developing a dengue vaccine: progress and future challengesstudy of a dengue vaccine (v180) in...

Ann. N.Y. Acad. Sci. ISSN 0077-8923

ANNALS OF THE NEW YORK ACADEMY OF SCIENCESIssue: Antimicrobial Therapeutics Reviews

Developing a dengue vaccine: progress and futurechallenges

Stephen J. ThomasWalter Reed Army Institute of Research, Silver Spring, Maryland

Address for correspondence: Stephen J. Thomas, M.D., Director, Viral Diseases Branch, Walter Reed Army Institute ofResearch, 503 Robert Grant Ave., Silver Spring, MD. [email protected]

Dengue is an expanding public health problem in the tropics and subtropical areas. Millions of people, most fromresource-constrained countries, seek treatment every year for dengue-related disease. Despite more than 70 yearsof effort, a safe and efficacious vaccine remains unavailable. Antidengue antiviral drugs also do not exist despiteattempts to develop or repurpose drug compounds. Gaps in the knowledge of dengue immunology, absence of avalidated animal or human model of disease, and suboptimal assay platforms to measure immune responses followinginfection or experimental vaccination are obstacles to drug and vaccine development efforts. The limited success ofone vaccine candidate in a recent clinical endpoint efficacy trial challenges commonly held beliefs regarding potentialcorrelates of protection. If a dengue vaccine is to become a reality in the near term, vaccine developers should expanddevelopment pathway explorations beyond those typically required to demonstrate safety and efficacy.

Keywords: dengue; vaccine; antiviral; immunology

Introduction

Dengue is the most important arboviral disease af-flicting the world today. Hundreds of millions ofinfections occur each year, of which more than90 million are clinically apparent.1 Mortality is re-portedly low compared to other vector borne dis-eases, but the brunt of severe dengue disease anddeath in many regions occurs disproportionatelyin children.2 In regions with endemic and hyper-endemic transmission, dengue absorbs significanthealthcare resources.3–17 Dengue season exacts ameasurable toll at the personal, community, andregional levels and is a leading cause of febrile, sys-temic illness in travelers.18–25 Deploying militarypopulations have confronted the potential and oc-currence of mission-disabling dengue epidemics forover a century.26–30

For decades, the global dengue burden has beentrending upward.31 Conditions favoring the closejuxtaposition of virus, vector, and susceptible host—a requirement for sustained transmission—arenumerous. Ecological conditions favoring vectorexpansion, population growth and increasing ur-

banization, and the ease of air travel have all con-tributed to the concentration, in time and space,of susceptible hosts, competent vectors, and dengueviruses (DENVs).32 A reversal of current epidemi-ologic trends without a strategic intervention isunlikely.

There is no licensed dengue vaccine and noprophylactic or therapeutic dengue drug (i.e., an-tiviral or anti-inflammatory). Vector control, evenwhen successful from an entomologic perspec-tive, does not always translate into a reductionin human infection or disease.33 The explorationof genetically modified mosquitoes and the po-tential to replace transmission-competent with in-competent mosquito populations is underway andrequires additional study.34–37 Personal protectivemeasures, such as wearing long sleeves and pants,use of bed nets, use of insecticides (i.e., N,N-diethyl-meta-toluamide, DEET), avoidance of vec-tors during prime feeding times, and reducing thenumber of man-made vector breeding sites (e.g.,standing water), are inconsistently applied and, asmore is learned more about vector-host interac-tions, may not be optimal.31,38 A safe and efficacious

doi: 10.1111/nyas.12413

140 Ann. N.Y. Acad. Sci. 1323 (2014) 140–159 Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Thomas Dengue vaccine

tetravalent dengue vaccine is the best strategy for re-ducing the global dengue burden.

Dengue vaccine development challenges

The dengue vaccine field is robust with numerouscandidates in preclinical and clinical development.Unfortunately, the challenges accompanying the de-velopment of a tetravalent dengue vaccine are alsonumerous: the existence of four DENV types, allcapable of causing disease and death, requires adengue vaccine capable of preventing clinical dis-ease caused by infection with any of the DENVs.Whether a vaccine will need to demonstrate DENVtype–specific efficacy for every type before licensureis a nuance developers and regulatory authoritiesare currently negotiating. DENV evolution and po-tential for jump of sylvatic dengue strains into asustained human transmission cycle raises the pos-sibility that new vaccine candidates will be requiredover time to address significant antigenic divergencefrom existing DENVs.39–41

The coordination of innate and adaptive im-mune responses that confer protection or contributeto pathogenesis following a DENV infection areincompletely understood.42 Consequently, vaccinedevelopers must extrapolate from natural infectiondata, animal studies, and other flavivirus vaccinedevelopment experiences (e.g., Yellow fever virusand Japanese encephalitis) to establish and pursueimmunogenicity benchmarks that could potentiallytranslate into clinical benefit (i.e., protective effi-cacy). There are examples of safe and efficaciousvaccines that were licensed and that experiencedwidespread use before their protective mechanismswere fully understood.43 A significant concern withdengue is the observation of enhanced dengue dis-ease when the convalescent immune response, es-tablished following a previous dengue infection withone DENV type, contributes to an immunopatho-logical response following infection with a dif-ferent DENV type. Greater numbers of infectedtargets cells (i.e., monocytes and macrophages), in-creased viral replication, and induction of a pro-inflammatory state result in a more severe clinicalphenotype that is marked by plasma leakage, co-agulation dysfunction, intravascular volume deple-tion and hemodynamic instability, and, potentially,death.42,44–47 Therefore, poorly performing denguevaccine candidates could place vaccine recipients at

increased risk for enhanced disease—a theoreticalrisk not yet demonstrated in field studies.48,49

Gaps in our understanding of dengue immunol-ogy complicate the process of defining an immunecorrelate of protection and this, in turn, complicatesvaccine clinical development plans and regulatorystrategies. Neutralizing antibody has been proposedas a likely immune correlate and may eventually bedefined as such, similar to vaccines for Yellow fevervirus and Japanese encephalitis virus.50–52 However,it is also possible that components of cellular im-mune responses (e.g., IFN-� secretion and cyto-toxic T cell activity) will be found to be correlatesof immune protection. Numerous methods are be-ing explored to measure both humoral and cellularimmune responses following natural infection andas immunogenicity assays to support vaccine devel-opment programs.53

Adversely affecting the exploration for an im-mune correlate of protection is the absence of adengue animal disease model. Humanized smallanimal models are being aggressively studied andhave shed light on various aspects of the immuno-logic and clinical responses to dengue infection.However, they do not currently appear to offer acomprehensive view of in vivo human dengue dis-ease pathology.54–57 Nonhuman primates developviremia and neutralizing antibody responses fol-lowing dengue infection, but there is a paucityof local or systemic clinical signs or biochemi-cal abnormalities.58–66 Variations in challenge viruspreparation or delivery may yield a more informa-tive model.

In addition to the absence of an animal modelthere is also no robust dengue human infectionmodel to support vaccine and drug developmentefforts, basic immunology studies, and the searchfor immune correlates. Currently, human infectionmodels expose healthy volunteers to wild-type orslightly attenuated pathogens to produce an uncom-plicated disease course. A human infection modelfor dengue could lower vaccine development risk byproviding an early indication of whether a vaccinecandidate has the potential for clinical benefit (ther-apeutic or protective), thereby reducing the risk ofexposing endemic populations to suboptimal vac-cine or drug candidates.

Experimental human dengue infections, docu-mented as early as 1902, have facilitated numeroussentinel discoveries in the field.67–78 The Walter Reed

141Ann. N.Y. Acad. Sci. 1323 (2014) 140–159 Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Dengue vaccine Thomas

Army Institute of Research (WRAIR) conductedtwo dengue human infection studies in the early2000s. The studies aimed to validate human in-fection model strains and then expose past exper-imental vaccine recipients to the best performingstrains.79–82 Those studies were the first documentedones in decades, and none have been completedsince. Following the limited success of the world’sfirst dengue vaccine efficacy study, there is renewedinterest in exploring human infection models fordengue.

Another issue influencing the dengue vaccinefield is the portfolio of assays used to measure vac-cine immunogenicity during preclinical and clinicaldevelopment activities. Experts in the field believeneutralizing antibodies have the greatest likelihoodof being a correlate of protection, and most assaydevelopment efforts supporting vaccine programshave been devoted to this readout.52 The classic as-say platform designed to measure neutralizing an-tibodies, the plaque reduction neutralization test(PRNT), has had significant interassay and interlabvariability and a low level of robustness.83–86 In addi-tion to reducing comparability of vaccine candidateperformance across different developers, interassayvariation (i.e., two- to threefold variability) withina single laboratory may considerably skew (posi-tive or negative) seroconversion rates and antibodytiters. And, thus, neutralizing antibody titers aroundsuch as assay cut-off may or may not represent trueimmunologic responses to vaccination. There arealso concerns regarding the cross-reactivity of neu-tralizing antibody platforms within the DENV typesand with other flaviviruses (e.g., Japanese encephali-tis virus, West Nile virus, and Yellow fever virus).Confusing data or misinformation misleads vaccinedevelopment decisions, such as vaccine candidatedown-selection and whether to advance develop-ment.

Variations of the PRNT are under developmentand more advanced programs have undergone qual-ification and validation procedures, improving as-say quality. Fortunately, there are numerous initia-tives to improve neutralizing assay platforms andto better capture conditions more representativeof the human in vivo experience. These includeusing human cell lines, cells bearing putative vi-ral receptors (i.e., Fc�R), execution of depletionsteps to improve measurement of homotypic versusheterotypic antibodies, employment of automated

platforms (e.g., fluorescence activated cell sorting(FACS)), and manipulation of control virus input(e.g., varying concentrations of control virus or re-porter virus particles).87–93

Assay platforms measuring cellular mediated im-munity (CMI) have also been applied to denguevaccine development programs.53 Readouts impli-cating a breadth of cellular responses have been eval-uated and compared to similar studies performedon samples from natural infection.94–100 Althoughdefining DENV type–specific responses has beena focus, this has been carried out without a clearunderstanding of what constitutes a bone fide im-munoprotective profile; thus, the data are difficult tointerpret. As more is learned about the complexitiesof the cellular immune response following denguevirus exposure, a potential correlate may emerge.

Dengue vaccine development efforts

GeneralIt is unclear whether a single dengue vaccine willmeet the diverse spectrum of global needs and re-quirements. Region-specific burden of disease dataand existing national immunization schedules aretwo of many factors that will drive dengue vaccina-tion policies and programs.101,102 Traveler and mili-tary populations also present unique circumstances.Elements of a desirable dengue vaccine target prod-uct profile (TPP) would include but not be limitedto (1) ability to protect against clinically relevantdengue disease of any severity caused by any DENVtype; (2) dosing schedules compatible with exist-ing national immunization (endemic areas) or trav-eler/military schedules (short lead time); (3) rapidoverall time to protection from time of first pri-mary vaccination (number of weeks or months);(4) durable immunity and a reasonable schedule ofbooster dose requirements if needed; and (5) abilityto store vaccine at a reasonable temperature for areasonable period of time.

