetravalent neutralizing

antibody response against four dengue serotypes by a single chimeric dengue envelope antigen.

Vaccine 2006;24:335–44.

[36] WHO Technical report series, No. 872, 1998.

[37] Guirakhoo F, Pugachev K, Zhang Z, Myers G, Levenbook I, Draper K, et al. Safety and efficacy of

chimeric yellow fever-dengue virus tetravalent vaccine formulations in nonhuman primates. J Virol

2004;78:4761–75.

[38] Shresta S, Kyle JL, Snider HM, Basavapatna M, Beatty PR, Harris E. Interferon-dependent

immunity is essential for resistance to primary dengue virus infection in mice, whereas T- and B-cell-

dependent immunity are less critical. J Virol 2004;78:2701–10.

[39] Shresta S, Sharar KL, Prigozhin DM, Beatty PR, Harris E. Murine model for dengue virus-induced

lethal disease with increased vascular permeability. J Virol 2006;80:10208–17.

[40] Libraty DH, Pichyangkul S, Ajariyakhajorn C, Endy TP, Ennis FA. Human dendritic cells are

activated by dengue virus infection: enhancement by gamma interferon and implications for disease

pathogenesis. J Virol 2001;75:3501–8.

[41] Palmer DR, Sun P, Celluzzi C, Bisbing J, Pang S, Sun W, et al. Differential effects of dengue virus on

infected and bystander dendritic cells. J Virol 2005;79(4):2432–9.

[42] Sanchez V, Hessler C, DeMonfort A, Lang J, Guy B. Comparison by flow cytometry of immune

changes induced in human monocyte-derived dendritic cells upon infection with dengue 2 live-

attenuated vaccine or 16681 parental strain. FEMS Immunol Med Microbiol 2006;46:113–23.

[43] Deauvieau F, Sanchez V, Balas C, Kennel A, de Montfort A, Lang J, et al. Innate immune responses

in human dendritic cells upon infection by chimeric Yellow fever Dengue vaccines serotype 1 to 4. Am

J Trop Med Hyg 2007;76:144–54.

[44] Higgs S, Vanlandingham DL, Klingler KA, McElroy KL, McGee CE, Harrington L, et al. Growth

characteristics of ChimeriVax-Den vaccine viruses in Aedes aegypti and Aedes albopictus from

Thailand. Am J Trop Med Hyg 2006;75:986–93.

ARTICLE IN PRESS


[45] Chanthavanich P, Luxemburger C, Sirivichayakul C, Lapphra K, Pengsaa K, Yoksan S, et al. Short

report: immune response and occurrence of dengue infection in thai children three to eight years after

vaccination with live attenuated tetravalent dengue vaccine. Am J Trop Med Hyg 2006;75:26–8.

[46] Rothman AL, Kanesa-thasan N, West K, Janus J, Saluzzo JF, Ennis FA. Induction of T lymphocyte

responses to dengue virus by a candidate tetravalent live attenuated dengue virus vaccine. Vaccine

2001;19:4694–9.

Documents

Towards a dengue vaccine: Progress to date and remaining challenges