Developing a dengue vaccine: progress and future challenges.

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

A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S Issue: Antimicrobial Therapeutics Reviews

Developing a dengue vaccine: progress and future challenges Stephen J. Thomas Walter Reed Army Institute of Research, Silver Spring, Maryland Address for correspondence: Stephen J. Thomas, M.D., Director, Viral Diseases Branch, Walter Reed Army Institute of Research, 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 from resource-constrained countries, seek treatment every year for dengue-related disease. Despite more than 70 years of effort, a safe and efficacious vaccine remains unavailable. Antidengue antiviral drugs also do not exist despite attempts to develop or repurpose drug compounds. Gaps in the knowledge of dengue immunology, absence of a validated animal or human model of disease, and suboptimal assay platforms to measure immune responses following infection or experimental vaccination are obstacles to drug and vaccine development efforts. The limited success of one vaccine candidate in a recent clinical endpoint efficacy trial challenges commonly held beliefs regarding potential correlates of protection. If a dengue vaccine is to become a reality in the near term, vaccine developers should expand development pathway explorations beyond those typically required to demonstrate safety and efficacy. Keywords: dengue; vaccine; antiviral; immunology

Introduction Dengue is the most important arboviral disease afflicting the world today. Hundreds of millions of infections occur each year, of which more than 90 million are clinically apparent.1 Mortality is reportedly low compared to other vector borne diseases, but the brunt of severe dengue disease and death in many regions occurs disproportionately in children.2 In regions with endemic and hyperendemic transmission, dengue absorbs significant healthcare resources.3–17 Dengue season exacts a measurable toll at the personal, community, and regional levels and is a leading cause of febrile, systemic illness in travelers.18–25 Deploying military populations have confronted the potential and occurrence of mission-disabling dengue epidemics for over a century.26–30 For decades, the global dengue burden has been trending upward.31 Conditions favoring the close juxtaposition of virus, vector, and susceptible host— a requirement for sustained transmission—are numerous. Ecological conditions favoring vector expansion, population growth and increasing ur-

banization, and the ease of air travel have all contributed to the concentration, in time and space, of susceptible hosts, competent vectors, and dengue viruses (DENVs).32 A reversal of current epidemiologic trends without a strategic intervention is unlikely. There is no licensed dengue vaccine and no prophylactic or therapeutic dengue drug (i.e., antiviral or anti-inflammatory). Vector control, even when successful from an entomologic perspective, does not always translate into a reduction in human infection or disease.33 The exploration of genetically modified mosquitoes and the potential to replace transmission-competent with incompetent mosquito populations is underway and requires additional study.34–37 Personal protective measures, such as wearing long sleeves and pants, use of bed nets, use of insecticides (i.e., N,Ndiethyl-meta-toluamide, DEET), avoidance of vectors during prime feeding times, and reducing the number of man-made vector breeding sites (e.g., standing water), are inconsistently applied and, as more is learned more about vector-host interactions, may not be optimal.31,38 A safe and efficacious

doi: 10.1111/nyas.12413 Ann. N.Y. Acad. Sci. xxxx (2014) 1–20 Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

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tetravalent dengue vaccine is the best strategy for reducing the global dengue burden. Dengue vaccine development challenges The dengue vaccine field is robust with numerous candidates in preclinical and clinical development. Unfortunately, the challenges accompanying the development of a tetravalent dengue vaccine are also numerous: the existence of four DENV types, all capable of causing disease and death, requires a dengue vaccine capable of preventing clinical disease caused by infection with any of the DENVs. Whether a vaccine will need to demonstrate DENV type–specific efficacy for every type before licensure is a nuance developers and regulatory authorities are currently negotiating. DENV evolution and potential for jump of sylvatic dengue strains into a sustained human transmission cycle raises the possibility that new vaccine candidates will be required over time to address significant antigenic divergence from existing DENVs.39–41 The coordination of innate and adaptive immune responses that confer protection or contribute to pathogenesis following a DENV infection are incompletely understood.42 Consequently, vaccine developers must extrapolate from natural infection data, animal studies, and other flavivirus vaccine development experiences (e.g., Yellow fever virus and Japanese encephalitis) to establish and pursue immunogenicity benchmarks that could potentially translate into clinical benefit (i.e., protective efficacy). There are examples of safe and efficacious vaccines that were licensed and that experienced widespread use before their protective mechanisms were fully understood.43 A significant concern with dengue is the observation of enhanced dengue disease when the convalescent immune response, established following a previous dengue infection with one DENV type, contributes to an immunopathological response following infection with a different DENV type. Greater numbers of infected targets cells (i.e., monocytes and macrophages), increased viral replication, and induction of a proinflammatory state result in a more severe clinical phenotype that is marked by plasma leakage, coagulation dysfunction, intravascular volume depletion and hemodynamic instability, and, potentially, death.42,44–47 Therefore, poorly performing dengue vaccine candidates could place vaccine recipients at 2

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increased risk for enhanced disease—a theoretical risk not yet demonstrated in field studies.48,49 Gaps in our understanding of dengue immunology complicate the process of defining an immune correlate of protection and this, in turn, complicates vaccine clinical development plans and regulatory strategies. Neutralizing antibody has been proposed as a likely immune correlate and may eventually be defined as such, similar to vaccines for Yellow fever virus and Japanese encephalitis virus.50–52 However, it is also possible that components of cellular immune responses (e.g., IFN-␥ secretion and cytotoxic T cell activity) will be found to be correlates of immune protection. Numerous methods are being explored to measure both humoral and cellular immune responses following natural infection and as immunogenicity assays to support vaccine development programs.53 Adversely affecting the exploration for an immune correlate of protection is the absence of a dengue animal disease model. Humanized small animal models are being aggressively studied and have shed light on various aspects of the immunologic and clinical responses to dengue infection. However, they do not currently appear to offer a comprehensive view of in vivo human dengue disease pathology.54–57 Nonhuman primates develop viremia and neutralizing antibody responses following dengue infection, but there is a paucity of local or systemic clinical signs or biochemical abnormalities.58–66 Variations in challenge virus preparation or delivery may yield a more informative model. In addition to the absence of an animal model there is also no robust dengue human infection model to support vaccine and drug development efforts, basic immunology studies, and the search for immune correlates. Currently, human infection models expose healthy volunteers to wild-type or slightly attenuated pathogens to produce an uncomplicated disease course. A human infection model for dengue could lower vaccine development risk by providing an early indication of whether a vaccine candidate has the potential for clinical benefit (therapeutic or protective), thereby reducing the risk of exposing endemic populations to suboptimal vaccine or drug candidates. Experimental human dengue infections, documented as early as 1902, have facilitated numerous sentinel discoveries in the field.67–78 The Walter Reed

