细胞自噬作用对提高乙型脑炎病毒疫苗长效保护性的研究

The Japanese encephalitis virus (JEV) mainly causes encephalitis in children whose age is under 10 years. In China, the large scale immunization of JEV vaccine had demonstrated success for preventing the disease in younger children. However, JEV infection in elder children and adult had been documented frequently, which implies the current JEV vaccine may not able to provide the protection longer enough. Recently studies had shown that autophagy, originally identified as a pathway for homeostasis, has been involved into the antigen presentation and plays the indispensable roles for maintain the memory B cell and secondary B cell immune response against virus. Although data up to now suggests that the B cell mediated response plays the key role for the JEV vaccine protection, it is not known whether autophagy could affect the B cell mediated secondary response after JEV vaccination. In this study, the role of autophagy played during B cell response after the JEV immunization will be investigated. The activation of B cell, titer of neutralizing antibody, duration of antibody, and secondary immune response of JEV vaccinated mice with or without autophagy enhancer/inhibitor will be compared to confirm previous speculation. Moreover, the autophagosome targeted JEV vaccine will be generated as well to confirm our findings. Completion of this project will provide new insight for future development of improved JEV vaccine which could provide the protection for long term.

乙型脑炎病毒（JEV）主要引起10岁以下儿童的脑炎。我国低龄儿童的JEV发病随着疫苗接种普及已大幅降低。而近年来大龄儿童和成人JEV感染持续增加，提示现有疫苗的长效保护不足。最新研究显示自噬（Autophagy）这一维持细胞代谢平衡的过程参与机体抗原加工，并是机体维持对病毒的记忆性B细胞和二次B细胞应答的关键。虽然记忆性B细胞对JEV疫苗的保护力起决定性的作用，但是对于自噬是否影响JEV疫苗诱导的B细胞体液免疫应答和免疫记忆则没有研究。本课题首先以JEV减毒疫苗免疫小鼠为基础，协同使用自噬增强剂（Rapamycin）和自噬抑制剂（3MA），确认自噬在JEV疫苗免疫应答中的作用。然后检测免疫后不同组小鼠的B细胞活化，JEV抗体滴度，持续时间以及二次免疫应答强度。并且更进一步构建JEV自噬靶向重组疫苗，探讨自噬靶向疫苗提供长效保护的可能，为提高现有JEV疫苗的长效保护提供新思路。

近年来尽管日本脑炎（Japanese encephalitis，JE）疫苗的研发本身在不断推进，由过去传统的灭活疫苗向减毒疫苗，重组疫苗的方向进一步发展。然而在查阅了近十年以来的国际和国内针对乙脑病毒的流行病学调查的基础之上发现日本脑炎疫苗的长效保护性在世界范围内均没有得到有效解决。自噬从被发现至今，自噬在病毒免疫过程中的多种促进或者抑制作用发现了自噬在抗病毒治疗和疫苗预防方面的巨大潜力。越来越多的证据也表明自噬与病原体感染过程中MHC I、MHC II对特定内源性合成肽的加工和递呈密切相关。有学者已经想到将抗原同自噬相关蛋白结合直接导向自噬通路，用以增强抗原特异性CD4+ T和CD8+ T淋巴细胞主导的免疫应答，另外自噬可以延长记忆型免疫细胞的存活寿命。相关实验只是发现了这些现象，但具体的机制问题仍未得到解决。本研究将自噬关键标志物LC3编码基因引入JE DNA疫苗，旨在明确自噬对JE DNA疫苗的免疫增强效应的影响及相关免疫机制，同时初步探索自噬对JE DNA疫苗免疫记忆及长效免疫保护的作用，为进一步研发新型高效JE DNA疫苗提供实验依据。该项研究主要内容围绕重组质粒DNA构建及鉴定、免疫应答、免疫记忆、保护性免疫，从JEV特异性IgG抗体、T细胞细胞因子分泌水平、CTL活性、淋巴细胞亚群等多方面系统分析自噬介导的JE DNA疫苗免疫增强效应特征，初步探究CD4+T细胞向Th1型分化的机制，最终评价疫苗诱导的免疫记忆及保护性免疫效力。.研究结果表明，pJME-LC3能够明显促进Th1型细胞因子IFN-γ、IL-12分泌水平，显著增强IgG2a为主的体液免疫应答，诱导Th1型细胞免疫应答；pJME-LC3能够增强JEV特异性CTL活性；pJME-LC3通过激活JAK2/STAT1信号通路上调转录因子T-bet的表达，促使CD4+T细胞向Th1细胞分化；pJME-LC3增加JEV特异性CD4/CD8TCM/TEM比例，促进JEV特异性中和抗体快速、持续生成，诱导长效免疫记忆和强效免疫保护。.本研究综合分析了细胞自噬在增强疫苗免疫效应、长效免疫保护方面的重要意义，为JE DNA疫苗的深入研究提供了实验依据。