慢加急性肝衰竭患者HBV表位变异对特异性细胞毒T淋巴细胞应答影响的研究

It is pivotal to clarify virus-immunity interaction in the management of severe hepatitis B. Hepattis B virus(HBV)-specific cytotoxic T lymphocytes (CTL) is a key factor in immune clearance and pathologic damage. Our previous study showed that compared to patients with acute or chronic hepatitis B, patients with acute-on-chronic failure had higher frequency of 8 sorts of HLA-A2-restricted CTL epitopes. The study is to clarify the impact of epitopic mutation of HBV-specific CTL on immune function. The study design is as follows. (1) To analyze affinity of various CTL epitopes using T2-binding test and epitopic peptide/HLA-A2 molecule refolding test. (2) To construct paired wild-type and variant epitope recombinant DNA vaccines to inoculate HLA-A2/HBV hybridized transgene mice, followed by monitoring of virologic and liver function impairment parameters. CTL frequency is detected by pentamer staining. CTL activities are examined by cell killing and other immune functional assays using mouse spleen T cells. (3) Peripheral blood mononuclear cells are prepared from 60 HLA-A2-positive ACLF patients with HBV/C infection under follow-up. Frequencies of selected wild-type and mutant epitope-specific CTL are dynamically measured and immune functional assays such as cell killing are performed in the CTL positive cases. Clinical implications of CTL epitopic mutations are analyzed in combination of clinical and laboratorial parameters. Our investigation group has set up a database for follow-up of ACLF patients and mastered relevant experimental techniques. The study will offer important help for clarifying pathogenesis of ACLF and facilitating optimal management for this lethal illness.

阐明病毒与宿主免疫相互作用机制是重症乙肝防治研究的要点和难点，特异性细胞毒T淋巴细胞（CTL）是免疫清除与病理损伤的要素。我们前期研究发现，HBV相关慢加急性肝衰竭（ACLF）患者有8个HLA-A2限制性CTL表位变异率显著高于急、慢性乙肝患者。本研究旨在明确CTL表位变异对免疫功能的影响。方案如下：用T2细胞结合等实验分析变异CTL表位亲和力变化；构建野生和变异表位基因重组DNA疫苗，分别免疫HLA-A2/HBV杂合转基因小鼠，监测病毒学和肝细胞损伤指标，五聚体染色检测CTL频率，取小鼠脾T细胞进行细胞杀伤等免疫功能实验检测CTL活性；随访60例A2阳性C型HBV感染的ACLF患者，动态检测外周血选定的表位特异性CTL频率，对阳性样本检测CTL杀伤等免疫活性；结合临床和实验指标分析意义。项目组已建立ACLF患者随访数据库和相关实验技术。研究将为ACLF发病机制阐明与优化防治策略提供帮助。

HBV感染相关慢加急性肝衰竭（ACLF）病死率高，发病机制复杂。阐明病毒与宿主免疫相互作用机制是防治研究的要点和难点。特异性细胞毒T 淋巴细胞（CTL）是免疫清除与病理损伤的要素。本课题旨在通过研究较大样本的ACLF患者HBV高变异频率HLA-A2限制性CTL表位，结合临床、动物与体外实验研究，明确了CTL表位变异对CTL免疫应答的影响，解析其对ACLF发生的影响机制。. 本课题取得的主要成果：1. 发现多种变异与乙肝重症化相关，包括3种HLA-A2限制性CTL表位变异发生率随着急性乙肝、慢性乙肝和ACLF的疾病进程呈阶梯性显著增加。2. 发现ACLF患者有12个HLA-A2限制性CTL表位的检出率与急性和慢性乙肝患者组相比有显著差异，多数突变表位可显著降低CTL亲和力。3. 通过临床随访动态分析HLA-A2阳性慢性HBV感染患者CTL表位变异，发现在长期核苷（酸）类药物治疗下，病毒学突破可能与env335-343的表位变化相关。4. 发现 HBV 前S缺失突变发生率随乙肝疾病进展升高，并与隐匿性HBV感染相关，其机制包括影响抗原分泌和缺失了CTL表位等重要抗原表位引起病毒免疫逃逸等。5.发现了新的HBV重组基因型并明确了其对疾病进展和免疫表位的影响。6. 创建的血清HLA-A2/A11/A24的PCR分型技术获得国家发明专利授权。 . 共发表学术论文17篇，包括SCI论文7篇（其中6篇标注本基金第一资助，SCI IF累计为20分），项目负责人均为通讯或共同通讯作者；在国际会议上交流研究报告4篇次（3篇次为发言）；获得国家发明专利授权1项，并转让北京科卫临床诊断试剂有限公司；培养研究生毕业3名、青年科研骨干1名。总体上较好完成了课题任务书指标。. 课题研究中存在的主要问题是有1篇论文投稿要补充较大样本量ACLF患者变异表位特异性CTL频率和功能分析数据，需要HLA-A2阳性C基因型HBV感染ACLF患者的较多量血细胞，样本来源不易，影响了该文发表进度。