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1.  The Human CD8+ T Cell Responses Induced by a Live Attenuated Tetravalent Dengue Vaccine Are Directed against Highly Conserved Epitopes 
Journal of Virology  2014;89(1):120-128.
ABSTRACT
The incidence of infection with any of the four dengue virus serotypes (DENV1 to -4) has increased dramatically in the last few decades, and the lack of a treatment or vaccine has contributed to significant morbidity and mortality worldwide. A recent comprehensive analysis of the human T cell response against wild-type DENV suggested an human lymphocyte antigen (HLA)-linked protective role for CD8+ T cells. We have collected one-unit blood donations from study participants receiving the monovalent or tetravalent live attenuated DENV vaccine (DLAV), developed by the U.S. National Institutes of Health. Peripheral blood mononuclear cells from these donors were screened in gamma interferon enzyme-linked immunosorbent spot assays with pools of predicted, HLA-matched, class I binding peptides covering the entire DENV proteome. Here, we characterize for the first time CD8+ T cell responses after live attenuated dengue vaccination and show that CD8+ T cell responses in vaccinees were readily detectable and comparable to natural dengue infection. Interestingly, whereas broad responses to structural and nonstructural (NS) proteins were observed after monovalent vaccination, T cell responses following tetravalent vaccination were, dramatically, focused toward the highly conserved NS proteins. Epitopes were highly conserved in a vast variety of field isolates and able to elicit multifunctional T cell responses. Detailed knowledge of the T cell response will contribute to the identification of robust correlates of protection in natural immunity and following vaccination against DENV.
IMPORTANCE The development of effective vaccination strategies against dengue virus (DENV) infection and clinically significant disease is a task of high global public health value and significance, while also being a challenge of significant complexity. A recent efficacy trial of the most advanced dengue vaccine candidate, demonstrated only partial protection against all four DENV serotypes, despite three subsequent immunizations and detection of measurable neutralizing antibodies to each serotype in most subjects. These results challenge the hypothesis that seroconversion is the only reliable correlate of protection. Here, we show that CD8+ T cell responses in vaccinees were readily detectable and comparable to natural dengue virus infection. Detailed knowledge of the T cell response may further contribute to the identification of robust correlates of protection in natural immunity and vaccination against DENV.
doi:10.1128/JVI.02129-14
PMCID: PMC4301095  PMID: 25320311
2.  Japanese encephalitis virus vaccine candidates generated by chimerization with dengue virus type 4 
Vaccine  2014;32(25):3010-3018.
Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis worldwide and vaccination is one of the most effective ways to prevent disease. A suitable live-attenuated JEV vaccine could be formulated with a live-attenuated tetravalent dengue vaccine for the control of these viruses in endemic areas. Toward this goal, we generated chimeric virus vaccine candidates by replacing the precursor membrane (prM) and envelope (E) protein structural genes of recombinant dengue virus type 4 (rDEN4) or attenuated vaccine candidate rDEN4Δ30 with those of wild-type JEV strain India/78. Mutations were engineered in E, NS3 and NS4B protein genes to improve replication in Vero cells. The chimeric viruses were attenuated in mice and some elicited modest but protective levels of immunity after a single dose. One particular chimeric virus, bearing E protein mutation Q264H, replicated to higher titer in tissue culture and was significantly more immunogenic in mice. The results are compared with live-attenuated JEV vaccine strain SA14-14-2.
doi:10.1016/j.vaccine.2014.03.062
PMCID: PMC4083695  PMID: 24699473
Japanese encephalitis virus; Chimeric virus; Live-attenuated vaccine
3.  Dual miRNA Targeting Restricts Host Range and Attenuates Neurovirulence of Flaviviruses 
PLoS Pathogens  2015;11(4):e1004852.
Mosquito-borne flaviviruses are among the most significant arboviral pathogens worldwide. Vaccinations and mosquito population control programs remain the most reliable means for flavivirus disease prevention, and live attenuated viruses remain one of the most attractive flavivirus vaccine platforms. Some live attenuated viruses are capable of infecting principle mosquito vectors, as demonstrated in the laboratory, which in combination with their intrinsic genetic instability could potentially lead to a vaccine virus reversion back to wild-type in nature, followed by introduction and dissemination of potentially dangerous viral strains into new geographic locations. To mitigate this risk we developed a microRNA-targeting approach that selectively restricts replication of flavivirus in the mosquito host. Introduction of sequences complementary to a mosquito-specific mir-184 and mir-275 miRNAs individually or in combination into the 3’NCR and/or ORF region resulted in selective restriction of dengue type 4 virus (DEN4) replication in mosquito cell lines and adult Aedes mosquitos. Moreover a combined targeting of DEN4 genome with mosquito-specific and vertebrate CNS-specific mir-124 miRNA can silence viral replication in two evolutionally distant biological systems: mosquitoes and mouse brains. Thus, this approach can reinforce the safety of newly developed or existing vaccines for use in humans and could provide an additional level of biosafety for laboratories using viruses with altered pathogenic or transmissibility characteristics.
