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Dengue remains one of the most important mosquito-borne diseases worldwide. Infection with one of the serologically related dengue viruses (DENVs) can lead to a wide range of clinical manifestations and severity. Severe dengue is characterized by plasma leakage and abnormal bleeding that can lead to shock and death. There is currently no specific treatment for severe dengue due to gaps in understanding of the underlying mechanisms. The transient period of vascular leakage is usually followed by a rapid recovery and is suggestive of the effects of short-lived biological mediators. Both the innate and the adaptive immune systems are activated in severe dengue and contribute to the cytokine production. We discuss the immunological events elicited during a DENV infection and identify candidate cytokines that may play a key role in the severe manifestations of dengue and possible interventions.

Mitochondria are highly dynamic organelles that undergo continuous cycles of fission and fusion to maintain essential cellular functions. An imbalance between these two processes can result in many pathophysiological outcomes. Dengue virus (DENV) interacts with cellular organelles, including mitochondria, to successfully replicate in cells. This study used live-cell imaging and found an increase in mitochondrial length and respiration during DENV infection. The level of mitochondrial fission protein, Dynamin-related protein 1 (Drp1), was decreased on mitochondria during DENV infection, as well as Drp1 phosphorylated on serine 616, which is important for mitochondrial fission. Viral proteins NS4b and NS3 were also associated with subcellular fractions of mitochondria. Induction of fission through uncoupling of mitochondria or overexpression of Drp1 wild-type and Drp1 with a phosphomimetic mutation (S616D) significantly reduced viral replication. These results demonstrate that DENV infection causes an imbalance in mitochondrial dynamics by inhibiting Drp1-triggered mitochondrial fission, which promotes viral replication.

Background Major histocompatibility complex class I chain–related (MIC) A and B (MICA and MICB) are polymorphic stress molecules recognized by natural killer cells. This study was performed to analyze MIC gene profiles in hospitalized Thai children with acute dengue illness. Methods MIC allele profiles were determined in a discovery cohort of patients with dengue fever or dengue hemorrhagic fever (DHF) (n = 166) and controls (n = 149). A replication cohort of patients with dengue (n = 222) was used to confirm specific MICB associations with disease. Results MICA*045 and MICB*004 associated with susceptibility to DHF in secondary dengue virus (DENV) infections (odds ratio [OR], 3.22; [95% confidence interval (CI), 1.18–8.84] and 1.99 [1.07–2.13], respectively), and MICB*002 with protection from DHF in secondary DENV infections (OR, 0.41; 95% CI, .21–.68). The protective effect of MICB*002 against secondary DHF was confirmed in the replication cohort (OR, 0.43; 95% CI, .22–.82) and was stronger when MICB*002 is present in individuals also carrying HLA-B*18, B*40, and B*44 alleles which form the B44 supertype of functionally related alleles (0.29, 95% CI, .14–.60). Conclusions Given that MICB*002 is a low expresser of soluble proteins, these data indicate that surface expression of MICB*002 with B44 supertype alleles on DENV-infected cells confer a protective advantage in controlling DENV infection using natural killer cells.

Prior exposure to dengue virus (DENV) has a profound impact on the outcome of infection, which varies according to the interval between infections. Antibodies secreted by B cells and cytokines secreted by T cells are thought to contribute both to protective immunity against DENV and the pathogenesis of dengue disease. We analyzed peripheral blood mononuclear cells (PBMC) collected from Thai children over a 5-year prospective cohort study to define the dynamics of DENV-specific memory B and T cell responses and the impact of symptomatic or subclinical DENV infections. To measure B cell responses, PBMC were stimulated with IL-2 plus R848 and culture supernatants were tested for DENV-binding antibodies by ELISA. To measure T cell responses, PBMC were stimulated in dual-color ELISPOT assays with overlapping peptide pools of structural and non-structural proteins from the four DENV types. B cell responses were low to one or more DENV types prior to symptomatic infection and increased with reactivity to all four types after infection. Subjects who had a subclinical infection or who did not experience a DENV infection during the study period showed strong memory B cell responses to all four DENV types. T cell responses to DENV peptides demonstrated a cytokine hierarchy of IFN-γ > IL-2 > IFN-γ/IL-2. T cell responses were low or absent prior to secondary infections. The trends in T cell responses to DENV peptides over 3 year post-infection were highly variable, but subjects who had experienced a secondary DENV1 infection showed higher cytokine responses compared to subjects who had experienced a secondary DENV2 or subclinical infection. The longitudinal nature of our study demonstrates persistent memory B cell responses over years and a lasting but variable impact of secondary DENV infection on DENV-specific T cell responses.

Heterogeneous exposure to mosquitoes determines an individual’s contribution to vector-borne pathogen transmission. Particularly for dengue virus (DENV), there is a major difficulty in quantifying human-vector contacts due to the unknown coupled effect of key heterogeneities. To test the hypothesis that the reduction of human out-of-home mobility due to dengue illness will significantly influence population-level dynamics and the structure of DENV transmission chains, we extended an existing modeling framework to include social structure, disease-driven mobility reductions, and heterogeneous transmissibility from different infectious groups. Compared to a baseline model, naïve to human pre-symptomatic infectiousness and disease-driven mobility changes, a model including both parameters predicted an increase of 37% in the probability of a DENV outbreak occurring; a model including mobility change alone predicted a 15.5% increase compared to the baseline model. At the individual level, models including mobility change led to a reduction of the importance of out-of-home onward transmission (R, the fraction of secondary cases predicted to be generated by an individual) by symptomatic individuals (up to -62%) at the expense of an increase in the relevance of their home (up to +40%). An individual’s positive contribution to R could be predicted by a GAM including a non-linear interaction between an individual’s biting suitability and the number of mosquitoes in their home (>10 mosquitoes and 0.6 individual attractiveness significantly increased R). We conclude that the complex fabric of social relationships and differential behavioral response to dengue illness cause the fraction of symptomatic DENV infections to concentrate transmission in specific locations, whereas asymptomatic carriers (including individuals in their pre-symptomatic period) move the virus throughout the landscape. Our findings point to the difficulty of focusing vector control interventions reactively on the home of symptomatic individuals, as this approach will fail to contain virus propagation by visitors to their house and asymptomatic carriers., Author summary Human mobility patterns can play an integral role in vector-borne disease dynamics by characterizing an individual’s potential contacts with disease-transmitting vectors. Dengue virus is transmitted by a sedentary vector, but human mobility allows individuals to have contact with mosquitoes at their home and other houses they frequent (their activity space). When accounting for the decreased mobility of symptomatic dengue cases in an agent-based simulation model, however, we found a severely diminished role of the activity space in onward transmission. Those who received the majority of their mosquito contacts outside their home experienced decreases in expected bites and onward transmission when mobility changes were accounted for. Onward transmission was driven by a synergistic relationship between the number of mosquitoes in an individual’s home and their biting suitability, where even those with the highest biting suitability would have limited contribution to transmission given a low number of household mosquitoes. Reactive vector control, which often targets symptomatic cases, could be effective for slowing onward transmission from these cases, but will fail to control virus transmission due to the disproportionate contribution of asymptomatic infections.

