School of Life Sciences, Zhengzhou University, Zhengzhou, 450001, Henan, China.
School of Life Sciences, Zhengzhou University, Zhengzhou, 450001, Henan, China.
Vet Microbiol. 2021 Aug;259:109131. doi: 10.1016/j.vetmic.2021.109131. Epub 2021 May 26.
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 M and M peptides each formed complexes with SLA-20502 and sβ2m, while M formed a complex with SLA-21201 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.
CD8+ 细胞毒性 T 淋巴细胞（CTLs）对于清除猪繁殖与呼吸综合征病毒（PRRSV）感染和调节宿主免疫反应至关重要。鉴定 SLA I 限制性 CD8+ CTL 表位将有助于 PRRSV 疫苗的开发。本研究通过 ELISPOT assay 从 PRRSV 免疫的大白猪（JXA1-R 株）外周血单核细胞（PBMCs）中分离的细胞中筛选出免疫优势 PRRSV-2 M 蛋白 T 细胞表位。在检测到的九个免疫优势表位中，有八个表位能引起个体猪之间和表位之间存在显著的 IFN-γ 分泌反应。为了预测哪些表位含有潜在的 CTL 表位，克隆和测序了大白猪的猪白细胞抗原（SLA）I 类基因，得到了 14 个不同的 SLA I 类基因序列。基于 ELISPOT 和 SLA 基因分型结果，预测了这 14 个预测的 I 类蛋白与从八个免疫优势表位衍生的肽段的 SLA 限制结合，并进行了计算机模拟分析。在评估了包含预测受限肽的 42 个 pET-肽-SLA-I-β2m 复合物后，通过 ELISA 检测了 33 个肽-SLA-I-β2m 复合物，检测到 4 个复合肽。通过体外复合重折叠实验评估了这四个肽，确认 M 和 M 肽分别与 SLA-20502 和 sβ2m 形成复合物，而 M 肽与 SLA-21201 和 sβ2m 形成复合物。ELISPOT 结果证实，这三个 9 肽潜在 CTL 表位在由这里指定的 SLA I 类分子呈递时能有效刺激 IFN-γ 分泌。本研究描述了用于未来研究猪细胞免疫的有效 CTL 表位鉴定方法，以开发基于 T 细胞的疫苗。