Author Summary
Despite advances in developing flavivirus live attenuated vaccine (LAV) candidates, a concern exists that they might not be safe in the environment due to their intrinsic genetic instability leading to potential reversion back to wild-type that could be associated with possible dissemination of these mutated viruses by mosquitoes. Here, we describe a miRNA targeting approach that can be adapted to support the design of environmentally-safe LAV restricted in their ability to infect and be transmitted by competent vectors, thereby limiting the possibility of subsequent viral evolution and unpredictable consequences. A combined co-targeting of flavivirus genome with mosquito- and vertebrate brain- specific miRNAs resulted in simultaneous restriction of dengue virus infection and replication in mosquitoes and in brains of newborn mice indicating that the miRNA-mediated approach for virus attenuation represents an alternative to non-specific strategies for the control of viral tissue tropism and pathogenesis in the vertebrate host and replicative efficacy in permissive vectors.
doi:10.1371/journal.ppat.1004852
PMCID: PMC4408003  PMID: 25906260
4.  Dengue Virus Infections and Maternal Antibody Decay in a Prospective Birth Cohort Study of Vietnamese Infants 
The Journal of infectious diseases  2009;200(12):1893-1900.
Dengue hemorrhagic fever can occur in primary dengue virus (DENV) infection of infants. The decay of maternally derived DENV immunoglobulin (Ig) G and the incidence of DENV infection were determined in a prospectively studied cohort of 1244 Vietnamese infants. Higher concentrations of total IgG and DENV-reactive IgG were found in cord plasma relative to maternal plasma. Maternally derived DENV-neutralizing and E protein–reactive IgG titers declined to below measurable levels in >90% of infants by 6 months of age. In contrast, IgG reactive with whole DENV virions persisted until 12 months of age in 20% of infants. Serological surveillance identified 10 infants with asymptomatic DENV infection for an incidence of 1.7 cases per 100 person-years. DENV-neutralizing antibodies remained measurable for ≥1 year after infection. These results suggest that whereas DENV infection in infants is frequently subclinical, there is a window between 4 and 12 months of age where virion-binding but nonneutralizing IgG could facilitate antibody-dependent enhancement.
doi:10.1086/648407
PMCID: PMC4340501  PMID: 19911991
5.  Mechanism and Significance of Cell Type-Dependent Neutralization of Flaviviruses 
Journal of Virology  2014;88(13):7210-7220.
ABSTRACT
The production of neutralizing antibodies (NAbs) is a correlate of protection for many human vaccines, including currently licensed vaccines against flaviviruses. NAbs are typically measured using a plaque reduction neutralization test (PRNT). Despite its extensive use, parameters that impact the performance of the PRNT have not been investigated from a mechanistic perspective. The results of a recent phase IIb clinical trial of a tetravalent dengue virus (DENV) vaccine suggest that NAbs, as measured using a PRNT performed with Vero cells, do not correlate with protection. This surprising finding highlights the importance of understanding how well the PRNT captures the complexity of the NAb response to DENV. In this study, we demonstrated that the structural heterogeneity of flaviviruses arising from inefficient virion maturation impacts the results of neutralization assays in a cell type-dependent manner. Neutralization titers of several monoclonal antibodies were significantly reduced when assayed on Vero cells compared to Raji cells expressing DC-SIGNR. This pattern can be explained by differences in the efficiency with which partially mature flaviviruses attach to each cell type, rather than a differential capacity of antibody to block infection. Vero cells are poorly permissive to the fraction of virions that are most sensitive to neutralization. Analysis of sera from recipients of live-attenuated monovalent DENV vaccine candidates revealed a strong correlation between the sensitivity of serum antibodies to the maturation state of DENV and cell type-dependent patterns of neutralization. Cross-reactive patterns of neutralization may be underrepresented by the “gold-standard” PRNT that employs Vero cells.
IMPORTANCE Cell type-dependent patterns of neutralization describe a differential capacity of antibodies to inhibit virus infection when assayed on multiple cellular substrates. In this study, we established a link between antibodies that neutralize infection in a cell type-dependent fashion and those sensitive to the maturation state of the flavivirus virion. We demonstrated that cell type-dependent neutralization reflects a differential capacity to measure neutralization of viruses that are incompletely mature. Partially mature virions that most efficiently bind maturation state-sensitive antibodies are poorly represented by assays typically used in support of flavivirus vaccine development. The selection of cellular substrate for neutralization assays may significantly impact evaluation of the neutralization potency of the polyclonal response. These data suggest that current assays do not adequately capture the full complexity of the neutralizing antibody response and may hinder the identification of correlates of protection following flavivirus vaccination.
doi:10.1128/JVI.03690-13
PMCID: PMC4054442  PMID: 24741083
6.  The live attenuated chimeric vaccine rWN/DEN4Δ30 is well-tolerated and immunogenic in healthy flavivirus-naïve adult volunteers 
Vaccine  2013;31(48):10.1016/j.vaccine.2013.07.064.
WNV has become the leading vector-borne cause of meningoencephalitis in the United States. Although the majority of WNV infections result in asymptomatic illness, approximately 20% of infections result in West Nile fever and 1% in West Nile neuroinvasive disease (WNND), which causes encephalitis, meningitis, or flaccid paralysis. The elderly are at particular risk for WNND, with more than half the cases occurring in persons older than sixty years of age. There is no licensed treatment for WNND nor is there any licensed vaccine for humans for the prevention of WNV infection. The Laboratory of Infectious Diseases at the National Institutes of Health has developed a recombinant live attenuated WNV vaccine based on chimerization of the wild-type WNV NY99 genome with that of the live attenuated DENV-4 candidate vaccine rDEN430. The genes encoding the prM and envelope proteins of DENV-4 were replaced with those of WNV NY99 and the resultant virus was designated rWN/DEN4Δ30. The vaccine was evaluated in healthy flavivirus-naïve adult volunteers age 18 – 50 years in two separate studies, both of which are reported here. The first study evaluated 103 or 104 PFU of the vaccine given as a single dose; the second study evaluated 105 PFU of the vaccine given as two doses 6 months apart. The vaccine was well-tolerated and immunogenic at all three doses, inducing seroconversion to WNV NY99 in 74% (103 PFU), 75% (104 PFU), and 55% (105 PFU) of subjects after a single dose. A second 105 PFU dose of rWN/DEN4Δ30 given 6 months after the first dose increased the seroconversion rate 89%. Based on the encouraging results from these studies, further evaluation of the candidate vaccine in adults older than 50 years of age is planned.
doi:10.1016/j.vaccine.2013.07.064
PMCID: PMC3833717  PMID: 23968769
West Nile virus (WNV); live attenuated WNV vaccine; clinical trial
7.  Vaccination of Volunteers with Low-Dose, Live-Attenuated, Dengue Viruses Leads to Serotype-specific Immunologic and Virologic Profiles 
Vaccine  2013;31(33):3347-3352.
