Mycobacterium tuberculosis antigen diversity

描述（由申请人提供）：结核分枝杆菌通过不完全表征的机制抵抗人体免疫应答的消除。为了确定结核分枝杆菌是否使用抗原变异来逃避T细胞应答的消除，我们验证了结核分枝杆菌产生人类T细胞识别的表位的逃逸突变的假设。我们比较了来自结核分枝杆菌复合体6个全球谱系的21株结核分枝杆菌中491个经实验验证的人类T细胞表位的序列，并令人惊讶地发现，468个（95%）已知表位在这些谱系中没有序列变异，这代表了祖先在3万年前分化的结核分枝杆菌菌株。为了确定观察到的T细胞表位的超保守是否仅仅是结核杆菌基因组序列多样性低的影响，我们将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比率进行了比较，发现已知表位的dN/dS在结核杆菌基因组中最低。观察到已知的人类T细胞表位是超保守的，这表明细菌实际上受益于T细胞识别，但这也与先前发现表位的努力倾向于发现保守的表位的可能性一致。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预测编码人类T细胞表位的可变序列。有了这组预测的表位，我们将检测T细胞对合成肽的反应，这些合成肽的序列与受试者感染分离物中的序列相匹配，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测试新发现的可变表位的序列变化是由于人类T细胞反应的选择的假设。在超出本应用范围的研究中，我们将使用选择的新发现的可变表位来验证人类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的假设。我们的研究可能会对人类结核病免疫领域产生巨大的整体影响，因为它们将显著扩展我们对结核分枝杆菌中人类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，并且它们可能为更有效的结核病疫苗提供途径。

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. PUBLIC HEALTH RELEVANCE: Tuberculosis afflicts nearly 1/3 of all humans, and kills nearly 2 million people every year. The work proposed in this project will reveal crucial new information on how the bacteria that cause tuberculosis interact with the human immune system. The findings will have an important impact on development of new vaccines against tuberculosis.