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.
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.
West Nile virus (WNV); live attenuated WNV vaccine; clinical trial
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.
Dengue virus; dengue vaccine; low dose; viremia; neutralizing antibodies; HID50
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.
human; B cells; hybridomas; dengue virus; antibodies; antibody-dependent enhancement; neutralization
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.
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.
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.
dengue vaccine; live attenuated tetravalent; clinical trial
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.
Dysregulated immune responses may contribute to the clinical complications that occur in some patients with dengue.
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.
The inability of prednisolone treatment to markedly attenuate the host immune response is instructive for planning future therapeutic strategies for dengue.
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.
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.
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.
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.
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.
Dengue virus (DENV); Sylvatic DENV; Endemic DENV; Phylogenetic and phenotypic analysis
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.
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.
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.
Flavivirus; West Nile virus; Dengue; Reporter virus; Antibody-mediated neutralization
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.
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.
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.
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).
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.
Dengue vaccine; DNA vaccine; vectored-vaccine; sub-unit protein vaccine
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.
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.
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.
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.
Dengue is a mosquito-borne viral disease of humans that has re-emerged in many parts of the world and has become an important international public health threat. Dengue incidence and geographical spread has dramatically increased in the last few decades and is now affecting most tropical and subtropical regions of the world. Despite extensive research efforts for several decades, no vaccines or therapeutics are currently available to prevent and treat dengue infections. One of the main obstacles to the development of countermeasures has been the lack of good animal models that recapitulate dengue pathogenesis in humans and reliably predict the safety and efficacy of countermeasures against dengue. In September 2008, the National Institute of Allergy and Infectious Diseases (NIAID) held a workshop to consider the current state-of-the-art developments in animal models for dengue and discuss strategies to accelerate progress in this field. This report summarizes the main discussions and recommendations that resulted from the meeting.
dengue; animal; models
Jamestown Canyon virus (JCV), family Bunyaviridae, is a mosquito-borne pathogen endemic in the United States and Canada that can cause encephalitis in humans and is considered an emerging threat to public health. The virus is genetically similar to Inkoo virus circulating in Europe, suggesting that much of the northern hemisphere contains JCV or similar variants.
We have completed the sequence of three isolates of JCV collected in geographically diverse locations over a 57 year time span. The nucleotide identity for the three strains is 90, 83, and 85% for the S, M, and L segments respectively whereas the percent identify for the predicted amino acid sequences of the N, NSS, M poly, GN, NSM, GC, and L proteins was 97, 91, 94, 98, 91, 94, and 97%, respectively. In Swiss Webster mice, each JCV isolate exhibits low neuroinvasiveness but high infectivity. Two of the three JCV isolates were highly neurovirulent after IC inoculation whereas one isolate, JCV/03/CT, exhibited low neurovirulence. In rhesus monkeys, JCV infection is accompanied by a low-titered viremia, lack of clinical disease, but a robust neutralizing antibody response.
The first complete sequence of JCV is reported for three separate isolates, and a relatively high level of amino acid sequence conservation was observed even for viruses isolated 57 years apart indicating that the virus is in relative evolutionary stasis. JCV is highly infectious for mice and monkeys, and these animals, especially mice, represent useful experimental hosts for further study.
Tahyna virus (TAHV) is a human pathogen of the California encephalitis virus (CEV) serogroup (Bunyaviridae) endemic to Europe, Asia, and Africa. TAHV maintains an enzootic life cycle with several species of mosquito vectors and hares, rabbits, hedgehogs, and rodents serving as small mammal amplifying hosts. Human TAHV infection occurs in summer and early fall with symptoms of fever, headache, malaise, conjunctivitis, pharyngitis, and nausea. TAHV disease can progress to CNS involvement, although unlike related La Crosse virus (LACV), fatalities have not been reported. Human infections are frequent with neutralizing antibodies present in 60-80% of the elderly population in endemic areas.
In order to determine the genomic sequence of wild-type TAHV, we chose three TAHV isolates collected over a 26-year period from mosquitoes. Here we present the first complete sequence of the TAHV S, M, and L segments. The three TAHV isolates maintained a highly conserved genome with both nucleotide and amino acid sequence identity greater than 99%. In order to determine the extent of genetic relatedness to other members of the CEV serogroup, we compared protein sequences of TAHV with LACV, Snowshoe Hare virus (SSHV), Jamestown Canyon virus (JCV), and Inkoo virus (INKV). By amino acid comparison, TAHV was most similar to SSHV followed by LACV, JCV, and INKV. The sequence of the GN protein is most conserved followed by L, N, GC, NSS, and NSM. In a weanling Swiss Webster mouse model, all three TAHV isolates were uniformly neurovirulent, but only one virus was neuroinvasive. In rhesus monkeys, the virus was highly immunogenic even in the absence of viremia. Cross neutralization studies utilizing monkey immune serum demonstrated that TAHV is antigenically distinct from North American viruses LACV and JCV.
Here we report the first complete sequence of TAHV and present genetic analysis of new-world viruses, LACV, SSHV, and JCV with old-world viruses, TAHV and INKV. Using immune serum generated in monkeys against TAHV, LACV, and JCV, we have demonstrated cross-neutralization within the CEV serogroup. Such cross reactivity may complicate virus identification, especially following JCV infection which elicited antibodies that cross neutralized both LACV and TAHV. These data also suggest that a single vaccine could generate a cross-neutralizing antibody response which may provide protection against CEV serogroup viruses from a wide geographic range.
Pseudoangiomatous stromal hyperplasia (PASH) is a rare benign proliferating breast condition. It was first reported in 1986 when Vuitch, Rosen, and Erlandson described nine cases of benign well-circumscribed, breast masses that simulated vascular lesions consisting of mammary stromal proliferations (Vuitch et al. (1986)). Since then there have been few reported cases of PASH in the literature (Taira et al. (2005)). We describe a large PASH, mimicking inflammatory carcinoma in a young lady that was excised with excellent cosmetic results.