There are currently no vaccines or therapeutics to prevent dengue disease which ranges in severity from asymptomatic infections to life-threatening illness. The National Institute of Allergy and Infectious Diseases (NIAID) Division of Intramural Research has developed live, attenuated vaccines to each of the four dengue serotypes (DENV-1 – DENV-4). Two doses (10 PFU and 1000 PFU) of three monovalent vaccines were tested in human clinical trials to compare safety and immunogenicity profiles. DEN4Δ30 had been tested previously at multiple doses. The three dengue vaccine candidates tested (DEN1Δ30, DEN2/4Δ30, and DEN3Δ30/31) were very infectious, each with a Human Infectious Dose 50% ≤ 10 PFU. Further, infectivity rates ranged from 90 −100% regardless of dose, excepting DEN2/4Δ30 which dropped from 100% at the 1000 PFU dose to 60% at the 10 PFU dose. Mean geometric peak antibody titers did not differ significantly between doses for DEN1Δ30 (92 ± 19 vs. 214 ± 97, p = 0.08); however, significant differences were observed between the 10 PFU and 1000 PFU doses for DEN2/4Δ30, 19 ± 9 vs. 102 ± 25 (p = 0.001), and DEN3Δ30/31, 119 ± 135 vs. 50 ± 50 (p=0.046). No differences in the incidences of rash, neutropenia, or viremia were observed between doses for any vaccines, though the mean peak titer of viremia for DEN1Δ30 was higher at the 1000 PFU dose (0.5 ± 0 vs. 1.1 ± 0.1, p = 0.007). These data demonstrate that atarget dose of 1000 PFU for inclusion of each dengue serotype into a tetravalent vaccine is likely to be safe and generate a balanced immune response for all serotypes.
doi:10.1016/j.vaccine.2013.05.075
PMCID: PMC3777849  PMID: 23735680
Dengue virus; dengue vaccine; low dose; viremia; neutralizing antibodies; HID50
8.  Human Monoclonal Antibodies Derived From Memory B Cells Following Live Attenuated Dengue Virus Vaccination or Natural Infection Exhibit Similar Characteristics 
The Journal of Infectious Diseases  2013;207(12):1898-1908.
The immunopathogenesis of severe dengue is poorly understood, but there is concern that induction of cross-reactive nonneutralizing antibodies by infection or vaccination may increase the likelihood of severe disease during a subsequent infection. We generated a total of 63 new human monoclonal antibodies to compare the B-cell response of subjects who received the National Institutes of Health live attenuated dengue vaccine rDEN1Δ30 to that of subjects following symptomatic primary infection with DENV1. Both infection and vaccination induced serum neutralizing antibodies and DENV1-reactive peripheral blood B cells, but the magnitude of induction was lower in vaccinated individuals. Serotype cross-reactive weakly neutralizing antibodies dominated the response in both vaccinated and naturally infected subjects. Antigen specificities were very similar, with a slightly greater percentage of antibodies targeting E protein domain I/II than domain III. These data shed light on the similarity of human B-cell response to live attenuated DENV vaccine or natural infection.
doi:10.1093/infdis/jit119
PMCID: PMC3654755  PMID: 23526830
human; B cells; hybridomas; dengue virus; antibodies; antibody-dependent enhancement; neutralization
9.  Investigation of Dengue and Japanese Encephalitis Virus Transmission in Hanam, Viet Nam 
This study investigated whether a large dengue epidemic that struck Hanoi in 2009 also affected a nearby semirural area. Seroconversion (dengue virus-reactive immunoglobulin G enzyme-linked immunosorbent assay) was high during 2009 compared with 2008, but neutralization assays showed that it was caused by both dengue virus and Japanese encephalitis virus infections. The findings highlight the importance of continued Japanese encephalitis virus vaccination and dengue surveillance.
doi:10.4269/ajtmh.13-0077
PMCID: PMC4015583  PMID: 24615123
10.  Replacement of conserved or variable sequences of the mosquito-borne dengue virus 3′ UTR with homologous sequences from Modoc virus does not change infectivity for mosquitoes 
The Journal of General Virology  2013;94(Pt 4):783-788.
The genus Flavivirus includes both vector-borne and no known vector (NKV) species, but the molecular determinants of transmission mode are not known. Conserved sequence differences between the two groups occur in 5′ and 3′ UTRs. To investigate the impact of these differences on transmission, chimeric genomes were generated, in which UTRs, UTRs+capsid, or the upper 3′ UTR stem–loop of mosquito-borne dengue virus (DENV) were replaced with homologous regions from NKV Modoc virus (MODV); the conserved pentanucleotide sequence (CPS) was also deleted from the DENV genome. Virus was not recovered following transfection of these genomes in three different cell types. However, DENV genomes in which the CPS or variable region (VR) of the 3′ UTR were replaced with MODV sequences were recovered and infected Aedes aegypti mosquitoes with similar efficiencies to DENV. These results demonstrate that neither vector-borne CPS nor VR is required for vector-borne transmission.
doi:10.1099/vir.0.046664-0
PMCID: PMC3709684  PMID: 23255623
11.  A Single Dose of Any of Four Different Live Attenuated Tetravalent Dengue Vaccines Is Safe and Immunogenic in Flavivirus-naive Adults: A Randomized, Double-blind Clinical Trial 
The Journal of Infectious Diseases  2013;207(6):957-965.