Background. The development of reagents to identify and characterize antigen-specific B cells has been challenging. Methods. We recently developed Alexa Fluor–labeled dengue viruses (AF DENVs) to characterize antigen-specific B cells in the peripheral blood of DENV-immune individuals. Results. In this study, we used AF DENV serotype 1 (AF DENV-1) together with AF DENV-2 on peripheral blood mononuclear cells (PBMCs) from children in Thailand with acute primary or secondary DENV-1 infections to analyze the phenotypes of antigen-specific B cells that reflected their exposure or clinical diagnosis. DENV serotype-specific and cross-reactive B cells were identified in PBMCs from all subjects. Frequencies of AF DENV+ class-switched memory B cells (IgD−CD27+ CD19+ cells) reached up to 8% during acute infection and early convalescence. AF DENV–labeled B cells expressed high levels of CD27 and CD38 during acute infection, characteristic of plasmablasts, and transitioned into memory B cells (CD38−CD27+) at the early convalescent time point. There was higher activation of memory B cells early during acute secondary infection, suggesting reactivation from a previous DENV infection. Conclusions. AF DENVs reveal changes in the phenotype of DENV serotype–specific and cross-reactive B cells during and after natural DENV infection and could be useful in analysis of the response to DENV vaccination.

Antibody-mediated humoral immunity is thought to play a central role in mediating the immunopathogenesis of acute DENV infection, but limited data are available on the diversity, specificity, and functionality of the antibody response at the molecular level elicited by primary or secondary DENV infection. In order to close this functional gap in our understanding of DENV-specific humoral immunity, we utilized high-throughput single cell RNA sequencing to investigate B cells circulating in both primary and secondary natural DENV infections. We captured full-length paired immunoglobulin receptor sequence data from 9,027 B cells from a total of 6 subjects, including 2,717 plasmablasts. In addition to IgG and IgM class-switched cells, we unexpectedly found a high proportion of the DENV-elicited plasmablasts expressing IgA, principally in individuals with primary DENV infections. These IgA class-switched cells were extensively hypermutated even in individuals with a serologically confirmed primary DENV infection. Utilizing a combination of conventional biochemical assays and high-throughput shotgun mutagenesis, we determined that DENV-reactive IgA class-switched antibodies represent a significant fraction of DENV-reactive Igs generated in response to DENV infection, and that they exhibit a comparable epitope specificity to DENV-reactive IgG antibodies. These results provide insight into the molecular-level diversity of DENV-elicited humoral immunity and identify a heretofore unappreciated IgA plasmablast response to DENV infection. Keywords: Dengue, Plasmablast, Single-cell RNA sequencing, IgA, Monoclonal antibody

Background Hyperendemic circulation of all four types of dengue virus (DENV-1-4) has expanded globally, fueling concern for increased incidence of severe dengue. While the majority of DENV infections are subclinical, epidemiologic studies suggest that type-cross-reactive immunity can influence disease outcome in subsequent infections. The mechanisms controlling these differential clinical outcomes remain poorly defined. Methodology/Principal findings Blood samples were collected from a cohort of school-aged Thai children who subsequently experienced a subclinical DENV infection or developed dengue illness. PBMC collected prior to infection were stimulated in vitro with DENV and the secretion of 30 cytokines was measured using a multiplexed, bead-based array. Significant differences were found in cytokine production based on both the type of DENV used for stimulation and the occurrence of clinical illness. Secretion of IL-15 and MCP-1 was significantly higher by PBMC of subjects who later developed symptomatic DENV infection. In addition, IL-6 was produced by PBMC from all subjects who subsequently developed symptomatic infection, versus 59% of subjects who had subclinical infection. Secretion of IL-12, IL-2R, MIP-1α, RANTES, GM-CSF, and TNFα was significantly lower by PBMC from subjects with symptomatic infection. Conclusions/Significance These data demonstrate significant differences in pre-existing immune responses to DENV associated with the clinical outcome of subsequent infection. The finding of higher levels of some cytokines in subjects with symptomatic infection and higher levels of other cytokines in subjects with subclinical infection supports the existence of both protective and pathologic immune profiles. Clinical-immunological correlations identified in the context of natural DENV infection may be useful for evaluating immune responses to dengue vaccines., Author summary Dengue is one of the most prevalent mosquito-borne infectious diseases worldwide. It is caused by one of four viruses, types 1–4, and ranges in severity from subclinical (mild or no symptoms) to dengue fever (febrile illness with headache and bone pain), or less frequently, dengue hemorrhagic fever, where patients experience leaky blood vessels, sometimes significant bleeding, and may be severe enough to cause death. While many risk factors have been associated with development of severe dengue, sequential infection with different virus types is a major factor, implying that the immune response generated after an initial infection is partly responsible for making subsequent exposure clinically worse. This study sought to identify profiles of immune markers that correlate with increased or decreased risk of dengue. Using samples from individuals ~5 months prior to dengue virus infection, who later experienced either subclinical infection or overt disease, we modeled virus exposure in vitro and compared the production of various immune proteins between the two outcome groups. Three of the proteins studied were produced at higher levels by subjects who subsequently had dengue, and six of the proteins were produced at higher levels by subjects who subsequently had subclinical infection. These results help define what constitutes beneficial versus potentially harmful immune responses, aiding in the design of effective dengue vaccines.

Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne pathogens that have a significant impact on human health. Immune sera, mAbs, and memory B cells (MBCs) isolated from patients infected with one DENV type can be cross-reactive with the other three DENV serotypes and even more distantly related flaviviruses such as ZIKV. Conventional ELISPOTs effectively measure Ab-secreting B cells but because they are limited to the assessment of a single Ag at a time, it is challenging to distinguish serotype-specific and cross-reactive MBCs in the same well. We developed a novel multifunction FluoroSpot assay using fluorescently labeled DENV and ZIKV (FLVs) that measures the cross-reactivity of Abs secreted by single B cells. Conjugation efficiency and recognition of FLVs by virus-specific Abs were confirmed by flow cytometry. Using a panel of DENV immune, ZIKV immune, and naive PBMC, FLVs were able to simultaneously detect DENV serotype-specific, ZIKV-specific, DENV serotype cross-reactive, and DENV/ZIKV cross-reactive Abs secreted by individual MBCs. Our findings indicate that the FLVs are sensitive and specific tools to detect specific and cross-reactive MBCs. These reagents will allow the assessment of the breadth as well as the durability of DENV/ZIKV B cell responses following vaccination or natural infection. This novel approach using FLVs in a FluoroSpot assay can be applied to other diseases such as influenza in which prior immunity with homosubtype- or heterosubtype-specific MBCs may influence subsequent infections.

BACKGROUND: Follicular helper T cells (T(FH)) are specialized CD4 T cells required for B-cell help and antibody production. METHODS: Given the postulated role of immune activation in dengue disease, we measured the expansion and activation of T(FH) in the circulation (peripheral T(FH) [pT(FH)]) collected from Thai children with laboratory-confirmed acute dengue virus (DENV) infection. RESULTS: We found significant expansion and activation of pT(FH) subsets during acute infection with the highest frequencies of activated pT(FH) (PD1(hi) pT(FH) and PD1(+)CD38(+) pT(FH)) detected during the critical phase of illness. Numbers of activated pT(FH) were higher in patients with secondary compared with primary infections and in patients with more severe disease. We also found a positive correlation between the frequencies of activated pT(FH) and the frequencies of plasmablasts. CONCLUSIONS: To our knowledge, this is the first ex vivo analysis of pT(FH) activation during acute DENV infection. Overall, our study supports the model that pT(FH) contribute to disease evolution during the critical stage of illness.

