The role of CD8 + T cells in dengue virus infection and subsequent disease manifestations is not fully understood. According to the original antigenic sin theory, skewing of T-cell responses induced by primary infection with one serotype causes less effective response upon secondary infection with a different serotype, predisposing individuals to severe disease. A comprehensive analysis of CD8 + responses in the general population from the Sri Lankan hyperendemic area, involving the measurement of ex vivo IFNγ responses associated with more than 400 epitopes, challenges the original antigenic sin theory. Although skewing of responses toward primary infecting viruses was detected, this was not associated with impairment of responses either qualitatively or quantitatively. Furthermore, we demonstrate higher magnitude and more polyfunctional responses for HLA alleles associated with decreased susceptibility to severe disease, suggesting that a vigorous response by multifunctional CD8 + T cells is associated with protection from dengue virus disease.

Prediction of HLA binding affinity is widely utilized to identify candidate T cell epitopes, and an affinity of 500 nM is routinely used as a threshold for peptide selection. However, the fraction (%) of peptides predicted to bind with affinities of 500 nM varies by allele. For example, of a large collection of about 30,000 dengue virus derived peptides only 0.3% were predicted to bind HLA A*0101, while nearly 5% were predicted for A*0201. This striking difference could not be ascribed to variation in accuracy of the algorithms utilized, as predicted values closely correlated with affinity measured in vitro with purified HLA molecules. These data raised the question whether different alleles would also vary in terms of epitope repertoire size, defined as the number of associated epitopes or, alternatively, whether alleles vary drastically in terms of the affinity threshold associated with immunogenicity. To address this issue, strains of HLA transgenic mice with wide (A*0201), intermediate (B*0702) or narrow (A*0101) repertoires were immunized with peptides of varying binding affinity and relative percentile ranking. The results show that absolute binding capacity is a better predictor of immunogenicity, and analysis of epitopes from the Immune Epitope Database (IEDB) revealed that predictive efficacy is increased using allele-specific affinity thresholds. Finally, we investigate the genetic and structural basis of the phenomenon. While no stringent correlate was defined, on average HLA B alleles are associated with significantly narrower repertoires than HLA A alleles.

The incidence of infection with any of the four dengue virus serotypes (DENV1 to -4) has increased dramatically in the last few decades, and the lack of a treatment or vaccine has contributed to significant morbidity and mortality worldwide. A recent comprehensive analysis of the human T cell response against wild-type DENV suggested an human lymphocyte antigen (HLA)-linked protective role for CD8 ؉ T cells. We have collected one-unit blood donations from study participants receiving the monovalent or tetravalent live attenuated DENV vaccine (DLAV), developed by the U.S. National Institutes of Health. Peripheral blood mononuclear cells from these donors were screened in gamma interferon enzyme-linked immunosorbent spot assays with pools of predicted, HLA-matched, class I binding peptides covering the entire DENV proteome. Here, we characterize for the first time CD8؉ T cell responses after live attenuated dengue vaccination and show that CD8 ؉ T cell responses in vaccinees were readily detectable and comparable to natural dengue infection. Interestingly, whereas broad responses to structural and nonstructural (NS) proteins were observed after monovalent vaccination, T cell responses following tetravalent vaccination were, dramatically, focused toward the highly conserved NS proteins. Epitopes were highly conserved in a vast variety of field isolates and able to elicit multifunctional T cell responses. Detailed knowledge of the T cell response will contribute to the identification of robust correlates of protection in natural immunity and following vaccination against DENV. IMPORTANCEThe development of effective vaccination strategies against dengue virus (DENV) infection and clinically significant disease is a task of high global public health value and significance, while also being a challenge of significant complexity. A recent efficacy trial of the most advanced dengue vaccine candidate, demonstrated only partial protection against all four DENV serotypes, despite three subsequent immunizations and detection of measurable neutralizing antibodies to each serotype in most subjects. These results challenge the hypothesis that seroconversion is the only reliable correlate of protection. Here, we show that CD8 ؉ T cell responses in vaccinees were readily detectable and comparable to natural dengue virus infection. Detailed knowledge of the T cell response may further contribute to the identification of robust correlates of protection in natural immunity and vaccination against DENV. Infections with dengue virus (DENV) occur with high incidence in more than 100 countries around the world. Recent reports estimate the number of annual infections with any of the four DENV serotypes (DENV1 to -4) to be as high as 390 million, of which 96 million manifest as clinically significant diseases, including life-threatening conditions such as dengue hemorrhagic fever and dengue shock syndrome (1). This constitutes an increasing public health problem in tropical and subtropical regions and underscores the urgent need f...

Background. Each year dengue virus (DENV) infects 400 million human but causes symptomatic disease in only a subset of patients, suggesting that host genetic factors may play a role. HLA molecules that restrict T-cell responses are one of the most polymorphic host factors in humans.Methods. Here we map HLA DRB1-restricted DENV-specific epitopes in individuals previously exposed to DENV, to identify the breadth and specificity of CD4 + T-cell responses. To investigate whether HLA-specific variations in the magnitude of response might predict associations between dengue outcomes and HLA-DRB1 alleles, we assembled samples from hospitalized patients with known severity of disease.Results. The capsid protein followed by nonstructural protein 3 (NS3), NS2A, and NS5 were the most targeted proteins. We further noticed a wide variation in magnitude of T-cell responses as a function of the restricting DRB1 allele and found several HLA alleles that showed trends toward a lower risk of hospitalized disease were associated with a higher magnitude of T-cell responses.Conclusions. Comprehensive identification of unique CD4 + T-cell epitopes across the 4 DENV serotypes allows the testing of T-cell responses by use of a simple, approachable technique and points to important implications for vaccine design.

