同时针对活动性及潜伏性结核，兼具预防与治疗功效的多阶段结核疫苗逐渐成为结核疫苗研发的重要方向，但还需在免疫原性及抗原基因选择等方面进一步深入研究。为增强疫苗免疫原性，项目组以能强效激活特异性CD4+ Th1和CD8+细胞免疫应答的减毒单增(LM)和绵羊李斯特菌(LI)作为疫苗载体，其中将LI作为疫苗载体为首创专利技术；在抗原基因选择上，首次将结核杆菌潜伏性感染和复发相关基因Rv2460c作为抗原基因组成融合基因构建新颖的LM/LI载体多阶段结核疫苗体系；采用包括BCG prime-LM/LI boost等多种策略免疫小鼠；除静脉免疫外，还探索呼吸道和胃肠道免疫途径的可行性和效果，重点系统研究疫苗诱导的预防性和治疗性免疫应答谱学特征及安全性，初步探讨疫苗的免疫保护和免疫治疗效果，为下一步临床前期试验提供充实可靠的数据，并为最终研发出安全、有效、符合多阶段疫苗理念的结核疫苗奠定坚实的研究基础。

Currently proposed multistage tuberculosis vaccine combining preventive and therapeutic effects against both active and latent tuberculosis, has become an important study subject in tuberculosis vaccine field. But the immunogenicity and antigenic gene constitution of currently reported multistage tuberculosis vaccine need to be further studied. In this project, attenuated Listeria monocytogenes (LM) and Listeria ivanovii (LI) which have confirmed effects on inducing robust specific CD4+ Th1 and CD8+ T cell immune response will be used as vaccine vector to construct vaccine in order to enhancing the vaccine immunogenicity. Notably, our team is the first to use LI as vaccine vector. Regarding antigenic gene constitution, a widely confirmed Mycobacterium tuberculosis pressure response gene Rv2460c, being relevant to latent infection and infection reaction is for the first time chosen as antigenic gene. A fusion antigenic gene fused with Rv2460c, Rv2660c, Rv3875 and Rv3804c will be used to construct a novel LM/LI based multistage tuberculosis vaccine system. To evaluate the immune responses induced by the multistage vaccine, the mice will be intravenously, intragastrically or intranasally administrated the above vaccine, via several immune strategies, including BCG prime and LM/LI boost strategy. After immunization, studies will focus on elucidating the preventive and therapeutic immune responses induced by vaccine and the biosafety of the vaccine. The protection against M. tuberculosis infection and therapy for tuberculosis will also be explored in this project. This work will supply substantial and reliable data for next preclinic trial, and is the basement for success in developing safe and efficacious multistage tuberculosis vaccines.