Ann. N.Y. Acad. Sci. xxxx (2014) 1–20 Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

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Army Institute of Research (WRAIR) conducted two dengue human infection studies in the early 2000s. The studies aimed to validate human infection model strains and then expose past experimental vaccine recipients to the best performing strains.79–82 Those studies were the first documented ones in decades, and none have been completed since. Following the limited success of the world’s first dengue vaccine efficacy study, there is renewed interest in exploring human infection models for dengue. Another issue influencing the dengue vaccine field is the portfolio of assays used to measure vaccine immunogenicity during preclinical and clinical development activities. Experts in the field believe neutralizing antibodies have the greatest likelihood of being a correlate of protection, and most assay development efforts supporting vaccine programs have been devoted to this readout.52 The classic assay platform designed to measure neutralizing antibodies, the plaque reduction neutralization test (PRNT), has had significant interassay and interlab variability and a low level of robustness.83–86 In addition to reducing comparability of vaccine candidate performance across different developers, interassay variation (i.e., two- to threefold variability) within a single laboratory may considerably skew (positive or negative) seroconversion rates and antibody titers. And, thus, neutralizing antibody titers around such as assay cut-off may or may not represent true immunologic responses to vaccination. There are also concerns regarding the cross-reactivity of neutralizing antibody platforms within the DENV types and with other flaviviruses (e.g., Japanese encephalitis virus, West Nile virus, and Yellow fever virus). Confusing data or misinformation misleads vaccine development decisions, such as vaccine candidate down-selection and whether to advance development. Variations of the PRNT are under development and more advanced programs have undergone qualification and validation procedures, improving assay quality. Fortunately, there are numerous initiatives to improve neutralizing assay platforms and to better capture conditions more representative of the human in vivo experience. These include using human cell lines, cells bearing putative viral receptors (i.e., Fc␥ R), execution of depletion steps to improve measurement of homotypic versus heterotypic antibodies, employment of automated

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platforms (e.g., fluorescence activated cell sorting (FACS)), and manipulation of control virus input (e.g., varying concentrations of control virus or reporter virus particles).87–93 Assay platforms measuring cellular mediated immunity (CMI) have also been applied to dengue vaccine development programs.53 Readouts implicating a breadth of cellular responses have been evaluated and compared to similar studies performed on samples from natural infection.94–100 Although defining DENV type–specific responses has been a focus, this has been carried out without a clear understanding of what constitutes a bone fide immunoprotective profile; thus, the data are difficult to interpret. As more is learned about the complexities of the cellular immune response following dengue virus exposure, a potential correlate may emerge. Dengue vaccine development efforts

General It is unclear whether a single dengue vaccine will meet the diverse spectrum of global needs and requirements. Region-specific burden of disease data and existing national immunization schedules are two of many factors that will drive dengue vaccination policies and programs.101,102 Traveler and military populations also present unique circumstances. Elements of a desirable dengue vaccine target product profile (TPP) would include but not be limited to (1) ability to protect against clinically relevant dengue disease of any severity caused by any DENV type; (2) dosing schedules compatible with existing national immunization (endemic areas) or traveler/military schedules (short lead time); (3) rapid overall time to protection from time of first primary vaccination (number of weeks or months); (4) durable immunity and a reasonable schedule of booster dose requirements if needed; and (5) ability to store vaccine at a reasonable temperature for a reasonable period of time. Efforts to develop a dengue vaccine have been documented as early as 1929, when scientists unsuccessfully attempted to produce an inactivated dengue virus vaccine using phenol, formalin, and bile.72 During World War II live attenuated virus (LAV) vaccines were explored by passing DENV-1 and DENV-2 strains in brains of suckling mice.75,77,103 However, mouse brain-derived candidates were eventually replaced with cell culture based vaccine candidates.