Background. Dengue virus (DENV) causes hundreds of millions of infections annually. Four dengue serotypes exist, and previous infection with one serotype increases the likelihood of severe disease with a second, heterotypic DENV infection.
Methods. In a randomized, placebo-controlled study, the safety and immunogenicity of 4 different admixtures of a live attenuated tetravalent (LATV) dengue vaccine were evaluated in 113 flavivirus-naive adults. Serum neutralizing antibody levels to all 4 dengue viruses were measured on days 0, 28, 42, and 180.
Results. A single dose of each LATV admixture induced a trivalent or better neutralizing antibody response in 75%–90% of vaccinees. There was no significant difference in the incidence of adverse events between vaccinees and placebo-recipients other than rash. A trivalent or better response correlated with rash and with non-black race (P < .0001). Black race was significantly associated with a reduced incidence of vaccine viremia.
Conclusions. TV003 induced a trivalent or greater antibody response in 90% of flavivirus-naive vaccinees and is a promising candidate for the prevention of dengue. Race was identified as a factor influencing the infectivity of the LATV viruses, reflecting observations of the effect of race on disease severity in natural dengue infection.
Clinical Trials Registration NCT01072786.
doi:10.1093/infdis/jis936
PMCID: PMC3571448  PMID: 23329850
dengue vaccine; live attenuated tetravalent; clinical trial
12.  The Human Immune Response to Dengue Virus Is Dominated by Highly Cross-Reactive Antibodies Endowed with Neutralizing and Enhancing Activity 
Cell host & microbe  2010;8(3):10.1016/j.chom.2010.08.007.
Summary
Antibodies protect against homologous Dengue virus (DENV) infection but can precipitate severe dengue by promoting heterotypic virus entry via Fcγ receptors (FcγR). We immortalized memory B cells from individuals after primary or secondary infection and analyzed anti-DENV monoclonal antibodies (mAbs) thus generated. MAbs to envelope (E) protein domain III (DIII) were either serotype specific or cross-reactive and potently neutralized DENV infection. DI/DII- or viral membrane protein prM-reactive mAbs neutralized poorly and showed broad cross-reactivity with the four DENV serotypes. All mAbs enhanced infection at subneutralizing concentrations. Three mAbs targeting distinct epitopes on the four DENV serotypes and engineered to prevent FcγR binding did not enhance infection and neutralized DENV in vitro and in vivo as postexposure therapy in a mouse model of lethal DENV infection. Our findings reveal an unexpected degree of cross-reactivity in human antibodies against DENV and illustrate the potential for an antibody-based therapy to control severe dengue.
doi:10.1016/j.chom.2010.08.007
PMCID: PMC3884547  PMID: 20833378
13.  Corticosteroids for Dengue – Why Don't They Work? 
Background
Dysregulated immune responses may contribute to the clinical complications that occur in some patients with dengue.
Findings
In Vietnamese pediatric dengue cases randomized to early prednisolone therapy, 81 gene-transcripts (0.2% of the 47,231 evaluated) were differentially abundant in whole-blood between high-dose (2 mg/kg) prednisolone and placebo-treated patients two days after commencing therapy. Prominent among the 81 transcripts were those associated with T and NK cell cytolytic functions. Additionally, prednisolone therapy was not associated with changes in plasma cytokine levels.
Conclusion
The inability of prednisolone treatment to markedly attenuate the host immune response is instructive for planning future therapeutic strategies for dengue.
Author Summary
Dengue is an acute, mosquito-borne febrile illness and around 390 million cases occur annually in more than 100 countries. A host pro-inflammatory immune response is widely believed to contribute to the clinical complications that occur in some patients with dengue. Synthetic glucocorticoids, which are immunomodulatory agents commonly used in medicine, have been suggested as a therapy for dengue. We recently performed a randomized, controlled trial of early oral glucocorticoid therapy in 225 dengue cases in Vietnam, comparing a three day regimen of high (2 mg/kg) or low (0.5 mg/kg) dose prednisolone with placebo. Here, we report on immunological changes occurring during prednisolone therapy with a view to understanding the lack of clinical benefit by glucocorticoid therapy and to guide future intervention strategies for dengue. In whole-blood gene expression arrays we found 81 transcripts from 64 genes differentially abundant between high-dose prednisolone and placebo treated patients. Prominent were the genes associated with T and NK cell cytolytic functions. These results are a reminder that the mechanisms causally behind some of the complications of dengue (e.g. altered capillary permeability) are very poorly understood and represent a major knowledge gap in our understanding of disease pathogenesis that also undermines attempts to improve clinical management.
doi:10.1371/journal.pntd.0002592
PMCID: PMC3861232  PMID: 24349598
14.  The Type-Specific Neutralizing Antibody Response Elicited by a Dengue Vaccine Candidate Is Focused on Two Amino Acids of the Envelope Protein 
PLoS Pathogens  2013;9(12):e1003761.
