Analysis of Murine CD8+ T-Cell Clones Specific for the Dengue Virus NS3 Protein: Flavivirus Cross-Reactivity and Influence of Infecting Serotype

The family Flaviviridae consists of over 65 arthropod-transmitted viruses that are known to infect humans. The dengue viruses, of which there are four serotypes, are the flaviviruses of greatest global health importance, causing an estimated 100 million infections each year (10, 28). Dengue hemorrhagic fever (DHF), the severe clinical form of dengue virus infection, has been shown to occur more frequently in individuals who have experienced a previous infection with a different dengue virus serotype (1, 37, 40), suggesting an immunopathologic basis for severe dengue illness. Members of our laboratory have been studying memory dengue virus-specific cytotoxic T-lymphocyte (CTL) responses in order to understand the potential protective and immunopathologic effects of these CTLs during secondary dengue virus infections. CD4+ and CD8+ CTL responses induced by primary dengue virus infection have been characterized with bulk cultures and at the clonal level in a small number of adult human volunteers who were infected under experimental conditions with live tissue culture-passaged dengue viruses (8, 17, 18, 23, 27). Both serotype-specific and serotype-cross-reactive CTLs have been isolated from most subjects. Most serotype-cross-reactive CTLs have been directed at epitopes on nonstructural proteins, particularly the NS1-NS2A and NS3 proteins. Several different patterns of cross-reactivity have been observed; some CTL clones have recognized several but not all dengue virus serotypes, other clones have recognized all four dengue virus serotypes, and other clones have also recognized closely related flaviviruses, such as West Nile virus (WNV) (14). The frequencies of different CTL specificities have varied among the subjects studied; however, it has not been possible to relate these differences to host factors, such as HLA type, or differences in the viruses causing primary infections. We previously reported that T-lymphocyte responses to primary dengue virus infection in mice are qualitatively similar to human T-lymphocyte responses to dengue virus in bulk cultures and at the clonal level (34–36). Both serotype-specific and serotype-cross-reactive dengue virus-specific CTL clones were isolated from BALB/c mice, and the serotype-cross-reactive clones recognized epitopes on the NS1-NS2A and NS3 proteins. One CD8+ CTL epitope, recognized in the context of H-2Kd, was mapped to amino acids 296 to 310 of the dengue type 2 virus NS3 protein (34). This epitope is completely conserved in dengue type 4 virus. To define further the potential importance of these memory dengue virus-specific CD8+ CTLs in secondary dengue virus infection, we analyzed in more detail the specificity of the NS3-specific CTL clones. As reported here, we noted that all four serotypes of dengue viruses as well as closely related flaviviruses showed a high degree of amino acid homology at the epitope mentioned above. Specific single amino acid differences in this epitope had different effects on recognition by different CTL clones, accounting for both dengue virus subcomplex-specific and dengue virus complex-cross-reactive patterns of responses. We also explored the effects of the infecting virus on the patterns of CTL responses generated.