HCV的高度变异是丙肝疫苗长期陷于困境的根本原因。HCV的细胞侵入由包膜蛋白介导，包膜蛋白E2氨基末端27个残基组成的肽段是HCV蛋白中变异频率最高的区域，被称为高变区1（HVR1），在HCV细胞侵入过程中起重要作用。HCV包膜蛋白中也存在高度保守的中和表位，然而，在HCV感染的急性期往往只能产生针对于HVR1的抗体而不能产生针对于保守表位的抗体。我们在多株HVR1中均鉴定出了唯一的中和表位，发现删除该中和表位可促成HCV颗粒表面形成和暴露保守中和表位，并增强包膜蛋白中保守表位的免疫原性。本课题利用果蝇S2细胞重组表达HCV核心、包膜E1和删除了HVR1中和表位的E2蛋白，制备HCV病毒样颗粒（VLP），分析该中和表位对VLP表面包膜蛋白抗原表位的形成、受体结合功能以及VLP在小动物和恒河猴诱导中和抗体的强度和广度的影响，旨在于研发成功一种能有效诱导广谱中和抗体的丙型肝炎预防性疫苗。

The biggest challenge of hepatitis C vaccine is high genetic heterogeneity of hepatitis C virus. HCV cell entry is mediated by its envelope proteins, therefore envelope proteins are the key targets for neutralizing antibodies. The hypervariable region 1 (HVR1) that comprises the first 27 amino acid residues of the E2 envelope glycoprotein is the most variable region within the HCV polyprotein, which plays a major role in both HCV cell entry and immune evasion. Increasing evidences demonstrated that high conserved neutralizing epitopes existed in envelope proteins across different HCV genotypes. However, in acute infection phase, individuals mainly produce antibodies against HVR1 but not to conserved epitopes in envelope protein outside of HVR1. We identified unique neutralizing epitope in HVR1 of several HCV strains, and found that removal of the neutralizing epitope significantly contribute the formation and exposure of conserved epitopes and enhance the immunogenicity of envelope proteins. To prepare a structure optimized HCV virus-like particles (VLP) vaccine, in the present study we will utilize Drosophila melanogaster Schneider 2 cells to express HCV structural proteins, including HCV core protein, envelope protein 1 and HVR1 neutralizing epitope deleted envelope protein 2 and then detect the production of VLP. The effect of the neutralizing epitope on formation of conserved epitope on VLP, receptor binding activity of VLP will be analyzed. The VLP will be use to immunize mice and rhesus monkeys to investigate its ability to induce cross-neutralizing antibodies against diverse HCV strains. The study will probably develop an efficient prophylactic hepatitis C vaccine that can elicit broad spectrum neutralizing antibodies against HCV infection.

HCV的高度变异是丙肝疫苗长期陷于困境的根本原因。HCV的细胞侵入由包膜蛋白介导，包膜蛋白E2氨基末端27个残基组成的肽段是HCV蛋白中变异频率最高的区域，被称为高变区1 （HVR1），在HCV细胞侵入过程中起重要作用。HCV包膜蛋白中也存在高度保守的中和表位 ，然而，在HCV感染的急性期往往只能产生针对于HVR1的抗体而不能产生针对于保守表位 的抗体。本课题在多株HVR1中均鉴定出了唯一的中和表位，发现删除该中和表位可促成HCV 颗粒表面形成和暴露保守中和表位，并增强包膜蛋白中保守表位的免疫原性。进一步利用 果蝇S2细胞重组表达HCV核心、包膜E1和删除了HVR1中和表位的E2蛋白，制备HCV病毒样颗 粒（VLP），分析该中和表位对VLP表面包膜蛋白抗原表位的形成、受体结合功能以及VLP 在小动物诱导中和抗体的强度和广度的影响，结果发现，删除HVR1的HCV VLP可直接与中和单抗高亲和力结合，且与HCV主要受体CD81的结合力增强，并在小鼠诱导高效价交叉中和抗体。本课题还分析了慢性HCV感染者的血清抗体反应特征，发现高HCV RNA载量血清的包膜蛋白抗体水平以及中和活性也相应较高，提示在慢性HCV感染者，包膜蛋白抗体不能有效抑制HCV复制。