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

Status Completed Active, not recruiting

Hawaii Biotech, Inc.a Inviragen Inc.b

Completed

Inviragen Inc.b

Recruiting

Inviragen Inc.b

Recruiting

Inviragen Inc.b

Active, not recruiting

Inviragen Inc.b,c

Active, not recruiting Recruiting

Merck Sharp & Dohme Corp. NIAIDd

Completed Completed

NIAID NIAID

Completed

NIAID

Recruiting

NIAID

Completed

NIAID

Recruiting

NIAID

Recruiting

NIAID

Completed

NIAID

Completed

NIAID

Recruiting

NIAID

Completed

NIAIDe

Completed

NIAIDe

Completed

NIAIDe

Completed

NIAIDe

Completed

NIAIDe

Completed

NIAIDe

Completed

NIAIDe

Study title

Identifier

Study of HBV-001 D1 in Healthy Adults

NCT00936429

Study to Investigate the Safety and Immunogenicity of a Tetravalent Chimeric Dengue Vaccine in Healthy Volunteers Between the Ages of 1.5–45 Years Safety and Immunogenicity Study to Assess DENVax, a Live Attenuated Tetravalent Vaccine for Prevention of Dengue Fever A Comparison of the Safety and Immunogenicity of Various Schedules of Dengue Vaccine in Healthy Adult Volunteers Phase 1b Study Investigating Safety & Immunogenicity of DENVax Given Intradermally by Needle or Needle Free PharmaJet Injector Impact of SQ vs IM Administration of DENVax on Safety and Immunogenicity Study of a Dengue Vaccine (V180) in Healthy Adults (V180-001 AM2) Phase II Trial to Evaluate Safety and Immunogenicity of a Dengue 1,2,3,4 (Attenuated) Vaccine Tetravalent Chimeric Dengue Vaccine Trial Safety and Immune Response to an Investigational Dengue Type 2 Vaccine Evaluation of the Safety and Immune Response to an Investigational Dengue Type 1 Vaccine Evaluating the Safety and Immune Response to Two Admixtures of a Tetravalent Dengue Virus Vaccine Safety of and Immune Response to DEN4 Vaccine Component Candidate for Dengue Virus Evaluating the Safety and Immune Response to Two Admixtures of a Tetravalent Dengue Virus Vaccine Evaluating the Safety and Immune Response to a Dengue Virus Vaccine in Healthy Adults Safety and Immune Response to Two Doses of rDEN2/4delta30 Dengue Vaccine Safety of and Immune Response to a Dengue Virus Vaccine (rDEN3–3’Ddelta30) in Healthy Adults Evaluating the Safety and Immune Response to Two Doses of a Dengue Virus Vaccine Administered 12 Months Apart Safety of and Immune Response to a Dengue Virus Vaccine (rDEN2/4delta30[ME]) in Healthy Adults Safety of and Immune Response to a Dengue Virus Vaccine (rDEN1delta30) in Healthy Adults Evaluation of the Safety and Immune Response of Five Admixtures of a Tetravalent Dengue Virus Vaccine Safety of and Immune Response to Two Different Dengue Virus Vaccines in Individuals Previously Immunized Against Dengue Virus Safety of and Immune Response to a Dengue Virus Vaccine (rDEN4delta30–200,201) in Healthy Adults Safety of and Immune Response to a Dengue Virus Vaccine (rDEN3delta30/31-7164) in Healthy Adults Safety of and Immune Response of a 2-dose Regimen of rDEN1delta30 Dengue Virus Vaccine

NCT01511250

NCT01224639

NCT01542632 NCT01765426

NCT01728792 NCT01477580 NCT01696422 NCT01110551 NCT01073306 NCT01084291 NCT01436422 NCT00919178 NCT01506570 NCT01931176 NCT00920517 NCT00712803 NCT01782300 NCT00094705 NCT00089908 NCT01072786 NCT00458120

NCT00270699 NCT00831012 NCT00473135

Continued 4


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Table 1. Continued Status

Vaccine candidate regulatory sponsor

Completed

NIAIDe

Completed

NIAIDe

Completed

Sanofi Pasteur

Completed

Sanofi Pasteur

Completed

Sanofi Pasteur


Sanofi Pasteur


Sanofi Pasteur

Completed

Sanofi Pasteur

Completed

Sanofi Pasteur

Completed

Sanofi Pasteur

Completed

Sanofi Pasteur


Sanofi Pasteur


Sanofi Pasteur


Sanofi Pasteur

Completed

Sanofi Pasteur


Sanofi Pasteur


Sanofi Pasteur

Active, not recruiting Active, not recruiting

Sanofi Pasteur Sanofi Pasteur

Enrolling by invitation

Sanofi Pasteur

Recruiting

Sanofi Pasteurf

Completed

Sanofi Pasteur


U.S. Army Medical Research and Materiel Command U.S. Army Medical Research and Materiel Commandg

Completed

Study title

Identifier

Safety of and Immune Response to a Dengue Virus Vaccine (rDEN4delta30-4995) in Healthy Adults Safety of and Immune Response to a Dengue Virus Vaccine (rDEN3/4delta30[ME]) in Healthy Adults A Study of Dengue Vaccine in Healthy Toddlers Aged 12 to 15 Months in the Philippines Immunogenicity and Safety of Sanofi Pasteur Pasteur’s CYD Dengue Vaccine in Healthy Children and Adolescents in Latin America Study of a Tetravalent Dengue Vaccine in Healthy Children Aged 2 to 11 Years in Malaysia Study of a Novel Tetravalent Dengue Vaccine in Healthy Children Aged 2 to 14 Years in Asia Study of a Novel Tetravalent Dengue Vaccine in Healthy Children and Adolescents Aged 9 to 16 Years in Latin America Study of ChimeriVaxTM Tetravalent Dengue Vaccine in Healthy Peruvian Children Aged 2 to 11 Years Study of ChimeriVaxTM Dengue Tetravalent Vaccine in Adult Subjects Immunogenicity and Safety of Three Formulations of Dengue Vaccines in Healthy Adults Aged 18 to 45 Years in the US Safety and Immunogenicity of Formulations of Dengue Vaccines in Healthy Flavivirus-Naive Adults Immune Response to Different Schedules of a Tetravalent Dengue Vaccine Given With or Without Yellow Fever Vaccine Study of Yellow Fever Vaccine Administered With Tetravalent Dengue Vaccine in Healthy Toddlers Study of a Booster Injection of PentaximTM Vaccine Administered With Dengue Vaccine in Healthy Toddlers Study of a Tetravalent Dengue Vaccine in Healthy Adults in Australia Study of Sanofi Pasteur Pasteur’s CYD Dengue Vaccine in Healthy Subjects in Singapore Study of a Tetravalent Dengue Vaccine in Healthy Adult Subjects Aged 18 to 45 Years in India Efficacy and Safety of Dengue Vaccine in Healthy Children Study of ChimeriVaxTM Tetravalent Dengue Vaccine in Healthy Subjects Long-Term Study of Hospitalized Dengue & Safety in Thai Children Included in a Tetravalent Dengue Vaccine Efficacy Study Immunologic Mechanisms of Immune Interference and/or Cross-Neutralizing Immunity After CYD Tetravalent Dengue Vaccine Study of CYD Dengue Vaccine in Healthy Children and Adolescents in South America Safety Study of a Vaccine (DENV-1 PIV) to Prevent Dengue Disease

