Most of the research performed on human T-cell cytotoxic responses to dengue viruses has been in Caucasian volunteers who received experimental live monovalent dengue virus vaccines. These individuals had no known prior exposure to dengue viruses and therefore received the dengue virus vaccine as a primary infection. However, the complications seen in dengue virus infection (DHF and DSS) are more common in patients who have preexisting immunity to one serotype of virus during infection with another serotype (secondary infection) of virus (8
). Virus-specific memory T cells have been implicated in contributing to the pathogenesis of severe dengue virus infection (16
). Analysis of T-cell responses in patients after natural secondary infections is therefore important because it may provide insights into the mechanisms of T-cell-mediated immunopathology. In this study, memory CTL responses were detected against dengue virus proteins in the PBMC of all four patients examined. This is the first report of dengue virus-specific CTLs after natural secondary dengue virus infections.
We have previously shown that nonstructural proteins, in particular NS3, are predominant targets for both CD4+
CTLs in vaccine recipients (17
). In the present study, we also found that three of four donors had CTL responses to NS3 and that the fourth donor had CTLs which recognized the nonstructural proteins NS1.2a. Proteins with a cytoplasmic intracellular localization have been noted to be the predominant source of cytotoxic T-cell antigenic peptides (18
). The NS3 protein constitutes approximately 25% of the cytoplasmic region of the polyprotein, which may explain why it is an immunodominant target for CTL recognition. Numerous other factors (rate of proteolysis, efficiency of transport, peptide stability, etc.) are also likely to influence why peptides from nonstructural proteins are preferentially presented on major histocompatibility complex molecules (1
). Since we have not tested vaccinia virus recombinants expressing NS5, it is possible that there are also epitopes on NS5.
Immune responsiveness is affected by the major histocompatibility complex, and the pathogenesis of some diseases has been associated with specific HLA alleles. In areas where DHF is endemic, only a small percentage of individuals exhibit severe disease, which suggests that host genetic factors may play a role in susceptibility to severe disease. One study found a positive association between HLA A2 and B blank and the development of DSS and a negative relationship for HLA B13 (4
Since it is known that there is diversity in both HLA and non-HLA gene loci between Southeast Asians and Caucasians (3
), it was important to analyze the recognition of dengue virus proteins by T cells in the context of HLA molecules in Thai patients. We previously isolated NS3-specific CD4+
CTL clones from Caucasian volunteers which were restricted by the HLA alleles B35 (17
), DR15 (12
), and DPw2 (14
). The NS1.2a-specific CTLs from patient KPP94-037 in the present study were B57 restricted and DR7 restricted; the NS3-specific CTLs from patient KPP94-024 were B7 restricted, and the NS3-specific CTLs from patient CHD94-020 were A11.1 restricted. These results indicate that despite differences in HLA alleles between Thais and Caucasians, peptides from dengue virus nonstructural proteins are also predominantly recognized by T cells from Thai children. These studies confirm our previous results of the dominance of nonstructural proteins as CTL targets in Caucasian dengue virus vaccine recipients. All of the T-cell lines isolated from patient KPP94-024 recognized autologous targets pulsed with aa 221 to 235 of the NS3 protein. The 15-mer which is lysed by all the B7 restricted CD8+
CTLs from this patient contains a proline at position 223. These results are entirely consistent with reports of antigenic peptides bearing a proline at position 2 and aromatic or hydrophobic residues at the C terminus, preferentially binding to HLA B7 and related class I alleles sharing the B7-like supertype (21
Our earlier work analyzing primary responses from PBMC of vaccine recipients, detected both serotype-specific and serotype-cross-reactive CTL responses against many proteins in individual donors (11
). We speculated that the serotype-cross-reactive memory T cells generated during the primary infection would be reactivated in a secondary infection and contribute to the immunopathology of DHF which is observed much more frequently in secondary infections. In this study, we have examined CTL responses after secondary infection in four Thai patients. All the NS1.2a-specific CTL lines generated from patient KPP94-037 and the NS3-specific CTLs from patient KPP94-024 in this study were cross-reactive with the other serotypes of dengue virus. The bulk culture CTLs from patient CHD94-020 were also cross-reactive with D2 and D3 virus NS3. We have not yet analyzed the primary CTL responses in Thai children; therefore, we cannot conclusively define the relationship between primary and secondary dengue virus infections. However, these results suggest that the CTL responses in secondary infections are predominantly due to reactivation of memory cross-reactive CTL from the primary infections, consistent with our hypothesis.
In conclusion, the CTL responses of Thai patients to natural secondary dengue virus infection are directed against nonstructural proteins and are mainly cross-reactive in nature.