A novel strategy for the identification of antigens that are recognised by bovine MHC class I restricted cytotoxic T cells in a protozoan infection using reverse vaccinology.
A novel strategy for the identification of antigens that are recognised by bovine MHC class I restricted cytotoxic T cells in a protozoan infection using reverse vaccinology.
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DOI：
10.1186/1745-7580-3-2
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发表时间：
2007-02-09
期刊：
影响因子：
--
通讯作者：
Taracha EL
中科院分区：
文献类型：
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作者：
Graham SP;Honda Y;Pellé R;Mwangi DM;Glew EJ;de Villiers EP;Shah T;Bishop R;van der Bruggen P;Nene V;Taracha EL
此前已证明，针对牛原生动物寄生虫的免疫是通过 I 类 MHC 限制性 CD8+ 细胞毒性 T 淋巴细胞裂解寄生虫感染的细胞来介导的。据推测，CTL 靶裂殖体抗原的鉴定将有助于亚单位疫苗的开发。我们利用完整基因组序列数据和生物信息学工具来识别编码分泌蛋白或膜锚定蛋白的基因，这些蛋白可能被感染细胞的 MHC I 类分子加工并呈递给 CTL。在由 T. parva 基因组 1 号染色体编码的 986 个预测开放阅读框 (ORF) 中，根据信号肽和/或跨膜螺旋结构域的存在选择了 55 个。 36 个选定的 ORF 成功克隆到真核表达载体中，瞬时转染到永生化牛皮肤成纤维细胞中，并使用 T. parva 特异性 CTL 进行体外筛选。使用 IFN-γ ELISpot 测定评估 CTL 对基因产物的识别。通过来自 4 只动物的 T. parva 特异性 CTL 鉴定出编码 174 个氨基酸蛋白质（称为 Tp2）的 525 个碱基对 ORF。这些 CTL 识别并裂解 Tp2 转染的皮肤成纤维细胞，并识别 4 个不同的表位。值得注意的是，在针对子孢子攻击的保护性免疫反应过程中观察到了 Tp2 特异性 CD8+ T 细胞反应。含有多个 CTL 表位的抗原的鉴定及其在保护性抗寄生虫反应期间的明显免疫优势使得 Tp2 成为评估其疫苗潜力的有吸引力的候选者。
Immunity against the bovine protozoan parasite Theileria parva has previously been shown to be mediated through lysis of parasite-infected cells by MHC class I restricted CD8+ cytotoxic T lymphocytes. It is hypothesized that identification of CTL target schizont antigens will aid the development of a sub-unit vaccine. We exploited the availability of the complete genome sequence data and bioinformatics tools to identify genes encoding secreted or membrane anchored proteins that may be processed and presented by the MHC class I molecules of infected cells to CTL. Of the 986 predicted open reading frames (ORFs) encoded by chromosome 1 of the T. parva genome, 55 were selected based on the presence of a signal peptide and/or a transmembrane helix domain. Thirty six selected ORFs were successfully cloned into a eukaryotic expression vector, transiently transfected into immortalized bovine skin fibroblasts and screened in vitro using T. parva-specific CTL. Recognition of gene products by CTL was assessed using an IFN-γ ELISpot assay. A 525 base pair ORF encoding a 174 amino acid protein, designated Tp2, was identified by T. parva-specific CTL from 4 animals. These CTL recognized and lysed Tp2 transfected skin fibroblasts and recognized 4 distinct epitopes. Significantly, Tp2 specific CD8+ T cell responses were observed during the protective immune response against sporozoite challenge. The identification of an antigen containing multiple CTL epitopes and its apparent immunodominance during a protective anti-parasite response makes Tp2 an attractive candidate for evaluation of its vaccine potential.