Dengue viruses are mosquito-borne flaviviruses that circulate in nature as four distinct serotypes (DENV1-4). These emerging pathogens are responsible for more than 100 million human infections annually. Severe clinical manifestations of disease are predominantly associated with a secondary infection by a heterotypic DENV serotype. The increased risk of severe disease in DENV-sensitized populations significantly complicates vaccine development, as a vaccine must simultaneously confer protection against all four DENV serotypes. Eliciting a protective tetravalent neutralizing antibody response is a major goal of ongoing vaccine development efforts. However, a recent large clinical trial of a candidate live-attenuated DENV vaccine revealed low protective efficacy despite eliciting a neutralizing antibody response, highlighting the need for a better understanding of the humoral immune response against dengue infection. In this study, we sought to identify epitopes recognized by serotype-specific neutralizing antibodies elicited by monovalent DENV1 vaccination. We constructed a panel of over 50 DENV1 structural gene variants containing substitutions at surface-accessible residues of the envelope (E) protein to match the corresponding DENV2 sequence. Amino acids that contribute to recognition by serotype-specific neutralizing antibodies were identified as DENV mutants with reduced sensitivity to neutralization by DENV1 immune sera, but not cross-reactive neutralizing antibodies elicited by DENV2 vaccination. We identified two mutations (E126K and E157K) that contribute significantly to type-specific recognition by polyclonal DENV1 immune sera. Longitudinal and cross-sectional analysis of sera from 24 participants of a phase I clinical study revealed a markedly reduced capacity to neutralize a E126K/E157K DENV1 variant. Sera from 77% of subjects recognized the E126K/E157K DENV1 variant and DENV2 equivalently (<3-fold difference). These data indicate the type-specific component of the DENV1 neutralizing antibody response to vaccination is strikingly focused on just two amino acids of the E protein. This study provides an important step towards deconvoluting the functional complexity of DENV serology following vaccination.
Author Summary
Despite decades of research, there remains a critical need for a dengue virus (DENV) vaccine. Vaccine development efforts are complicated by a requirement to protect against four DENV serotypes (DENV1-4), and incomplete immunity as a risk factor for severe disease. Antibodies play a major protective role against DENV. However, they also have been implicated in severe clinical manifestations of DENV infection. The antibody response to DENV is composed of antibodies that neutralize only the infecting DENV serotype (type-specific), as well as those that are cross-reactive. Cross-reactive antibodies are hypothesized to contribute to severe dengue following heterologous infections. Identifying DENV epitopes that are targets of type-specific neutralizing antibodies may facilitate vaccine development and the identification of correlates of protection. In this study, we identified amino acids on DENV1 recognized by type-specific neutralizing antibodies elicited by DENV1 vaccination. Our results indicate that the type-specific DENV1 response is remarkably focused on just two regions of the DENV1 envelope protein. Furthermore, a significant contribution of antibodies with this specificity was a common feature among vaccine recipients. This study identifies targets of neutralizing antibodies elicited by DENV1 vaccination and provides an important first step toward identifying epitopes recognized by each component of a tetravalent vaccine.
doi:10.1371/journal.ppat.1003761
PMCID: PMC3857832  PMID: 24348242
15.  Dengue Research Opportunities in the Americas 
The Journal of Infectious Diseases  2012;206(7):1121-1127.
Dengue is a systemic arthropod-borne viral disease of major global public health importance. At least 2.5 billion people who live in areas of the world where dengue occurs are at risk of developing dengue fever (DF) and its severe complications, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Repeated reemergences of dengue in sudden explosive epidemics often cause public alarm and seriously stress healthcare systems. The control of dengue is further challenged by the lack of effective therapies, vaccines, and point-of-care diagnostics. Despite years of study, even its pathogenic mechanisms are poorly understood. This article discusses recent advances in dengue research and identifies challenging gaps in research on dengue clinical evaluation, diagnostics, epidemiology, immunology, therapeutics, vaccinology/clinical trials research, vector biology, and vector ecology. Although dengue is a major global tropical pathogen, epidemiologic and disease control considerations in this article emphasize dengue in the Americas.
doi:10.1093/infdis/jis351
PMCID: PMC3499110  PMID: 22782946
16.  Genetic and phenotypic characterization of sylvatic dengue virus type 2 strains 
Virology  2008;377(2):296-307.
The four serotypes of endemic dengue viruses (DENV) circulate between humans and peridomestic Aedes mosquitoes. At present endemic DENV infect 100 million people per year, and a third of the global population is at risk. In contrast, sylvatic DENV strains are maintained in a transmission cycle between nonhuman primates and sylvatic Aedes species, and are evolutionarily and ecologically distinct from endemic DENV strains. Phylogenetic analyses place sylvatic strains basal to each of the endemic serotypes, supporting the hypothesis that each of the endemic DENV serotypes emerged independently from sylvatic ancestors. We utilized complete genome analyses of both sylvatic and endemic DENV serotype 2 (DENV-2) to expand our understanding of their genetic relationships. A high degree of conservation was observed in both the 5′- and 3′-untranslated genome regions, whereas considerable differences at the nucleotide and amino acid levels were observed within the open reading frame. Additionally, replication of the two genotypes was compared in cultured cells, where endemic DENV strains produced a significantly higher output of progeny in human liver cells, but not in monkey kidney or mosquito cells. Understanding the genetic relationships and phenotypic differences between endemic and sylvatic DENV genotypes may provide valuable insight into DENV emergence and guide monitoring of future outbreaks.
doi:10.1016/j.virol.2008.04.044
PMCID: PMC3612928  PMID: 18570968
Dengue virus (DENV); Sylvatic DENV; Endemic DENV; Phylogenetic and phenotypic analysis
17.  A Novel Approach for the Rapid Mutagenesis and Directed Evolution of the Structural Genes of West Nile Virus 
Journal of Virology  2012;86(7):3501-3512.
