Construction of safe and effective live tuberculosis vaccines

描述（由申请人提供）：本提案旨在更详细地了解结核分枝杆菌阻断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菌株。先前的研究发现了多个参与阻断受感染宿主细胞凋亡的分枝杆菌基因，这与病原体阻止MHC i类抗原呈递的能力密切相关。广泛的初步工作还发现了结核分枝杆菌中干扰MHC II类抗原呈递的基因，以及阻断关键细胞因子产生的基因。本研究的主要目标是构建安全的减毒结核分枝杆菌菌株，其中特定的免疫逃避基因已被删除，从而产生更有效的抗分枝杆菌免疫启动疫苗。该方法包括从一组四种候选基因中鉴定出最有效的抗凋亡基因，这些基因已经部分表征，并将这些基因中的突变与强减弱的缺陷突变相结合，以消除毒力。利用由专门的转导噬菌体介导的等位基因交换，将在感兴趣的基因中产生精确的突变，这种方法在PI及其合作者的实验室中得到了很好的建立。候选疫苗株的效力将通过在干扰MHC II类呈递或干扰IL-12p70或TNF产生的基因中加入额外的突变而进一步增强。候选疫苗株对CD4和CD8 T细胞启动的免疫学研究将在小鼠模型中进行，从而确定最有利的特定基因缺失组合，从而推进啮齿动物的临床前疫苗接种研究。候选疫苗株诱导稳定T细胞记忆的研究将在小鼠中进行，并且还将启动分枝杆菌蛋白增强初级反应的影响。总的来说，拟议的研究将大大提高我们对结核病宿主-病原体相互作用的理解，并直接有助于预防和控制这一主要人类疾病的疫苗开发。

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. PUBLIC HEALTH RELEVANCE: This proposal aims to understand in greater detail the mechanisms by which Mycobacterium tuberculosis evades host immunity to cause serious disease and mortality. The information gained will be applied to the design and construction of better vaccines for the prevention of tuberculosis.