Efforts to develop a dengue vaccine havebeen documented as early as 1929, when sci-entists unsuccessfully attempted to produce aninactivated dengue virus vaccine using phenol,formalin, and bile.72 During World War II live atten-uated virus (LAV) vaccines were explored by passingDENV-1 and DENV-2 strains in brains of suck-ling mice.75,77,103 However, mouse brain-derivedcandidates were eventually replaced with cell cul-ture based vaccine candidates.



Table 1. Current vaccine trial entries in ClinicalTrials.gov (search terms were “dengue” and “vaccine”), arranged bysponsor (accessed Dec. 1, 2013)

Status

Vaccine candidateregulatory

sponsor Study title Identifier

Completed Hawaii Biotech,Inc.a

Study of HBV-001 D1 in Healthy Adults NCT00936429

Active, not recruiting Inviragen Inc.b Study to Investigate the Safety and Immunogenicity of aTetravalent Chimeric Dengue Vaccine in Healthy VolunteersBetween the Ages of 1.5–45 Years

NCT01511250

Completed Inviragen Inc.b Safety and Immunogenicity Study to Assess DENVax, a LiveAttenuated Tetravalent Vaccine for Prevention of DengueFever

NCT01224639

Recruiting Inviragen Inc.b A Comparison of the Safety and Immunogenicity of VariousSchedules of Dengue Vaccine in Healthy Adult Volunteers

NCT01542632

Recruiting Inviragen Inc.b Phase 1b Study Investigating Safety & Immunogenicity ofDENVax Given Intradermally by Needle or Needle FreePharmaJet Injector

NCT01765426

Active, not recruiting Inviragen Inc.b,c Impact of SQ vs IM Administration of DENVax on Safety andImmunogenicity

NCT01728792

Active, not recruiting Merck Sharp &Dohme Corp.

Study of a Dengue Vaccine (V180) in Healthy Adults(V180-001 AM2)

NCT01477580

Recruiting NIAIDd Phase II Trial to Evaluate Safety and Immunogenicity of aDengue 1,2,3,4 (Attenuated) Vaccine

NCT01696422

Completed NIAID Tetravalent Chimeric Dengue Vaccine Trial NCT01110551Completed NIAID Safety and Immune Response to an Investigational Dengue

Type 2 VaccineNCT01073306

Completed NIAID Evaluation of the Safety and Immune Response to anInvestigational Dengue Type 1 Vaccine

NCT01084291

Recruiting NIAID Evaluating the Safety and Immune Response to TwoAdmixtures of a Tetravalent Dengue Virus Vaccine

NCT01436422

Completed NIAID Safety of and Immune Response to DEN4 Vaccine ComponentCandidate for Dengue Virus

NCT00919178

Recruiting NIAID Evaluating the Safety and Immune Response to TwoAdmixtures of a Tetravalent Dengue Virus Vaccine

NCT01506570

Recruiting NIAID Evaluating the Safety and Immune Response to a DengueVirus Vaccine in Healthy Adults

NCT01931176

Completed NIAID Safety and Immune Response to Two Doses ofrDEN2/4delta30 Dengue Vaccine

NCT00920517

Completed NIAID Safety of and Immune Response to a Dengue Virus Vaccine(rDEN3–3’Ddelta30) in Healthy Adults

NCT00712803

Recruiting NIAID Evaluating the Safety and Immune Response to Two Doses of aDengue Virus Vaccine Administered 12 Months Apart

NCT01782300

Completed NIAIDe Safety of and Immune Response to a Dengue Virus Vaccine(rDEN2/4delta30[ME]) in Healthy Adults

NCT00094705

Completed NIAIDe Safety of and Immune Response to a Dengue Virus Vaccine(rDEN1delta30) in Healthy Adults

NCT00089908

Completed NIAIDe Evaluation of the Safety and Immune Response of FiveAdmixtures of a Tetravalent Dengue Virus Vaccine

NCT01072786

Completed NIAIDe Safety of and Immune Response to Two Different DengueVirus Vaccines in Individuals Previously ImmunizedAgainst Dengue Virus

NCT00458120

Completed NIAIDe Safety of and Immune Response to a Dengue Virus Vaccine(rDEN4delta30–200,201) in Healthy Adults

NCT00270699

Completed NIAIDe Safety of and Immune Response to a Dengue Virus Vaccine(rDEN3delta30/31-7164) in Healthy Adults

NCT00831012

Completed NIAIDe Safety of and Immune Response of a 2-dose Regimen ofrDEN1delta30 Dengue Virus Vaccine

NCT00473135

Continued



Table 1. Continued

StatusVaccine candidateregulatory sponsor Study title Identifier

Completed NIAIDe Safety of and Immune Response to a Dengue Virus Vaccine(rDEN4delta30-4995) in Healthy Adults

NCT00322946

Completed NIAIDe Safety of and Immune Response to a Dengue Virus Vaccine(rDEN3/4delta30[ME]) in Healthy Adults

NCT00375726

Completed Sanofi Pasteur A Study of Dengue Vaccine in Healthy Toddlers Aged 12 to 15Months in the Philippines

NCT01064141

Completed Sanofi Pasteur Immunogenicity and Safety of Sanofi Pasteur Pasteur’s CYDDengue Vaccine in Healthy Children and Adolescents inLatin America

NCT00993447

Completed Sanofi Pasteur Study of a Tetravalent Dengue Vaccine in Healthy ChildrenAged 2 to 11 Years in Malaysia

NCT01254422

Active, not recruiting Sanofi Pasteur Study of a Novel Tetravalent Dengue Vaccine in HealthyChildren Aged 2 to 14 Years in Asia

NCT01373281

Active, not recruiting Sanofi Pasteur Study of a Novel Tetravalent Dengue Vaccine in HealthyChildren and Adolescents Aged 9 to 16 Years in LatinAmerica

NCT01374516

Completed Sanofi Pasteur Study of ChimeriVaxTM Tetravalent Dengue Vaccine inHealthy Peruvian Children Aged 2 to 11 Years

NCT00788151

Completed Sanofi Pasteur Study of ChimeriVaxTM Dengue Tetravalent Vaccine in AdultSubjects

NCT00730288

Completed Sanofi Pasteur Immunogenicity and Safety of Three Formulations of DengueVaccines in Healthy Adults Aged 18 to 45 Years in the US

NCT00617344

Completed Sanofi Pasteur Safety and Immunogenicity of Formulations of DengueVaccines in Healthy Flavivirus-Naive Adults

NCT00740155

Active, not recruiting Sanofi Pasteur Immune Response to Different Schedules of a TetravalentDengue Vaccine Given With or Without Yellow FeverVaccine

NCT01488890

Active, not recruiting Sanofi Pasteur Study of Yellow Fever Vaccine Administered With TetravalentDengue Vaccine in Healthy Toddlers

NCT01436396

Active, not recruiting Sanofi Pasteur Study of a Booster Injection of PentaximTM VaccineAdministered With Dengue Vaccine in Healthy Toddlers

NCT01411241

Completed Sanofi Pasteur Study of a Tetravalent Dengue Vaccine in Healthy Adults inAustralia

NCT01134263

Active, not recruiting Sanofi Pasteur Study of Sanofi Pasteur Pasteur’s CYD Dengue Vaccine inHealthy Subjects in Singapore

NCT00880893

Active, not recruiting Sanofi Pasteur Study of a Tetravalent Dengue Vaccine in Healthy AdultSubjects Aged 18 to 45 Years in India

NCT01550289

Active, not recruiting Sanofi Pasteur Efficacy and Safety of Dengue Vaccine in Healthy Children NCT00842530Active, not recruiting Sanofi Pasteur Study of ChimeriVaxTM Tetravalent Dengue Vaccine in

Healthy SubjectsNCT00875524

Enrolling by invitation Sanofi Pasteur Long-Term Study of Hospitalized Dengue & Safety in ThaiChildren Included in a Tetravalent Dengue Vaccine EfficacyStudy

NCT01983553

Recruiting Sanofi Pasteurf Immunologic Mechanisms of Immune Interference and/orCross-Neutralizing Immunity After CYD TetravalentDengue Vaccine

NCT01943825

Completed Sanofi Pasteur Study of CYD Dengue Vaccine in Healthy Children andAdolescents in South America

NCT01187433

Active, not recruiting U.S. Army MedicalResearch andMaterielCommand

Safety Study of a Vaccine (DENV-1 PIV) to Prevent DengueDisease

NCT01502735

Completed U.S. Army MedicalResearch andMaterielCommandg

A Trial of a Walter Reed Army Institute of Research (WRAIR)Live Attenuated Virus Tetravalent Dengue Vaccine inHealthy US Adults

NCT00239577

Continued



Table 1. Continued

Status

Vaccine candidateregulatory

sponsor Study title Identifier

Completed U.S. Army MedicalResearch andMaterielCommandh

A Phase I/II Trial of Tetravalent Live Attenuated DengueVaccine in Flavivirus Antibody Naive Infants

NCT00322049


A Phase I/II Trial of a Tetravalent Live Attenuated DENVaccine in Flavivirus Antibody Naive Children

NCT01843621


A Phase II Trial of a Walter Reed Army Institute of Research(WRAIR) Live Attenuated Virus Tetravalent DengueVaccine in Healthy Adults in Thailand

NCT00370682


A Study of Two Doses of WRAIR Dengue VaccineAdministered Six Months Apart to Healthy Adults andChildren

NCT00468858

Recruiting U.S. Army MedicalResearch andMaterielCommandi

A Two-dose Primary Vaccination Study of a TetravalentDengue Virus Purified Inactivated Vaccine vs. Placebo inHealthy Adults (in Puerto Rico)

NCT01702857

Active, not recruiting U.S. Army MedicalResearch andMaterielCommandi

A Two-dose Primary Vaccination Study of a TetravalentDengue Virus Purified Inactivated Vaccine vs. Placebo inHealthy Adults

NCT01666652


Follow-Up Study of Thai Children From Dengue-003 andEvaluation of a Booster Dose of Dengue Vaccine

NCT00318916


A Phase I/II Trial of a Tetravalent Live Attenuated DengueVaccine in Flavivirus Antibody Naive Children

NCT00384670


A Phase II Trial of a WRAIR Live Attenuated Virus TetravalentDengue Vaccine in Healthy US Adults

NCT00350337

Active, not recruiting U.S. Army MedicalResearch andMaterielCommandj

Evaluation of the Safety and the Ability of a DNA Vaccine toProtect Against Dengue Disease

NCT01502358

Completed U.S. Army Officeof the SurgeonGeneralk

Safety Study of a Dengue Virus DNA Vaccine NCT00290147

aHawaii Biotech Inc. dengue vaccine program acquired by Merck and Company (2010).bInviragen acquired by Takeda Pharmaceutical Company Limited (2013).cNCT01728792 being executed in cooperation with the Walter Reed Army Institute of Research and the State University of New York,Upstate Medical University.dStudy sponsor is Butantan Institute.eCollaborator listed as Johns Hopkins Bloomberg School of Public Health.fCollaborator listed as U.S. Department of Defense.gStudy Sponsor listed as GlaxoSmithKline.hCollaborator listed as GlaxoSmithKline.iCollaborators listed as GlaxoSmithKline and the Walter Reed Army Institute of Research.jCollaborators listed as Vical, the Walter Reed Army Institute of Research (WRAIR), and the Naval Medical Research Center.kCollaborator listed as the U.S. Army Medical Research and Materiel Command.



