Identification of H-2K(b)-restricted T-cell epitopes within the nucleocapsid protein of Hantaan virus and establishment of cytotoxic T-cell clones.

Although neutralizing antibodies against Hantaan virus (HTV) can protect hosts from viral infection, T-cell responses to HTV are also important in host defense against HTV. However, much less is known about cytotoxic T lymphocyte (CTL) responses to HTV. To identify CTL epitopes in the HTV nucleocapsid protein (NP), we selected 7 H-2K(b)-motif-fitting peptides. Of these peptides, 3 peptides (NP3, NP4, and NP7) were recognized by CTL responses derived from HTV-immunized mouse splenocytes. NP3 and NP4 peptides were also recognized by HTV-immunized splenocytes after secondary in vitro stimulation with the relevant peptide, but NP7 could not be recognized after in vitro stimulation. These results agree well with peptide immunization studies showing that peptide-specific CTL responses could be induced with NP3 and NP4 but not with NP7 peptide. Furthermore, CTL activity assay using targets, prepared to express the antigen (NP) endogenously, demonstrated that NP3 and NP4 peptides could be presented endogenously. CTL elicited with NP4 peptide retained some cross-reactivity and was difficult to long-term culture. However, NP3-elicited CTL was very specific for NP3 peptide and was stable enough to be cloned. Among many CTL lines elicited with HTV or HTV NP peptides, 6 NP3-specific CTL clones were established and have been maintained more than 2 years. All 6 CTL clones were characterized to be CD3+, CD4-, CD8+, CD25+, CD62L-, and NK1.1-, and to use TCR Vbeta6. This preferential usage of TCR Vbeta6 indicates that TCR Vbeta6 regions are important for recognition of the HTV NP3 epitope (NP221-228, SVIGFLAL) on H-2K(b) molecule. Our data demonstrate the definition of mouse CTL epitopes in HTV and the generation of HTV-specific mouse CTL clones.