Molecular clone technology has proven to be a powerful tool for investigating the life cycle of flaviviruses, their interactions with the host, and vaccine development. Despite the demonstrated utility of existing molecular clone strategies, the feasibility of employing these existing approaches in large-scale mutagenesis studies is limited by the technical challenges of manipulating relatively large molecular clone plasmids that can be quite unstable when propagated in bacteria. We have developed a novel strategy that provides an extremely rapid approach for the introduction of mutations into the structural genes of West Nile virus (WNV). The backbone of this technology is a truncated form of the genome into which DNA fragments harboring the structural genes are ligated and transfected directly into mammalian cells, bypassing entirely the requirement for cloning in bacteria. The transfection of cells with this system results in the rapid release of WNV that achieves a high titer (∼107 infectious units/ml in 48 h). The suitability of this approach for large-scale mutagenesis efforts was established in two ways. First, we constructed and characterized a library of variants encoding single defined amino acid substitutions at the 92 residues of the “pr” portion of the precursor-to-membrane (prM) protein. Analysis of a subset of these variants identified a mutation that conferred resistance to neutralization by an envelope protein-specific antibody. Second, we employed this approach to accelerate the identification of mutations that allow escape from neutralizing antibodies. Populations of WNV encoding random changes in the E protein were produced in the presence of a potent monoclonal antibody, E16. Viruses resistant to neutralization were identified in a single passage. Together, we have developed a simple and rapid approach to produce infectious WNV that accelerates the process of manipulating the genome to study the structure and function of the structural genes of this important human pathogen.
doi:10.1128/JVI.06435-11
PMCID: PMC3302547  PMID: 22258236
18.  Development and clinical evaluation of multiple investigational monovalent DENV vaccines to identify components for inclusion in a live attenuated tetravalent DENV vaccine 
Vaccine  2011;29(42):7242-7250.
The Laboratory of Infectious Diseases at the National Institute of Allergy and Infectious Diseases, National Institutes of Health has been engaged in an effort to develop a safe, efficacious, and affordable live attenuated tetravalent dengue vaccine (LATV) for more than ten years. Numerous recombinant monovalent DENV vaccine candidates have been evaluated in the SCID-HuH-7 mouse and in rhesus macaques to identify those candidates with a suitable attenuation phenotype. In addition, the ability of these candidates to infect and disseminate in Aedes mosquitoes had also been determined. Those candidates that were suitably attenuated in SCID-HuH-7 mice, rhesus macaques, and mosquitoes were selected for further evaluation in humans. This review will describe the generation of multiple candidate vaccines directed against each DENV serotype, the preclinical and clinical evaluation of these candidates, and the process of selecting suitable candidates for inclusion in a LATV dengue vaccine.
doi:10.1016/j.vaccine.2011.07.023
PMCID: PMC3170437  PMID: 21781997
19.  Temperature-dependent production of pseudoinfectious dengue reporter virus particles by complementation 
Virology  2008;381(1):67-74.
Dengue virus (DENV) is a mosquito-borne flavivirus responsible for 50 to 100 million human infections each year, highlighting the need for a safe and effective vaccine. In this study, we describe the production of pseudoinfectious DENV reporter virus particles (RVPs) using two different genetic complementation approaches, including the creation of cell lines that release reporter viruses in an inducible fashion. In contrast to studies with West Nile virus (WNV), production of infectious DENV RVPs was temperature-dependent; the yield of infectious DENV RVPs at 37 °C is significantly reduced in comparison to experiments conducted at lower temperatures or with WNV. This reflects both a significant reduction in the rate of infectious DENV RVP release over time, and the more rapid decay of infectious DENV RVPs at 37 °C. Optimized production approaches allow the production of DENV RVPs with titers suitable for the study of DENV entry, assembly, and the analysis of the humoral immune response of infected and vaccinated individuals.
doi:10.1016/j.virol.2008.08.021
PMCID: PMC3428711  PMID: 18801552
Flavivirus; West Nile virus; Dengue; Reporter virus; Antibody-mediated neutralization
20.  Primary Vaccination with Low Dose Live Dengue 1 Virus Generates a Proinflammatory, Multifunctional T Cell Response in Humans 
The four dengue virus serotypes (DENV-1–DENV-4) have a large impact on global health, causing 50–100 million cases of dengue fever annually. Herein, we describe the first kinetic T cell response to a low-dose DENV-1 vaccination study (10 PFU) in humans. Using flow cytometry, we found that proinflammatory cytokines, IFNγ, TNFα, and IL-2, were generated by DENV-1-specific CD4+ cells 21 days post-DENV-1 exposure, and their production continued through the latest time-point, day 42 (p<0.0001 for all cytokines). No statistically significant changes were observed at any time-points for IL-10 (p = 0.19), a regulatory cytokine, indicating that the response to DENV-1 was primarily proinflammatory in nature. We also observed little T cell cross-reactivity to the other 3 DENV serotypes. The percentage of multifunctional T cells (T cells making ≥2 cytokines simultaneously) increased with time post-DENV-1 exposure (p<0.0001). The presence of multifunctional T cells together with neutralizing antibody data suggest that the immune response generated to the vaccine may be protective. This work provides an initial framework for defining primary T cell responses to each DENV serotype and will enhance the evaluation of a tetravalent DENV vaccine.
