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

Tingyu Luo, Chang Xin, Hongyi Liu, Changwen Li, Hongyan Chen, Changyou Xia, Caixia Gao
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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.
从 PRRSV 膜蛋白中发现的潜在 SLA Hp-4.0 单倍型限制 CTL 表位可诱导细胞免疫反应
猪白细胞抗原(SLA)I类分子限制性T细胞表位可诱导细胞毒性T淋巴细胞(CTL)反应,在清除猪繁殖与呼吸综合征病毒(PRRSV)和开发高效保护性疫苗方面发挥着关键作用。SLA-1*04:01:01、SLA-2*04:01 和 SLA-3*04:01 等位基因被分配为 Hp-4.0 单倍型,它们在所有猪种中都非常普遍,而且通常都存在。然而,PRRSV 结构膜(M)蛋白中的 SLA Hp-4.0 单倍型限制性 CTL 表位仍然未知。在本研究中,我们使用 CTL 表位预测工具预测了 M 蛋白中可能存在的 27 个与 SLA-1*04:01:01 结合得分较高的 9 聚体表位肽。我们在体外共构建了45个SLA I类复合物,包括预测的多肽、SLA-I分子的胞外区域和β2-微球蛋白,分别检测了这些多肽与SLA-1*04:01:01(27个复合物)、SLA-2*04:01(9个复合物)和SLA-3*04:01(9个复合物)的特异性结合。结果显示,M27(T91WKFITSRC)、M39(N130HAFVVRRP)和 M49(G158RKAVKQGV)肽分别与 SLA-1*04:01:01、SLA-2*04:01 和 SLA-3*04:01 特异性结合。随后,我们利用从接种了商品化 PRRSV HuN4-F112 株疫苗的同型 Hp-4.0 和 Hp-26.0 单倍型仔猪体内分离的外周血单核细胞(PBMCs),用细胞计数试剂盒-8 测定了这 27 种潜在多肽刺激其增殖的能力,并用 ELISpot 法和实时 qPCR 法分别测定了它们分泌和表达γ干扰素(IFN-γ)的能力。因此,当 M27、M39 和 M49 有效诱导 SLA Hp-4.0 单倍型仔猪 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,M27、M39 和 M49 在 1998 年至 2019 年收集的 248 株基因型 II PRRSV 株系中高度保守。这些发现有助于了解 PRRSV 的细胞介导免疫应答机制。我们的研究还为鉴定和确认潜在的 SLA 单倍型限制性 CTL 表位提供了一种新策略,这些表位可用于开发新型的基于肽的猪病疫苗。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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