Identification of human T-cell epitopes on Mycobacterium tuberculosis small molecular weight proteins and development of DNA vaccines based on the epitopes

CD8~+细胞毒性T淋巴细胞(CTL)在抗结核分枝杆菌感染中起着关键作用。我们在HLAA^0201转基因HHD小鼠中发现了一个新的结核分枝杆菌保护性抗原MPT51的CTL表位。用基因枪法将编码MPT51基因的DNA免疫HHD小鼠，检测免疫小鼠脾细胞产生干扰素-γ的情况。对于合成的覆盖成熟MPT51序列的重叠多肽文库，只有一种多肽p51-70刺激脾细胞产生干扰素-γ。细胞表面CD4和CD8染色的三色流式细胞术分析表明，p51-70肽含有一个免疫优势的CD8~+T细胞表位。使用计算机辅助算法的进一步分析允许识别出真正的T细胞表位P53-62。用T2细胞进行MHC-I类稳定实验证实，该表位由人类白细胞抗原A^*0201呈递。观察到MPT51、P53-62/HLA0201四聚体能与免疫HHD脾细胞的CD8+T细胞结合。此外，在结核菌素皮肤试验阳性的正常人中还发现了MPT51、P53-62特异的记忆性CD8^+T细胞。该HLAA^0201限制性CD8+T细胞表位可用于分析MPT51特异性T细胞在保护性免疫中的作用，并用于未来抗结核分枝杆菌感染的疫苗设计。

CD8^+ cytotoxic T lymphocytes (CTL) play a pivotal role in protection against Mycobacterium tuberculosis infection. We identified a novel CTL epitope on a protective antigen of M. tuberculosis, MPT51, in HLA-A^*0201-transgenic HHD mice. HHD mice were immunized with plasmid DNA encoding MPT51 using gene gun and interferon (IFN)-y production from the immune splenocytes was analyzed. In response to synthetic overlapping peptide library covering the mature MPT51 sequence, only one peptide, p51-70, stimulated the splenocytes to produce IFN-y. Three-color flow cytometric analysis of cell-surface CD4 and CD8 staining revealed that p51-70 peptide contains an immunodominant CD8^+ T-cell epitope. Further analysis using computer-assisted algorithms permitted identification of a bona fide T-cell epitope, p53-62. MHC class I stabilization assay using T2 cells confirmed that the epitope is presented by HLA-A^*0201. MPT51 p53-62/HLA-A^*0201 tetramer was observed to bind to the specific CD8^+ T cells of immune HHD splenocytes. In addition, MPT51 p53-62-specific memory CD8^+ T cells were found in tuberculin skin test-positive HLA-A^*0201^+ healthy individuals. This HLA-A^*0201-restricted CD8^+ T-cell epitope would be feasible for analysis of the role of MPT51-specific T cells in protective immunity and for future vaccine design against M. tuberculosis infection.