Dengue vaccine development has made significant strides, but a better understanding of how vaccine-induced immune responses correlate with vaccine efficacy can greatly accelerate development, testing, and deployment as well as ameliorate potential risks and safety concerns. Advances in basic immunology knowledge and techniques have already improved our understanding of cell-mediated immunity of natural dengue virus infection and vaccination. We conclude that the evidence base is adequate to argue for inclusion of assessments of cell-mediated immunity as part of clinical trials of dengue vaccines, although further research to identify useful correlates of protective immunity is needed.

Dengue is a rapidly expanding global health problem. Development of a safe and efficacious tetravalent vaccine along with strategic application of vector control activities represents a promising approach to reducing the global disease burden. Although many vaccine development challenges exist, numerous candidates are in clinical development and one has been tested in three clinical endpoint studies. The results of these studies have raised numerous questions about how we measure vaccine immunogenicity and how these readouts are associated with clinical outcomes in vaccine recipients who experience natural infection. In this review the authors discuss the dengue vaccine pipeline, development challenges, the dengue vaccine-immunologic profiling intersection, and research gaps.

International audience; Dengue is of public health importance in tropical and sub-tropical regions. Dengue virus (DENV) transmission dynamics was studied in Kamphaeng Phet Province, Thailand, using an enhanced spatiotemporal surveillance of 93 hospitalized subjects with confirmed dengue (initiates) and associated cluster individuals (associates) with entomologic sampling. A total of 438 associates were enrolled from 208 houses with household members with a history of fever, located within a 200-m radius of an initiate case. Of 409 associates, 86 (21%) had laboratory-confirmed DENV infection. A total of 63 (1.8%) of the 3,565 mosquitoes collected were dengue polymerase chain reaction positive (PCR+). There was a significant relationship between spatial proximity to the initiate case and likelihood of detecting DENV from associate cases and Aedes mosquitoes. The viral detection rate from human hosts and mosquito vectors in this study was higher than previously observed by the study team in the same geographic area using different methodologies. We propose that the sampling strategy used in this study could support surveillance of DENV transmission and vector interactions.

BACKGROUND Human leukocyte antigen (HLA) supertypes are groups of functionally related alleles that present structurally similar antigens to the immune system. OBJECTIVES To analyze HLA class I supertype associations with clinical outcome in hospitalized Thai children with acute dengue illness. METHODS Seven hundred sixty-two patients and population-matched controls recruited predominantly in Bangkok were HLA-A and -B typed. HLA supertype frequencies were compared and tested for significant dengue disease associations using logistic regression analyses. Multivariable models were built by conducting forward stepwise selection procedures. RESULTS In the final logistic regression model, the HLA-B44 supertype was protective against dengue hemorrhagic fever (DHF) in secondary infections (odds ratio [OR] = 0.46, 95% confidence interval [CI], .30-.72), while the HLA-A02 supertype (OR = 1.92, 95% CI, 1.30-2.83) and the HLA-A01/03 supertype (OR = 3.01, 95% CI, 1.01-8.92) were associated with susceptibility to secondary dengue fever. The B07 supertype was associated with susceptibility to secondary DHF in the univariate analysis (OR = 1.60, 95% CI, 1.05-2.46), whereas that was not retained in the final model. CONCLUSIONS As the HLA-B44 supertype is predicted to target conserved epitopes in dengue, our results suggest that B44 supertype-restricted immune responses to highly conserved regions of the dengue proteome may protect against secondary DHF.

The ideal dengue vaccine will provide protection against all serotypes of dengue virus and will be economical and uncomplicated in its administration. To determine the ability of a single dose of the live attenuated tetravalent dengue vaccine TV003 to induce a suitable neutralizing antibody response, a placebo-controlled clinical trial was performed in 48 healthy adults who received 2 doses of vaccine or placebo administered 12 months apart. Evaluation of safety, vaccine viremia, and neutralizing antibody response after each dose indicated that the first dose of vaccine was capable of preventing infection with the second dose, thus indicating that multiple doses are unnecessary.

As with many pathogens, most dengue infections are subclinical and therefore unobserved 1 . Coupled with limited understanding of the dynamic behaviour of potential serological markers of infection, this observational problem has wide-ranging implications, including hampering our understanding of individual- and population-level correlates of infection and disease risk and how these change over time, between assay interpretations and with cohort design. Here we develop a framework that simultaneously characterizes antibody dynamics and identifies subclinical infections via Bayesian augmentation from detailed cohort data (3,451 individuals with blood draws every 91 days, 143,548 haemagglutination inhibition assay titre measurements)2,3. We identify 1,149 infections (95% confidence interval, 1,135–1,163) that were not detected by active surveillance and estimate that 65% of infections are subclinical. After infection, individuals develop a stable set point antibody load after one year that places them within or outside a risk window. Individuals with pre-existing titres of ≤1:40 develop haemorrhagic fever 7.4 (95% confidence interval, 2.5–8.2) times more often than naive individuals compared to 0.0 times for individuals with titres >1:40 (95% confidence interval: 0.0–1.3). Plaque reduction neutralization test titres ≤1:100 were similarly associated with severe disease. Across the population, variability in the size of epidemics results in large-scale temporal changes in infection and disease risk that correlate poorly with age. Analyses of antibody dynamics and subclinical infections show that across the population, variability in the infection strength of dengue viruses results in large-scale temporal changes in infection and disease risk that correlate poorly with age.

The global burden of dengue and its geographic distribution have increased over the past several decades. The introduction of dengue in new areas has often been accompanied by high case-fatality rates. Drawing on the experience in managing dengue cases at the Queen Sirikit National Institute of Child Health in Bangkok, Thailand, this article provides the authors' perspectives on key clinical lessons to improve dengue-related outcomes. Parallels between this clinical experience and outcomes reported in randomized controlled trials, results of efforts to disseminate practice recommendations, and suggestions for areas for further research are also discussed.

Advances in the field of T cell immunology have contributed to the understanding that cross-reactivity is an intrinsic characteristic of the T cell receptor (TCR), and that each TCR can potentially interact with many different T cell epitopes. To better define the potential for TCR cross-reactivity between epitopes derived from the human genome, the human microbiome, and human pathogens, we developed a new immunoinformatics tool, JanusMatrix, that represents an extension of the validated T cell epitope mapping tool, EpiMatrix. Initial explorations, summarized in this synopsis, have uncovered what appear to be important differences in the TCR cross-reactivity of selected regulatory and effector T cell epitopes with other epitopes in the human genome, human microbiome, and selected human pathogens. In addition to exploring the T cell epitope relationships between human self, commensal and pathogen, JanusMatrix may also be useful to explore some aspects of heterologous immunity and to examine T cell epitope relatedness between pathogens to which humans are exposed (Dengue serotypes, or HCV and Influenza, for example). In Hand-Foot-Mouth disease (HFMD) for example, extensive enterovirus and human microbiome cross-reactivity (and limited cross-reactivity with the human genome) seemingly predicts immunodominance. In contrast, more extensive cross-reactivity with proteins contained in the human genome as compared to the human microbiome was observed for selected Treg epitopes. While it may be impossible to predict all immune response influences, the availability of sequence data from the human genome, the human microbiome, and an array of human pathogens and vaccines has made computationally–driven exploration of the effects of T cell epitope cross-reactivity now possible. This is the first description of JanusMatrix, an algorithm that assesses TCR cross-reactivity that may contribute to a means of predicting the phenotype of T cells responding to selected T cell epitopes. Whether used for explorations of T cell phenotype or for evaluating cross-conservation between related viral strains at the TCR face of viral epitopes, further JanusMatrix studies may contribute to developing safer, more effective vaccines.