NCT00322946

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

NCT00375726 NCT01064141 NCT00993447

NCT01254422 NCT01373281 NCT01374516

NCT00788151 NCT00730288 NCT00617344 NCT00740155 NCT01488890

NCT01436396 NCT01411241 NCT01134263 NCT00880893 NCT01550289 NCT00842530 NCT00875524 NCT01983553

NCT01943825

NCT01187433 NCT01502735

NCT00239577

Continued Ann. N.Y. Acad. Sci. xxxx (2014) 1–20 Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

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Table 1. Continued Vaccine candidate regulatory sponsor

Status Completed

Completed

Completed

Completed

Recruiting


Completed

Completed

Completed


Completed

U.S. Army Medical Research and Materiel Commandh U.S. Army Medical Research and Materiel Commandh U.S. Army Medical Research and Materiel Commandh U.S. Army Medical Research and Materiel Commandh U.S. Army Medical Research and Materiel Commandi U.S. Army Medical Research and Materiel Commandi U.S. Army Medical Research and Materiel Commandh U.S. Army Medical Research and Materiel Commandh U.S. Army Medical Research and Materiel Commandh U.S. Army Medical Research and Materiel Commandj U.S. Army Office of the Surgeon Generalk

Study title

Identifier

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

NCT00322049

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

NCT01843621

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

NCT00370682

A Study of Two Doses of WRAIR Dengue Vaccine Administered Six Months Apart to Healthy Adults and Children

NCT00468858

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

NCT01702857

A Two-dose Primary Vaccination Study of a Tetravalent Dengue Virus Purified Inactivated Vaccine vs. Placebo in Healthy Adults

NCT01666652

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

NCT00318916

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

NCT00384670

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

NCT00350337

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

NCT01502358

Safety Study of a Dengue Virus DNA Vaccine

NCT00290147

a Hawaii

Biotech Inc. dengue vaccine program acquired by Merck and Company (2010). acquired by Takeda Pharmaceutical Company Limited (2013). c NCT01728792 being executed in cooperation with the Walter Reed Army Institute of Research and the State University of New York, Upstate Medical University. d Study sponsor is Butantan Institute. e Collaborator listed as Johns Hopkins Bloomberg School of Public Health. f Collaborator listed as U.S. Department of Defense. g Study Sponsor listed as GlaxoSmithKline. h Collaborator listed as GlaxoSmithKline. i Collaborators listed as GlaxoSmithKline and the Walter Reed Army Institute of Research. j Collaborators listed as Vical, the Walter Reed Army Institute of Research (WRAIR), and the Naval Medical Research Center. k Collaborator listed as the U.S. Army Medical Research and Materiel Command. b Inviragen

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Multiple different vaccine approaches have been tested in human clinical trials; a single candidate is in phase III, and there are numerous candidates in preclinical development (Table 1). Developers have taken unique approaches to targeting different regions of the DENV genome to include full genome LAV using cell passage, directed mutagenesis, and chimeric technology; inactivated virus with aluminum hydroxide or proprietary adjuvant systems; recombinant proteins; Yellow fever virus backbone with DENV premembrane (prM) and E substitution for the Yellow fever virus sequences; and DNA-based candidates. Each approach makes assumptions about what will constitute a protective immune response (e.g., neutralizing antibody to the E gene, requirement for CMI directed against nonstructural (NS) proteins, both, or others) and how to best stimulate this response in a DENV type– specific manner. Safety measurements during dengue vaccine trials include assessment of local and systemic solicited and unsolicited symptoms, and objective clinical findings during vaccination and during subacute and long-term follow up periods (dengue endemic regions). Clinical laboratory abnormalities and vaccine-induced viremia are also evaluated. During studies in dengue-endemic regions and during efficacy trials, the phenotype (dengue, severe dengue) of natural DENV infections that occur in placebo/control recipients and breakthrough cases in experimental vaccine recipients is also monitored during vaccination periods (between doses), as well as remote from vaccination. More severe phenotypes in vaccine recipients (statistically significant increases) compared to control recipients would be concerning for vaccine breakthrough and contribution to more severe disease.104,105 Expected local reactions include pain, redness, and induration/swelling. Expected systemic symptoms would mimic those seen in natural DENV infections, including fever, headache, muscle pain, bone pain, pain with eye movement, nausea, vomiting, and fatigue. Clinical signs would include objective fever, rash, hepatomegaly, lymphadenopathy, and evidence of bleeding. Each sign and symptom is graded on a severity scale, and duration is documented. Laboratory measures of specific concern include abnormalities in the complete blood count (CBC), especially absolute neutrophil counts (ANC) and platelet decrements. Liver-associated

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enzymes (ALT, AST) elevations are also routinely assessed. Qualitative and quantitative peripheral viremia measurements following vaccination with replicating vaccines not only confirm vaccine take but also may indicate immunologic enhancement in flavivirus-primed populations (i.e., significantly higher viremia following dose 2 or following dose 1 in primed volunteers with or without symptoms). The immune readout measured by all vaccine developers is the neutralizing antibody. There are numerous platforms currently in use to measure this endpoint to include the classic PRNT, a microneutralization platform, a microplaque reduction neutralization test (␮PRN), a microfocus reduction neutralization test (mFRNT), and FACSbased assays.84,106–108 Antibody measurements are made at baseline (day 0) and then on the day of each vaccination and 1 month following each vaccination. In small trials, all volunteers are sampled at each time point, while in larger studies, subsets are sampled and the results are extrapolated to the cohort. This schedule of measurement provides insight into vaccine immunogenicity. Sample collection at time points remote from vaccination provides an understanding of immune response durability over time, kinetics of immune response maturation, and possible neutralizing antibody set points following a period of waning. Immune responses at the cohort level are characterized by the percent of volunteers who seroconvert to each DENV type (i.e., antibody response below the assay cutoff before vaccination and then above the cutoff following vaccination). Geometric mean titer calculations are used to quantitatively represent cohort level immune responses to each DENV type. Antibody profiles are used to approximate whether individuals and cohorts develop what is presumed to be a protective response. Desired profiles include antibody responses measured to all or at least three DENV types, that is, tetravalent and trivalent profiles, respectively. Nonresponders are considered susceptible to dengue infection and disease, and monovalent responders are considered to be higher risk for severe disease with natural infection (secondary infection). The common thinking is that a balanced antibody response is desired. Another proposal (advocated by the present author, STJ) is that the development goal should be to achieve a DENV type–specific neutralizing antibody response above the required


