敲除抑制自噬基因增强卡介苗介导的自噬对结核病的预防和机制

Mycobacterium bovis BCG is the only licensed vaccine current used for the prevention of tuberculosis (TB). The protection mechanism is based on CD4+Th1 responses by the route of phagosome-lysosome and antigen presentation by MHC II molecules, which is effective for children primary TB while only lasting for 10-15 years and poor protection for adult TB as post-primary infection. By comparative genomics and bioinformatics, we can conclude that BCG has an anti-autophagy gene. Importantly, the protective efficacy of BCG knocked out of this gene vaccinated mice against M. tuberculosis aerosol challenge is much more significantly enhanced, when compared to wild type BCG. In order to analyze the mechanism of enhanced protection, we propose the following hypothesis: anti-autophagy gene knockout BCG, relative to wild type BCG, could present antigens not only through traditional ways of phagosomes and lysosomes, but also through autophagosome and lysosomal pathway, and bind the degradation of the peptide to MHC II molecules, resulting in a strong and durable activation of CD4+ Th1-type responses, enhancing its anti-infection protection, and prolonging the protection period. Therefore, we firstly need to identify that this gene is an anti-autophagy gene presented in the genome of BCG. Then, in order to clarify the effects of anti-autophagy gene on the immunological mechanisms of BCG, we want to compare the differential antigen presentation pathway between anti-autophagy gene knockout BCG and wild type BCG, and verify its long-term protective immune response and anti-TB infection in animal experiments. Our results will provide a scientific basis for the development of more effective TB vaccine.

卡介苗(BCG)是结核病(TB)唯一的预防疫苗，感染抗原递呈细胞可经吞噬体-溶酶体途径活化CD4+Th1型应答，有效预防儿童原发重症TB，但保护期仅10-15年，对成人肺结核病的保护性差。经生物信息学分析，推断BCG具有抑制自噬基因，且证实敲除该基因的BCG可显著增强免疫小鼠抗TB感染的短期保护性。为了深入分析增强保护性的机制，我们提出以下假说：抑制自噬基因敲除型BCG相对于野生型BCG，既通过传统的吞噬体-溶酶体途径，又通过新发现的自噬小体-溶酶体途径，同时将降解产生的肽段结合MHC II类分子，导致活化的CD4+Th1型应答强大持久、增强保护性和延长抗感染保护期。因此，拟鉴定该基因的抑制自噬功能，分别比较二者经自噬小体及吞噬体途径提呈抗原的差异，并经动物实验验证其抗TB感染的免疫效应和长期保护性，阐明敲除该基因对BCG保护性的影响和机制，为发展更为有效的TB疫苗提供科学依据。

背景：牛分枝杆菌卡介苗（BCG）是预防结核病（TB)唯一的一种（减毒活）疫苗，但对TB的保护效应不够充分，其机制尚不清楚。我们假设卡介苗继承了牛分枝杆菌亲代毒株的免疫逃避策略，本研究旨在鉴定相关的毒力基因并评估其对BCG疫苗效力的影响。.方法：首先通过生物信息学分析获得并鉴定了与免疫逃逸相关的三个基因，即BCG_3174、BCG_1782和BCG_2432c，然后通过聚合酶链反应（PCR）和DNA测序在牛分枝杆菌毒株基因组和12种商用BCG疫苗亚株中确认其存在。通过噬菌体基因敲除系统破坏这些基因分别产生不同的突变菌株，进一步体外和体内试验与卡介苗疫苗比较，评价这些基因对自噬的影响及其对BCG保护效力的效应。.结果：与卡介苗疫苗相比，在三个已鉴定的基因中，只有BCG_2432c的破坏，即ΔBCG_2432c免疫小鼠，对鼻内接种小鼠的结核分枝杆菌毒株具有更强的保护作用。ΔBCG_2432c在感染的THP-1细胞中表现出更强的触发细胞内ROS介导的完全自噬流的能力，从而导致更强的抗原呈递。增强的保护作用可归因于ΔBCG_2432c免疫小鼠的脾脏和肺中早期和增加的IFN-γ+CD4+TEM和IL-2+CD4+TCM细胞。.结论：卡介苗的预防效应不充分是由于卡介苗具有重要的自噬抑制基因BCG_2432c，阻断了抗原呈递的自噬体-溶酶体途径。ΔBCG_2432c可为替代现有的BCG疫苗或开发更有效的结核病疫苗提供了一个有希望的平台。