Mycobacterium tuberculosis antigen diversity

描述（申请方提供）：结核分枝杆菌通过不完全表征的机制抵抗人体免疫反应的消除。确定M.结核分枝杆菌利用抗原变异逃避T细胞应答的消除，我们检验了M.结核病产生人T细胞识别的表位的逃逸突变体。我们比较了21株M.结核分枝杆菌的六个全球谱系。结核菌复合体，并令人惊讶地发现，468（95%）的已知表位在这些谱系中没有表现出序列变异，这些谱系代表了M.结核病的祖先在3万年前就分道扬镳了。为了确定所观察到的T细胞表位的高度保守性是否仅仅是M.结核分枝杆菌基因组中的表位的非同义和同义单核苷酸多态性（dN/dS）与实验确定的必需基因和非必需基因的dN/dS的比率进行比较，发现结核分枝杆菌中已知表位的dN/dS最低。结核病基因组已知的人类T细胞表位是高度保守的这一观察结果表明，细菌实际上受益于T细胞识别，但这也与先前在表位发现方面的努力倾向于发现保守表位的可能性一致。为了验证M.结核病，我们将测序180个不同基因的M。结核病，并确定最多样化的地区的M。结核病基因组然后，我们将使用广泛的计算机分析来鉴定预测编码人T细胞表位的那些可变序列。利用这组预测的表位，我们将测定T细胞对具有与受试者的感染分离物中的序列相匹配的序列的合成肽的应答，以鉴定哪些预测的表位是人类T细胞识别的真正靶标。由于我们预计，这些努力将导致一个大的可变表位的发现，我们描述了进一步的努力，以测试的假设，即在新发现的可变表位的序列变异是由于人类T细胞反应的选择。在超出本申请范围的研究中，我们将使用选定的新发现的可变表位来检验以下假设：人T细胞对可变表位的识别比对先前发现的超保守表位的识别与保护性免疫更密切相关。我们的研究很可能对结核病的人类免疫领域产生巨大的整体影响，因为它们将显着扩大我们对M中人类T细胞靶点的认识。它们将使目前不可能的新研究和发现成为可能，并且它们可能提供一种获得更有效的结核疫苗的途径。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis resists elimination by human immune responses through incompletely- characterized mechanisms. To determine whether M. tuberculosis uses antigenic variation to evade elimination by T cell responses, we tested the hypothesis that M. tuberculosis generates escape mutants of the epitopes recognized by human T cells. We compared the sequences of 491 experimentally-verified human T cell epitopes in 21 strains of M. tuberculosis from the six global lineages of the M. tuberculosis complex, and made the surprising discovery that 468 (95%) of the known epitopes exhibit no sequence variation across these lineages, which represent strains of M. tuberculosis whose ancestors diverged >30,000 years ago. To determine whether the observed hyperconservation of T cell epitopes was simply the effect of low sequence diversity in the M. tuberculosis genome, we compared the ratio of the rates of nonsynonymous and synonymous single- nucleotide polymorphisms (dN/dS) in the epitopes compared with that in the experimentally-determined essential and nonessential genes, and found that the dN/dS of the known epitopes is the lowest in the M. tuberculosis genome. The observation that the known human T cell epitopes are hyperconserved suggests that the bacteria actually benefit from T cell recognition, but it is also consistent with the possibility that prior efforts at epitope discovery were skewed toward discovery of conserved epitopes. To test the hypothesis that there are variable T cell epitopes of M. tuberculosis, we will sequence 180 phylogenetically-diverse strains of M. tuberculosis, and identify the most diverse regions of the M. tuberculosis genome. We will then use extensive in silico analyses to identify those variable sequences that are predicted to encode human T cell epitopes. With that set of predicted epitopes, we will assay T cell responses to synthetic peptides having sequences matching those in the subject's infecting isolate, to identify which of the predicted epitopes are true targets of human T cell recognition. Since we anticipate that these efforts will lead to the discovery of a large set of variable epitopes, we describe further efforts to test the hypothesis that sequence variations in the newly-discovered variable epitopes are due to selection by human T cell responses. In studies beyond the scope of this application, we will use selected newly-discovered variable epitopes to test the hypothesis that human T cell recognition of variable epitopes is more closely associated with protective immunity than is recognition of the previously-discovered hyperconserved epitopes. Our studies are likely to have a large overall impact on the field of human immunity to tuberculosis, as they will markedly expand our knowledge of targets of human T cells in M. tuberculosis, they will enable novel studies and discoveries that are not currently possible, and they are likely to provide a pathway to more efficacious TB vaccines.