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protective threshold for each individual DENV type. It is likely that the protective threshold varies for each DENV type—even, perhaps, for each genotype within a given type—and pursuing a balanced response could underestimate the required response to protect against one or more types. Unfortunately, at this time a protective threshold has not been defined for any DENV type. Cellular immune responses (i.e., DENV type– specific cytotoxtic T cell and INF-␥ responses, polyfunctional T cells, etc.) to vaccination and other potential immune response measures (i.e., quantitating DENV type–specific memory B cell populations, antibody avidity and affinity, etc.) are under assessment, but their investigation and application are not standard across development programs.

Vaccine candidates in clinical development Flaviviral LAV vaccines (e.g., against Yellow fever (YF) virus and Japanese encephalitis virus) have proved to be safe and durably efficacious.109–112 LAV vaccines replicate in the recipient at a relatively low and controlled manner yet present all viral antigens in the vaccine construct and elicit both antibody and 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. Investigators at Mahidol University in Bangkok were among the first groups to attenuate DENVs by passing viral strains in dog (PDK) and nonhuman primate (PGMK) cell lines.113–119 And while that effort was successful initially, achieving a safe and immunogenic (i.e., neutralizing antibody responses to each DENV type) tetravalent formulation proved difficult.49,94,120,121 The Walter Reed Army Institute of Research (WRAIR) also developed multivalent LAV candidates by serial PDK cell passage followed by a final passage in fetal rhesus lung (FRhL) cells. Early development efforts identified promising formulations by combining variations of attenuation and DENV antigen concentrations.122–130 A single formulation tested in Thai schoolchildren and toddlers was well tolerated and sufficiently immunogenic to pursue continued development.131,132 Newly derived vaccine lots were tested in adults in the United States, Thailand, and in Puerto Rico across a broad age range (12 months to 50 years). In the U.S. study, tetravalent response rates to new vaccine formula8

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tions were between 60% and 66.7% in dengue naive (unprimed) subjects, following two doses delivered at study days 0 and 180; a third dose did not increase tetravalent antibody rates.133 The Thailand study cohort was largely primed to dengue (88– 98%) at study initiation. All dengue unprimed subjects and at least 97.1% of primed subjects experienced tetravalent seroconversion when measured 1 month following dose 2 (unpublished data, SJT). Data from the Puerto Rico study are pending. Available data from the three phase II studies indicate no overt safety problems in over 300 vaccine recipients and moderate to high seroconversion rates. Further development of these candidates has been indefinitely suspended in search of an improved TPP. The U.S. National Institutes of Health (NIH) constructed cDNA clones of a DENV-4 candidate and induced a 30-nucleotide deletion in the 3 untranslated region (rDEN430; Fig. 1 and Table 2).134 The monovalent candidate was immunogenic and induced lower viremia than the parent strain. Additional candidates were tested in phase I clinical trials to identify optimal components for tetravalent formulations.135–145 Tetravalent candidates were prepared and tested in numerous phase I clinical trials.146 A panel of human monoclonal antibodies (mAbs) was made from subjects who received an rDEN130 candidate and from subjects who were naturally infected with DENV-1. The results demonstrated a high degree of similarity in the induced memory profile (high frequency of B cells encoding serotype cross-reactive, weakly neutralizing antibodies).147 Dose ranging trials exploring human infectious dose 50% (ID50 ) parameters yielded a target dose of 1000 plaque forming units (PFU) for each DENV type formulated into a tetravalent vaccine.148 A lead candidate (admixture TV003) was identified that induced 74% tetravalent seroconversion in flavivirus-naive vaccines, and 92% seroconverted to 3 serotypes following a single dose. A TV003 dose of 1000 PFU per DENV component was also explored in flavivirus-primed individuals. A single dose was provided at time 0 and a second dose (“challenge,” per the authors) provided at 6 months. Sixty percent of all vaccinees had at least one vaccine virus recovered from the blood following dose 1, there was no viremia following dose 2, and there was no significant difference in the adverse event profile. Dose 1 induced a tetravalent neutralizing antibody profile in


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Table 2. Current vaccine candidate sponsor, name, description, dosing schedule, and phase of development based on clinicaltrials.gov (accessed Feb. 3, 2014) Current vaccine candidate regulatory sponsor Inviragen Inc.b

Current vaccine name DENVax

Merck Sharp & Dohme V180 Corp.

Description

Current dosing schedulea

Development phase

Live attenuated virus DENV-2 PDK-53 backbone DENV-1/2, -3/2, and -4/2 chimeras

Dose 1–Day 0 Dose 2–Day 90

2

Recombinant protein 80% of DENV-1–4 E protein

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

1

National Institute of Allergy and Infectious Diseases (NIAID) Sanofi Pasteur

TetraVax-DV-TV003 (TV003)

Live virus vaccine Attenuated by directed mutagenesis (nucleotide deletions) and development of chimeras

Dose 1–Day 0

2

CYD Dengue Vaccine

Live attenuated virus Yellow fever 17D virus backbone with preM and E removed and replaced with DENV-1–4 preM and E proteins (CYD = chimeric-Yellow fever-dengue)

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

3

U.S. Army Medical Research and Materiel Command (Walter Reed Army Institute of Research) U.S. Army Medical Research and Materiel Command (U.S. Naval Medical Research Center)

TDEN-PIV

Purified inactivated vaccine

Dose 1–Day 0 Dose 2–Day 28

1

Tetravalent Dengue Vaccine (TVDV)

