Conserved amino acids 193-324 of non-structural protein 3 are a dominant source of peptide determinants for CD4+ and CD8+ T cells in a healthy Japanese encephalitis virus-endemic cohort.

Our earlier identification of the non-structural protein 3 (NS3) of Japanese encephalitis virus (JEV) as a dominant CD4+ as well as CD8+ T cell-eliciting antigen in a healthy JEV-endemic cohort with a wide HLA distribution implied the presence of several epitopes dispersed over the length of the protein. Use of various truncated versions of NS3 in lymphocyte stimulation and interferon (IFN)-gamma secretion assays revealed that amino acids (aa) 193-324 of NS3 were comparable with, if not superior to, the full-length protein in evoking Th1 responses. The potential of this 14.4 kDa stretch to stimulate IFN-gamma production from both subtypes of T cells in a manner qualitatively and quantitatively similar to the 68 kDa parent protein suggested the presence within it of both class I and II epitopes and demonstrated that the entire immunogenicity of NS3 was focused on aa 193-324. Interestingly, this segment contained five of the eight helicase motifs of NS3. Analysis of variability of the NS3 protein sequence across 16 JEV isolates revealed complete identity of aa 219-318, which is contained within the above segment, suggesting that NS3-specific epitopes tend to cluster in relatively conserved regions that harbour functionally critical domains of the protein.