Abstract
Vaccination with live attenuated classical swine fever virus (CSFV) vaccines induces a rapid onset of protection which has been associated with virus-specific CD8 T cell IFN-γ responses. In this study, we assessed the specificity of this response, by screening a peptide library spanning the CSFV C-strain vaccine polyprotein to identify and characterise CD8 T cell epitopes. Synthetic peptides were pooled to represent each of the 12 CSFV proteins and used to stimulate PBMC from four pigs rendered immune to CSFV by C-strain vaccination and subsequently challenged with the virulent Brescia strain. Significant IFN-γ expression by CD8 T cells, assessed by flow cytometry, was induced by peptide pools representing the core, E2, NS2, NS3 and NS5A proteins. Dissection of these antigenic peptide pools indicated that, in each instance, a single discrete antigenic peptide or pair of overlapping peptides was responsible for the IFN-γ induction. Screening and titration of antigenic peptides or truncated derivatives identified the following antigenic regions: core&sub2;&sub4;&sub1;₋&sub2;₅₅ PESRKKLEKALLAWA and NS3&sub1;₉&sub0;&sub2;₋&sub1;₉&sub1;&sub2; VEYSFIFLDEY, or minimal length antigenic peptides: E2₉₉₆₋&sub1;&sub0;&sub0;&sub3; YEPRDSYF, NS2&sub1;&sub2;&sub2;&sub3;₋&sub1;&sub2;&sub3;&sub0; STVTGIFL and NS5A&sub3;&sub0;₇&sub0;₋&sub3;&sub0;₇₈ RVDNALLKF. The epitopes are highly conserved across CSFV strains and variable sequence divergence was observed with related pestiviruses. Characterisation of epitope-specific CD8 T cells revealed evidence of cytotoxicity, as determined by CD107a mobilisation, and a significant proportion expressed TNF-α in addition to IFN-γ. Finally, the variability in the antigen-specificity of these immunodominant CD8 T cell responses was confirmed to be associated with expression of distinct MHC class I haplotypes. Moreover, recognition of NS&sub1;&sub2;&sub2;&sub3;₋&sub1;&sub2;&sub3;&sub0; STVTGIFL and NS3&sub1;₉&sub0;&sub2;₋&sub1;₉&sub1;&sub2; VEYSFIFLDEY by a larger group of C-strain vaccinated animals showed that these peptides could be restricted by additional haplotypes. Thus the antigenic regions and epitopes identified represent attractive targets for evaluation of their vaccine potential against CSFV.