Plasmid DNA vaccine

Dose 1–Day 0 Dose 2–Day 30 Dose 3–Day 90

1

a b

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

85% of vaccines and a trivalent or better response in 100% of recipients.149 Phase II trials in endemic populations and across a diverse age range are poised to begin in Thailand and Brazil. The vaccine has been exclusively licensed to the Instituto Butantan (Sao Paulo, Brazil). St. Louis University Health Sciences Center created a Yellow fever (YF)-dengue chimeric vaccine candidate by inserting dengue preM and E genes into the cDNA backbone of the YF 17D vaccine. The construct was further developed by Acambis, Inc. and then licensed to Sanofi Pasteur (Fig. 2 and Table 2 (CYD Dengue vaccine)).150–154 Clinical trials of monovalent and tetravalent vaccine preparations demonstrated excellent safety and immunogenicity across volunteers of various ages, genetic backgrounds, and flavivirus priming status (i.e., to individuals with pre-existing immunity to Yellow fever virus, dengue virus, and Japanese

encephalitis virus).155–161 Sanofi’s chimeric Yellow fever–dengue virus (CYD; ChimeriVax) was the first dengue vaccine candidate tested in a clinical end-point efficacy study. The phase IIb trial was an observer-blinded, randomized, controlled, single center trial in healthy Thai schoolchildren (N = 4002) aged 4–11 years who were randomly assigned (2:1) to receive three injections of dengue vaccine or control (rabies vaccine or placebo) at 0, 6, and 12 months. All subjects were followed for dengue illness. The primary objective was to assess protective efficacy against virologically confirmed, symptomatic dengue, irrespective of severity or DENV type, occurring 1 month or longer after the third injection. Although the vaccine was safe and neutralizing antibody responses were moderate to high, overall efficacy was 30.2% (95% CI 13.4– 56.6), and differed by serotype.48 Sequencing of circulating DENVs compared to vaccine strains and


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Figure 1. National Institute of Allergy and Infectious Diseases (NIAID) TetraVax-DV-TV003 candidate (TV003). Recombinant attenuated DENV vaccine candidates were constructed by deletion of nucleotides from the 3 UTR (Δ30 and Δ30/31) or by chimerization of genomic regions from different serotypes (preM and E genes from DENV-2 chimerized into DENV-430). Provided courtesy of NIAID.

cross-neutralization experiments with vaccine volunteer sera failed to prove wild-type strains escaped vaccine-induced immunity.162,163 The results of this trial were disappointing and challenged the PRNT assay platform and neutralizing antibody readout as a correlate of protection. Further studies of phase IIb and phase III trials in Latin America and Asia (total enrollment >25,000) continue with results expected in 2014. The U.S. Centers for Disease Control and Prevention (CDC) developed a tetravalent chimeric dengue vaccine candidate by introducing DENV-1, DENV-3, and DENV-4 prM and E genes into cDNA derived from an attenuated LAV DENV-2 component (Fig. 3 and Table 2 (DENVax)).164–168 DENVDENV chimeras were formulated as a DENV-1/ DENV-2, DENV-3/DENV-2, and DENV-4/ DENV-2 tetravalent vaccines and licensed to Inviragen, Inc., which was recently acquired by Takeda Pharmaceutical Company Limited.169,170 The DENVax candidate had an acceptable safety and immunogenicity profile in small phase I studies of flavivirus-naive adults following subcutaneous or intradermal delivery. A phase II age de-escalation trial is underway in four endemic countries to assess the safety and immunogenicity of two subcutaneous doses (days 0 and 90) in 1.5–45 year olds. Thus far, vaccination has been well tolerated, with mostly mild and transient local or systemic reactions. A preliminary analysis revealed that after one or two doses 98.8% of subjects had a trivalent or better antibody profile, and 87.2% had a tetravalent profile when measured 1 month after dose 2.171 Tetravalent formulations with variations

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in the DENV-4 viral concentration (delivery of two doses at day 0 with or without additional doses at day 90) and variation in delivery method (intramuscular, intradermal, needle, needless device delivery) are all being explored.172 Inactivated whole virus or viral subunit vaccines have potential advantages, including the inability to revert to a more pathogenic phenotype, lack of immune interference when combined in a tetravalent formulation, and, theoretically, fewer acute safety issues. Inactivated flaviviral vaccines have been licensed and are in wide use to prevent Japanese encephalitis and tick-borne encephalitis.173–175 Potential disadvantages include inducing antibodies to only a portion of the structural proteins and deviation from wild-type structural antigen conformations. High antigen concentrations and multiple doses may also be required. There is potential for an adverse response following natural infection, such as was observed with the occurrence of atypical measles and respiratory syncytial virus (RSV).176,177 Merck & Co. is developing a dengue vaccine candidate produced in a Drosophila S2 cell expression system (Fig. 4 and Table 2 (V180)).178–180 Nonhuman primate studies assessing persistence of antibody responses and impact of DENV priming on immunogenicity are being completed. A dose ranging study of the candidate (V180) in humans is underway in Australia. Combining three doses (months 0, 1, and 2) of V180 with a range of doses of ISCOMATRIXTM adjuvant is being evaluated in the same study.181 The WRAIR developed a purified inactivate virus (PIV) dengue vaccine candidate by inactivation of