Multiple different vaccine approaches have beentested in human clinical trials; a single candidate isin phase III, and there are numerous candidatesin preclinical development (Table 1). Developershave taken unique approaches to targeting differ-ent regions of the DENV genome to include fullgenome LAV using cell passage, directed mutagene-sis, and chimeric technology; inactivated virus withaluminum hydroxide or proprietary adjuvant sys-tems; recombinant proteins; Yellow fever virus back-bone with DENV premembrane (prM) and E sub-stitution for the Yellow fever virus sequences; andDNA-based candidates. Each approach makes as-sumptions about what will constitute a protectiveimmune response (e.g., neutralizing antibody to theE gene, requirement for CMI directed against non-structural (NS) proteins, both, or others) and howto best stimulate this response in a DENV type–specific manner.

Safety measurements during dengue vaccine tri-als include assessment of local and systemic so-licited and unsolicited symptoms, and objectiveclinical findings during vaccination and during sub-acute and long-term follow up periods (dengueendemic regions). Clinical laboratory abnormali-ties and vaccine-induced viremia are also evaluated.During studies in dengue-endemic regions and dur-ing efficacy trials, the phenotype (dengue, severedengue) of natural DENV infections that occur inplacebo/control recipients and breakthrough casesin experimental vaccine recipients is also monitoredduring vaccination periods (between doses), as wellas remote from vaccination. More severe pheno-types in vaccine recipients (statistically significantincreases) compared to control recipients would beconcerning for vaccine breakthrough and contribu-tion to more severe disease.104,105

Expected local reactions include pain, redness,and induration/swelling. Expected systemic symp-toms would mimic those seen in natural DENVinfections, including fever, headache, muscle pain,bone pain, pain with eye movement, nausea,vomiting, and fatigue. Clinical signs would includeobjective fever, rash, hepatomegaly, lymphadenopa-thy, and evidence of bleeding. Each sign and symp-tom is graded on a severity scale, and duration isdocumented. Laboratory measures of specific con-cern include abnormalities in the complete bloodcount (CBC), especially absolute neutrophil counts(ANC) and platelet decrements. Liver-associated

enzymes (ALT, AST) elevations are also routinelyassessed. Qualitative and quantitative peripheralviremia measurements following vaccination withreplicating vaccines not only confirm vaccine takebut also may indicate immunologic enhancementin flavivirus-primed populations (i.e., significantlyhigher viremia following dose 2 or following dose 1in primed volunteers with or without symptoms).

The immune readout measured by all vaccinedevelopers is the neutralizing antibody. There arenumerous platforms currently in use to measurethis endpoint to include the classic PRNT, a mi-croneutralization platform, a microplaque reduc-tion neutralization test (�PRN), a microfocus re-duction neutralization test (mFRNT), and FACS-based assays.84,106–108 Antibody measurements aremade at baseline (day 0) and then on the day ofeach vaccination and 1 month following each vacci-nation. In small trials, all volunteers are sampled ateach time point, while in larger studies, subsets aresampled and the results are extrapolated to the co-hort. This schedule of measurement provides insightinto vaccine immunogenicity. Sample collection attime points remote from vaccination provides anunderstanding of immune response durability overtime, kinetics of immune response maturation, andpossible neutralizing antibody set points followinga period of waning.

Immune responses at the cohort level are char-acterized by the percent of volunteers who sero-convert to each DENV type (i.e., antibody responsebelow the assay cutoff before vaccination and thenabove the cutoff following vaccination). Geomet-ric mean titer calculations are used to quantitativelyrepresent cohort level immune responses to eachDENV type. Antibody profiles are used to approxi-mate whether individuals and cohorts develop whatis presumed to be a protective response. Desiredprofiles include antibody responses measured to allor at least three DENV types, that is, tetravalentand trivalent profiles, respectively. Nonrespondersare considered susceptible to dengue infection anddisease, and monovalent responders are consideredto be higher risk for severe disease with natural in-fection (secondary infection).

The common thinking is that a balanced antibodyresponse is desired. Another proposal (advocatedby the present author, STJ) is that the developmentgoal should be to achieve a DENV type–specificneutralizing antibody response above the required



protective threshold for each individual DENV type.It is likely that the protective threshold varies foreach DENV type—even, perhaps, for each geno-type within a given type—and pursuing a balancedresponse could underestimate the required responseto protect against one or more types. Unfortunately,at this time a protective threshold has not been de-fined for any DENV type.

Cellular immune responses (i.e., DENV type–specific cytotoxtic T cell and INF-� responses, poly-functional T cells, etc.) to vaccination and otherpotential immune response measures (i.e., quanti-tating DENV type–specific memory B cell popula-tions, antibody avidity and affinity, etc.) are underassessment, but their investigation and applicationare not standard across development programs.

Vaccine candidates in clinical developmentFlaviviral LAV vaccines (e.g., against Yellow fever(YF) virus and Japanese encephalitis virus) haveproved to be safe and durably efficacious.109–112 LAVvaccines replicate in the recipient at a relatively lowand controlled manner yet present all viral antigensin the vaccine construct and elicit both antibodyand T cell responses. A tetravalent vaccine approach(i.e., DENV-1 + DENV-2 + DENV-3 + DENV-4)is designed to induce a multivalent, DENV type–specific convalescent immune response. Investiga-tors at Mahidol University in Bangkok were amongthe first groups to attenuate DENVs by passingviral strains in dog (PDK) and nonhuman pri-mate (PGMK) cell lines.113–119 And while that ef-fort was successful initially, achieving a safe and im-munogenic (i.e., neutralizing antibody responses toeach DENV type) tetravalent formulation proveddifficult.49,94,120,121

The Walter Reed Army Institute of Research(WRAIR) also developed multivalent LAV candi-dates by serial PDK cell passage followed by a finalpassage in fetal rhesus lung (FRhL) cells. Early devel-opment efforts identified promising formulationsby combining variations of attenuation and DENVantigen concentrations.122–130 A single formulationtested in Thai schoolchildren and toddlers was welltolerated and sufficiently immunogenic to pursuecontinued development.131,132 Newly derived vac-cine lots were tested in adults in the United States,Thailand, and in Puerto Rico across a broad agerange (12 months to 50 years). In the U.S. study,tetravalent response rates to new vaccine formula-

tions were between 60% and 66.7% in dengue naive(unprimed) subjects, following two doses deliveredat study days 0 and 180; a third dose did not in-crease tetravalent antibody rates.133 The Thailandstudy cohort was largely primed to dengue (88–98%) at study initiation. All dengue unprimed sub-jects and at least 97.1% of primed subjects expe-rienced tetravalent seroconversion when measured1 month following dose 2 (unpublished data, SJT).Data from the Puerto Rico study are pending. Avail-able data from the three phase II studies indicate noovert safety problems in over 300 vaccine recipientsand moderate to high seroconversion rates. Furtherdevelopment of these candidates has been indefi-nitely suspended in search of an improved TPP.

The U.S. National Institutes of Health (NIH)constructed cDNA clones of a DENV-4 candidateand induced a 30-nucleotide deletion in the 3′

untranslated region (rDEN4�30; Fig. 1 and Ta-ble 2).134 The monovalent candidate was immuno-genic and induced lower viremia than the parentstrain. Additional candidates were tested in phaseI clinical trials to identify optimal components fortetravalent formulations.135–145 Tetravalent candi-dates were prepared and tested in numerous phaseI clinical trials.146 A panel of human monoclonalantibodies (mAbs) was made from subjects whoreceived an rDEN1�30 candidate and from sub-jects who were naturally infected with DENV-1.The results demonstrated a high degree of similar-ity in the induced memory profile (high frequencyof B cells encoding serotype cross-reactive, weaklyneutralizing antibodies).147 Dose ranging trials ex-ploring human infectious dose 50% (ID50) param-eters yielded a target dose of 1000 plaque formingunits (PFU) for each DENV type formulated into atetravalent vaccine.148 A lead candidate (admixtureTV003) was identified that induced 74% tetrava-lent seroconversion in flavivirus-naive vaccines, and92% seroconverted to �3 serotypes following a sin-gle dose. A TV003 dose of 1000 PFU per DENVcomponent was also explored in flavivirus-primedindividuals. A single dose was provided at time 0and a second dose (“challenge,” per the authors)provided at 6 months. Sixty percent of all vacci-nees had at least one vaccine virus recovered fromthe blood following dose 1, there was no viremiafollowing dose 2, and there was no significantdifference in the adverse event profile. Dose 1 in-duced a tetravalent neutralizing antibody profile in



Table 2. Current vaccine candidate sponsor, name, description, dosing schedule, and phase of development basedon clinicaltrials.gov (accessed Feb. 3, 2014)

Current vaccinecandidate regulatorysponsor

Current vaccinename

Description Current dosingschedulea

Developmentphase

Inviragen Inc.b DENVax Live attenuated virusDENV-2 PDK-53 backboneDENV-1/2, -3/2, and -4/2 chimeras

Dose 1–Day 0Dose 2–Day 90

2

Merck Sharp & DohmeCorp.

V180 Recombinant protein80% of DENV-1–4 E protein

Dose 1–Month 0Dose 2–Month 1Dose 3–Month 2

1

National Institute ofAllergy andInfectious Diseases(NIAID)

TetraVax-DV-TV003(TV003)

Live virus vaccineAttenuated by directed mutagenesis (nucleotidedeletions) and development of chimeras

Dose 1–Day 0 2

Sanofi Pasteur CYD Dengue Vaccine Live attenuated virusYellow fever 17D virus backbone with preM

and E removed and replaced withDENV-1–4 preM and E proteins(CYD = chimeric-Yellow fever-dengue)

Dose 1–Month 0Dose 2–Month 6Dose 3–Month 12

3

U.S. Army MedicalResearch andMateriel Command(Walter Reed ArmyInstitute ofResearch)

TDEN-PIV Purified inactivated vaccine Dose 1–Day 0Dose 2–Day 28

1

U.S. Army MedicalResearch andMateriel Command(U.S. Naval MedicalResearch Center)

Tetravalent DengueVaccine (TVDV)

Plasmid DNA vaccine Dose 1–Day 0Dose 2–Day 30Dose 3–Day 90

1

aAs listed in most recent clinicatrials.gov entry.bInviragen acquired by Takeda Pharmaceutical Company Limited (2013).

85% of vaccines and a trivalent or better responsein 100% of recipients.149 Phase II trials in endemicpopulations and across a diverse age range are poisedto begin in Thailand and Brazil. The vaccine hasbeen exclusively licensed to the Instituto Butantan(Sao Paulo, Brazil).