This study provides new insight into differential serotype-specific immunogenicity of DENV proteins. It further provides a potentially valuable tool for future investigations of CD8(+) T-cell responses in the typically small sample volumes available from patients with acute fever and children without requiring prior knowledge of either infecting DENV serotype or HLA type.

The frequency of dengue virus (DENV) infection has increased dramatically in the last few decades, and the lack of a vaccine has led to significant morbidity and mortality worldwide. To date, a convenient murine system to study human T cell responses to DENV has not been available. Mice transgenic for human leukocyte antigens (HLA) are widely used to model human immune responses and it has been shown that mouse-passaged DENV is able to replicate to significant levels in IFN-α/βR−/− mice. To cover a wide range of HLA phenotypes, we backcrossed IFN-α/βR−/− mice with HLA A*0201, A*0101, A*1101, B*0702 and DRB1*0101 transgenic mice. A DENV proteome-wide screen identified a total of 42 epitopes across all HLA-transgenic IFN-α/βR−/− strains tested. In contrast only 8 of these elicited responses in the corresponding IFN-α/βR+/+ mice. We were able to identify T cell epitopes from 9 out of the 10 DENV proteins. However, the majority of responses were derived from the highly conserved nonstructural proteins NS3 and NS5. The relevance of this model is further demonstrated by the fact that most of the epitopes identified in our murine system are also recognized by PBMC from DENV exposed human donors, and a dominance of HLA B*0702 restricted responses has been detected in both systems. Our results provide new insights into HLA-restricted T cell responses against DENV, and we herein describe a novel murine model, which allows the investigation of T cell-mediated immune mechanisms relevant to vaccine design.

Dengue virus (DENV) is the causative agent of dengue fever (DF). This disease can be caused by any of four DENV serotypes (DENV1 to -4) which share 67 to 75% sequence homology with one another. The effect of subsequent infections with different serotypes on the T cell repertoire is not fully understood. We utilized mice transgenic for human leukocyte antigens (HLA) lacking the alpha/beta interferon (IFN-␣/␤) receptor to study responses to heterologous DENV infection. First, we defined the primary T cell response to DENV3 in the context of a wide range of HLA molecules. The primary DENV3 immune response recognized epitopes derived from all 10 DENV proteins, with a significant fraction of the response specific for structural proteins. This is in contrast to primary DENV2 infection, in which structural proteins are a minor component of the response, suggesting differential antigen immunodominance as a function of the infecting serotype. We next investigated the effect of secondary heterologous DENV infection on the T cell repertoire. In the case of both DENV2/3 and DENV3/2 heterologous infections, recognition of conserved/cross-reactive epitopes was either constant or expanded compared to that in homologous infection. Furthermore, in heterologous infection, previous infection with a different serotype impaired the development of responses directed to serotype-specific but not conserved epitopes. Thus, a detrimental effect of previous heterotypic responses might not be due to dysfunctional and weakly cross-reactive epitopes dominating the response. Rather, responses to the original serotype might limit the magnitude of responses directed against epitopes that are either cross-reactive to or specific for the most recently infecting serotype. IMPORTANCE DENV transmission occurs in more than

Dengue virus (DENV) is responsible for growing numbers of infections worldwide and has proven to be a significant challenge for vaccine development. We previously demonstrated that CD8 ϩ T cell responses elicited by a dengue live attenuated virus (DLAV) vaccine resemble those observed after natural infection. In this study, we screened peripheral blood mononuclear cells (PBMCs) from donors vaccinated with a tetravalent DLAV vaccine (TV005) with pools of dengue virusderived predicted major histocompatibility complex (MHC) class II binding peptides. The definition of CD4 ϩ T cell responses after live vaccination is important because CD4 ϩ T cells are known contributors to host immunity, including cytokine production, help for CD8 ϩ T and B cells, and direct cytotoxicity against infected cells. While responses to all antigens were observed, DENV-specific CD4 ϩ T cells were focused predominantly on the capsid and nonstructural NS3 and NS5 antigens. Importantly, CD4 ϩ T cell responses in vaccinees were similar in magnitude and breadth to those after natural infection, recognized the same antigen hierarchy, and had similar profiles of HLA restriction. We conclude that TV005 vaccination has the capacity to elicit CD4 ϩ cell responses closely mirroring those observed in a population associated with natural immunity.IMPORTANCE The development of effective vaccination strategies against dengue virus infection is of high global public health interest. Here we study the CD4 T cell responses elicited by a tetravalent live attenuated dengue vaccine and show that they resemble responses seen in humans naturally exposed to dengue virus. This is an important issue, since it is likely that optimal immunity induced by a vaccine requires induction of CD4 ϩ responses against the same antigens as those recognized as dominant in natural infection. Detailed knowledge of the T cell response may further contribute to the identification of robust correlates of protection against dengue virus.