结核亚单位疫苗诱导的记忆性T细胞亚型特征、分化调控及保护效应研究

We and other labs have found that tuberculosis subunit vaccine could provide effective protection against Mycobacterium tuberculosis infection in mice and other animal models. The strength and duration of protection induced by tuberculosis vaccine may be related to sub-populations of memory T cells induced by vaccine. However, the characteristics of memory T cell subsets induced by tuberculosis subunit vaccine are not yet clear, especially for the protective efficacy and regulation of central memory T cells. We have constructed a subunit vaccine EAMMH (ESAT6-Ag85B-MPT64<190-198>Mtb8.4-HspX), which was proven to be highly protective against Mycobacterium tuberculosis infection in mice. This project is intended to study the number, characteristics, and protective effect of effector memory T cells (TEM) and central memory T cells (TCM) induced by subunit vaccine EAMMH. Furthermore, we are going to promote the differentiation of T cells to TCM during EAMMH vaccination by regulating the doses of EAMMH, and adding the treatment of rapamycin and cytokines IL-7/IL-15. Then the protective efficacy is to be evaluated so as to learn the effect of TCM. In addition, the expression of transcriptional factors with the treatment of rapamycin and IL-7/IL-15 during vaccination is to be analyzed, and the transcriptional regulation mechanism of TCM differentiation is to be investigated too. Through this study, we are to determine the number, characteristics and protective effect of memory T cell subsets induced by tuberculosis subunit vaccine, especially to pay attention to the role of TCM in protection against Mycobacterium tuberculosis and learn how to control the differentiation of TCM. The knowledge on memory T cell subsets will be helpful to design novel subunit vaccine and to improve the protective efficacy of tuberculosis subunit vaccine.

结核亚单位疫苗能在动物体内诱导抗结核分枝杆菌免疫保护力,其强弱和维持时间与疫苗诱导的记忆性T细胞亚型有关。然而，目前结核亚单位疫苗诱导的记忆性T细胞亚型特征及其功能尚不清晰，尤其对存活时间长的中央记忆T细胞的抗感染作用及调控机制了解较少。本课题拟以我们前期筛选到的有较高免疫保护力的亚单位疫苗EAMMH（ESAT6-Ag85B-MPT64<190-198>-Mtb8.4-HspX）为基础，研究结核亚单位疫苗激活的效应记忆T细胞（TEM）和中央记忆T细胞（TCM）的数量和特性，并通过调节抗原剂量，以及应用雷帕霉素和细胞因子IL-7/IL-15促进TCM分化，进而分析TCM的抗感染保护作用，同时深入研究促进TCM分化的转录调控机制。通过本研究将明确结核亚单位疫苗诱导的记忆性T细胞亚型的特征和保护作用，尤其阐明TCM的保护效果和分化调控机制，对疫苗设计及研究提高结核疫苗免疫保护力的策略有重要意义。

结核病疫苗卡介苗不能提供终身免疫保护力可能与其诱导的记忆性T细胞寿命较短有关。理想的疫苗应该能诱导长期的免疫保护力。本课题较为系统地研究了结核亚单位疫苗诱导的免疫记忆特征，探讨了细胞因子与转录因子对记忆性T细胞发育分化的影响。主要研究内容及结果如下： （1）亚单位疫苗剂量及疫苗免疫方案对T细胞免疫记忆的影响。研究了融合蛋白LT69不同剂量（50 ug，10 ug，2ug）的免疫保护作用。结果显示末次免疫24周后低剂量组淋巴细胞产生IFN-γ的水平及保护效率明显高于高剂量组（Plos One, 2015）。采用融合蛋白LT70和MH,研究了疫苗接种不同时间间隔（0-3-6w、0-4-12w和0-4-24w）对免疫记忆的影响。结果表明0-4-12w免疫方案诱导产生了更多的中央记忆T细胞（TCM），免疫保护水平也相应增强。在此研究过程中建立了TCM检测方法（Tuberculosis, 2018）。（2）细胞因子IL-7和IL-15对T细胞免疫记忆的影响。构建重组腺病毒rAd-IL-7、rAd-IL-15、rAd-IL-7-IL-15（Linker），联合结核亚单位疫苗LT70和MH免疫小鼠，发现rAd-IL-7-IL-15（Linker）联合亚单位疫苗能够更有效地促进TCM的形成，提高TCM介导的免疫应答和保护水平。（3）雷帕霉素等药物对T细胞免疫记忆的影响。发现雷帕霉素、二甲双胍和吡嗪酰胺可以诱导更多的TCM，增强T细胞的长期免疫记忆反应。（4）TCM形成过程中转录因子的改变及转录因子干预TCM分化的研究。发现应用慢病毒包装Blimp1，与亚单位疫苗LT70+MH联合应用，可以显著提高TCM介导的免疫应答。（5）进一步研究了抗原持续刺激导致记忆性T细胞功能耗竭及对骨髓造血细胞的影响，发现活化T细胞产生的细胞因子IFN-γ影响造血细胞的分化，抗原持续刺激可导致造血细胞及T细胞功能障碍，IL-2具有一定的治疗作用。总之，本项目我们围绕结核亚单位疫苗免疫过程中记忆性T细胞的分化发育及干预因素展开了较为系统的研究，明确了TCM细胞在结核疫苗保护效应中的重要作用，建立了检测TCM的方法，研究确定了疫苗剂量、免疫方案、细胞因子等对TCM的影响，对于结核亚单位疫苗研究具有重要的指导意义。