St. Louis University Health Sciences Center cre-ated a Yellow fever (YF)-dengue chimeric vaccinecandidate by inserting dengue preM and E genesinto the cDNA backbone of the YF 17D vaccine.The construct was further developed by Acambis,Inc. and then licensed to Sanofi Pasteur (Fig. 2and Table 2 (CYD Dengue vaccine)).150–154 Clin-ical trials of monovalent and tetravalent vaccinepreparations demonstrated excellent safety and im-munogenicity across volunteers of various ages,genetic backgrounds, and flavivirus priming sta-tus (i.e., to individuals with pre-existing immunityto Yellow fever virus, dengue virus, and Japanese

encephalitis virus).155–161 Sanofi’s chimeric Yellowfever–dengue virus (CYD; ChimeriVax) was thefirst dengue vaccine candidate tested in a clini-cal end-point efficacy study. The phase IIb trialwas an observer-blinded, randomized, controlled,single center trial in healthy Thai schoolchildren(N = 4002) aged 4–11 years who were randomlyassigned (2:1) to receive three injections of denguevaccine or control (rabies vaccine or placebo) at0, 6, and 12 months. All subjects were followedfor dengue illness. The primary objective was toassess protective efficacy against virologically con-firmed, symptomatic dengue, irrespective of sever-ity or DENV type, occurring 1 month or longer afterthe third injection. Although the vaccine was safeand neutralizing antibody responses were moderateto high, overall efficacy was 30.2% (95% CI 13.4–56.6), and differed by serotype.48 Sequencing of cir-culating DENVs compared to vaccine strains and



Figure 1. National Institute of Allergy and Infectious Diseases (NIAID) TetraVax-DV-TV003 candidate (TV003). Recombinantattenuated DENV vaccine candidates were constructed by deletion of nucleotides from the 3′ UTR (Δ30 and Δ30/31) or bychimerization of genomic regions from different serotypes (preM and E genes from DENV-2 chimerized into DENV-4�30).Provided courtesy of NIAID.

cross-neutralization experiments with vaccine vol-unteer sera failed to prove wild-type strains escapedvaccine-induced immunity.162,163 The results of thistrial were disappointing and challenged the PRNTassay platform and neutralizing antibody readout asa correlate of protection. Further studies of phase IIband phase III trials in Latin America and Asia (totalenrollment >25,000) continue with results expectedin 2014.

The U.S. Centers for Disease Control andPrevention (CDC) developed a tetravalent chimericdengue vaccine candidate by introducing DENV-1,DENV-3, and DENV-4 prM and E genes into cDNAderived from an attenuated LAV DENV-2 compo-nent (Fig. 3 and Table 2 (DENVax)).164–168 DENV-DENV chimeras were formulated as a DENV-1/DENV-2, DENV-3/DENV-2, and DENV-4/DENV-2 tetravalent vaccines and licensed toInviragen, Inc., which was recently acquired byTakeda Pharmaceutical Company Limited.169,170

The DENVax candidate had an acceptable safetyand immunogenicity profile in small phase Istudies of flavivirus-naive adults following sub-cutaneous or intradermal delivery. A phase II agede-escalation trial is underway in four endemiccountries to assess the safety and immunogenicityof two subcutaneous doses (days 0 and 90) in1.5–45 year olds. Thus far, vaccination has been welltolerated, with mostly mild and transient local orsystemic reactions. A preliminary analysis revealedthat after one or two doses 98.8% of subjects had atrivalent or better antibody profile, and 87.2% hada tetravalent profile when measured 1 month afterdose 2.171 Tetravalent formulations with variations

in the DENV-4 viral concentration (delivery oftwo doses at day 0 with or without additionaldoses at day 90) and variation in delivery method(intramuscular, intradermal, needle, needlessdevice delivery) are all being explored.172

Inactivated whole virus or viral subunit vaccineshave potential advantages, including the inability torevert to a more pathogenic phenotype, lack of im-mune interference when combined in a tetravalentformulation, and, theoretically, fewer acute safetyissues. Inactivated flaviviral vaccines have been li-censed and are in wide use to prevent Japanese en-cephalitis and tick-borne encephalitis.173–175 Poten-tial disadvantages include inducing antibodies toonly a portion of the structural proteins and de-viation from wild-type structural antigen confor-mations. High antigen concentrations and multi-ple doses may also be required. There is potentialfor an adverse response following natural infection,such as was observed with the occurrence of atypicalmeasles and respiratory syncytial virus (RSV).176,177

Merck & Co. is developing a dengue vaccine can-didate produced in a Drosophila S2 cell expressionsystem (Fig. 4 and Table 2 (V180)).178–180 Nonhu-man primate studies assessing persistence of an-tibody responses and impact of DENV primingon immunogenicity are being completed. A doseranging study of the candidate (V180) in humansis underway in Australia. Combining three doses(months 0, 1, and 2) of V180 with a range of dosesof ISCOMATRIXTM adjuvant is being evaluated inthe same study.181

The WRAIR developed a purified inactivate virus(PIV) dengue vaccine candidate by inactivation of



Figure 2. Sanofi Pasteur CYD (chimeric-Yellow fever-dengue) Dengue Vaccine. The CYD TDV candidate is composed of fourrecombinant, live, attenuated vaccines (CYD-1–4) based on a yellow fever vaccine 17D (YFV 17D) backbone, each expressing thepremembrane and envelope genes of one of the four dengue virus serotypes.

each of the DENV types with formalin.180,182–184

A phase I trial of a monovalent DENV-1 PIVadjuvanted with alum was completed in the UnitedStates in a small number of flavivirus-naive vol-unteers. The candidate vaccine had an acceptablesafety profile with low to moderate immunogenic-ity following two doses administered on days 0and 28 (TDEN-PIV, Table 2). There was minimalcross-reactive antibody and titers were short lived;cellular responses are currently being assessed (un-published data, SJT). The U.S. Army granted an ex-clusive license to GlaxoSmithKline Vaccines (GSK)and the two are codeveloping, with the OswaldoCruz Foundation (FioCruz), a tetravalent PIV for-mulation (DPIV) adjuvanted with aluminum hy-droxide (AlOH) and GSK’s proprietary AdjuvantSystems (AS). Phase I trials in the United Statesand Puerto Rico are exploring DPIV candidatesadjuvanted with AlOH, AS01E, and AS03B. Datathrough study day 56 (dose 1 at day 0, dose 2 at day28) in the U.S. trial indicate a well-tolerated vac-cine. Day 56 neutralizing antibody measurementsin dengue-naive volunteers determined by 50% MNassay demonstrated 53%, 94%, 92%, and 100% ofsubjects who received two doses of 1 �g DPIV pertype + AlOH, 4 �g + AlOH, 1 �g + AS01E, and 1 �g+AS03B, respectively, experienced tetravalent sero-conversions. DENV-1–4 type–specific GMTs werehigher in the 1 �g + AS03B (526, 341, 468, and 406)and 1 �g + AS01E groups (411, 485, 540, and 307).Analysis of cell-mediated immune responses andneutralizing antibody determinations remote from

vaccination (i.e., >6 months) are ongoing.108 Allvaccinations have been administered in Puerto Ricoand are pending analysis.

DNA vaccines consist of a plasmid (or plasmids)containing DENV genes reproduced in bacteria suchas Escherichia coli (Fig. 5 and Table 2 (TVDV)). Theplasmid contains a eukaryotic promoter and termi-nation sequence to drive transcription and presen-tation to the immune system. Potential advantagesof DNA-based vaccine include ease of production,stability, ability to add new genes, and ability toimmunize against multiple pathogens with a singleconstruct.185 A DENV-1 monovalent DNA vaccinetrial enrolled 22 flavivirus–naive U.S. volunteers andadministered three doses at months 0, 1, and 5. Noneof the low-dosage recipients and only 5 of 11 high-dosage recipients developed neutralizing antibodies.The safety profile was acceptable and supported ex-ploration of a tetravalent adjuvanted DNA vaccinecandidate; final results are pending.99

Unanswered questions and future directionsThe dengue vaccine field is confronting numerousquestions, especially in the aftermath of CYD’s lim-ited success in phase IIb. The exploration of DENVevolution in time and space introduces the con-cern that even minor degrees of antigen mismatchbetween vaccine and circulating wild-type virusescould significantly affect DENV type–specific andoverall efficacy. This was a leading hypothesis forCYD’s phase IIb performance (DENV-2 efficacy�9%), but currently available data would not



Figure 3. Inviragen Inc., DENVax. Formulations of chimeric dengue vaccine (DENVax) viruses containing the premembrane(prM) and envelope (E) genes of serotypes 1–4 expressed in the context of the attenuated DENV-2 PDK-53 genome.

support this conclusion. Regardless, there is an ex-tensive and growing body of data describing DENVepidemiologic trends and the potential that vari-ations in circulating genotypes may influence ob-served clinical phenotypes.186–190 There are indica-tions that only a few strategically placed mutationsin the DENV genome may be necessary to have sig-nificant impacts on its ability to be neutralized.191

The most significant implication would be the re-curring requirement for developers to update theirlicensed vaccines with contemporary DENV strains(e.g., similar to influenza but with a more protractedtimeline). Epidemiologic observations of prolongedhomotypic protection, perhaps lifelong, followinginfection would argue against this possibility. How-ever, the immune profile imparted by sequential nat-ural infections with full DENV genomes is (at leastto the present author) likely to be different than thatgenerated by a simultaneous exposure to four at-tenuated vaccine DENV strains. Largescale studiesevaluating the association of genotype and clinicalillness severity would be informative. Postlicensurestudies of DENV evolution measured in a definedtime and space, and the impact of large-scale vacci-nation on the same, are prudent.

The development, optimization, and applicationof assay platforms to measure vaccine immuno-genicity require additional work. It is clear fromthe CYD study that in vitro neutralizing antibodiesmeasured in a vaccinated subgroup did not predictthe overall in vivo clinical experience following in-fection; measured neutralizing antibody titers didnot predict protection, especially for DENV-2. It ispossible there were issues with how the immuno-genicity subgroup was selected, and that their im-munologic experience following vaccination or risk

of infection and disease did not reflect that of thelarger cohort.192–194 It is imperative that the risk ofinfection and potential for disease experienced bythe subgroup must both accurately represent the en-tire cohort’s and be balanced between vaccine andplacebo/control recipients. The numerous factorsthat contribute to these risks, such as pre-existingimmunologic background, living conditions (i.e.,water source), transmission trends throughout theyear (high and low vector and transmission activ-ity), and daily living activities (indoor vs. outdoor),make this a difficult task requiring well conceivedenrollment strategies.

Another potential reason for the apparentdisconnect between immunogenicity and protec-tion may be the measurement and interpretationof heterotypic cross-reactive nonneutralizingantibody as homotypic DENV type–specific anti-body. The former may do nothing, attenuate, orcontribute to increased disease severity followingwild-type DENV exposure, while the latter isexpected to provide decades of protection fromdisease following infection with the same DENVtype.74,195–197 Unfortunately, it is very difficultto discern between the two using conventionalassay platforms following secondary and repeatedDENV exposures. Assays incorporating heterotypicantibody depletion steps, using constant serumconcentration with varied viral input (log neutral-ization index), or incorporating human cell lines orcell lines bearing Fc�R may improve the signal (ho-motypic) to noise (heterotypic) ratio.50,87,88,198,199

Requirements for sample volume, throughput,automation potential, and cost will influenceapplicability of new assay platforms into large-scaletrials.