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Figure 2. Sanofi Pasteur CYD (chimeric-Yellow fever-dengue) Dengue Vaccine. The CYD TDV candidate is composed of four recombinant, live, attenuated vaccines (CYD-1–4) based on a yellow fever vaccine 17D (YFV 17D) backbone, each expressing the premembrane 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 PIV adjuvanted with alum was completed in the United States in a small number of flavivirus-naive volunteers. The candidate vaccine had an acceptable safety profile with low to moderate immunogenicity following two doses administered on days 0 and 28 (TDEN-PIV, Table 2). There was minimal cross-reactive antibody and titers were short lived; cellular responses are currently being assessed (unpublished data, SJT). The U.S. Army granted an exclusive license to GlaxoSmithKline Vaccines (GSK) and the two are codeveloping, with the Oswaldo Cruz Foundation (FioCruz), a tetravalent PIV formulation (DPIV) adjuvanted with aluminum hydroxide (AlOH) and GSK’s proprietary Adjuvant Systems (AS). Phase I trials in the United States and Puerto Rico are exploring DPIV candidates adjuvanted with AlOH, AS01E , and AS03B . Data through study day 56 (dose 1 at day 0, dose 2 at day 28) in the U.S. trial indicate a well-tolerated vaccine. Day 56 neutralizing antibody measurements in dengue-naive volunteers determined by 50% MN assay demonstrated 53%, 94%, 92%, and 100% of subjects who received two doses of 1 ␮g DPIV per type + AlOH, 4 ␮g + AlOH, 1 ␮g + AS01E , and 1 ␮g +AS03B , respectively, experienced tetravalent seroconversions. DENV-1–4 type–specific GMTs were higher 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 and neutralizing antibody determinations remote from

vaccination (i.e., >6 months) are ongoing.108 All vaccinations have been administered in Puerto Rico and are pending analysis. DNA vaccines consist of a plasmid (or plasmids) containing DENV genes reproduced in bacteria such as Escherichia coli (Fig. 5 and Table 2 (TVDV)). The plasmid contains a eukaryotic promoter and termination sequence to drive transcription and presentation to the immune system. Potential advantages of DNA-based vaccine include ease of production, stability, ability to add new genes, and ability to immunize against multiple pathogens with a single construct.185 A DENV-1 monovalent DNA vaccine trial enrolled 22 flavivirus–naive U.S. volunteers and administered three doses at months 0, 1, and 5. None of the low-dosage recipients and only 5 of 11 highdosage recipients developed neutralizing antibodies. The safety profile was acceptable and supported exploration of a tetravalent adjuvanted DNA vaccine candidate; final results are pending.99

Unanswered questions and future directions The dengue vaccine field is confronting numerous questions, especially in the aftermath of CYD’s limited success in phase IIb. The exploration of DENV evolution in time and space introduces the concern that even minor degrees of antigen mismatch between vaccine and circulating wild-type viruses could significantly affect DENV type–specific and overall efficacy. This was a leading hypothesis for CYD’s phase IIb performance (DENV-2 efficacy 9%), but currently available data would not


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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 extensive and growing body of data describing DENV epidemiologic trends and the potential that variations in circulating genotypes may influence observed clinical phenotypes.186–190 There are indications that only a few strategically placed mutations in the DENV genome may be necessary to have significant impacts on its ability to be neutralized.191 The most significant implication would be the recurring requirement for developers to update their licensed vaccines with contemporary DENV strains (e.g., similar to influenza but with a more protracted timeline). Epidemiologic observations of prolonged homotypic protection, perhaps lifelong, following infection would argue against this possibility. However, the immune profile imparted by sequential natural infections with full DENV genomes is (at least to the present author) likely to be different than that generated by a simultaneous exposure to four attenuated vaccine DENV strains. Largescale studies evaluating the association of genotype and clinical illness severity would be informative. Postlicensure studies of DENV evolution measured in a defined time and space, and the impact of large-scale vaccination on the same, are prudent. The development, optimization, and application of assay platforms to measure vaccine immunogenicity require additional work. It is clear from the CYD study that in vitro neutralizing antibodies measured in a vaccinated subgroup did not predict the overall in vivo clinical experience following infection; measured neutralizing antibody titers did not predict protection, especially for DENV-2. It is possible there were issues with how the immunogenicity subgroup was selected, and that their immunologic experience following vaccination or risk

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of infection and disease did not reflect that of the larger cohort.192–194 It is imperative that the risk of infection and potential for disease experienced by the subgroup must both accurately represent the entire cohort’s and be balanced between vaccine and placebo/control recipients. The numerous factors that contribute to these risks, such as pre-existing immunologic background, living conditions (i.e., water source), transmission trends throughout the year (high and low vector and transmission activity), and daily living activities (indoor vs. outdoor), make this a difficult task requiring well conceived enrollment strategies. Another potential reason for the apparent disconnect between immunogenicity and protection may be the measurement and interpretation of heterotypic cross-reactive nonneutralizing antibody as homotypic DENV type–specific antibody. The former may do nothing, attenuate, or contribute to increased disease severity following wild-type DENV exposure, while the latter is expected to provide decades of protection from disease following infection with the same DENV type.74,195–197 Unfortunately, it is very difficult to discern between the two using conventional assay platforms following secondary and repeated DENV exposures. Assays incorporating heterotypic antibody depletion steps, using constant serum concentration with varied viral input (log neutralization index), or incorporating human cell lines or cell lines bearing Fc␥ R may improve the signal (homotypic) to noise (heterotypic) ratio.50,87,88,198,199 Requirements for sample volume, throughput, automation potential, and cost will influence applicability of new assay platforms into large-scale trials.


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Figure 4. Merck Sharp & Dohme Corp., V180. The truncated dengue envelope proteins (DEN-80E) for all four dengue virus types are expressed in the Drosophila S2 cell expression at high levels and have been shown to maintain native-like conformation.