Author Summary
40% of the world's population is at risk for developing dengue fever, an acute febrile illness caused by the 4 serotypes of dengue viruses (DENV). Though most of the 50–100 million annual DENV infections resolve without medical intervention, approximately 500,000 cases are severe and require hospitalization. Supportive care is currently the only available treatment for dengue disease. As a result, DENV infections cause strain on healthcare systems and economic burden in endemic countries. Much of the research in the dengue field has focused on understanding the mechanism of severe dengue disease. To better understand human adaptive immune responses to asymptomatic or mild DENV infections, we used longitudinal specimens collected following low dose vaccination with a live DENV-1 candidate vaccine. We found that CD4+ T cells made the proinflammatory cytokines, IFNγ, TNFα and IL-2, 3 weeks following exposure to DENV-1. IFNγ and TNFα production continued for 6 weeks post-vaccination, our final time-point. T cell responses were predominantly multifunctional: T cells produced ≥2 cytokines simultaneously. Lastly, we observed little cross-reactivity in T cell responses. This work helps establish the kinetics and characteristics of a primary adaptive immune response to DENV and aids in the development of a tetravalent vaccine against DENV.
doi:10.1371/journal.pntd.0001742
PMCID: PMC3398956  PMID: 22816004
21.  Replacement of the 3′ untranslated variable region of mosquito-borne dengue virus with that of tick-borne Langat virus does not alter vector specificity 
The Journal of General Virology  2011;92(Pt 4):841-848.
The four major flavivirus clades are transmitted by mosquitoes, ticks, directly between vertebrates or directly between arthropods, respectively, but the molecular determinants of mode of transmission in flaviviruses are unknown. To assess the role of the UTRs in transmission, we generated chimeric genomes in which the 5′ UTR, capsid and/or 3′ UTR of mosquito-borne dengue virus serotype 4 (rDENV-4) were replaced, separately or in combination, with those of tick-borne Langat virus (rLGTV). None of the chimeric genomes yielded detectable virus following transfection. Replacement of the variable region (VR) in the rDENV-4 3′ UTR with that of rLGTV generated virus rDENV-4-rLGTswapVR, which showed lower replication than its wild-type parents in mammalian but not mosquito cells in culture and was able to infect mosquitoes in vivo. Neither rDENV-4 nor rDENV-4-rLGTswapVR could infect larval Ixodes scapularis ticks immersed in virus, while rLGTV was highly infectious via this route.
doi:10.1099/vir.0.026997-0
PMCID: PMC3133702  PMID: 21216984
22.  Heterotypic Dengue Infection with Live Attenuated Monotypic Dengue Virus Vaccines: Implications for Vaccination of Populations in Areas Where Dengue Is Endemic 
The Journal of Infectious Diseases  2011;203(3):327-334.
Background. Because infection with any of the 4 Dengue virus serotypes may elicit both protective neutralizing antibodies and nonneutralizing antibodies capable of enhancing subsequent heterotypic Dengue virus infections, the greatest risk for severe dengue occurs during a second, heterotypic Dengue virus infection. It remains unclear whether the replication of live attenuated vaccine viruses will be similarly enhanced when administered to Dengue-immune individuals.
Methods. We recruited 36 healthy adults who had previously received a monovalent live Dengue virus vaccine 0.6–7.4 years earlier. Participants were assigned to 1 of 4 cohorts and were randomly chosen to receive placebo or a heterotypic vaccine. The level of replication, safety, and immunogenicity of the heterotypic vaccine virus was compared with that of Dengue virus immunologically naive vaccinees.
Results. Vaccine virus replication and reactogenicity after monovalent Dengue virus vaccination in naive and heterotypically immune vaccinees was similar. In contrast to naive vaccinees, the antibody response in heterotypically immune vaccinees was broadly neutralizing and mimicked the response observed by natural secondary Dengue virus infection.
Conclusions. Enhanced replication of these live attenuated Dengue virus vaccines was minimal in heterotypically immune vaccinees and suggests that the further evaluation of these candidate vaccines in populations with preexisting DENV immunity can proceed safely.
Clinical trials registration: NCT00458120 (http://www.clinicaltrials.gov/ct2/show/NCT00458120).
doi:10.1093/infdis/jiq059
PMCID: PMC3071099  PMID: 21208923
23.  Next-Generation Dengue Vaccines: Novel Strategies Currently Under Development 
Viruses  2011;3(10):1800-1814.
Dengue has become the most important arboviral infection worldwide with more than 30 million cases of dengue fever estimated to occur each year. The need for a dengue vaccine is great and several live attenuated dengue candidate vaccines are proceeding through clinical evaluation. The need to induce a balanced immune response against all four DENV serotypes with a single vaccine has been a challenge for dengue vaccine developers. A live attenuated DENV chimeric vaccine produced by Sanofi Pasteur has recently entered Phase III evaluation in numerous dengue-endemic regions of the world. Viral interference between serotypes contained in live vaccines has required up to three doses of the vaccine be given over a 12-month period of time. For this reason, novel DENV candidate vaccines are being developed with the goal of achieving a protective immune response with an immunization schedule that can be given over the course of a few months. These next-generation candidates include DNA vaccines, recombinant adenovirus vectored vaccines, alphavirus replicons, and sub-unit protein vaccines. Several of these novel candidates will be discussed.
