全长HCV Core抑制HBsAg诱导的免疫应答及机制研究

HCV Core protein contains several well-characterized B-cell and cytotoxic T-lymphocyte (CTL) epitopes and thus is regarded as candidate HCV vaccines. However, the immune response induced by the HCV Core in DNA vaccination is always weak. Moreover, the wild-type HCV Core protein itself could inhibit the priming of immune responses in the course of a DNA vaccination. Our preliminary data has shown that the full-length HCV core inhibits the induction of immune response to the heterogeneous HBsAg, indicating that the full-length HCV Core exerts its immunosuppressive action more generously. Therefore,it will be very meaningful to study the inhibitory mechanism of HBsAg-induced immune response by full-length HCV core. In this project, the constructs expressing the full-length or the truncated HCV core, and the HBsAg or recombinant HBsAg protein will be applied in mice using DNA vaccination strategies. And the specific T cell and B cell immune responses to HBsAg will be assessed to characterize the interference effect. Furthermore, the key sequence on HCV Core in immunosuppression also will be checked. In vitro, dendritic cells (DC) will be isolated from mice and the pivotal cytokines and surface molecules to initiate the adaptive immune response will be analyzed to check the influence of HCV Core on the function of DC. The Toll-like receptor2 (TLR2) signaling pathway will be detected to clear the molecular mechanism of immunosuppression. Finally, a HBV replication-competent plasmid will be administrated to mice via hydrodynamic injection to test whether the protection provided by HBsAg could be affected by the interference of HCV core with the priming of immune response to HBsAg. This project will provide implications in the design of HCV vaccines or certain vaccine combinations and allow us to get new insights into the pathogenesis, prevention and therapy of HBV/HCV coinfection.

HCV核心蛋白因含有丰富的T、B细胞表位而成为HCV疫苗的候选抗原。但研究发现，该蛋白并不诱导高水平的免疫反应，且能抑制同源抗原诱导的免疫应答。我们初步研究发现全长HCV核心蛋白能抑制HBsAg诱导的免疫应答，提示该蛋白的免疫抑制可能具有广谱性，深入解析其抑制机制无疑具有重要意义。本项目将采用全长或截短的HCV核心蛋白表达质粒，与HBsAg表达质粒或重组蛋白以不同策略免疫小鼠，分析特异性T和B细胞应答，回答核心蛋白抑制异源抗原诱导免疫反应的特点，获得发挥免疫抑制作用的关键序列；分析DC细胞用于起始适应性免疫应答过程的关键细胞因子和表面分子，回答核心蛋白对DC功能的影响；分析TLR2及其信号通路，明确其分子抑制机制；利用HBV尾静脉高压水注射模型，回答核心蛋白对HBsAg疫苗保护效应的影响。项目将为HCV疫苗的研制及疫苗的应用策略提供重要参考，为解释HBV/HCV共感染致病机制提供新线索。

HCV核心蛋白因含有丰富的T、B 细胞表位而成为HCV 疫苗的候选抗原。但研究发现，该蛋白并不诱导高水平的免疫反应，且能抑制同源抗原诱导的免疫应答。我们前期研究发现全长HCV 核心蛋白能抑制HBsAg 诱导的免疫应答，提示该蛋白的免疫抑制可能具有广谱性。本课题基于前期结果提出“HCV Core 对于HBV 感染是否具有影响”这一关键科学问题，深入研究了全长HCV Core 抑制HBsAg诱导的免疫应答特点及机制。通过采用全长或截短的HCV 核心蛋白表达质粒，与HBsAg 表达质粒或重组蛋白以不同策略免疫小鼠，分析特异性T 和B 细胞应答，发现全长HCV Core对HBsAg 免疫应答的负调节作用类型是免疫抑制；只有当两种质粒在同一位点进行免疫时才能抑制HBsAg诱导的免疫应答，表明全长HCV 核心蛋白抑制HBsAg诱导的免疫应答是作用于局部的；全长HCV 核心蛋白主要干扰由质粒DNA所诱导的免疫应答，而不干扰重组HBsAg蛋白诱导的应答，表明全长HCV Core在基因水平抑制HBsAg诱导的免疫应答；利用小鼠尾静脉高压水注射模型证实，HCV核心蛋白削弱HBsAg疫苗的保护效果，还抑制HBV复制和基因表达。研究成果回答了项目提出的关键科学问题，提前完成了预期目标，为研究HCV/HBV混合感染的致病机制和治疗策略提供了新线索（见已发表的PLOS ONE文章）。.鉴于项目提前顺利完成，我们同时开展了新的研究方向。HBV因为抗病毒治疗以及免疫压力等因素导致其以较为复杂的群体“准种形式”存在于机体。HBV准种是否在肝病重症化过程中发挥作用仍不清楚，我们提出“HBV准种间的相互作用可能是导致HBV感染重症化的关键机制”这一科学假说。我们从肝衰竭患者血清中获得两个独特的HBV毒株SH-DPS和SH。SH-DPS在病毒基因组前S区存在两段缺失突变；SH序列含有A1762T、G1764A、G1896A等经典BCP区突变和HBeAg突变。SH-DPS和SH以4:1的比例共存。将HBV-SH与HBV-SH-DPS以1:4的比例共转，病毒复制明显增强；并在小鼠中诱导异常增强的宿主免疫反应。该结果首次揭示不同HBV变异毒株的共存可能通过调节宿主免疫反应程度来促进免疫介导的肝损伤，对于了解HBV准种间相互作用及致肝炎重症化机制具有重要参考意义（已发表在Journal of Virology)。