Figure 4. Merck Sharp & Dohme Corp., V180. The truncateddengue envelope proteins (DEN-80E) for all four dengue virustypes are expressed in the Drosophila S2 cell expression at highlevels and have been shown to maintain native-like conforma-tion.

The immune response following natural DENVexposure or following vaccination are kinetic andmature from a cross-reactive, and for a period ofmonths cross-protective, response to a monotypicand DENV type–specific response. Measurement ofimmune responses 1 month following exposure toa tetravalent dengue vaccine may inform developersabout acute immunogenicity, but not potential forefficacy. In practical terms, a subgroup’s neutralizingantibody profile measured 1 month following vacci-nation may predict high efficacy against a particularDENV type, but if measured 6 months followingvaccination and after a period of maturation, theefficacy assessment may be significantly different.One recommendation would be for developers tocollect blood samples required to complete acute(1 month), subacute (6–9 months), and longerterm (1 year and greater) analyses that as-sociate immunogenicity with clinical end-pointdata.

The limited success of the CYD candidate in thephase IIb trial emphasizes the need to continueand explore contributions of cellular immunity toimmuno-protective profiles. Many of the CD4+ andCD8+ T cell epitopes are located in the NS proteins(NS1, 3, 5).42 Vaccine candidates without DENVNS proteins, or NS proteins representative of onlyone DENV type, may fail to activate the compre-hensive immune response required to protect thevaccine recipient from dengue disease caused by anyDENV type. Quantitative and qualitative/functional

assay platforms (e.g., intracellular cytokine stain-ing (ICS), Luminex, ELISA, and ELISPOT) are be-ing used early in the vaccine development processto assess DENV type–specific immunogenicity andcomplement neutralizing antibody data. How theinformation is being used to support developmentdecisions is unclear. Interpreting a vaccine can-didate’s effect on cellular immunity in flavivirus-primed volunteers is challenging, and contributionsof anti-DENV cellular immunity to the overall hu-moral immune response are less well described.43

Continued exploration and deconstruction of cel-lular immune responses following natural DENVinfections (primary and subsequent), with compar-ison to the same responses following vaccination, isrequired. Prospective collection of adequate bloodsamples is required to associate preillness cellularimmune profiles with clinical endpoint and out-come data in natural infection and vaccine studies.

Live virus vaccines are subject to immune in-terference following administration and suboptimalimmune responses. Each DENV type has unique vi-rologic (i.e., infection, replication) properties thatmay be sustained following attenuation and inclu-sion into tetravalent dengue vaccine200,201 formula-tions. This combined with baseline flavivirus immu-nity, from previous infection with dengue, Japaneseencephalitis or Yellow fever viruses or by vaccina-tion, may shape vaccine immunogenicity such thatone or two DENV types dominate the infection,antigen replication process, and subsequent overallimmune response. Certain DENV vaccine strainswill be efficacious, while others will offer some orno protection. While some vaccine developers ad-vance candidate vaccines composed of equal viralconcentrations for each DENV type, others manip-ulate the degree of attenuation and viral concen-tration of each DENV component.127 As discussedabove, the concept of DENV type–specific protec-tion threshold versus quantitative balance shouldbe considered (personal view, SJT). Comprehensivedose ranging–studies that evaluate the interplay be-tween the DENV types are necessary early in vaccinedevelopment programs, especially with replicatingvaccine candidates.

Central to many of the challenges and unan-swered questions in dengue vaccinology is the ab-sence of a known correlate of protection or validatedanimal disease model. Although work continueson the development of small animal or nonhuman



Figure 5. U.S. Army Medical Research and Materiel Command/Naval Medical Research Center Tetravalent Dengue Vaccine(TVDV). Regulatory elements (CMV promoter, CMV intron A, BGH terminator) are indicated by shaded boxes. Coding sequences(prM/E, Kan-r) and their orientation are indicated by arrows. The PCR insert was subcloned using the indicated 3′ BamHI restrictionsite and a hybrid XhoI/SalI site at the 5′ terminus of the inserted prM/E gene (not shown). Provided courtesy of the U.S. NavalMedical Research Center.

primate models that more accurately approximatehuman in vivo infection and disease experience,prospects in the near term remain limited. Conse-quently, a dengue human infection model (DHIM)is receiving increasing attention. As of 2013 over650 human infection experiments have been de-scribed. A well-characterized and consistently per-forming DHIM could help reduce risks associatedwith vaccine development and allow for a glimpseof potential efficacy early in development. Early ter-mination of vaccine candidates unable to protectrecipients from challenge with DHIM strains wouldprevent exposing large numbers of people in clini-cal endpoint trials to poor vaccine candidates. Theabsence of a DENV-specific antiviral agent raises

ethical questions and the need for an informed riskassessment.

Conclusion

The global dengue burden is increasing, and ev-ery indication is consistent with the conclusionthat this trend will continue to worsen unless aneffective vaccine is found. Unfortunately, numerousobstacles hamper the development and licensure ofa safe and efficacious vaccine. A number of candi-dates are in preclinical and clinical development rep-resenting diverse approaches. A recent efficacy trialyielded disappointing results and challenged longheld beliefs regarding associations between vac-cine immunogenicity and potential efficacy. Dengue



virus evolution, measurement of homotypic versusheterotypic antibody responses, durable immunity,and animal and human disease models all requireexpanded exploration and study.

Conflicts of interest

SJT is an employee of the U.S. Government. Hisassigned duties include working with vaccine devel-opers listed in this manuscript. Disclosure is madein the spirit of transparency not because a conflictof interest is believed to exist. The opinions or as-sertions contained herein are the private views ofthe author and are not to be construed as reflect-ing the official views of the U.S. Army or the U.S.Department of Defense.

References

1. Bhatt, S. et al. 2013. The global distribution and burden ofdengue. Nature 496: 504–507.

2. Murray, N.E., M.B. Quam & A. Wilder-Smith. 2013. Epi-demiology of dengue: past, present and future prospects.Clin Epidemiol 5: 299–309.

3. Shepard, D.S., E.A. Undurraga & Y.A. Halasa. 2013. Eco-nomic and disease burden of dengue in southeast Asia.PLoS Negl Trop Dis 7: e2055.

4. Wettstein, Z.S. et al. 2012. Total economic cost and burdenof dengue in Nicaragua: 1996–2010. Am J Trop Med Hyg87: 616–622.

5. Tam, P.T. et al. 2012. High household economic burdencaused by hospitalization of patients with severe denguefever cases in Can Tho province, Vietnam. Am J Trop MedHyg 87: 554–558.

6. Halasa, Y.A., D.S. Shepard & W. Zeng. 2012. Economic costof dengue in Puerto Rico. Am J Trop Med Hyg 86: 745–752.

7. Gubler, D.J. 2012. The economic burden of dengue. Am JTrop Med Hyg 86: 743–744.

8. Shepard, D.S. et al. 2011. Economic impact of dengue illnessin the Americas. Am J Trop Med Hyg 84: 200–207.

9. Lee, B.Y. et al. 2011. Economic value of dengue vaccine inThailand. Am J Trop Med Hyg 84: 764–772.

10. Carrasco, L.R. et al. 2011. Economic impact of dengueillness and the cost-effectiveness of future vaccination pro-grams in Singapore. PLoS Negl Trop Dis 5: e1426.

11. Beatty, M.E. et al. 2011. Health economics of dengue: asystematic literature review and expert panel’s assessment.Am J Trop Med Hyg 84: 473–488.

12. Garg, P. et al. 2008. Economic burden of dengue infectionsin India. Trans R Soc Trop Med Hyg 102: 570–577.

13. Canyon, D.V. 2008. Historical analysis of the economic costof dengue in Australia. J Vector Borne Dis 45: 245–248.

14. Armien, B. et al. 2008. Clinical characteristics and nationaleconomic cost of the 2005 dengue epidemic in Panama. AmJ Trop Med Hyg 79: 364–371.

15. Torres, J.R. & J. Castro. 2007. The health and economicimpact of dengue in Latin America. Cadernos de saudepublica 23(Suppl. 1): S23–S31.

16. Harving, M.L. & F.F. Ronsholt. 2007. The economic impactof dengue hemorrhagic fever on family level in SouthernVietnam. Dan Med Bull 54: 170–172.

17. Clark, D.V. et al. 2005. Economic impact of denguefever/dengue hemorrhagic fever in Thailand at the fam-ily and population levels. Am J Trop Med Hyg 72:786–791.

18. Sanchez-Vegas, C. et al. 2013. Prevalence of dengue virusinfection in US travelers who have lived in or traveled todengue-endemic countries. J Travel Med 20: 352–360.

19. Jensenius, M. et al. 2013. Acute and potentially life-threatening tropical diseases in western travelers—aGeoSentinel multicenter study, 1996–2011. Am J Trop MediHyg 88: 397–404.

20. Wattal, C. & N. Goel. 2012. Infectious disease emergenciesin returning travelers: special reference to malaria, denguefever, and chikungunya. Med Clin North Am 96: 1225–1255.

21. Mizuno, Y. et al. 2012. Imported malaria and dengue feverin returned travelers in Japan from 2005 to 2010. TravelMed Infect Dis 10: 86–91.

22. Hynes, N.A. 2012. Dengue: a reemerging concern for trav-elers. Cleve Clin J Med 79: 474–482.

23. Chen, L.H. & M.E. Wilson. 2012. Dengue and chikungunyain travelers: recent updates. Curr Opin Infect Dis 25: 523–529.

24. Chen, L.H. & M.E. Wilson. 2010. Dengue and chikun-gunya infections in travelers. Curr Opin Infect Dis 23:438–444.

25. Marano, C. & D.O. Freedman. 2009. Global health surveil-lance and travelers’ health. Curr Opin Infect Dis 22: 423–429.

26. Gibbons, R.V. et al. 2012. Dengue and US military op-erations from the Spanish-American War through today.Emerg Infect Dis 18: 623–630.

27. Trofa, A.F. et al. 1997. Dengue fever in US military person-nel in Haiti. JAMA 277: 1546–1548.

28. Defraites, R. et al. 1995. Dengue fever among US militarypersonnel—Haiti, September- November, 1994 (reprintedfrom MMWR, vol 43, pg 845–848, 1994). JAMA 273: 14–15.

29. Centers for Disease, C. & Prevention. 1994. Denguefever among U.S. military personnel—Haiti, September-November, 1994. MMWR. Morbid Mortal Weekly Rep 43:845–848.

30. Hayes, C.G. et al. 1989. Dengue fever in American militarypersonnel in the Philippines: clinical observations on hos-pitalized patients during a 1984 epidemic. Southeast AsianJ Trop Med Public Health 20: 1–8.

31. Dengue Guidelines for Diagnosis, Treatment, Preventionand Control. Geneva: World Health Organization. 2009.http://whqlibdoc.who.int/publications/2009/9789241547871_eng.pdf?ua=1. Accessed Feb 24, 2014

32. Gubler, D.J. 2011. Dengue, urbanization and globalization:the unholy trinity of the 21(st) century. Trop Med Health39: 3–11.

33. Ooi, E.E., K.T. Goh & D.J. Gubler. 2006. Dengue preventionand 35 years of vector control in Singapore. Emerg InfectDis 12: 887–893.

