HBc衣壳样颗粒展示CVB3VP1蛋白及免疫原性研究

Coxsackievirus B3 (CVB3) causes viral myocarditis and can ultimately result in dilated cardiomyopathy. However, there is no vaccine available for clinical use. The major capsid protein VP1 has been shown to be immunogenic.Our former studies constructed genetically engineering vaccines to express VP1 applying vectors such as plasmid or replication defect adenovirus.We also achieved puried protein VP1 expressed in E.coli cells.Yet, despite encouraging early results, the level of specific immunity induced by these approches has generally been insufficient to afford complete protection the mice from lethal CVB3 challenge.The core protein of Hepatits B virus (HBc) can be recombinantly expressed in E. coli which spontaneously assembles to icosahedral shaped capsid-like particles (CLPs) of extreme immunogenicty. This immunogenicity can be assigned to foreign proteins especially if they are inserted into the surface exposed but centrally located c/e1-epitope of the HBc. The presentation of selected full-length proteins has already been established. However, many other foreign sequences impair the formation of CLPs.To solve this problem,some researchers designed a system where the HBc is splited within the c/e1-epitope into two fragments, CoreN and CoreC. These so called SplitCore CLPs have now two artificial surface exposed termini that can be used for further protein fusions. When co-expressed in E. coli the two fragments can self-complement each other and are able to assemble to CLPs that are undistinguishable from wild type CLPs in electronmicroscopy. All the described features of SplitCore CLPs offer entirely new possibilities to use the concept of recombinant HBc particles as a carrier of protein VP1 of CVB3.Thus, the aim of this study is to present the innovative prophylactic vaccination strategy based on HBc-CLPs expressing CVB3 VP1. Followed immunization, mice are challenged with CVB3 infection to evaluate the immunological effect of the chimeric CLPs.As a result of the project we expect to generate a recombinant CLPs which induce vigorous humoral and cellular immune response and can be used as a prophylactic vaccine againast CVB3 infection.

柯萨奇病毒B3(CVB3)是病毒性心肌炎的主要病原，衣壳蛋白VP1是CVB3主要中和抗原。本课题组前期以VP1基因构建了核酸疫苗、腺病毒载体疫苗和用大肠杆菌表达了VP1蛋白，免疫小鼠显示，VP1蛋白的免疫效果明显优于前两种疫苗，但不能完全保护动物抵抗致死量病毒感染。有效提高VP1蛋白的免疫原性是制备亚单位疫苗的关键。乙肝病毒核心抗原（HBc）用大肠杆菌表达能自我组装成衣壳样颗粒(CLPs),将外源短肽插入其MIR区的c/e1表位能形成嵌合CLPs，并使外源短肽的免疫原性明显增强。但只有少数N端与C端靠近的大分子蛋白质插入c/e1部位能形成嵌合CLPs。采用SplitCore方法可以克服这一不足，使N端与C端距离较远的大分子蛋白以天然构象展示在HBc颗粒表面。我们拟用该方法以HBcCLPs展示CVB3VP1蛋白，免疫小鼠，观察其免疫原性和对病毒攻击的免疫保护作用，为研制CVB3疫苗奠定基础。

病毒性疾病严重危害人类健康，研制有效的疫苗预防病毒感染具有重要的理论和现实意义。传统的灭活疫苗或减毒活疫苗存在灭活不彻底或毒力恢复的安全隐患。蛋白疫苗仅含有一种或几种保护性抗原，而不含有病原体的其他遗传信息，因此发展成为一类安全有效的疫苗用以预防多种病毒性疾病。但一些蛋白疫苗的免疫原性较差，需要联合应用佐剂或多次免疫等其他途径才可达到理想保护效果。病毒样颗粒(Virus Like Particles, VLPs)疫苗含有病毒的结构蛋白成分，但不含有病毒核酸，可以重复且高密度的展示抗原表位，成为一种理想的蛋白疫苗。研发VLPs的包装策略以及提高VLPs的表达产量是该类疫苗的研究重点和难点。本课题拟以HBV作为载体，表面嵌合柯萨奇病毒病毒B3(Coxsackievirus B3,CVB3)的衣壳蛋白VP1，探讨该策略在大肠杆菌内包装嵌合型VLPs的可行性以及探讨该VLPs的免疫原性。依据原技术路线，课题组构建了相关表达质粒，转化大肠杆菌，经蛋白表达条件和表达时间等条件的优化，均未能成功包装出嵌合型VLPs。杆状病毒昆虫细胞表达系统是包装VLPs的另一常用系统，故课题组尝试了在该系统内包装该病毒样颗粒。为了进一步探讨病毒样颗粒的包装策略，在该系统内同时进行了流感病毒以及中东呼吸综合征冠状病毒（Middle East respiratory syndrome coronavirus, MERS-CoV）VLPs的包装，结果证明该系统可成功包装出流感病毒以及MERS-CoV的VLPs，而不能包装出CVB3的VLPs,这表明病毒样颗粒的成功包装可能与病毒本身性质以及病毒抗原成分密切相关。