Dengue virus (DENV) is a rapidly spreading pathogen with unusual pathogenesis, and correlates of protection from severe dengue disease and vaccine efficacy have not yet been established. Although DENV-specific CD8 + T-cell responses have been extensively studied, the breadth and specificity of CD4 + T-cell responses remains to be defined. Here we define HLA-restricted CD4 + T-cell epitopes resulting from natural infection with dengue virus in a hyperepidemic setting. Ex vivo flow-cytometric analysis of DENVspecific CD4+ T cells revealed that the virus-specific cells were highly polarized, with a strong bias toward a CX3CR1 A ll four of the dengue virus serotypes (DENV 1-4) have spread rapidly within countries and across regions in the past few decades, resulting in an increased frequency of epidemics and severe dengue disease. Multiple serotypes circulate simultaneously in many tropical countries, and recent outbreaks have been reported in Europe and the continental United States (1, 2). These circumstances make dengue the most prevalent and rapidly spreading mosquito-borne viral disease in humans (3). Recent reports estimate that 390 million infections occur each year, with ∼25% of cases resulting in symptomatic disease (2).All four dengue serotypes can cause a spectrum of disease, ranging from self-limiting dengue fever to potentially lethal severe dengue disease, such as states of dengue hemorrhagic fever and Dengue shock syndrome, which are associated with the plasma leakage syndromes leading to visceral organ injury (4). It is not yet fully understood why only a subset of people infected with DENV progresses to severe disease. One risk factor for severe disease is the acquisition of DENV-reactive Abs before secondary infection with a different serotype (heterologous infection). These Abs can either be acquired from a previous infection with a different serotype or, in the case of infants, acquired from an immune mother (5, 6). It has been shown that subneutralizing levels of DENV-specific Abs exacerbate disease in a phenomenon termed Ab-dependent enhancement of infection (7,8). In brief, dengue-specific crossreactive Abs produced after an initial DENV infection combine with those produced after a second viral infection to form immune complexes that perpetuate infection by increasing the number of infected cells and, therefore, viral output per cell (6).The observation that only a minority of patients develops severe disease suggests that host genetic factors may play an important role in disease severity. Relatedly, a role for T cells in control of disease has been suggested by several studies that correlate the expression of certain HLA molecules with susceptibility to or protection from DENV disease (9-15). HLA molecules are one of the most polymorphic host factors in humans, with several thousand variants thus far known (16,17). Each HLA variant is present with variable frequency, depending on ethnic lineage and geographic locality. For HLA class I MHC restricted responses, it has been recently shown tha...

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...

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.

Dengue disease is a large public health problem that mainly afflicts tropical and subtropical regions. Understanding of the correlates of protection against dengue virus (DENV) is poor and hinders the development of a successful human vaccine. The present study aims to define DENV-specific CD8 ؉ T cell responses in general and those of HLA alleles associated with dominant responses in particular. In human blood donors in Nicaragua, we observed a striking dominance of HLA B-restricted responses in general and of the allele B*35:01 in particular. Comparing these patterns to those in the general population of Sri Lanka, we found a strong correlation between restriction of the HLA allele and the breadth and magnitude of CD8 ؉ T cell responses, suggesting that HLA genes profoundly influence the nature of responses. The majority of gamma interferon (IFN-␥) responses were associated with effector memory phenotypes, which were also detected in non-B*35:01-expressing T cells. However, only the B*35:01 DENV-specific T cells were associated with marked expression of the programmed death 1 protein (PD-1). These cells did not coexpress other inhibitory receptors and were able to proliferate in response to DENV-specific stimulation. Thus, the expression of particular HLA class I alleles is a defining characteristic influencing the magnitude and breadth of CD8 responses, and a distinct, highly differentiated phenotype is specifically associated with dominant CD8 ؉ T cells. These results are of relevance for both vaccine design and the identification of robust correlates of protection in natural immunity. IMPORTANCEDengue is an increasingly significant public health problem as its mosquito vectors spread over greater areas; no vaccines against the virus have yet been approved. An important step toward vaccine development is defining protective immune responses; toward that end, we here characterize the phenotype of the immunodominant T cell responses. These DENV-reactive T cells express high levels of the receptor programmed death 1 protein (PD-1), while those from disease-susceptible alleles do not. Not only does this represent a possible correlate of immunodominance, but it raises the hypothesis that PD-1 might be a regulator that prevents excessive damage while preserving antiviral function. Further, as this study employs distinct populations (Nicaraguan and Sri Lankan donors), we also confirmed that this pattern holds despite geographic and ethnic differences. This finding indicates that HLA type is the major determinant in shaping T cell responses.

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