doi:10.3390/v3101800
PMCID: PMC3205382  PMID: 22069516
Dengue vaccine; DNA vaccine; vectored-vaccine; sub-unit protein vaccine
24.  A Single Dose of the DENV-1 Candidate Vaccine rDEN1Δ30 Is Strongly Immunogenic and Induces Resistance to a Second Dose in a Randomized Trial 
Dengue is an emerging infectious disease that has become the most important arboviral infection worldwide. There are four serotypes of dengue virus, DENV-1, DENV-2, DENV-3, and DENV-4, each capable of causing the full spectrum of disease. rDEN1Δ30 is a live attenuated investigational vaccine for the prevention of DENV-1 illness and is also a component of an investigational tetravalent DENV vaccine currently in Phase I evaluation. A single subcutaneous dose of rDEN1Δ30 was previously shown to be safe and immunogenic in healthy adults. In the current randomized placebo-controlled trial, 60 healthy flavivirus-naive adults were randomized to receive 2 doses of rDEN1Δ30 (N = 50) or placebo (N = 10), either on study days 0 and 120 (cohort 1) or 0 and 180 (cohort 2). We sought to evaluate the safety and immunogenicity of this candidate vaccine in 50 additional vaccinees and to test whether the humoral immune response could be boosted by a second dose administered 4 or 6 months after the first dose. The first dose of vaccine was well tolerated, infected 47/50 vaccinees and induced seroconversion in 46/50 vaccinees. Irrespective of dosing interval, the second dose of vaccine was also well tolerated but did not induce any detectable viremia or ≥4-fold rise in serum neutralizing antibody titer.Only five subjects had an anamnestic antibody response detectable by ELISA following a second dose of vaccine, demonstrating that the vaccine induced sterilizing humoral immunity in most vaccinees for at least six months following primary vaccination.The promising safety and immunogenicity profile of this vaccine confirms its suitability for inclusion in a tetravalent dengue vaccine.
Author Summary
Globally, dengue fever has become the most common clinically significant mosquito-transmitted viral illness. Dengue viruses exist as four serotypes, and increasingly several serotypes co-circulate in the same region. Infection with one serotype increases the risk of severe illness following infection with a second serotype. Therefore, any dengue virus vaccine needs to protect against all four serotypes. We and others are working to develop a live-attenuated tetravalent dengue vaccine that contains four monovalent vaccine viruses. Since two or more doses of such a vaccine are thought to be necessary for induction of long-lasting protective immunity, a feasible dose interval needs to be determined. Here, boosting with a second dose of a monovalent dengue type 1 (DENV-1) vaccine at four months or six months was compared in flavivirus-naïve healthy adult subjects with regard to safety, infectivity, and immunogenicity. We found that both doses of the vaccine were safe and well tolerated. While the first dose infected 92% of recipients, the second dose was neither infectious nor immunogenic, irrespective of the dose interval. These findings indicate that in most subjects, a single dose of this monovalent vaccine confers sterilizing humoral immunity against a second dose for at least six months.
doi:10.1371/journal.pntd.0001267
PMCID: PMC3149013  PMID: 21829748
25.  A Dynamic Landscape for Antibody Binding Modulates Antibody-Mediated Neutralization of West Nile Virus 
PLoS Pathogens  2011;7(6):e1002111.
Neutralizing antibodies are a significant component of the host's protective response against flavivirus infection. Neutralization of flaviviruses occurs when individual virions are engaged by antibodies with a stoichiometry that exceeds a required threshold. From this “multiple-hit” perspective, the neutralizing activity of antibodies is governed by the affinity with which it binds its epitope and the number of times this determinant is displayed on the surface of the virion. In this study, we investigated time-dependent changes in the fate of West Nile virus (WNV) decorated with antibody in solution. Experiments with the well-characterized neutralizing monoclonal antibody (MAb) E16 revealed a significant increase in neutralization activity over time that could not be explained by the kinetics of antibody binding, virion aggregation, or the action of complement. Additional kinetic experiments using the fusion-loop specific MAb E53, which has limited neutralizing activity because it recognizes a relatively inaccessible epitope on mature virions, identified a role of virus “breathing” in regulating neutralization activity. Remarkably, MAb E53 neutralized mature WNV in a time- and temperature-dependent manner. This phenomenon was confirmed in studies with a large panel of MAbs specific for epitopes in each domain of the WNV envelope protein, with sera from recipients of a live attenuated WNV vaccine, and in experiments with dengue virus. Given enough time, significant inhibition of infection was observed even for antibodies with very limited, or no neutralizing activity in standard neutralization assays. Together, our data suggests that the structural dynamics of flaviviruses impacts antibody-mediated neutralization via exposure of otherwise inaccessible epitopes, allowing for antibodies to dock on the virion with a stoichiometry sufficient for neutralization.
Author Summary
Neutralizing antibodies are a critical aspect of protection from flavivirus infection. The primary targets of neutralizing antibodies are the envelope (E) proteins incorporated into virions. The neutralizing activity of antibodies is determined by the affinity with which they interact with the virion, and the total number of sites available for binding. In this study, we investigate the impact of dynamic motion of the viral E proteins on antibody-mediated neutralization. Using panels of monoclonal antibodies and immune sera, we demonstrate that the dynamic motion of virions significantly impacts antibody-mediated neutralization of West Nile and dengue viruses by modulating epitope accessibility. Increasing the length of the interactions between antibody and virus resulted in increased neutralization reflecting engagement of epitopes that are not exposed on the surface of the virion in its average state, but instead become accessible through the dynamic motion of E proteins. While examples of the impact of structural dynamics on antibody binding have been described previously, our data suggests this phenomenon plays a role in neutralization by all antibodies that bind the array of E proteins on the virion. Our data identifies epitope accessibility as a critical, yet dynamic, factor that governs the neutralizing activity of anti-flavivirus antibodies.
doi:10.1371/journal.ppat.1002111
PMCID: PMC3128118  PMID: 21738473

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