The immune response to dengue virus (DENV) infection is complex and not fully understood. Using longitudinal data from 181 children with dengue in Thailand who were followed for up to 3 years, we describe neutralizing antibody kinetics following symptomatic DENV infection. We observed that antibody titers varied by serotype, homotypic vs heterotypic responses, and primary versus postprimary infections. The rates of change in antibody titers over time varied between primary and postprimary responses. For primary infections, titers increased from convalescence to 6 months. By comparing homotypic and heterotypic antibody titers, we saw an increase in type specificity from convalescence to 6 months for primary DENV3 infections but not primary DENV1 infections. In postprimary cases, there was a decrease in titers from convalescence up until 6 months after infection. Beginning 1 year after both primary and postprimary infections, there was evidence of increasing antibody titers, with greater increases in children with lower titers, suggesting that antibody titers were boosted due to infection and that higher levels of neutralizing antibody may be more likely to confer a sterilizing immune response. These findings may help to model virus transmission dynamics and provide baseline data to support the development of vaccines and therapeutics.

Dengue, caused by the four serotypes of dengue virus (DENV), represents an expanding global health challenge. The potential for serotype-cross-reactive antibodies to exacerbate disease during a secondary infection with a heterologous DENV serotype has driven efforts to study human DENV-specific antibodies. Most DENV-specific antibodies generated in humans are serotype-cross-reactive, weakly neutralizing, and directed against the immature pre-membrane (prM), envelope (E), and nonstructural 1 (NS1) proteins. To broaden the characterization of human DENV-specific antibodies, we assessed B-cell responses by ELISpot assays and isolated B cells from the peripheral blood of a human subject with previous DENV infection. Forty-eight human IgG monoclonal antibodies (hMAbs) were initially characterized by their potential to bind to an inactivated lysate of DENV-infected cells. Subsequently, most DENV-specific hMAbs were found to bind soluble, recombinant E protein (rE). Two hMAbs were unable to bind rE, despite strongly binding to the DENV-infected cell lysate. Further analyses showed that these two hMAbs bound conformation-dependent, reduction-sensitive epitopes on E protein. These data shed light on the breadth of DENV-specific hMAbs generated within a single immune donor.

Objective To determine the temporal intervals at which spatial clustering of dengue hospitalisations occurs. Methods Space-time analysis of 262 people hospitalised and serologically confirmed with dengue virus infections in Kamphaeng Phet, Thailand was performed. The cases were observed between 1 January 2009 and 6 May 2011. Spatial coordinates of each patient’s home were captured using the Global Positioning System. A novel method based on the Knox test was used to determine the temporal intervals between cases at which spatial clustering occurred. These intervals are indicative of the length of time between successive illnesses in the chain of dengue virus transmission. Results The strongest spatial clustering occurred at the 15–17-day interval. There was also significant spatial clustering over short intervals (2–5 days). The highest excess risk was observed within 200 m of a previous hospitalised case and significantly elevated risk persisted within this distance for 32–34 days. Conclusions Fifteen to seventeen days are the most likely serial interval between successive dengue illnesses. This novel method relies only on passively detected, hospitalised case data with household locations and provides a useful tool for understanding region-specific and outbreak-specific dengue virus transmission dynamics. Objectif: Determiner les intervalles temporels au cours desquels survient le regroupement spatial des hospitalisations pour dengue. Methodes: L’’analyse spatio-temporelle de 262 personnes hospitalisees et confirmees par la serologie pour des infections au virus de la dengue a Kamphaeng Phet, en Thailande a ete realisee. Les cas ont ete observes entre le 1er janvier 2009 et le 6 mai 2011. Les coordonnees spatiales du domicile de chaque patient ont ete capturees a l’aide du Systeme de Positionnement Global. Une nouvelle methode basee sur le test de Knox a ete utilisee pour determiner les intervalles temporels entre les cas au cours desquels le regroupement spatial a eu lieu. Ces intervalles sont des indicateurs de la duree de temps entre les maladies successives dans la chaine de transmission de virus de la dengue. Resultats: Le plus important regroupement spatial a eu lieu dans l’intervalle de 15 a 17 jours. Il y avait egalement un regroupement spatial significatif sur des intervalles courts (2 a 5 jours). Le risque en exces le plus eleve a ete observea endeans 200 m d’un cas precedent hospitalise et un risque significativement eleve a persiste dans cette zone durant 32 a 34 jours. Conclusions: L’intervalle de series de 15 a 17 jours est le plus probable entre les maladies successives de la dengue. Cette nouvelle methode repose uniquement sur les donnees de cas hospitalises, detectes passivement avec les emplacements des menages et constitue un outil utile pour comprendre la dynamique de la transmission du virus de la dengue specifique a la region et specifique a l’epidemie. Objetivo: Determinar los intervalos temporales en los que ocurren conglomerados espaciales de hospitalizacion por dengue. Metodos: Se realizo un analisis espacio temporal a 262 personas hospitalizadas y con confirmacion serologica de infeccion por el virus del dengue en Kamphaeng Phet, Tailandia. Los casos se observaron entre el 1 de Enero del 2009 y el 6 de Mayo del 2011. Las coordenadas espaciales del hogar de cada paciente fueron capturadas utilizando un Sistema de Posicionamiento Global. Se utilizo un nuevo metodo, basado en la prueba de Knox, para determinar los intervalos temporales entre los casos en los que habia conglomerados espaciales. Estos intervalos son indicativos del lapso de tiempo entre enfermedades sucesivas en la cadena de transmision del virus del dengue. Resultados: El mayor conglomerado espacial ocurrio en un intervalo de 15 a 17 dias. Tambien habia un conglomerado espacial significativo en intervalos cortos (2–5 dias). El mayor exceso de riesgo se observo dentro de un radio de 200 m de un caso previamente hospitalizado y un riesgo significativamente elevado persistia, dentro de esta distancia, durante 32–34 dias. Conclusiones: Los 15–17 dias son el intervalo mas probable entre dos casos sucesivos de dengue. Este nuevo metodo depende solamente de la disponibilidad de datos de casos hospitalizados, detectados de forma pasiva y con hogares localizables, y brinda una herramienta util para entender la dinamica de transmision del virus del dengue, region-especifica y especifica de brotes.

