EV71 VP1"食品级"乳酸菌活载体疫苗及异源抗原联合免疫诱导黏膜和系统免疫的研究

Hand food and mouth disease (HFMD) induced by enterovirus 71 (EV71) often associated with severe neurological damage, particularly in young children. In recent years, there were repeatedly outbreaks of EV71 with obstinately higher disability and fatality in the Asia-Pacific region, including China. But no effective vaccines or antiviral therapies are currently available for prevention or treatment of HFMD caused by EV71. Therefore, the development of therapeutic and preventive strategies against HFMD is of growing importance. Because EV71 mainly transmit by mucosal routes, effective mucosal immunity will prevent the virus infection at the first step. In this study, two respects were studied to look for effective ways inducing protective mucosal immune response. At first, codon optimized VP1 recombinant L.lactis live vector vaccines based on food-grade NICE expression system were constructed to explore safe and efficient means inducing protective mucosal and systemic immunity against EV71. Secondly, a heterologous antigen prime-boost immunization strategy using codon optimized EV71 VP1 DNA vaccine combined with VP1 L.lactis live vector vaccines or VP1 protein / EV71 inactivated vaccine was exploited to search out potent priming function of mucosal immunity by non-mucosal vaccination. This study will be meaningful for the development of novel EV71 candidate vaccines applying to young children and eventual elimination of its infection.

肠道病毒71型（EV71）常导致严重的神经症状，致残、致死率较高，但缺乏预防控制措施。研发安全有效的疫苗迫在眉睫。EV71主要通过黏膜途径接触传播，保护性黏膜免疫反应对于早期阻断EV71感染至关重要。本研究拟从两个方面探寻有效激发机体黏膜免疫反应的途径和方法：首先，采用安全高效的NICE诱导表达系统，设计、构建"食品级"密码子优化的VP1 L. lactis活载体疫苗，研究其激发机体黏膜和系统免疫应答的能力，为开发更加安全、有效和适用于婴幼儿的EV71疫苗、并最终消除其感染奠定基础。其次，利用密码子优化的EV71 VP1 DNA疫苗初免+VP1 L. lactis活载体疫苗或VP1蛋白/EV71灭活疫苗加强的异源抗原prime-boost策略，探讨非粘膜途径接种可否对粘膜免疫反应产生有效的初免作用，从而诱导更好的粘膜和系统免疫反应，为EV71疫苗的设计和开发提供新的思路。

肠道病毒 71 型（EV71）导致的手足口病常引起婴幼儿严重的神经症状，致残、致死率较高。从中国疾病预防控制中心的统计数据发现，近十年来手足口病的发病率和死亡率居高不下，依然是严重威胁婴幼儿的重要传染病之一，研发安全有效的疫苗迫在眉睫。因EV71 主要通过黏膜途径接触传播，保护性黏膜免疫反应对于早期阻断 EV71 感染至关重要。本课题原计划从探讨有效激发机体黏膜免疫反应的途径和方法出发，采用具有“公认的安全性”的乳酸菌为载体，设计密码子优化的 VP1 “NICE 诱导表达系统” L. lactis 活载体疫苗。但由于Nisin诱导表达系统不稳定，蛋白表达水平不高，经口腔黏膜免疫难以克服“口服耐受”的巨大障碍等原因，导致疫苗免疫效果较差。 由于EV71重症感染及死亡病例主要导致脑炎和神经性肺水肿，而对于EV71如何导致脑部和肺部严重感染的机制并不清楚。鉴于VP1/乳酸菌疫苗表达系统结果的不确定性，为了进一步探讨EV71病毒感染的机制，同时为了丰富研究结果，本课题扩展做了VP1对小鼠肺和脑组织毒性的研究，并发现VP1蛋白可以通过改变组织细胞连接蛋白的表达水平、增加血管通透性以及增加相关趋化因子的表达水平而促进中性粒细胞浸润，诱导局部炎症反应和组织损伤。