Mycobacterium tuberculosis antigen diversity

描述(申请人提供)：结核分枝杆菌通过不完全特征化的机制抵抗人类免疫反应的消除。为了确定结核分枝杆菌是否使用抗原变异来逃避T细胞反应的消除，我们测试了结核分枝杆菌产生人类T细胞识别表位的逃逸突变的假设。我们比较了21株结核分枝杆菌的491个经实验验证的人类T细胞表位的序列，这些菌株来自结核分枝杆菌复合体的六个全球谱系，并发现468个已知表位(95%)在这些谱系中没有显示出序列变化，这些谱系代表的是结核分枝杆菌的菌株，其祖先在30,000年前发生了分歧。为了确定观察到的T细胞表位的高度保守性是否仅仅是结核分枝杆菌基因组低序列多样性的影响，我们比较了T细胞表位中非同义和同义单核苷酸多态(Dn/DS)的比率与实验确定的必要和非必要基因中的比率，发现已知表位的Dn/DS是结核分枝杆菌基因组中最低的。已知的人类T细胞表位高度保守的观察表明，细菌实际上受益于T细胞识别，但这也与之前发现表位的努力偏向于发现保守表位的可能性一致。为了验证结核分枝杆菌存在可变T细胞表位的假设，我们将对180株系统发育不同的结核分枝杆菌菌株进行测序，并确定结核分枝杆菌基因组中最多样化的区域。然后，我们将使用广泛的计算机分析来识别那些被预测为编码人类T细胞表位的变量序列。有了这组预测的表位，我们将测试T细胞对合成肽的反应，这些合成肽的序列与受试者的感染分离株中的序列匹配，以确定哪些预测的表位是人类T细胞识别的真正目标。由于我们预计这些努力将导致大量可变表位的发现，我们描述了进一步的努力来检验新发现的可变表位的序列变化是由于人类T细胞反应的选择这一假设。在超出这一应用范围的研究中，我们将使用选定的新发现的可变表位来检验这一假设，即人类T细胞识别可变表位与保护性免疫的关系比识别先前发现的高度保守的表位更密切。我们的研究可能会对人类对结核病的免疫领域产生巨大的整体影响，因为它们将显著扩大我们对结核分枝杆菌人类T细胞靶标的了解，它们将使目前无法实现的新研究和发现成为可能，它们可能提供一条获得更有效的结核病疫苗的途径。

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.