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Abstract：
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.
摘要：
猪白细胞抗原（SLA）I类分子限制性T细胞表位，诱导细胞毒性T淋巴细胞（CTL）反应，在猪繁殖与呼吸综合征病毒(PRRSV)的清除和高效保护性疫苗的开发中发挥关键作用。SLA-1*04:01:01、SLA-2*04:01和SLA-3*04:01等位基因，分配了Hp-4.0单倍型，非常普遍，通常存在于所有猪品种中。然而,PRRSV结构膜（M）蛋白中SLAHp-4.0单倍型限制性CTL表位仍未知。在这项研究中,我们使用CTL表位预测工具预测了M蛋白中27种可能的9聚体表位肽，其SLA-1*04:01:01的结合得分较高。总的来说,45SLAI类配合物，包含预测的肽，SLA-I分子的胞外区，和β2-微球蛋白，在体外构建以检测这些肽与SLA-1*04:01:01(27个复合物)的特异性结合，SLA-2*04:01（9个配合物），和SLA-3*04:01(9个复合物)，分别。我们的结果表明，M27(T91WKFITSRC)，M39(N130HAFVVRRP),和M49(G158RKAVKQGV)肽分别与SLA-1*04:01、SLA-2*04:01和SLA-3*04:01特异性结合。随后，使用从接种商业PRRSVHuN4-F112菌株的纯合Hp-4.0和Hp-26.0单倍型仔猪中分离的外周血单核细胞（PBMC），我们用细胞计数试剂盒-8确定了这27种潜在肽刺激其增殖的能力，并通过ELISpot测定和实时qPCR确定了干扰素γ（IFN-γ）的分泌和表达。分别。因此，当M27，M39和M49有效诱导具有普遍的SLAHp-4.0单倍型仔猪的PBMC增殖和IFN-γ分泌时，它们的免疫活性得到了证实。氨基酸序列比对显示，在1998年至2019年收集的248株基因型II型PRRSV中，M27，M39和M49高度保守。这些发现有助于理解细胞介导的对PRRSV的免疫应答的机制。我们的研究还提供了一种新的策略，用于鉴定和确认潜在的SLA单倍型限制性CTL表位，可用于开发针对猪疾病的新型基于肽的疫苗。
