氧化压力抗性基因mazG缺失提高卡介苗保护效力的免疫机制研究

A growing body of evidence suggests that intracellular bacteria use antioxidant systems down-modulating immune responses, of both the innate and acquired type. Whilst this can be advantageous to the pathogen, it is contrary to requirements of a tuberculosis vaccine. Indeed, evidence suggests that some of the deficiencies of the current BCG vaccine can be attributed to an increased capacity to produce antioxidants that has been acquired during vaccine derivation. The effect could be countered by experimental deletion of genes encoding SigH which reduced antioxidant activity and secretion of superoxide dismutase and resulted in a stronger protective immune response to the vaccine. Our own studies revealed that mycobacterial MazG is a potent nucleoside triphosphate pyrophosphohydrolase (NTPPPase) and this activity is required to maintain the full capacity of the mycobacteria to respond to oxidative stress. Deletion of mazG in Mtb leads to a mutator phenotype under oxidative stress and reduced survival in activated macrophages and in the spleen of infected mice. These findings bring us to postulate that deletion of mazG in BCG could also impair antioxidant activity and thus enhance BCG-mediated immunity and protection against Mtb infection. Preliminary experiments have demonstrated the impaired antioxidant phenotype and enhanced immunogenicity and protective efficacy against virulent Mtb H37Rv infection when compared with the wild-type BCG vaccination (see the detail in the Preliminary Results Section); however, the detailed immune mechanism still remains elusive. In this proposal, we intend to use flow cytometry techniques such as surface staining, intracellular cytokine staining, CFSE staining etc., to analyze multifunctional T cell responses, T cell proliferation, and to further define the immune correlates between the immune responses, anti-TB protection and mazG-mediated antioxidant activity in vitro, in vivo and in animal model. This may be possible to explain a novel mechanism of immune suppression of BCG vaccine, and be also critical for better evaluation or enhancement of immune protection and rational development of new vaccines against TB infection.

胞内细菌可通过抗氧化系统下调宿主的固有/适应性免疫而利于在胞内的生存，有证据显示卡介苗(BCG)在体外传代过程中获得了提高的抗氧化能力而使其子代菌株较之早期疫苗株的抗结核保护效力下降。我们推测在BCG中敲除氧化压力抗性基因mazG可能通过解除这种氧化压力抗性介导的免疫抑制而提高其免疫力及保护效果。为此，我们构建了mazG缺失的BCG突变株并初步证实了其缺陷的抗氧化表型及提高的抗结核保护效力，但其保护机制尚未阐明。因此，我们拟利用流式胞内因子染色、CFSE染色等技术手段综合分析免疫小鼠在感染前后的多功能T细胞频率、T细胞增殖潜力等免疫学指标，分别在体外、细胞与动物水平中系统研究mazG介导产生的抗氧化水平及其诱导产生的免疫应答、免疫记忆特征与功能变化与抗结核免疫保护之间的关系，以阐明mazG介导的免疫抑制机制，可为增强BCG效力提供一种新思路，并对结核疫苗的合理设计提供一定的指导。

胞内细菌可通过抗氧化系统下调宿主的固有/适应性免疫而利于在胞内的生存，有证据显示卡介苗(BCG)在体外传代过程中获得了提高的抗氧化能力而使其子代菌株较之早期疫苗株的抗结核保护效力下降。分枝杆菌MazG作为一种强活性的核苷三磷酸焦磷酸水解酶(NTP-PPase)，是分枝杆菌应对活性氧压力的必需组分。因而我们推测在BCG中敲除mazG可能通过解除这种氧化压力抗性介导的免疫抑制而提高其免疫力及保护效果。为此，我们构建了mazG缺失的BCG突变株，并进一步分析了突变株包括抗氧化表型在内的生物学功能及其抗结核保护性免疫水平。本研究综合利用流式胞内因子染色、CFSE染色等技术手段综合分析免疫小鼠在感染前后的多功能T细胞频率、T细胞增殖潜力等免疫学指标，分别在体外、细胞与动物水平中系统研究mazG介导产生的抗氧化水平及其诱导产生的免疫应答、免疫记忆特征与功能变化与抗结核免疫保护之间的关系，证实了mazG突变株可通过抑制巨噬细胞凋亡等抗菌途径，增强其胞内和体内生存能力，从而诱导更强的CD4 T细胞免疫应答及其抗结核保护性免疫，为增强BCG效力提供一种新思路，并对结核疫苗的合理设计提供一定的指导。