Potential SLA Hp-4.0 haplotype-restricted CTL epitopes identified from the membrane protein of PRRSV induce cell...

Swine leukocyte antigen (SLA) class I molecule-restricted T-cell epitopes, which induce cytotoxic T lymphocyte (CTL) responses, play a critical role in the clearance of porcine reproductive and respiratory syndrome virus (PRRSV) and the development of efficient protective vaccines. The SLA-1*04:01:01, SLA-2*04:01, and SLA-3*04:01 alleles, assigned the Hp-4.0 haplotype, are highly prevalent and usually present in all pig breeds. However, the SLA Hp-4.0 haplotype-restricted CTL epitopes in the structural membrane (M) protein of PRRSV are still unknown. In this study, we predicted 27 possible 9-mer epitope peptides in M protein with high binding scores for SLA-1*04:01:01 using CTL epitope prediction tools. In total, 45 SLA class I complexes, comprising the predicted peptide, extracellular region of the SLA-I molecules, and β2-microglobulin, were constructed in vitro to detect the specific binding of these peptides to SLA-1*04:01:01 (27 complexes), SLA-2*04:01 (9 complexes), and SLA-3*04:01 (9 complexes), respectively. Our results showed that the M27 (T₉₁WKFITSRC), M39 (N₁₃₀HAFVVRRP), and M49 (G₁₅₈RKAVKQGV) peptides bind specifically to SLA-1*04:01:01, SLA-2*04:01, and SLA-3*04:01, respectively. Subsequently, using peripheral blood mononuclear cells (PBMCs) isolated from the homozygous Hp-4.0 and Hp-26.0 haplotype piglets vaccinated with commercial PRRSV HuN4-F112 strain, we determined the capacities of these 27 potential peptides to stimulate their proliferation with a Cell Counting Kit-8 and their secretion and expression of interferon gamma (IFN-γ) with an ELISpot assay and real-time qPCR, respectively. The immunological activities of M27, M39, and M49 were therefore confirmed when they efficiently induced PBMC proliferation and IFN-γ secretion in PBMCs from piglets with the prevalent SLA Hp-4.0 haplotype. The amino acid sequence alignment revealed that M27, M39, and M49 are highly conserved among 248 genotype II PRRSV strains collected between 1998 and 2019. These findings contribute to the understanding of the mechanisms of cell-mediated immune responses to PRRSV. Our study also provides a novel strategy for identifying and confirming potential SLA haplotype-restricted CTL epitopes that could be used to develop novel peptide-based vaccines against swine diseases.