SUMMARY. To evaluate T cell immunity in advanced liver disease, antigen-specific lymphoproliferative (LP) responses were prospectively studied in the context of the Hepatitis C Antiviral Long-term Treatment against Cirrhosis trial. Peripheral blood responses to hepatitis C virus (HCV), tetanus and Candida protein antigens were measured at baseline, month 12 (M12), M24, M36 and M48 in 186 patients randomized to either low-dose peginterferon-alfa-2a (PEG-IFN) onlyorobservation.Liverhistologywasevaluatedatbaseline, M24 and M48. Patients with cirrhosis (Ishak 5‐6) were less likely to have positive LP responses to HCV at baseline than patients with fibrosis (15% vs 29%, P = 0.03) and had lower levels of HCV c100 responses at baseline, M24 and M48 (P = 0.11, P = 0.05, P = 0.02, respectively). For 97 patients with complete longitudinal data, the frequency of positive LP responses to HCV, tetanus and Candida antigens declined over time (P < 0.003), and the slope of this decline was greater in the PEG-IFN treatment group than the observation group (P < 0.02). Lower levels of tetanus LP responses were associatedwithfibrosisprogressionandclinicaloutcomes(P= 0.009). Poorer CD4+ T cell proliferative function was associated with more advanced liver disease in chronic hepatitis C and may be further affected by long-term PEG-IFN treatment.

Dengue is a mosquito-borne viral disease of expanding geographical range and incidence. The existence of four viral serotypes and the association of prior dengue virus infection with an increased risk for more severe disease have presented significant obstacles to vaccine development. An increased understanding of the adaptive immune response to natural dengue virus infection and candidate dengue vaccines has helped to define the specific antibody and T cell responses that are associated with either protective or pathological immunity during dengue infection. Further characterization of immunological correlates of disease outcome and the validation of these findings in vaccine trials will be invaluable for developing effective dengue vaccines.

Background Early recognition of dengue, particularly patients at risk for plasma leakage, is important to clinical management. The objective of this study was to build predictive models for dengue, dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS) using structural equation modelling (SEM), a statistical method that evaluates mechanistic pathways. Methods/Findings We performed SEM using data from 257 Thai children enrolled within 72 h of febrile illness onset, 156 with dengue and 101 with non-dengue febrile illnesses. Models for dengue, DHF, and DSS were developed based on data obtained three and one day(s) prior to fever resolution (fever days -3 and -1, respectively). Models were validated using data from 897 subjects who were not used for model development. Predictors for dengue and DSS included age, tourniquet test, aspartate aminotransferase, and white blood cell, % lymphocytes, and platelet counts. Predictors for DHF included age, aspartate aminotransferase, hematocrit, tourniquet test, and white blood cell and platelet counts. The models showed good predictive performances in the validation set, with area under the receiver operating characteristic curves (AUC) at fever day -3 of 0.84, 0.67, and 0.70 for prediction of dengue, DHF, and DSS, respectively. Predictive performance was comparable using data based on the timing relative to enrollment or illness onset, and improved closer to the critical phase (AUC 0.73 to 0.94, 0.61 to 0.93, and 0.70 to 0.96 for dengue, DHF, and DSS, respectively). Conclusions Predictive models developed using SEM have potential use in guiding clinical management of suspected dengue prior to the critical phase of illness., Author summary Dengue virus infection is one of the most critical public health issues, particularly in tropical and subtropical regions. This study developed statistical predictive models using the data obtained from 257 Thai children for dengue, dengue hemorrhagic fever, and dengue shock syndrome using structural equation modelling (SEM). We performed SEM based on clinical and laboratory factors on three and one day(s) prior to fever resolution. Our SEM models showed that age, tourniquet test, aspartate aminotransferase, and white blood cell, % lymphocytes, and platelet counts on three days prior to fever resolution were important risk factors for dengue and dengue hemorrhagic fever. Age, aspartate aminotransferase, hematocrit, tourniquet test, and white blood cell and platelet counts were important risk factors for dengue shock syndrome. Our predictive models showed good performances in the validation subjects (n = 897) who were not used for SEM, and thus we concluded that our predictive models can be practically used to guide clinical management of suspected dengue patients. Our study also showed that SEM can be used to predict the developments or severities of other illnesses.

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 sub-tropical 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 virus infection causes a spectrum of clinical manifestations, usually classified according to the World Health Organization (WHO) guidelines into dengue fever (DF) and dengue hemorrhagic fever (DHF). The ability of these guidelines to categorize severe dengue illness has recently been questioned.We evaluated dengue case definitions in a prospective study at a pediatric hospital in Bangkok, Thailand, during 1994-2005. One thousand thirteen children were enrolled within the first 3 days after onset of fever and observed with standardized data collection. Cases were classified on the basis of application of the strict WHO criteria. All dengue virus infections were laboratory confirmed. We retrospectively grouped patients on the basis of whether they received significant intervention based on fluid replacement and/or requirements for blood transfusion.Eighty-five (58%) of 150 persons with DHF, 40 (15%) of 264 with DF, and 73 (12%) of 599 with other febrile illnesses (OFIs) received significant intervention. Sixty-eight percent of dengue cases requiring intervention met strict WHO criteria for DHF. In contrast, only 1% of OFI cases met WHO criteria for DHF. Plasma leakage and thrombocytopenia were the 2 components contributing to the specificity of the WHO case definition and identified dengue cases that required intervention. Hemorrhagic tendency did not reliably differentiate DF and DHF. In DF cases, thrombocytopenia and bleeding were associated with severity.Dengue illness is heterogeneous in severity, and severe clinical features occurred in patients whose cases were not characterized as DHF. The WHO case definition of DHF demonstrated sensitivity of 62% and specificity of 92% for identification of dengue illness requiring intervention, without the need for laboratory confirmation of dengue virus infection, in an area of endemicity.

The available evidence suggests that plasma leakage in dengue hemorrhagic fever is caused by the action of circulating mediators on the vascular endothelium, and that the host immune response is a critical determinant of this response. We have been exploring the role of dengue virus-specific T lymphocytes in the immunopathogenesis of plasma leakage. Memory dengue virus-specific T cells induced during a primary dengue virus infection are reactivated by the heterologous viral serotype during a secondary infection to expand to high levels and produce a skewed cytokine profile. Investigations are underway to identify the immunologic profiles associated with increased or decreased risk for severe disease.

Management of yellow fever is focused on the prevention of illness by the use of the yellow fever virus (YFV) 17D vaccine. The role of neutralizing antibodies in protection is generally accepted with YFV-specific T cells likely contributing to the control of viral replication. We studied CD8(+) T-cell responses to four defined human leucocyte antigen-B35-restricted epitopes in YFV vaccine recipients as a model of the kinetics of cytotoxic T-lymphocyte responses to an acute human viral infection. Multiple features of these epitope-specific responses were analysed after vaccination including magnitude, cytokine production, phenotype and T-cell receptor repertoire. Peak peptide-specific interferon-gamma (IFN-gamma) responses of almost 1% of CD8(+) T cells were seen as early as 2 weeks post-vaccination; however, dominant responses varied between donors. Peptide-specific responses were still detectable at 54 months post-vaccination. Tetramer-positive cells, at high frequencies, were detected as early as 7-9 days, before detectable IFN-gamma-producing cells, suggesting a defect in the functional capacity of some antigen-specific cells early post-vaccination. The predominant memory phenotype of the tetramer-positive population was a differentiated effector (CD45RA(+) CCR7(-) CD62L(-)) phenotype. The T-cell receptor Vbeta analysis revealed a diverse oligoclonal repertoire in tetramer-positive T-cell populations in two individuals. These characteristics of the YFV-specific T-cell response could contribute to vaccine effectiveness.