34. McMeniman, C.J. et al. 2009. Stable introduction of a life-shortening Wolbachia infection into the mosquito Aedesaegypti. Science 323: 141–144.



35. Iturbe-Ormaetxe, I., T. Walker & O.N. SL. 2011. Wol-bachia and the biological control of mosquito-borne dis-ease. EMBO Rep 12: 508–518.

36. Harris, A.F. et al. 2011. Field performance of engineeredmale mosquitoes. Nat Biotechnol 29: 1034–1037.

37. James, S., C.P. Simmons & A.A. James. 2011. Ecology.Mosquito trials. Science 334: 771–772.

38. Gratz, N.G. & S.B. Halstead. 2008. “The control of denguevectors.” In Dengue, Vol. 5. S.B. Halstead, Ed.: 361–387.London: Imperial College Press.

39. Cardosa, J. et al. 2009. Dengue virus serotype 2 from asylvatic lineage isolated from a patient with dengue hem-orrhagic fever. PLoS Negl Trop Dis 3: e423.

40. Vasilakis, N. et al. 2011. Fever from the forest: prospectsfor the continued emergence of sylvatic dengue virus andits impact on public health. Nature reviews. Microbiology9: 532–541.

41. Weaver, S.C. & N. Vasilakis. 2009. Molecular evolution ofdengue viruses: contributions of phylogenetics to under-standing the history and epidemiology of the preeminentarboviral disease. Infect Genet Evol 9: 523–540.

42. Rothman, A.L. 2011. Immunity to dengue virus: a tale oforiginal antigenic sin and tropical cytokine storms. Nat RevImmunol 11: 532–543.

43. Thakur, A., L.E. Pedersen & G. Jungersen. 2012. Immunemarkers and correlates of protection for vaccine inducedimmune responses. Vaccine 30: 4907–4920.

44. Srikiatkhachorn, A. et al. 2012. Dengue viral RNA levels inperipheral blood mononuclear cells are associated with dis-ease severity and preexisting dengue immune status. PloSOne 7: e51335.

45. Rothman, A.L. 2004. Dengue: defining protective versuspathologic immunity. J Clin Invest 113: 946–951.

46. Halstead, S.B. & E.J. O’Rourke. 1977. Antibody-enhanceddengue virus infection in primate leukocytes. Nature 265:739–741.

47. Ubol, S. & S.B. Halstead. 2010. How innate immune mech-anisms contribute to antibody-enhanced viral infections.Clin Vacc Immunol 17: 1829–1835.

48. Sabchareon, A. et al. 2012. Protective efficacy of the recom-binant, live-attenuated, CYD tetravalent dengue vaccine inThai schoolchildren: a randomised, controlled phase 2btrial. Lancet 380: 1559–1567.

49. Chanthavanich, P. et al. 2006. Short report: immune re-sponse and occurrence of dengue infection in thai childrenthree to eight years after vaccination with live attenuatedtetravalent dengue vaccine. Am J Trop Med Hyg 75: 26–28.

50. Mason, R.A. et al. 1973. Yellow fever vaccine: direct chal-lenge of monkeys given graded doses of 17D vaccine. ApplMicrobiol 25: 539–544.

51. Hombach, J. et al. 2005. Report on a WHO consultation onimmunological endpoints for evaluation of new Japaneseencephalitis vaccines, WHO, Geneva, 2–3 September, 2004.Vaccine 23: 5205–5211.

52. Hombach, J. et al. 2007. Scientific consultation on im-munological correlates of protection induced by denguevaccines report from a meeting held at the World HealthOrganization 17–18 November 2005. Vaccine 25: 4130–4139.

53. Thomas, S.J., J. Hombach & A. Barrett. 2009. Scien-tific consultation on cell mediated immunity (CMI) indengue and dengue vaccine development. Vaccine 27:355–368.

54. Cassetti, M.C. et al. 2010. Report of an NIAID workshopon dengue animal models. Vaccine 28: 4229–4234.

55. Zompi, S. & E. Harris. 2012. Animal models of denguevirus infection. Viruses 4: 62–82.

56. Akkina, R. 2013. New generation humanized mice for virusresearch: comparative aspects and future prospects. Virol-ogy 435: 14–28.

57. Akkina, R. 2013. Human immune responses and poten-tial for vaccine assessment in humanized mice. Curr OpinImmunol 25: 403–409.

58. Harrison, V.R. et al. 1977. Virulence and immunogenicity ofa temperature-sensitive dengue-2 virus in lower primates.Infect Immun 18: 151–156.

59. Rosen, L. 1958. Experimental infection of New World mon-keys with dengue and yellow fever viruses. Am J Trop MedHyg 7: 406–410.

60. Halstead, S.B. et al. 1973. Studies on the immunization ofmonkeys against dengue. I: protection derived from singleand sequential virus infections. Am J Trop Med Hyg 22:365–374.

61. Halstead, S.B. & N.E. Palumbo. 1973. Studies on the im-munization of monkeys against dengue. II: protection fol-lowing inoculation of combinations of viruses. Am J TropMed Hyg 22: 375–381.

62. Halstead, S.B., H. Shotwell & J. Casals. 1973. Studies on thepathogenesis of dengue infection in monkeys. I: clinicallaboratory responses to primary infection. J Infect Dis 128:7–14.

63. Halstead, S.B., H. Shotwell & J. Casals. 1973. Studies on thepathogenesis of dengue infection in monkeys. II: clinicallaboratory responses to heterologous infection. J Infect Dis128: 15–22.

64. Marchette, N.J. & S.B. Halstead. 1973. Survival of Denguevirus in post mortem samples of tissues from experimen-tally infected Rhesus monkeys. Am J Trop Med Hyg 22:242–243.

65. Marchette, N.J. et al. 1973. Studies on the pathogenesis ofdengue infection in monkeys. 3: sequential distribution ofvirus in primary and heterologous infections. J Infect Dis128: 23–30.

66. Marchette, N.J. et al. 1973. Studies on the pathogenesis ofdengue infection in monkeys. III: sequential distributionof virus in primary and heterologous infections. J Infect Dis128: 23–30.

67. Graham, H. 1902. Dengue: a study of its mode of propaga-tion and pathology. Med Rec NY 61: 204–207.

68. Graham, H. 1903. The dengue: a study of its pathology andmode of propagation. J. Trop. Med. 209–214.

69. Ashburn, P.M. & C.F. Craig. 1907. Experimental investiga-tions regarding the etiology of dengue fever. J Infect Dis 4:440–475.

70. Siler, J.F., M.W. Hall & A.P. Hitchens. 1926. Dengue: Itshistory, epidemilogy, mechanism of transmission, etiology,clinical manifestations, immunity, and prevention. PhilippJ Sci 29: 1–304.



71. Cleland, J.B., B. Bradley & W. Macdonald. 1919. Furtherexperiments in the etiology of dengue fever. J Hyg 18: 217–254.

72. Simmons, J.S., J.H. St John & F.H.K. Reynolds. 1931. Ex-perimental studies of dengue. Philipp J Sci 44: 1–252.

73. Sawada, T., H. Sato & S. Sai. 1943. On the experimentaldengue infection in man. Nippon Igaku 3325: 529–531.

74. Sabin, A.B. 1952. Research on dengue during World WarII. Am J Trop Med Hyg 1: 30–50.

75. Sabin, A.B. & R.W. Schlesinger. 1945. Production of im-munity to dengue with virus modified by propagation inmice. Science 101: 640–642.

76. McCoy, O.R. & A.B. Sabin. 1946. “Dengue.” In PreventiveMedicine in World War II. Communicable Diseases. Arthro-podborne Diseases Other Than Malaria, Vol. VII. J.B. Coates,E.C. Hoff & P.M. Hoff, Eds.: 29–62. Washington, DC: Officeof the Surgeon General.

77. Hotta, S. 1952. Experimental studies on dengue. I: isolation,identification and modification of the virus. J Infect Dis 90:1–9.

78. Yaoi, H. 1958. A summary of our studies on dengue. YokohMed Bull 9: 1–20.

79. Mammen, M.P., A. Lyons, B.L. Innis, et al. 2014. Evaluationof dengue virus strains for human challenge studies. Vaccine32: 1488–1494.

80. Sun, W. et al. 2013. Experimental dengue virus challenge ofhuman subjects previously vaccinated with live attenuatedtetravalent dengue vaccines. J Infect Dis 207: 700–708.

81. Statler, J. et al. 2008. Sonographic findings of healthy vol-unteers infected with dengue virus. J Clin Ultrasound 36:413–417.

82. Gunther, V.J. et al. 2011. A human challenge model fordengue infection reveals a possible protective role for sus-tained interferon gamma levels during the acute phase ofillness. Vaccine 29: 3895–3904.

83. Roehrig, J.T., J. Hombach & A.D. Barrett. 2008. Guidelinesfor plaque-reduction neutralization testing of human anti-bodies to dengue viruses. Viral Immunol 21: 123–132.

84. Russell, P.K. et al. 1967. A plaque reduction test for denguevirus neutralization antibodies. J Immunol 99: 285–290.

85. Thomas, S.J. et al. 2009. Dengue plaque reduction neu-tralization test (PRNT) in primary and secondary denguevirus infections: how alterations in assay conditions impactperformance. Am J Trop Med Hyg 81: 825–833.

86. Rainwater-Lovett, K. et al. 2012. Variation in dengue virusplaque reduction neutralization testing: systematic reviewand pooled analysis. BMC Infect Dis 12: 233.

87. Chawla, T. et al. 2013. Dengue virus neutralization in cellsexpressing Fc gamma receptors. PloS One 8: e65231.

88. Wu, R.S. et al. 2012. Neutralization of dengue virus inthe presence of Fc receptor-mediated phagocytosis distin-guishes serotype-specific from cross-neutralizing antibod-ies. Antiviral Res 96: 340–343.

89. Rodrigo, W.W. et al. 2009. Dengue virus neutralization ismodulated by IgG antibody subclass and Fcgamma recep-tor subtype. Virology 394: 175–182.

90. Rodrigo, W.W. et al. 2009. An automated dengue virusmicroneutralization plaque assay performed in human

Fc{gamma} receptor-expressing CV-1 cells. Am J Trop MedHyg 80: 61–65.

91. de Alwis, R. et al. 2012. Identification of human neutral-izing antibodies that bind to complex epitopes on denguevirions. Proc Natl Acad Sci USA 109: 7439–7444.

92. Putnak, J.R. et al. 2008. Comparative evaluation of threeassays for measurement of dengue virus neutralizing anti-bodies. Am J Trop Med Hyg 79: 115–122.

93. Martin, N.C. et al. 2006. An immunocytometric assaybased on dengue infection via DC-SIGN permits rapidmeasurement of anti-dengue neutralizing antibodies. J Vi-rol Methods 134: 74–85.

94. Balas, C. et al. 2011. Different innate signatures inducedin human monocyte-derived dendritic cells by wild-typedengue 3 virus, attenuated but reactogenic dengue 3 vaccinevirus, or attenuated nonreactogenic dengue 1–4 vaccinevirus strains. J Infect Dis 203: 103–108.

