HLA-A*02:01限制性 CD8 +T细胞免疫反应对HCV清除和病毒逃逸突变的影响

Approximately, 20% of hepatitis C virus (HCV) infected individuals spontaneously clear the virus, while the rests progress to chronic infection which is a major risk for liver cirrhosis and hepatocellular carcinoma. However, the mechanism underlying chronic HCV infection is not fully elucidated. In our previous study, we found that HLA-A*02:01 is the only HLA class I gene which associates with HCV spontaneous clearance. Besides, high variations in NS5B2972-2980 amino acid sequences were observed among HLA-A*02:01 carriers who were chronically infected with HCV 6a. We therefore hypothesize that the HLA-A*02:01 restricted CD8+ T cell response plays a critical role in HCV spontaneous clearance and the mutation of HCV epitopes leads to immune escape. By owning the advantage of HCV “window period” infection individuals and the massive cases of HCV spontaneous clearance from our blood center, this study aims to compare the extent of T cell immune response stimulated by HLA-A*02:01 restricted HCV epitopes between HCV cleared and chronically infected donors, as well as to analyze the impact on the host cellular immune response by the mutations within A*02:01 restricted HCV epitopes of chronic HCV infection. In addition, with “window period” infected cases, we aim to understand the mechanism underlying HCV spontaneous clearance or chronic infection.

HCV感染人体后，约20%个体能够自发清除病毒，其余转为慢性，部分感染者发展成肝硬化或肝癌。目前HCV感染慢性化的机制尚未完全清楚。我们前期发现HLA-A*02:01是唯一与机体自发清除HCV相关联的HLA-Ⅰ类基因，且HLA-A*02:01阳性的HCV6a慢性感染者NS5B2972-2980氨基酸序列存在较大的变异，推测HLA-A*02:01限制性CD8+T细胞反应在机体清除HCV和HCV抗原表位突变所致的免疫逃逸中起重要作用。本项目拟利用血液中心能够获得“窗口期”HCV感染者和大量自发清除HCV献血者标本的优势，比较慢性感染和自发清除HCV者对HLA-A*02:01限制性HCV表位多肽刺激的特异性T细胞免疫反应；分析慢性HCV感染者A*02:01限制性HCV抗原表位的变异及其对细胞免疫反应的影响，并以“窗口期”感染者为跟踪检测研究队列，揭示机体清除病毒或慢性转归的免疫机制。

丙型肝炎是一种严重危害人类健康的常见病和多发病，由丙型肝炎病毒（Hepatitis C Virus，HCV）引起，主要经血源传播。HCV感染人体后，约20%个体能够自发清除病毒，其余转为慢性，部分感染者发展成肝硬化或肝癌。由于HCV序列高度变异，目前尚未研制出预防HCV感染的疫苗。为了研究HCV特异性T细胞抗原表位及其HLA-A*02:01限制性，本项目根据广州地区流行的两种主要HCV病毒株，即HCV 1b和6a的氨基酸序列设计重叠肽段798条。通过肽段库或单条多肽刺激HCV感染者和健康献血者的PBMC，ELISPOT检测T细胞应答水平，获得了5条HCV特异性T细胞抗原新表位。我们还比较了HLA-A*02:01+组和HLA-A*02:01-组的HCV氨基酸序列，发现2组之间的突变频率存在差异，这可能和病毒的免疫逃逸相关；对HCV感染献血者细胞免疫和体液免疫反应进行分析，发现慢性HCV感染者NK细胞亚群CD56dim NK中更多地表达抑制型受体（NKG2A+ NKG2C-），而自发清除组则更多地表达激活型受体（NKG2A- NKG2C+）；检测HCV慢性感染者和自发清除者血浆细胞因子，发现HCV自发清除组的促炎症反应的细胞因子IL-1α、IL-2、IL-9和TNF-α高于慢性感染组，而抑制炎症反应的细胞因子IL-10和IP-10低于慢性感染组；比较HCV慢性感染者和自发清除者T 细胞反应，发现自发清除组T细胞有更强的分泌IL-6（PMA刺激）和IL-10（HCV重组蛋白刺激）能力。我们还对广州地区流行的主要HCV毒株进行了分子进化分析。我们的研究结果将为HCV疫苗的研制和HCV预防策略的制定提供帮助。

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