Severe dengue virus (DENV) infection is characterized by a cascade of cytokine production, including the production of tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin-alpha (LT-alpha). We have analyzed a variety of polymorphisms in the TNF and LTA genes of 435 ethnic Thais who had subclinical DENV infection, primary or secondary dengue fever (DF), or primary or secondary dengue hemorrhagic fever (DHF). The TNF -238A polymorphism marking the TNF-4,LTA-3 haplotype occurred in a significantly greater number of patients with secondary DHF (20 [15.2%] of 132) than patients with secondary DF (7 [4.1%] of 169) (P.001; P corrected by use of Bonferroni adjustment, .022; odds ratio, 4.13 [95% confidence interval, 1.59-11.17]). In a subset of patients, the LTA-3 haplotype was associated with in vivo intracellular production of LT-alpha and TNF-alpha during the acute viremic phase of infection. Two extended human major histocompatibility complex (MHC) haplotypes containing TNF-4 and LTA-3, together with HLA-B48, HLA-B57, and HLA-DPB1*0501, were detected only in patients with secondary DHF. These observations indicate that polymorphism in functionally distinct MHC-encoded proteins contributes to the risk of developing severe secondary DENV infection and warrants further investigation.

Immunization with vaccinia virus (VACV) resulted in long-lasting protection against smallpox and successful global eradication of the disease. VACV elicits strong cellular and humoral immune responses. Although neutralizing antibody is essential for protection, cellular immunity seems to be more important for recovery from infection in humans. We analyzed the immunodominance hierarchy of 73 previously identified VACV human CD8(+) T-cell epitopes restricted by HLA-A1, -A2, -A3, -A24, -B7, or -B44 alleles or the alleles belonging to one of these supertypes in 56 donors after primary VACV immunization. With the exception of the responses to HLA-A24 supertype-restricted epitopes, there were no consistent patterns of epitope immunodominance among donors sharing the same HLA alleles or supertypes, which is in sharp contrast with the mouse studies. However, we identified 12 epitopes that were recognized by >or=20% of donors sharing the same HLA allele; 6 of these epitopes contributed >or=20% of the total VACV-specific T-cell response in at least one individual. VACV-specific CD8(+) T-cell responses targeted a group of epitopes, "relatively dominant" epitopes, without a strong immunodominance hierarchy in humans, which may be advantageous to humans to prevent the emergence of T-cell escape mutants.

Background Human metapneumovirus (hMPV) is a newly discovered paramyxovirus that causes acute respiratory illness. Despite apparent near-universal exposure during early childhood, immunity is transient.

This report describes the safety observations following administration of a polyvalent DNA prime–protein boost HIV-1 vaccine formulated with adjuvant QS21. Local injection site reactions were the most common (65% of subjects), and included type IV delayed-type hypersensitivity (DTH) reactions at prior DNA inoculation sites in 12 of 28 (43%) subjects following protein vaccination. Systemic reactions revealed two cases of vasculitis temporally related to inoculation with recombinant Env protein + QS21 adjuvant. Questions remain regarding the cause of the vasculitis, but the unique DTH observation may have contributed to the high level of immune responses previously reported for this vaccine.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes an acute febrile disease in humans, characterized by musculoskeletal pain, headache, rash and leukopenia. The cause of myalgia during DENV infection is still unknown. To determine whether DENV can infect primary muscle cells, human muscle satellite cells were exposed to DENV in vitro. The results demonstrated for the first time high-efficiency infection and replication of DENV in human primary muscle satellite cells. Changes in global gene expression were also examined in these cells following DENV infection using Affymetrix GeneChip analysis. The differentially regulated genes belonged to two main functional categories: cell growth and development, and antiviral type I interferon (IFN) response genes. Increased expression of the type I IFN response genes for tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), melanoma-derived antigen 5 (MDA-5), IFN-γ-inducible protein 10 (IP-10), galectin 3 soluble binding protein (LGals3BP) and IFN response factor 7 (IRF7) was confirmed by quantitative RT-PCR. Furthermore, higher levels of cell-surface-bound intracellular adhesion molecule-1 (ICAM-1) and soluble ICAM-1 in the cell-culture medium were detected following DENV infection. However, DENV infection impaired the ability of the infected cells in the culture medium to upregulate cell-surface expression of MHC I molecules, suggesting a possible mechanism of immune evasion by DENV. The findings of this study warrant further clinical research to identify whether muscle cells are targets for DENV infection during the acute stage of the disease in vivo.

Thirty adults were tested for humoral and cellular immune responses following immunization with the trivalent inactivated influenza vaccine. Modest but significant inverse correlations between the baseline and the fold changes in the number of IFNgamma-producing cells and the levels of neutralizing antibodies were observed. Specific increases in proliferative responses in the CD8 CD45RA+ population were noted after vaccination. Minimal correlations between neutralizing antibody titers and the number of IFNgamma-producing cells in terms of prevaccination levels or fold increases were observed. These results show specific increases in a CD8 T cell subset and discordant T and B responses induced by the trivalent inactivated influenza vaccine.

Levels of the soluble form of the interleukin-1 receptor-like 1 protein (IL-1RL-1/ST2) are elevated in the serum of patients with diseases characterized by an inflammatory response. The objective of this study was to determine the concentration of soluble ST2 (sST2) in dengue infected patients during the course of the disease. Twenty-four patients with confirmed dengue infection, classified as dengue fever, and 11 patients with other febrile illness (OFI) were evaluated. Levels of sST2 in serum and laboratory variables usually altered during dengue infections were measured. Dengue infected patients had higher serum sST2 levels than OFI at the end of the febrile stage and at defervescence (p = 0.0088 and p = 0.0004, respectively). Patients with secondary dengue infections had higher serum sST2 levels compared with patients with primary dengue infections (p = 0.047 at last day of fever and p = 0.030 at defervescence). Furthermore, in dengue infected patients, we found a significant negative correlation of sST2 with platelet and WBC counts, and positive correlation with thrombin time and transaminases activity. We suggest that sST2 could be a potential marker of dengue infection, could be associated with severity or could play a role in the immune response in secondary dengue virus infection.

Dengue fever is an important tropical illness for which there is currently no virus-specific treatment. To shed light on mechanisms involved in the cellular response to dengue virus (DV), we assessed gene expression changes, using Affymetrix GeneChips (HG-U133A), of infected primary human cells and identified changes common to all cells. The common response genes included a set of 23 genes significantly induced upon DV infection of human umbilical vein endothelial cells (HUVECs), dendritic cells (DCs), monocytes, and B cells (analysis of variance,P< 0.05). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), one of the common response genes, was identified as a key link between type I and type II interferon response genes. We found that DV induces TRAIL expression in immune cells and HUVECs at the mRNA and protein levels. The induction of TRAIL expression by DV was found to be dependent on an intact type I interferon signaling pathway. A significant increase in DV RNA accumulation was observed in anti-TRAIL antibody-treated monocytes, B cells, and HUVECs, and, conversely, a decrease in DV RNA was seen in recombinant TRAIL-treated monocytes. Furthermore, recombinant TRAIL inhibited DV titers in DV-infected DCs by an apoptosis-independent mechanism. These data suggest that TRAIL plays an important role in the antiviral response to DV infection and is a candidate for antiviral interventions against DV.