95. Guy, B. 2009. Immunogenicity of sanofi pasteur tetravalentdengue vaccine. J Clin Virol 46(Suppl. 2): S16–S19.

96. Guy, B. et al. 2008. Cell-mediated immunity induced bychimeric tetravalent dengue vaccine in naive or flavivirus-primed subjects. Vaccine 26: 5712–5721.

97. Deauvieau, F. et al. 2007. Innate immune responses in hu-man dendritic cells upon infection by chimeric yellow-feverdengue vaccine serotypes 1–4. Am J Trop Med Hyg 76: 144–154.

98. Sanchez, V. et al. 2006. Comparison by flow cytometryof immune changes induced in human monocyte-deriveddendritic cells upon infection with dengue 2 live-attenuatedvaccine or 16681 parental strain. FEMS Immunol Med Mi-crobiol 46: 113–123.

99. Beckett, C.G. et al. 2011. Evaluation of a prototype dengue-1 DNA vaccine in a phase 1 clinical trial. Vaccine 29: 960–968.

100. Rothman, A.L. et al. 2001. Induction of T lymphocyte re-sponses to dengue virus by a candidate tetravalent live at-tenuated dengue virus vaccine. Vaccine 19: 4694–4699.

101. Torresi, J., R. Tapia-Conyer & H. Margolis. 2013. Preparingfor dengue vaccine introduction: recommendations fromthe 1st Dengue v2V International Meeting. PLoS Negl TropDis 7: e2261.

102. Mahoney, R. et al. 2011. Dengue vaccines regulatory path-ways: a report on two meetings with regulators of develop-ing countries. PLoS Med 8: e1000418.

103. Dorrance, W.R. et al. 1956. Clinical and serologic responseof man to immunization with attenuated dengue and yel-low fever viruses. J Immunol 77: 352–364.

104. Live Dengue Vaccines Technical Consultation Reporting, G.et al. 2013. Long-term safety assessment of live attenuatedtetravalent dengue vaccines: deliberations from a WHOtechnical consultation. Vaccine 31: 2603–2609.

105. Edelman, R. & J. Hombach. 2008. “Guidelines for the clin-ical evaluation of dengue vaccines in endemic areas”: sum-mary of a World Health Organization Technical Consulta-tion. Vaccine 26: 4113–4119.

106. Vorndam, V. & M. Beltran. 2002. Enzyme-linked im-munosorbent assay-format microneutralization test fordengue viruses. Am J Trop Med Hyg 66: 208–212.



107. Jirakanjanakit, N. et al. 1997. The micro-focus reductionneutralization test for determining dengue and Japaneseencephalitis neutralizing antibodies in volunteers vacci-nated against dengue. Trans R Soc Trop Med Hyg 91: 614–617.

108. Schmidt A et al. 2013. Phase 1 study of a tetravalent denguepurified inactivated virus (DPIV) vaccine in healthy U.S.adults. In American Society of Tropical Medicine and Hy-giene Annual Meeting. Washington, DC.

109. Pulendran, B. 2009. Learning immunology from the yel-low fever vaccine: innate immunity to systems vaccinology.Nature reviews. Immunology 9: 741–747.

110. Querec, T.D. et al. 2009. Systems biology approach predictsimmunogenicity of the yellow fever vaccine in humans. NatImmunol 10: 116–125.

111. Ohrr, H. et al. 2005. Effect of single dose of SA 14–14–2 vaccine 1 year after immunisation in Nepalese childrenwith Japanese encephalitis: a case-control study. Lancet 366:1375–1378.

112. Yu, Y. 2010. Phenotypic and genotypic characteristics ofJapanese encephalitis attenuated live vaccine virus SA14–14–2 and their stabilities. Vaccine 28: 3635–3641.

113. Halstead, S.B. & N.J. Marchette. 2003. Biologic propertiesof dengue viruses following serial passage in primary dogkidney cells: studies at the University of Hawaii. Am J TropMed Hyg 69: 5–11.

114. Bhamarapravati, N. et al. 1987. Immunization with a liveattenuated dengue-2-virus candidate vaccine (16681-PDK53): clinical, immunological and biological responses inadult volunteers. Bull World Health Org 65: 189–195.

115. Bhamarapravati, N. & S. Yoksan. 2000. Live attenu-ated tetravalent dengue vaccine. Vaccine 18(Suppl. 2):44–47.

116. Kanesa-thasan, N. et al. 2001. Safety and immunogenicityof attenuated dengue virus vaccines (Aventis Pasteur) inhuman volunteers. Vaccine 19: 3179–3188.

117. Bhamarapravati, N. & S. Yoksan. 1989. Study of bivalentdengue vaccine in volunteers. Lancet 1: 1077.

118. Sabchareon, A. et al. 2002. Safety and immunogenicity oftetravalent live-attenuated dengue vaccines in Thai adultvolunteers: role of serotype concentration, ratio, and mul-tiple doses. Am J Trop Med Hyg 66: 264–272.

119. Hombach, J. et al. 2005. Review on flavivirus vaccine devel-opment. Proceedings of a meeting jointly organised by theWorld Health Organization and the Thai Ministry of PublicHealth, 26–27 April 2004, Bangkok, Thailand. Vaccine 23:2689–2695.

120. Kitchener, S. et al. 2006. Immunogenicity and safety of twolive-attenuated tetravalent dengue vaccine formulations inhealthy Australian adults. Vaccine 24: 1238–1241.

121. Sanchez, V. et al. 2006. Innate and adaptive cellular im-munity in flavivirus-naive human recipients of a live-attenuated dengue serotype 3 vaccine produced in Verocells (VDV3). Vaccine 24: 4914–4926.

122. Sabin, A.B. 1955. Recent advances in our knowledge ofdengue and sandfly fever. Am J Trop Med Hyg 4: 198–207.

123. Innis, B.L. et al. 1988. Virulence of a live dengue virusvaccine candidate: a possible new marker of dengue virusattenuation. J Infect Dis 158: 876–880.

124. McKee, K.T., Jr. et al. 1987. Lack of attenuation of a candi-date dengue 1 vaccine (45AZ5) in human volunteers. Am JTrop Med Hyg 36: 435–442.

125. Hoke, C.H., Jr. et al. 1990. Preparation of an attenuateddengue 4 (341750 Carib) virus vaccine. II: safety and im-munogenicity in humans. Am J Trop Med Hyg 43: 219–226.

126. Edelman, R. et al. 1994. A live attenuated dengue-1 vaccinecandidate (45AZ5) passaged in primary dog kidney cellculture is attenuated and immunogenic for humans. J InfectDis 170: 1448–1455.

127. Edelman, R. et al. 2003. Phase I trial of 16 formulations of atetravalent live-attenuated dengue vaccine. Am J Trop MedHyg 69: 48–60.

128. Kanesa-Thasan, N. et al. 2003. Phase 1 studies of Wal-ter Reed Army Institute of Research candidate attenuateddengue vaccines: selection of safe and immunogenic mono-valent vaccines. Am J Trop Med Hyg 69: 17–23.

129. Sun, W. et al. 2003. Vaccination of human volunteers withmonovalent and tetravalent live-attenuated dengue vaccinecandidates. Am J Trop Med Hyg 69: 24–31.

130. Sun, W. et al. 2009. Phase 2 clinical trial of three for-mulations of tetravalent live-attenuated dengue vaccine inflavivirus-naive adults. Hum Vacc 5: 33–40.

131. Watanaveeradej, V. et al. 2011. Safety and immunogenicityof a tetravalent live-attenuated dengue vaccine in flavivirus-naive infants. Am J Trop Med Hyg 85: 341–351.

132. Simasathien, S. et al. 2008. Safety and immunogenicity ofa tetravalent live-attenuated dengue vaccine in flavivirusnaive children. Am J Trop Med Hyg 78: 426–433.

133. Thomas, S.J. et al. 2013. A phase II, randomized, safetyand immunogenicity study of a re-derived, live-attenuateddengue virus vaccine in healthy adults. Am J Trop Med Hyg88: 73–88.

134. Blaney, J.E., Jr. et al. 2001. Chemical mutagenesis of denguevirus type 4 yields mutant viruses which are temperaturesensitive in Vero cells or human liver cells and attenuatedin mice. J Virol 75: 9731–9740.

135. Lai, C.J. et al. 1991. Infectious RNA transcribed from stablycloned full-length cDNA of dengue type 4 virus. Proc NatAcad Sci USA 88: 5139–5143.

136. Bray, M. & C.J. Lai. 1991. Dengue virus premembrane andmembrane proteins elicit a protective immune response.Virology 185: 505–508.

137. Men, R. et al. 1996. Dengue type 4 virus mutants containingdeletions in the 3’ noncoding region of the RNA genome:analysis of growth restriction in cell culture and alteredviremia pattern and immunogenicity in rhesus monkeys. JVirol 70: 3930–3937.

138. Durbin, A.P. et al. 2005. rDEN4delta30, a live attenuateddengue virus type 4 vaccine candidate, is safe, immuno-genic, and highly infectious in healthy adult volunteers. JInfect Dis 191: 710–718.

139. Blaney, J.E., Jr. et al. 2006. Development of a live attenuateddengue virus vaccine using reverse genetics. Viral Immunol19: 10–32.

140. McArthur, J.H. et al. 2008. Phase I clinical evaluation ofrDEN4Delta30–200,201: a live attenuated dengue 4 vaccinecandidate designed for decreased hepatotoxicity. Am J TropMed Hyg 79: 678–684.



141. Durbin, A.P. et al. 2006. The live attenuated dengueserotype 1 vaccine rDEN1Delta30 is safe and highly im-munogenic in healthy adult volunteers. Hum Vacc 2: 167–173.

142. Durbin, A.P. et al. 2006. rDEN2/4Delta30(ME), a live at-tenuated chimeric dengue serotype 2 vaccine is safe andhighly immunogenic in healthy dengue-naive adults. HumVacc 2: 255–260.

143. Durbin, A.P. et al. 2011. Development and clinical evalu-ation of multiple investigational monovalent DENV vac-cines to identify components for inclusion in a live attenu-ated tetravalent DENV vaccine. Vaccine 29: 7242–7250.

144. Durbin, A.P. et al. 2011. Heterotypic dengue infection withlive attenuated monotypic dengue virus vaccines: implica-tions for vaccination of populations in areas where dengueis endemic. J Infect Dis 203: 327–334.

145. Durbin, A.P. et al. 2011. A single dose of the DENV-1candidate vaccine rDEN1Delta30 is strongly immunogenicand induces resistance to a second dose in a randomizedtrial. PLoS Negl Trop Dis 5: e1267.

146. Durbin, A.P. et al. 2013. A single dose of any of four dif-ferent live attenuated tetravalent dengue vaccines is safeand immunogenic in flavivirus-naive adults: a randomized,double-blind clinical trial. J Infect Dis 207: 957–965.

147. Smith, S.A. et al. 2013. Human monoclonal antibodies de-rived from memory B cells following live attenuated denguevirus vaccination or natural infection exhibit similar char-acteristics. J. Infect. Dis. 207: 1898–1908.

