沙门菌交叉保护性抗原的发掘及其诱导保护性免疫的影响因素

沙门菌各血清型间免疫保护力微弱，但特定沙门菌减毒株可诱导中等的交叉保护效应，提示交叉保护性免疫可在一定条件下被诱导，但发挥效用的抗原及其激发保护应答的机制均不清楚。本申请拟通过反向疫苗学手段筛选2-4种交叉保护性抗原，将它们稳定表达于减毒鼠伤寒沙门菌载体。通过HbpD等信号分子调控靶抗原的空间位置（细菌外膜、细菌胞外、宿主细胞质等）；通过PpagC等启动子及不同拷贝数质粒调控抗原的表达时相（组成性或阶段性表达）和表达量；通过缺失干扰抗原递呈通路的Ⅲ型分泌系统2效应分子基因优化疫苗载体。然后以免疫学方法解析这些因素对树突状细胞抗原递呈效率、载体疫苗激发的适应性免疫水平和交叉免疫保护力的影响，证实靶抗原空间位置、表达时相、表达量及特定效应分子基因缺失在诱导沙门菌交叉保护性免疫中的协同作用机制，并确定免疫保护相关因子。本研究将推进沙门菌病广谱疫苗的成功研制，为我国沙门菌病防控提供新武器。

The protective cross-immunity between Salmonella serovars is usually quite limited; however, a few live attenuated Salmonella strains can induce moderate cross-protective immunity, indicating the presence of cross-protective antigens between serovars which can stimulate protective immunity under certain conditions. The mechanism underlying this phenomenon is not clear. We will screen 2 to 4 cross-protective antigens via reverse vaccinology, and then construct S. Typhimurium strains with regulated delayed attenuation to deliver selected target antigens in diverse expression patterns. The signal sequences (SseF, HbpD, β-lactamase and fliC, etc), promoters (Ptrc, PpagC、PfliC、Psaf), and plasmids with different copy numbers will be utilized to regulate the localization (bacterial cytoplasm, outer membrane, secretion and host cell cytoplasm), expression time (constitutive or temporal expression) and expression level of antigens, respectively. Furthermore, the effector genes of Salmonella pathogenicity island 2 (SPI-2) T3SS which have a critical role in inhibiting antigen presentation will also be deleted in recombinant S. Typhimurium strains. All constructed recombinant vaccine strains will infect dendritic cells in vitro or immunize BALB/c mice to evaluate the effects of antigen localization, expression time and level, and the deletion of SPI-2 T3SS effector genes on the capacity of antigen presentation, the level of adaptive immune responses and cross-protective efficacies induced by vaccine strains. Finally, the study will demonstrate the synergistic roles of localization, expression time and levels of target antigens and certain T3SS effector gene mutations in induction of cross-protective immunity, and determine the correlates of protection. Our study will promote the successful construction of effective recombinant S. Thyphimurium vaccine with broad serovar coverage, which will provide a new tool for prevention of salmonellosis.