Construction of safe and effective live tuberculosis vaccines

描述(由申请人提供)：这项提案试图更详细地了解结核分枝杆菌阻止有效宿主免疫启动的机制，最终目标是利用这些信息创造更有效的活疫苗株。以前的工作发现了多个分枝杆菌基因参与阻止受感染宿主细胞的凋亡，这与病原体阻止MHC I类提呈其抗原的能力密切相关。广泛的前期工作还发现了结核分枝杆菌中干扰MHC II类抗原提呈的基因，以及阻止关键细胞因子产生的基因。这一建议的一个主要目标是构建安全的结核分枝杆菌减毒株，其中特定的免疫逃避基因已被删除，从而创造出更有效的疫苗来启动抗分枝杆菌免疫。该方法包括从一组已经部分确定特征的四个候选基因中识别最有效的抗凋亡基因，并将这些基因的突变与强烈减弱营养缺乏症的突变相结合，以消除毒力。目标基因的精确突变将通过特殊的转导噬菌体介导的等位基因交换来创建，这种方法在PI及其合作者的实验室中得到了很好的确立。通过在干扰MHC II类递呈的基因中加入额外的突变，或者干扰IL-12p70或肿瘤坏死因子的产生，候选疫苗株的效力将进一步增强。候选疫苗株对CD4和CD8 T细胞的免疫学研究将在小鼠模型中进行，从而确定特定基因缺失的最有利组合，以推进啮齿动物的临床前疫苗研究。候选疫苗株诱导稳定的T细胞记忆的研究将在小鼠身上进行，分支杆菌蛋白增强初级反应的影响也将启动。总体而言，拟议的研究将极大地促进我们对结核病宿主-病原体相互作用的理解，并直接有助于研制预防和控制这一重大人类疾病的疫苗。

DESCRIPTION (provided by applicant): This proposal seeks to understand in greater detail the mechanisms by which Mycobacterium tuberculosis blocks the priming of effective host immunity, with the ultimate goal of using this information to create more effective live vaccine strains. Previous work identified multiple mycobacterial genes involved in blocking apoptosis of infected host cells, which is intimately linked to the ability of the pathogen to prevent presentation of its antigens by MHC class I. Extensive preliminary work has also identified genes in M. tuberculosis that interfere with MHC class II antigen presentation, and genes that block production of key cytokines. A major goal of this proposal is to construct safely attenuated strains of M. tuberculosis in which specific immune evasion genes have been deleted, thus creating more effective vaccines for priming of anti- mycobacterial immunity. The approach involves identifying the most potent anti- apoptotic genes from a group of four candidates that have already been partially characterized, and combining mutations in these with strongly attenuating auxotrophy mutations to eliminate virulence. Precise mutations in the genes of interest will be created using allelic exchange mediated by specialized transducing phages, a methodology that is well established in the laboratory of the PI and his collaborators. The potency of candidate vaccine strains will be further enhanced by incorporating additional mutations in genes that interfere with MHC class II presentation, or with production of IL-12p70 or TNF. Immunological studies of CD4 and CD8 T cell priming by candidate vaccine strains will be carried out in mouse models, allowing identification of the most favorable combinations of specific gene deletions to advance into preclinical vaccination studies in rodents. Studies of the induction of stable T cell memory by candidate vaccine strains will be performed in mice, and the impact of boosting primary responses with mycobacterial proteins will also be initiated. Overall, the proposed studies will significantly advance our understanding of the host-pathogen interaction in tuberculosis, and contribute directly to vaccine development for prevention and control of this major human disease.