The immune response following natural DENV exposure or following vaccination are kinetic and mature from a cross-reactive, and for a period of months cross-protective, response to a monotypic and DENV type–specific response. Measurement of immune responses 1 month following exposure to a tetravalent dengue vaccine may inform developers about acute immunogenicity, but not potential for efficacy. In practical terms, a subgroup’s neutralizing antibody profile measured 1 month following vaccination may predict high efficacy against a particular DENV type, but if measured 6 months following vaccination and after a period of maturation, the efficacy assessment may be significantly different. One recommendation would be for developers to collect blood samples required to complete acute (1 month), subacute (6–9 months), and longer term (1 year and greater) analyses that associate immunogenicity with clinical end-point data. The limited success of the CYD candidate in the phase IIb trial emphasizes the need to continue and explore contributions of cellular immunity to immuno-protective profiles. Many of the CD4+ and CD8+ T cell epitopes are located in the NS proteins (NS1, 3, 5).42 Vaccine candidates without DENV NS proteins, or NS proteins representative of only one DENV type, may fail to activate the comprehensive immune response required to protect the vaccine recipient from dengue disease caused by any DENV type. Quantitative and qualitative/functional

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assay platforms (e.g., intracellular cytokine staining (ICS), Luminex, ELISA, and ELISPOT) are being used early in the vaccine development process to assess DENV type–specific immunogenicity and complement neutralizing antibody data. How the information is being used to support development decisions is unclear. Interpreting a vaccine candidate’s effect on cellular immunity in flavivirusprimed volunteers is challenging, and contributions of anti-DENV cellular immunity to the overall humoral immune response are less well described.43 Continued exploration and deconstruction of cellular immune responses following natural DENV infections (primary and subsequent), with comparison to the same responses following vaccination, is required. Prospective collection of adequate blood samples is required to associate preillness cellular immune profiles with clinical endpoint and outcome data in natural infection and vaccine studies. Live virus vaccines are subject to immune interference following administration and suboptimal immune responses. Each DENV type has unique virologic (i.e., infection, replication) properties that may be sustained following attenuation and inclusion into tetravalent dengue vaccine200,201 formulations. This combined with baseline flavivirus immunity, from previous infection with dengue, Japanese encephalitis or Yellow fever viruses or by vaccination, may shape vaccine immunogenicity such that one or two DENV types dominate the infection, antigen replication process, and subsequent overall immune response. Certain DENV vaccine strains will be efficacious, while others will offer some or no protection. While some vaccine developers advance candidate vaccines composed of equal viral concentrations for each DENV type, others manipulate the degree of attenuation and viral concentration of each DENV component.127 As discussed above, the concept of DENV type–specific protection threshold versus quantitative balance should be considered (personal view, SJT). Comprehensive dose ranging–studies that evaluate the interplay between the DENV types are necessary early in vaccine development programs, especially with replicating vaccine candidates. Central to many of the challenges and unanswered questions in dengue vaccinology is the absence of a known correlate of protection or validated animal disease model. Although work continues on the development of small animal or nonhuman


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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 restriction site and a hybrid XhoI/SalI site at the 5 terminus of the inserted prM/E gene (not shown). Provided courtesy of the U.S. Naval Medical Research Center.

primate models that more accurately approximate human in vivo infection and disease experience, prospects in the near term remain limited. Consequently, a dengue human infection model (DHIM) is receiving increasing attention. As of 2013 over 650 human infection experiments have been described. A well-characterized and consistently performing DHIM could help reduce risks associated with vaccine development and allow for a glimpse of potential efficacy early in development. Early termination of vaccine candidates unable to protect recipients from challenge with DHIM strains would prevent exposing large numbers of people in clinical endpoint trials to poor vaccine candidates. The absence of a DENV-specific antiviral agent raises 14

ethical questions and the need for an informed risk assessment. Conclusion The global dengue burden is increasing, and every indication is consistent with the conclusion that this trend will continue to worsen unless an effective vaccine is found. Unfortunately, numerous obstacles hamper the development and licensure of a safe and efficacious vaccine. A number of candidates are in preclinical and clinical development representing diverse approaches. A recent efficacy trial yielded disappointing results and challenged long held beliefs regarding associations between vaccine immunogenicity and potential efficacy. Dengue


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virus evolution, measurement of homotypic versus heterotypic antibody responses, durable immunity, and animal and human disease models all require expanded exploration and study. Conflicts of interest SJT is an employee of the U.S. Government. His assigned duties include working with vaccine developers listed in this manuscript. Disclosure is made in the spirit of transparency not because a conflict of interest is believed to exist. The opinions or assertions contained herein are the private views of the author and are not to be construed as reflecting 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 of dengue. Nature 496: 504–507. 2. Murray, N.E., M.B. Quam & A. Wilder-Smith. 2013. Epidemiology of dengue: past, present and future prospects. Clin Epidemiol 5: 299–309. 3. Shepard, D.S., E.A. Undurraga & Y.A. Halasa. 2013. Economic 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 burden of dengue in Nicaragua: 1996–2010. Am J Trop Med Hyg 87: 616–622. 5. Tam, P.T. et al. 2012. High household economic burden caused by hospitalization of patients with severe dengue fever cases in Can Tho province, Vietnam. Am J Trop Med Hyg 87: 554–558. 6. Halasa, Y.A., D.S. Shepard & W. Zeng. 2012. Economic cost of dengue in Puerto Rico. Am J Trop Med Hyg 86: 745–752. 7. Gubler, D.J. 2012. The economic burden of dengue. Am J Trop Med Hyg 86: 743–744. 8. Shepard, D.S. et al. 2011. Economic impact of dengue illness in the Americas. Am J Trop Med Hyg 84: 200–207. 9. Lee, B.Y. et al. 2011. Economic value of dengue vaccine in Thailand. Am J Trop Med Hyg 84: 764–772. 10. Carrasco, L.R. et al. 2011. Economic impact of dengue illness and the cost-effectiveness of future vaccination programs in Singapore. PLoS Negl Trop Dis 5: e1426. 11. Beatty, M.E. et al. 2011. Health economics of dengue: a systematic 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 infections in India. Trans R Soc Trop Med Hyg 102: 570–577. 13. Canyon, D.V. 2008. Historical analysis of the economic cost of dengue in Australia. J Vector Borne Dis 45: 245–248. 14. Armien, B. et al. 2008. Clinical characteristics and national economic cost of the 2005 dengue epidemic in Panama. Am J Trop Med Hyg 79: 364–371. 15. Torres, J.R. & J. Castro. 2007. The health and economic impact of dengue in Latin America. Cadernos de saude publica 23(Suppl. 1): S23–S31.

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