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.
rDEN4Δ30-4995 is a live attenuated dengue virus type 4 (DENV4) vaccine candidate specifically designed as a further attenuated derivative of the rDEN4Δ30 parent virus. In a previous study, 5 of 20 vaccinees who received 105 plaque-forming units (PFU) of rDEN4Δ30 developed a transient elevation of the serum alanine aminotransferase (ALT) level and an asymptomatic maculopapular rash developed in 10 of 20. In the current study, 28 healthy adult volunteers were randomized to receive 105 PFU of rDEN4Δ30-4995 (20) or placebo (8) as a single subcutaneous injection. The vaccine was safe, well-tolerated, and immunogenic. An asymptomatic generalized maculopapular rash and elevations in ALT levels were observed in 10% of the rDEN4Δ30-4995 vaccinees. None of the rDEN4Δ30-4995 vaccinees became viremic, yet 95% developed a four-fold or greater increase in neutralizing antibody titers. Thus, rDEN4Δ30-4995 was demonstrated to be safe, highly attenuated, and immunogenic. However, an asymptomatic localized erythematous rash at the injection site was seen in 17/20 rDEN4Δ30-4995 vaccinees. Therefore, alternative DENV4 vaccine strains were selected for further clinical development.
Infants account for a small proportion of the overall dengue case burden in endemic countries but can be clinically more difficult to manage. The clinical and laboratory features in infants with dengue have not been extensively characterised.
This prospective, cross-sectional descriptive study of infants hospitalized with dengue was conducted in Vietnam from November 2004 to December 2007. More than two-thirds of 303 infants enrolled on clinical suspicion of dengue had a serologically confirmed dengue virus (DENV) infection. Almost all were primary dengue infections and 80% of the infants developed DHF/DSS. At the time of presentation and during hospitalization, the clinical signs and symptoms in infants with dengue were difficult to distinguish from those with other febrile illnesses, suggesting that in infants early laboratory confirmation could assist appropriate management. Detection of plasma NS1 antigen was found to be a sensitive marker of acute dengue in infants with primary infection, especially in the first few days of illness.
Collectively, these results provide a systematic description of the clinical features of dengue in infants and highlight the value of NS1 detection for diagnosis.
Dengue is a major public health problem in tropical and subtropical countries, including Vietnam. Dengue cases occur in children and young adults; however, severe dengue also occurs in infants less than 1 year of age. Prompt recognition of dengue is important for appropriate case management, particularly in infants in whom febrile illness from other causes is common. We describe the clinical picture, virological and immunological characteristics of infants with dengue admitted to three hospitals in southern Vietnam, compared with infants admitted with fever not due to dengue. We show that infants with dengue are difficult to distinguish from those with other febrile illnesses based on signs and symptoms at presentation, and so laboratory tests to confirm dengue virus infection may be useful for diagnosis and management. Conventional diagnostic methods for dengue have low sensitivity early in infection, and we show that an alternative antigen-detection assay that has demonstrated good sensitivity and specificity in older age groups also performs well in infants. This study will help to inform the diagnosis and management of dengue in infants.
To develop a live attenuated virus vaccine against St. Louis encephalitis virus (SLE), two antigenic chimeric viruses were generated by replacing the membrane precursor and envelope protein genes of dengue virus type 4 (DEN4) with those from SLE with or without a 30 nucleotide deletion in the DEN4 3′ untranslated region of the chimeric genome. Chimeric viruses were compared with parental wild-type SLE for level of neurovirulence and neuroinvasiveness in mice and for safety, immunogenicity, and protective efficacy in rhesus monkeys. The resulting viruses, SLE/DEN4 and SLE/DEN4Δ30, had greatly reduced neuroinvasiveness in immunodeficient mice but retained neurovirulence in suckling mice. Chimerization of SLE with DEN4 resulted in only moderate restriction in replication in rhesus monkeys, whereas the presence of the Δ30 mutation led to over-attenuation. Introduction of previously described attenuating paired charge-to-alanine mutations in the DEN4 NS5 protein of SLE/DEN4 reduced neurovirulence in mice and replication in rhesus monkeys. Two modified SLE/DEN4 viruses, SLE/DEN4-436,437 clone 41 and SLE/DEN4-654,655 clone 46, have significantly reduced neurovirulence in mice and conferred protective immunity in monkeys against SLE challenge. These viruses may be considered for use as SLE vaccine candidates and for use as diagnostic reagents with reduced virulence.
St. Louis encephalitis virus; chimeric virus; vaccine
With the steady rise in tick-borne encephalitis virus (TBEV) infections in Europe, development of a live attenuated vaccine that will generate long-lasting immunity would be of considerable benefit. A chimeric flavivirus, designated LGT/DEN4, was previously constructed to have a genome containing the prM and E protein genes of Langat virus (LGT), a naturally attenuated member of the TBEV complex, and the remaining genetic sequences derived from dengue 4 virus (DEN4). LGT/DEN4 was highly attenuated in rodents and non-human primates, and clinical trials in humans were initiated. Twenty-eight healthy seronegative adult volunteers were randomly assigned in a 4:1 ratio to receive 103 PFU of LGT/DEN4 or placebo. Volunteers were closely monitored for clinical responses and for blood chemistry and hematological changes, and the level of viremia and the magnitude and duration of the neutralizing antibody response were determined. The LGT/DEN4 vaccine was safe and viremia was seen in only one vaccinee. Infection induced a neutralizing antibody response to wild-type LGT in 80% of volunteers with a geometric mean titer (GMT) of 1:63 present on day 42 post-immunization; however the antibody response against TBEV was both much less frequent (35%) and lower in magnitude (GMT = 1:9). To assess the response to a booster dose, 21 of the original 28 volunteers were re-randomized to receive a second dose of either 103 PFU of vaccine or placebo given 6 to 18 months after the first dose. The immunogenicity against either LGT or TBEV was not significantly enhanced after the second dose of vaccine. Thus, chimerization of LGT with DEN4 yielded a vaccine virus that was highly attenuated yet infectious in humans. The level of replication was sufficiently restricted to induce only a weak cross-reactive antibody response to TBEV. To provide a sufficient level of immunity to widely prevalent, highly neurovirulent strains of TBEV in humans, vaccine candidates will likely need to be based on the TBEV structural protein genes.