Background Preexisting dengue virus (DV)-specific antibodies from prior heterologous DV infection may have several effects in secondary DV infection. These antibodies may mediate protective effects by means of antibody-dependent cellular cytotoxicity (ADCC), in which virus-specific antibodies bind to the surface of heterologous DV-infected cells and mediate natural killer cell lysis. In the present study, we examined the ability of plasma obtained before secondary DV infection to induce ADCC of DV-infected cells. Methods Plasma samples were obtained before DV2 or DV3 infection in a prospective cohort study of Thai schoolchildren. The ADCC activity in the plasma samples was measured by (51)Cr-release assay, using persistently DV2- or DV3-infected Raji cells as targets. Results ADCC activity in plasma obtained before secondary infection directly correlated with neutralizing antibody titers, anti-DV immunoglobulin G1 levels, and a multitypic 50% plaque reduction neutralization test pattern. ADCC activity in pre-secondary DV3 infection plasma samples inversely correlated with plasma viremia levels, but no such correlation was seen in pre-secondary DV2 infection plasma samples. ADCC activity did not correlate with disease severity in subsequent secondary DV2 or DV3 infection but was lowest in plasma from patients with dengue hemorrhagic fever due to secondary DV3 infection. Conclusions ADCC may contribute to the early control of secondary DV3 viremia in vivo.

Background: Although plasma leakage is the major cause of mortality and morbidity in patients with dengue hemorrhagic fever (DHF), a detailed assessment of the natural course of this process is still lacking. We employed serial ultrasound examination to delineate the locations and the timing of plasma leakage and to evaluate the usefulness of ultrasound in detecting plasma leakage in DHF. Method: Daily ultrasound examinations of the abdomen and right thorax were performed in 158 suspected dengue cases to detect ascites, thickened gall bladder wall and pleural effusions. Cases were classified into dengue fever (DF), DHF or other febrile illness (OFI) based on serology and evidence of plasma leakage including hemoconcentration and pleural effusion detected by chest radiograph. Results: Ultrasonographic evidence of plasma leakage was detected in DHF cases starting from 2 days before defervescence and was detected in some cases within 3 days after fever onset. Pleural effusion was the most common ultrasonographic sign of plasma leakage (62% of DHF cases one day after defervescence). Thickening of the gallbladder wall and ascites were detected less frequently (43% and 52% of DHF cases respectively) and resolved more rapidly than pleural effusions. The size of pleural effusions, ascites and gall bladder wall thickness in DHF grade I and II were smaller than those of grade III patients. Ultrasound detected plasma leakage in 12 of 17 DHF cases who did not meet the criteria for significant hemoconcentration. Conclusions: Ultrasound examinations detected plasma leakage in multiple body compartments around the time of defervescence. Ultrasonographic signs of plasma leakage were detectable before changes in hematocrits. Ultrasound is a useful tool for detecting plasma leakage in dengue infection.

Background The effect of prior dengue virus (DENV) exposure on subsequent heterologous infection can be beneficial or detrimental depending on many factors including timing of infection. We sought to evaluate this effect by examining a large database of DENV infections captured by both active and passive surveillance encompassing a wide clinical spectrum of disease. Methods We evaluated datasets from 17 years of hospital-based passive surveillance and nine years of cohort studies, including clinical and subclinical DENV infections, to assess the outcomes of sequential heterologous infections. Chi square or Fisher’s exact test was used to compare proportions of infection outcomes such as disease severity; ANOVA was used for continuous variables. Multivariate logistic regression was used to assess risk factors for infection outcomes. Results Of 38,740 DENV infections, two or more infections were detected in 502 individuals; 14 had three infections. The mean ages at the time of the first and second detected infections were 7.6 ± 3.0 and 11.2 ± 3.0 years. The shortest time between sequential infections was 66 days. A longer time interval between sequential infections was associated with dengue hemorrhagic fever (DHF) in the second detected infection (OR 1.3, 95% CI 1.2-1.4). All possible sequential serotype pairs were observed among 201 subjects with DHF at the second detected infection, except DENV-4 followed by DENV-3. Among DENV infections detected in cohort subjects by active study surveillance and subsequent non-study hospital-based passive surveillance, hospitalization at the first detected infection increased the likelihood of hospitalization at the second detected infection. Conclusions Increasing time between sequential DENV infections was associated with greater severity of the second detected infection, supporting the role of heterotypic immunity in both protection and enhancement. Hospitalization was positively associated between the first and second detected infections, suggesting a possible predisposition in some individuals to more severe dengue disease. Electronic supplementary material The online version of this article (doi:10.1186/s12889-015-1590-z) contains supplementary material, which is available to authorized users.

The continued emergence of dengue virus infection and its severe disease manifestation, dengue hemorrhagic fever, is a growing public health problem. The majority of severe infections occur upon secondary encounters with heterologous dengue virus serotypes, suggesting an immune-mediated process.Significant findings in the past year include a greater understanding of dengue virus interactions with target cells such as dendritic cells, hepatocytes and endothelial cells. Infection of these cells results in the production of immune mediators that then shape the adaptive humoral and cellular immune response. The circulation of high levels of secreted NS1 in the presence of pre-existing heterologous non-neutralizing antibody may mediate complement activation and trigger plasma leakage. The role of enhancing antibodies in disease pathogenesis remains unclear. Recent studies demonstrate low avidity crossreactive T cells, which may produce an altered profile of cytokines leading to plasma leakage. Ongoing prospective studies that include epidemiological, virological and immunological risk factors are crucial to our understanding of the mechanisms of immunopathogenesis of dengue hemorrhagic fever.The immune mechanisms that lead to dengue hemorrhagic fever are complex and need to be elucidated further for the development of therapeutics as well as safe and efficacious dengue vaccines.

An infusion of funding, talent and new scientific techniques in virology and immunology has recently yielded major gains in the field of dengue research, with promising developments in the basic un...

The interplay of different inflammatory cytokines induced during a dengue (DEN) virus infection plays a role in either protection or increased disease severity. We measured the frequencies and characterized the cytokine responses of DEN virus-specific memory CD4+ T cells in PBMC of six volunteers who received experimental live attenuated monovalent DEN vaccines. IFN-γ and TNF-α responses to inactivated DEN Ags were detected in up to 0.54 and 1.17% of total circulating CD4+ T cells, respectively. Ags from the homologous serotype elicited the highest IFN-γ response. The ratio of TNF-α- to IFN-γ-producing CD4+ T cells was higher after stimulation with Ags from heterologous DEN serotypes. Peptide-specific CD4+ T cell frequencies of up to 0.089% were detected by direct staining using HLA class II tetramers. IFN-γ and TNF-α responses to individual HLA class II-restricted peptide epitopes were detected in up to 0.05 and 0.27% of CD4+ T cells, respectively. Peptide sequences from the homologous serotype elicited a variety of cytokine response patterns. TNF-α- to IFN-γ-positive CD4+ T cell ratios varied between peptides, but the ratio of the sum of responses was highest against heterologous serotypes. These results demonstrate epitope sequence-specific differences in T cell effector function. These patterns of effector responses may play a role in the immunopathogenesis of DEN hemorrhagic fever.

