Identification of potential SLA-I-restricted CTL epitopes within the M protein of porcine reproductive and respiratory syndrome virus

CD8+ cytotoxic T lymphocytes (CTLs), are essential for clearance of porcine reproductive and respiratory syndrome virus (PRRSV) infection and regulation of host immune responses. Identification of SLA I-restricted CD8+ CTL epitopes would facilitate PRRSV vaccine development. Here, cells isolated from peripheral blood mononuclear cells (PBMCs) of PRRSV-immunized Large White pigs (JXA1-R strain) were screened for immunodominant PRRSV-2 M protein T cell epitopes via ELISPOT assay. Of nine immunodominant epitopes detected, eight elicited significant IFN-γ secretion responses that varied among individual pigs and according to epitope. To predict which epitopes harbored potential CTL epitopes, swine leukocyte antigen (SLA) class I genes of Large White pigs were cloned and sequenced, yielding fourteen distinct SLA class I gene sequences. Based on ELISPOT and SLA genotyping results, SLA-restricted binding of the fourteen predicted class I proteins to peptides derived from the eight immunodominant epitopes were predicted in-silico. After evaluation of 42 pET-peptide-SLA-I-β2m complexes containing predicted restricted peptides, extracellular SLA class I domains and β2m, ELISA testing of 33 peptide-SLA-I-β2m complexes detected four complexed peptides. These four peptides were evaluated using in vitro complex refolding assays that confirmed that M2-5 and M6-1 peptides each formed complexes with SLA-2*0502 and sβ2m, while M9-1 formed a complex with SLA-2*1201 and sβ2m. ELISPOT results confirmed these three 9-mer potential CTL epitopes efficiently stimulated IFN-γ secretion when presented by SLA class I molecules specified here. This study describes effective CTL epitope identification methods for use in future investigations of swine cellular immunity toward T cell-based vaccine development.