148. Lindow, J.C. et al. 2013. Vaccination of volunteers withlow-dose, live-attenuated, dengue viruses leads to serotype-specific immunologic and virologic profiles. Vaccine 31:3347–3352.

149. Durbin, A.P. et al. 2013. The live attenuated tetravalentdengue candidate vaccine TV003 is well tolerated andhighly immunogenic in flavivirus-experienced subjects. InAmerican Society of Tropical Medicine and Hygiene An-nual Meeting. Washington, DC.

150. Rice, C.M. et al. 1989. Transcription of infectious yellowfever RNA from full-length cDNA templates produced byin vitro ligation. N Biol 1: 285–296.

151. Chambers, T.J. et al. 1999. Yellow fever/Japanese encephali-tis chimeric viruses: construction and biological properties.J Virol 73: 3095–3101.

152. Guirakhoo, F. et al. 2000. Recombinant chimeric yellowfever-dengue type 2 virus is immunogenic and protectivein nonhuman primates. J Virol 74: 5477–5485.

153. Lai, C.J. & T.P. Monath. 2003. Chimeric flaviviruses: novelvaccines against dengue fever, tick-borne encephalitis, andJapanese encephalitis. Adv Virus Res 61: 469–509.

154. Guirakhoo, F. et al. 2001. Construction, safety, and im-munogenicity in nonhuman primates of a chimeric yellowfever-dengue virus tetravalent vaccine. J Virol 75: 7290–7304.

155. Monath, T.P. et al. 2002. Clinical proof of principle forChimeriVax: recombinant live, attenuated vaccines againstflavivirus infections. Vaccine 20: 1004–1018.

156. Guirakhoo, F. et al. 2006. Live attenuated chimeric yellowfever dengue type 2 (ChimeriVax-DEN2) vaccine. Phase Iclinical trial for safety and immunogenicity: effect of yel-

low fever pre-immunity in induction of cross neutralizingantibody responses to all 4 dengue serotypes. Hum Vacc 2:60–67.

157. Morrison, A.C. et al. 2010. Epidemiology of dengue virusin Iquitos, Peru 1999 to 2005: interepidemic and epidemicpatterns of transmission. PLoS Negl Trop Dis 4: e670.

158. Poo, J., F. Galan, R. Forrat, et al. 2011. Live-attenuatedtetravalent dengue vaccine in dengue-naive children, ado-lescents, and adults in Mexico City: randomized controlledphase 1 trial of safety and immunogenicity. Pediatr. Infect.Dis. J. 30: e9–e17.

159. Capeding, R.Z. et al. 2011. Live-attenuated, tetravalentdengue vaccine in children, adolescents and adults in adengue endemic country: randomized controlled phase Itrial in the Philippines. Vaccine 29: 3863–3872.

160. Guy, B., J. Almond & J. Lang. 2011. Dengue vaccineprospects: a step forward. Lancet 377: 381–382.

161. Guy, B., M. Saville & J. Lang. 2010. Development of SanofiPasteur tetravalent dengue vaccine. Hum. Vaccin. 6 [Epubahead of print: Sept 16].

162. Hu, B. et al. 2013. Neutralization of wild-type dengue virusisolates by antibodies elicited after immunization with atetravalent dengue vaccine. In American Society of TropicalMedicine and Hygiene Annual Meeting. Washington, DC.

163. Girerd-Chambaz, Y. et al. 2013. Sequence analysis of theDENV2 strains isolated in the Phase IIb CYD vaccine effi-cacy trial in Ratchaburi, Thailand. In American Society ofTropical Medicine and Hygiene Annual Meeting. Washing-ton, DC.

164. Kinney, R.M. et al. 1997. Construction of infectious cDNAclones for dengue 2 virus: strain 16681 and its attenuatedvaccine derivative, strain PDK-53. Virology 230: 300–308.

165. Huang, C.Y. et al. 2000. Chimeric dengue type 2 (vaccinestrain PDK-53)/dengue type 1 virus as a potential candidatedengue type 1 virus vaccine. J Virol 74: 3020–3028.

166. Butrapet, S. et al. 2000. Attenuation markers of a candi-date dengue type 2 vaccine virus, strain 16681 (PDK-53),are defined by mutations in the 5’ noncoding region andnonstructural proteins 1 and 3. J Virol 74: 3011–3019.

167. Huang, C.Y. et al. 2003. Dengue 2 PDK-53 virus as achimeric carrier for tetravalent dengue vaccine develop-ment. J Virol 77: 11436–11447.

168. Butrapet, S., R.M. Kinney & C.Y. Huang. 2006. Determin-ing genetic stabilities of chimeric dengue vaccine candi-dates based on dengue 2 PDK-53 virus by sequencing andquantitative TaqMAMA. J Virol Methods 131: 1–9.

169. Huang, C.Y. et al. 2013. Genetic and phenotypic charac-terization of manufacturing seeds for a tetravalent denguevaccine (DENVax). PLoS Negl Trop Dis 7: e2243.

170. Osorio, J.E. et al. 2011. Development of DENVax: achimeric dengue-2 PDK-53-based tetravalent vaccine forprotection against dengue fever. Vaccine 29: 7251–7260.

171. Gordon, G.S. et al. 2013. A phase 2 age-de-escalation clini-cal trial of a recombinant live attenuated tetravalent denguevaccine (DENVax) in healthy volunteers from endemiccountries. In American Society of Tropical Medicine andHygiene Annual Meeting. Washington, DC.

172. Gordon, G.S. et al. 2013. Effect of formulation ratios anddosing schedules on the safety and immunogenicity of a



recombinant live attenuated tetravalent dengue vaccine(DENVax) in healthy adult volunteers. In American So-ciety of Tropical Medicine and Hygiene Annual Meeting.Washington, DC.

173. Hoke, C.H., Jr. et al. 1988. Protection against Japanese en-cephalitis by inactivated vaccines. N Engl J Med 319: 608–614.

174. Kaltenbock, A. et al. 2009. Safety and immunogenicityof concomitant vaccination with the cell-culture basedJapanese Encephalitis vaccine IC51 and the hepatitis Avaccine HAVRIX1440 in healthy subjects: a single-blind,randomized, controlled Phase 3 study. Vaccine 27: 4483–4489.

175. Craig, S.C. et al. 1999. An accelerated schedule for tick-borne encephalitis vaccine: the American military experi-ence in Bosnia. Am J Trop Med Hyg 61: 874–878.

176. Fulginiti, V.A. et al. 1967. Altered reactivity to measles virus.Atypical measles in children previously immunized withinactivated measles virus vaccines. JAMA 202: 1075–1080.

177. Polack, F.P. 2007. Atypical measles and enhanced respira-tory syncytial virus disease (ERD) made simple. Pediatr Res62: 111–115.

178. Coller, B.A. et al. 2011. The development of recombinantsubunit envelope-based vaccines to protect against denguevirus induced disease. Vaccine 29: 7267–7275.

179. Clements, D.E. et al. 2010. Development of a recombinanttetravalent dengue virus vaccine: immunogenicity and effi-cacy studies in mice and monkeys. Vaccine 28: 2705–2715.

180. Robert Putnak, J. et al. 2005. An evaluation of dengue type-2inactivated, recombinant subunit, and live-attenuated vac-cine candidates in the rhesus macaque model. Vaccine 23:4442–4452.

181. Coller, B.-A. et al. 2013. Preclinical and clinical testing ofa recombinant subunit vaccine for dengue. In AmericanSociety of Tropical Medicine and Hygiene Annual Meeting.Washington, DC.

182. Putnak, R. et al. 1996. Development of a purified, inacti-vated, dengue-2 virus vaccine prototype in Vero cells: im-munogenicity and protection in mice and rhesus monkeys.J Infect Dis 174: 1176–1184.

183. Putnak, R. et al. 1996. Immunogenic and protective re-sponse in mice immunized with a purified, inactivated,dengue-2 virus vaccine prototype made in fetal rhesus lungcells. Am J Trop Med Hyg 55: 504–510.

184. Eckels, K.H. & R. Putnak. 2003. Formalin-inactivatedwhole virus and recombinant subunit flavivirus vaccines.Adv Virus Res 61: 395–418.

185. Danko, J.R., C.G. Beckett & K.R. Porter. 2011. Developmentof dengue DNA vaccines. Vaccine 29: 7261–7266.

186. Anez, G., M.E. Morales-Betoulle & M. Rios. 2011. Circula-tion of different lineages of dengue virus type 2 in Central

America, their evolutionary time-scale and selection pres-sure analysis. PloS One 6: e27459.

187. Vasilakis, N. & S.C. Weaver. 2008. The history and evolutionof human dengue emergence. Adv Virus Res 72: 1–76.

188. Holmes, E.C. 2006. The evolutionary biology of denguevirus. Novartis Foundation Symp 277: 177–187; discussion187–192, 251–173.

189. Rico-Hesse, R. 2003. Microevolution and virulence ofdengue viruses. Adv Virus Res 59: 315–341.

190. Chen, S.P. 2012. Molecular evolution and epidemiology offour serotypes of dengue virus in Thailand from 1973 to2007. Epidemiology and infection1–6.

191. VanBlargan LA et al. 2013. The type-specific neutralizingantibody response elicited by a dengue vaccine candidateis focused on two amino acids of the envelope protein.In American Society of Tropical Medicine and HygeineAnnual Meeting. Washington, DC.

192. Sabchareon, A. et al. 2013. Efficacy of tetravalent denguevaccine in Thai schoolchildren—Authors’ reply. Lancet381: 1094–1095.

193. Moi, M.L., T. Takasaki & I. Kurane. 2013. Efficacy of tetrava-lent dengue vaccine in Thai schoolchildren. Lancet 381:1094.

194. McKibben, L. 2013. Efficacy of tetravalent dengue vaccinein Thai schoolchildren. Lancet 381: 1094.

195. Endy, T.P. et al. 2011. Determinants of inapparent andsymptomatic dengue infection in a prospective study ofprimary school children in Kamphaeng Phet, Thailand.PLoS Negl Trop Dis 5: e975.

196. Endy, T.P. et al. 2004. Relationship of preexisting denguevirus (DV) neutralizing antibody levels to viremia andseverity of disease in a prospective cohort study of DVinfection in Thailand. J Infect Dis 189: 990–1000.

197. Halstead, S.B. 2009. Antibodies determine virulence indengue. Ann NY Acad Scie 1171(Suppl. 1): E48–E56.

198. Beltramello, M. et al. 2010. The human immune responseto dengue virus is dominated by highly cross-reactiveantibodies endowed with neutralizing and enhancing ac-tivity. Cell Host Microbe 8: 271–283.

199. Moi, M.L. et al. 2010. Discrepancy in dengue virus neutral-izing antibody titers between plaque reduction neutralizingtests with Fcgamma receptor (FcgammaR)-negative andFcgammaR-expressing BHK-21 cells. Clin Vacc Immunol17: 402–407.

200. Anderson, K.B. et al. 2011. Interference and facilita-tion between dengue serotypes in a tetravalent livedengue virus vaccine candidate. J Infect Dis 204:442–450.

201. Guy, B. et al. 2009. Evaluation of interferences betweendengue vaccine serotypes in a monkey model. Am J TropMed Hyg 80: 302–311.


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