There is evidence that severe dengue disease is associated with alterations of the microvascular endothelium. We examined the hypothesis that activation and damage of microvascular endothelial cells (EC) could be induced by inflammatory mediators present in dengue patient's sera. We cultured human microvascular EC (HMEC-1) in vitro with sera from patients with acute dengue infection. Sera from patients with acute dengue induced an increase in ICAM-1 expression on HMEC-1. This effect was greater with samples from the acute febrile phase than with samples from the convalescent phase of the disease. Acute dengue sera had elevated levels of TNF-alpha and the endothelial activating effect of acute dengue sera was inhibited up to 80% by pre-treatment with monoclonal antibodies against TNF-alpha. Furthermore, acute dengue sera induced apoptosis in HMEC-1. These findings support the pathophysiologic significance of microvascular EC and serum inflammatory mediators in dengue.

Chronic hepatitis C virus (HCV) infection is characterized by extensive infiltration of inflammatory cells in the liver, where there is a compartmentalization of HCV-reactive T lymphocytes. Previous studies have demonstrated a broad intrahepatic TCR repertoire; however, there is little information regarding the stability of this intrahepatic T cell population. We studied the T cell repertoires in sequential liver biopsy samples from five individuals with chronic HCV infection using TCR spectratype analysis; four subjects had been treated with IFN-alpha during the interval between biopsies. Transcripts from most TCRBV families were detectable in the liver tissues, and 25-85% of these had skewed spectratype profiles indicative of T cell clonal expansions. Most of the intrahepatic T cell expansions were not evident in an analysis of peripheral blood T cells collected at the same time as the liver biopsy. Detailed analysis using TCRBJ-primed run-off reactions revealed that the intrahepatic TCR repertoires were not stable within an individual, although some TCR clonotypes were maintained for at least 45 months.

Sin Nombre virus (SNV) causes hantavirus pulmonary syndrome (HPS), with a high rate of mortality in humans who are infected by the transmission of virus from the natural rodent host. In humans, cytotoxic T lymphocytes (CTL) specific for SNV appear to play an important role in the pathogenicity of HPS. There is a correlation between the frequencies of SNV-specific CTLs and the severity of HPS disease. In order to create a mouse model to study the role of SNV-specific T cells in vivo, T cell responses to SNV nucleocapsid (N) protein in B6.PL Thy1 a/Cy mice (H-2b) immunized with plasmid DNA or recombinant vaccinia virus expressing SNV N protein were examined. Four peptides, NC94–101, NC175–189, NC217–231 and NC331–345, were recognized by CD8+ T cells in CTL and ELISPOT assays in SNV N-immunized mice. Interestingly, two of these epitopes are located in the central region of the SNV N protein, where several human CD8+ T-cell epitopes have been defined in Puumala virus and SNV. CTL lines specific for these four epitopes were cross-reactive to corresponding Puumala virus peptides, but only one of them was cross-reactive to Hantaan virus peptides. These results will enable the analysis of the roles of CTL in immunopathology of HPS in experimental mouse models of HPS.

Endothelial cells are permissive to dengue virus (DV) infection in vitro, although their importance as targets of DV infection in vivo remains a subject of debate. To analyze the virus-host interaction, we studied the effect of DV infection on gene expression in human umbilical vein endothelial cells (HUVECs) by using differential display reverse transcription-PCR (DD-RTPCR), quantitative RT-PCR, and Affymetrix oligonucleotide microarrays. DD identified eight differentially expressed cDNAs, including inhibitor of apoptosis-1, 2′-5′ oligoadenylate synthetase (OAS), a 2′-5′ OAS-like (OASL) gene, galectin-9, myxovirus protein A (MxA), regulator of G-protein signaling, endothelial and smooth muscle cell-derived neuropilin-like protein, and phospholipid scramblase 1. Microarray analysis of 22,000 human genes confirmed these findings and identified an additional 269 genes that were induced and 126 that were repressed more than fourfold after DV infection. Broad functional responses that were activated included the stress, defense, immune, cell adhesion, wounding, inflammatory, and antiviral pathways. These changes in gene expression were seen after infection of HUVECs with either laboratory-adapted virus or with virus isolated directly from plasma of DV-infected patients. Tumor necrosis factor alpha, OASL, and MxA and h-IAP1 genes were induced within the first 8 to 12 h after infection, suggesting a direct effect of DV infection. These global analyses of DV effects on cellular gene expression identify potentially novel mechanisms involved in dengue disease manifestations such as hemostatic disturbance.

Immunization with vaccinia virus resulted in long-lasting protection against smallpox and was the approach used to eliminate natural smallpox infections worldwide. Due to the concern about the potential use of smallpox virus as a bioweapon, smallpox vaccination is currently being reintroduced. Severe complications from vaccination were associated with congenital or acquired T cell deficiencies, but not with congenital agammaglobulinemia, suggesting the importance of T cell immunity in recovery from infection. In this report, we identified two CD8+ T cell epitopes restricted by the most common human major histocompatibility complex (MHC) class I allele, HLA-A*0201. Both epitopes are highly conserved in vaccinia and variola viruses. The frequency of vaccinia-specific CD8+ T cell responses to these epitopes measured by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay and HLA/peptide tetramer staining peaked 2 wk after primary immunization and then declined, but were still detectable 1 to 3 yr after primary immunization. 2 wk after immunization, IFN-γ–producing cells specific to these two epitopes were 14% of total vaccinia virus-specific IFN-γ–producing cells in one donor, 35% in the second donor, and 6% in the third donor. This information will be useful for studies of human T cell memory and for the design and analyses of the immunogenicity of experimental vaccinia vaccines.

Little is known of the role of classical HLA-A and -B class I alleles in determining resistance, susceptibility, or the severity of acute viral infections. Appropriate paradigms for immunogenetic studies of acute viral infections are dengue fever (DF) and dengue hemorrhagic fever (DHF). Both primary and secondary infections with dengue virus (DEN) serotypes 1, 2, 3 or 4, can result in either clinically less severe DF or the more severe DHF. In secondary exposures, a memory response is induced in immunologically primed individuals, which can both clear the infecting dengue virus and contribute to its pathology. In a case-control study of 263 ethnic Thai patients infected with either DEN-1, -2, -3 or -4, we detected HLA class I associations with secondary infections, but not in immunologically naive patients with primary infections. HLA-A*0203 was associated with the less severe DF, regardless of the secondary infecting virus serotype. By contrast, HLA-A*0207 was associated with susceptibility to the more severe DHF in patients with secondary DEN-1 and DEN-2 infections only. Conversely, HLA-B*51 was associated with the development of DHF in patients with secondary infections, and HLA-B*52 was associated with DF in patients with secondary DEN-1 and DEN-2 infections. Moreover, HLA-B44, B62, B76 and B77 also appeared to be protective against developing clinical disease after secondary dengue virus infection. These results confirm that classical HLA class I alleles are associated with the clinical outcome of exposure to dengue virus, in previously exposed and immunologically primed individuals.