Infection with one of the four serotypes of dengue virus (DENV1–4) can result in a range of clinical manifestations in humans, from dengue fever to the more serious dengue hemorrhagic fever/dengue shock syndrome. Although T cells have been implicated in the immunopathogenesis of secondary infections with heterologous DENV serotypes, the role of T cells in protection against DENV is unknown. In this study, we used a mouse-passaged DENV2 strain, S221, to investigate the role of CD8+ T cells in the immune response to primary DENV infection. S221 did not replicate well in wild-type mice, but did induce a CD8+ T cell response, whereas viral replication and a robust CD8+ T cell response were observed after infection of IFN-α/βR−/− mice. Depletion of CD8+ T cells from IFN-α/βR−/− mice before infection resulted in significantly higher viral loads compared with undepleted mice. Mapping the specificity of the CD8+ T cell response led to the identification of 12 epitopes derived from 6 of the 10 DENV proteins, with a similar immunodominance hierarchy observed in wild-type and IFN-α/βR−/− mice. DENV-specific CD8+ T cells produced IFN-γ, TNF-α, expressed cell surface CD107a, and exhibited cytotoxic activity in vivo. Finally, immunization with four of the immunodominant CD8+ T cell epitopes enhanced viral clearance. Collectively, our results reveal an important role for CD8+ T cells in the host defense against DENV and demonstrate that the anti-DENV CD8+ T cell response can be enhanced by immunization, providing rationale for designing DENV-specific vaccines that induce cell-mediated immunity.

The major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), is recognized by Toll-like receptor 2 (TLR2), TLR4, and CD14. In these studies, mice deficient in CD14, TLR2, TLR4, and the TLR-associated adaptor protein, MyD88, were utilized to investigate the contribution of TLRs and CD14 to in vivo host defenses against C. neoformans. MyD88 ؊/؊ mice had significantly reduced survival compared with wild-type C57BL/6 mice after intranasal (i.n.) and intravenous (i.v.) infection with live C. neoformans. CD14؊/؊ mice had reduced survival when infected i.v., while TLR2 ؊/؊ mice died significantly earlier after i.n. infection. Mortality was similar comparing TLR4 mutant C3H/HeJ mice and control C3H/HeOuJ mice following i.v. or i.n. challenge with C. neoformans. The course of pulmonary cryptococcosis was studied in more detail in the CD14 ؊/؊ , TLR2 ؊/؊ , and MyD88 ؊/؊ mice. MyD88 ؊/؊ mice infected i.n. had higher numbers of CFU in the lungs as well as higher GXM levels in the sera and lungs 7 days after infection than wild-type mice did. Surprisingly, there were no major differences in the levels of tumor necrosis factor alpha, interleukin-4 (IL-4), IL-10, IL-12p70, or gamma interferon in the lungs of C. neoformans-infected knockout mice compared with wild-type mice. Histopathologic analysis of the lungs on day 7 postinfection revealed minimal inflammation in all mouse groups. These studies demonstrate a major role for MyD88 and relatively minor roles for CD14 and TLR2 in the response to cryptococcal infection, with the decreased survival of MyD88 ؊/؊ mice correlating with increased numbers of lung CFU and serum and lung GXM levels.

The contribution of T cells to the host response to dengue virus (DENV) infection is not well understood. We previously demonstrated a protective role for CD8+ T cells during primary DENV infection using a mouse-passaged DENV strain and IFN-α/βR−/− C57BL/6 mice, which are susceptible to DENV infection. In this study, we examine the role of CD4+ T cells during primary DENV infection. Four I-Ab–restricted epitopes derived from three of the nonstructural DENV proteins were identified. CD4+ T cells expanded and were activated after DENV infection, with peak activation occurring on day 7. The DENV-specific CD4+ T cells expressed intracellular IFN-γ, TNF, IL-2, and CD40L, and killed peptide-pulsed target cells in vivo. Surprisingly, depletion of CD4+ T cells before DENV infection had no effect on viral loads. Consistent with this observation, CD4+ T cell depletion did not affect the DENV-specific IgG or IgM Ab titers or their neutralizing activity, or the DENV-specific CD8+ T cell response. However, immunization with the CD4+ T cell epitopes before infection resulted in significantly lower viral loads. Thus, we conclude that whereas CD4+ T cells are not required for controlling primary DENV infection, their induction by immunization can contribute to viral clearance. These findings suggest inducing anti-DENV CD4+ T cell responses by vaccination may be beneficial.

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.

T he four serotypes of dengue virus (DENV1 to -4) belong to the Flaviviridae family, which also includes West Nile virus (WNV), St. Louis encephalitis virus (SLEV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV). More than 2.5 billion people live in areas where DENV is endemic, and approximately 50 million people are infected each year (17). DENV generally manifests as an acute systemic infection that is cleared within 14 days (46). In most cases, infected individuals develop a febrile illness (dengue fever [DF]) characterized by flu-like symptoms, often accompanied by retro-orbital pain, ostealgia, and maculopapular rash. In nearly 500,000 cases per year, patients develop severe vascular leakage, which may result in hypovolemic shock (dengue hemorrhagic fever/dengue shock syndrome [DHF/ DSS]), ultimately leading to fatality rates as high as 10 to 15% in some countries or more than 25,000 deaths worldwide each year (15,17). In a subset of both DF and DHF/DSS cases (16), DENV infection was linked to encephalitis and signs of encephalopathy, including lethargy, confusion, seizure, and coma, as well as delayed neurological symptoms, such as paralysis of extremities, loss of sensation, and psychosis (2,21,23,30,36,38,43). DENVinduced neurological disease is being increasingly recognized as an important component of dengue disease in humans independent of DF and DHF/DSS (24). The prevalence of DENV-induced neurological disease has been estimated to be 4.2% of DENV cases (44) and as high as 18% of undiagnosed suspected viral central nervous system (CNS) infections in regions where DENV is endemic (21, 23).In the last decade, DENV has increasingly been linked to neurological disease in both the presence and absence of DF. Viruses of the Flaviviridae family generally cluster in a phylogenetic pattern that mirrors the typical disease manifestations they cause (13); for example, encephalitic flaviviruses cluster together. DENV, however, usually causes hemorrhagic illness but is genetically more homologous to the encephalitic viruses, including WNV, SLEV, and JEV, than it is to other hemorrhagic flaviviruses, such as YFV, based on several analyses of the nonstructural (NS) proteins NS3 and NS5 (11, 13). The close relationship of DENV to encephalitic flaviviruses may help explain the capacity of DENV to cause neurological disease and, possibly, infection of the CNS in humans (6, 37). Additionally, DENV-induced paralysis has been reported in numerous mouse models (4, 49), but only a few studies have investigated this phenomenon (1,18,20). Presently, whether DENV infects the CNS in natural human infections continues to be a subject of debate (25,30,32).Mice lacking the alpha/beta interferon and gamma interferon receptors (IFN-␣/␤R and IFN-␥R) in the 129/Sv genetic background (AG129) are highly susceptible to disease resembling human DHF/DSS, as well as paralysis, following infection with mouse-passaged variants of DENV2 clinical isolate PL046 (5,35,40,51). In contrast, congenic mice lacking only IFN-␣/␤R (A12...