Tick-Borne Encephalitis; Live Virus Vaccine; Human
The dengue virus type 3 (DENV-3) vaccine candidate, rDEN3Δ30, was previously found to be under-attenuated in both SCID-HuH-7 mice and rhesus monkeys. Herein, two strategies have been employed to generate attenuated rDEN3 vaccine candidates which retain the full complement of structural and nonstructural proteins of DENV-3 and thus are able to induce humoral or cellular immunity to each of the DENV-3 proteins. First, using the predicted secondary structure of the 3’ untranslated region (3’-UTR) of DENV-3 to design novel deletions, nine deletion mutant viruses were engineered and found to be viable. Four of nine deletion mutants replicated efficiently in Vero cells and were genetically stable. Second, chimeric rDENV-3 viruses were generated by replacement of the 3’-UTR of the rDENV-3 cDNA clone with that of rDENV-4 or rDEN4Δ30 yielding the rDEN3-3’D4 and rDEN3-3’D4Δ30 viruses, respectively. Immunization of rhesus monkeys with either of two deletion mutant viruses, rDEN3Δ30/31 and rDEN3Δ86, or with rDEN3-3’D4Δ30 resulted in infection without detectable viremia, with each virus inducing a strong neutralizing antibody response capable of conferring protection from DENV-3 challenge. The rDEN3Δ30/31 virus showed a strong host range restriction phenotype with complete loss of replication in C6/36 mosquito cells despite robust replication in Vero cells. In addition, rDEN3Δ30/31 had reduced replication in Toxorynchites mosquitoes following intrathoracic inoculation. The results are discussed in the context of vaccine development and the physical structure of the DENV 3’-UTR.
The rDEN4Δ30-200,201 is a live attenuated DENV-4 vaccine candidate specifically designed to further attenuate the rDEN4Δ30 parent virus. In the present study, 28 healthy adult volunteers were randomized to receive either 105 plaque-forming unit (PFU) of vaccine (20) or placebo (8) as a single subcutaneous injection. Volunteers were evaluated for safety every other day for 16 days. Serum neutralizing antibody titer against DEN4 was determined at study day 28, 42, and 180. The vaccine infected all vaccinees and was well tolerated without inducing alanine aminotransferase (ALT) elevations. Although virus was not recovered from the serum of any vaccinee, moderate levels of neutralizing antibody were induced in all volunteers. Thus the restricted replication of rDEN4Δ30-200,201 previously documented in animal models was confirmed in humans. The rDEN4Δ30-200,201 is a promising candidate and can be considered for inclusion in a tetravalent dengue virus (DENV) vaccine.
West Nile virions incorporate 180 envelope (E) proteins that orchestrate the process of virus entry and are the primary target of neutralizing antibodies. The E proteins of newly synthesized West Nile virus (WNV) are organized into trimeric spikes composed of pre-membrane (prM) and E protein heterodimers. During egress, immature virions undergo a protease-mediated cleavage of prM that results in a reorganization of E protein into the pseudo-icosahedral arrangement characteristic of mature virions. While cleavage of prM is a required step in the virus life cycle, complete maturation is not required for infectivity and infectious virions may be heterogeneous with respect to the extent of prM cleavage. In this study, we demonstrate that virion maturation impacts the sensitivity of WNV to antibody-mediated neutralization. Complete maturation results in a significant reduction in sensitivity to neutralization by antibodies specific for poorly accessible epitopes that comprise a major component of the human antibody response following WNV infection or vaccination. This reduction in neutralization sensitivity reflects a decrease in the accessibility of epitopes on virions to levels that fall below a threshold required for neutralization. Thus, in addition to a role in facilitating viral entry, changes in E protein arrangement associated with maturation modulate neutralization sensitivity and introduce an additional layer of complexity into humoral immunity against WNV.
West Nile virus (WNV) virions incorporate 180 envelope (E) proteins that are the primary target of neutralizing antibodies. As newly formed WNV virions are released from infected cells, the E proteins undergo a significant organizational change associated with maturation into an infectious virus. However, this process is not always efficient, as populations of infectious WNV include virions that did not complete the maturation process and may be heterogeneous with respect to the arrangement of E proteins on the virion. In this study, we found that neutralization by antibodies specific for epitopes commonly recognized in vivo is strongly impacted by the maturation state of WNV. Our studies suggest that maturation of WNV reduces the accessibility of some, but not all, epitopes on the virion for antibody binding. Virions that retain some immature character can be neutralized by monoclonal antibodies that fail to block infection of populations of WNV composed solely of mature virions. Similar results were found using polyclonal human serum obtained from volunteers of two clinical trials of candidate WNV vaccines. These studies identify unappreciated aspects of the antigenic complexity of WNV and highlight the importance of understanding the heterogenous forms of WNV that may be introduced into or replicating within the host.