乙肝S抗原为载体的HCV嵌合病毒样颗粒预防疫苗的初步实验研究

HCV infection is one of the leading causes of chronic liver disease, which is associated with a high risk of cirrhosis and hepatocellular carcinoma. HCV infection is a major public health problem because of no prophylactic vaccine. We have studied the immune responses induced by HCV mulit-antigen epitopes in mice and preparation of the virus-like particles (VLPs) chimericed with HCV neutralized antigen epitope. Base on these works, we select several confirmed cross neutralized epitopes of HCV and series of these multi-epitopes are jointed to extracellular region of HBV S gene. The chimeric HCV-HBV VLPs expressed multi-epitopes will be prepared. At the same time, HCV E1, E2 will be amplified and jointed to extracellular region of HBV S gene to prepare chimeric HCV-HBV VLPs expressed HCV envelope protein. New Zealand rabbits will be immunized with these two kinds of purificated VLPs and the difference of the serum neutralized antibodies levels will be analysised. We want to know whether the antiserum will inhibit infection of HCVpp and HCVcc to Huh-7.5 cells. Finally, stable production of the chimeric HCV-HBV VLPs CHO cell line will be screeninged for next use of pilot scale production. Our design may be a novel strategy for development of HCV prophylactic vaccine and HCV-HBV bivalent prophylactic vaccine, and we can provid reliable results for further researches.

丙型肝炎病毒感染后极易慢性化和发展为肝硬化及肝癌，严重危害公众健康，迄今尚无预防疫苗。本课题在以往HCV多表位抗原诱导的免疫应答研究，以及HCV中和抗原表位嵌合病毒样颗粒制备的基础上，结合HCV中和抗原表位研究进展，选择具有广泛交叉中和活性的中和表位串联，采用乙肝病毒S抗原氨基端胞外区嵌合方式，制备嵌合HCV多中和抗原表位的HCV-HBV病毒样颗粒（VLPs）；同时将HCV膜蛋白E1、E2嵌合于HBV S抗原氨基端胞外区，制备嵌合HCV包膜蛋白的HCV-HBV VLPs；纯化的嵌合HCV-HBV VLPs免疫新西兰兔，观察两种VLPs免疫后诱导中和抗体水平的差异；观察免疫抗血清对HCVpp和HCVcc感染HuH-7.5细胞的抑制作用；筛选建立可大量制备的稳定表达嵌合VLPs的CHO细胞系。该研究结果可为HCV预防性疫苗以及HBV-HCV双价疫苗的发展提供新的思路和可靠的实验依据。

丙型肝炎病毒（HCV）感染后极易慢性化和发展为肝硬化及肝癌，迄今尚无预防疫苗。发展HCV预防疫苗迫切需要。本课题将保守的中和表位串联后嵌合于HBV S抗原氨基端，制备HCV多中和抗原表位的HBV S抗原病毒样颗粒（VLPs）。结果获得纯化的VLPs。纯化的VLPs与HCV感染患者血清进行反应，结果发现HCV RNA阳性患者血清中有一定水平的中和抗体存在；用纯化的VLPs免疫新西兰兔，测定免疫兔体内的中和抗体，结果在免疫兔体内产生一定水平的中和抗体。观察免疫兔血清对HCVpp及HCVcc感染Huh-7.5细胞的抑制作用；结果表明免疫兔体内产生的中和抗体能抑制不同型别的HCVpp和HCVcc对Huh7.5的感染。研究结果表明HCV多中和抗原表位的VLPs能诱导产生具有保护作用的中和抗体，该实验结果为发展HCV预防性嵌合VLPs疫苗提供了实验依据。该课题对研发HCV预防疫苗有较好指导意义。课题已发表相关论文，申请了专利。