乙型肝炎病毒前S/S基因突变对抗HBV免疫应答的影响及其临床意义

Hepatitis B virus (HBV) preS/S gene mutations are associated with immune escape, affacting diesase progression and therapeutic efficacy. In addition, the mutations may cause misdiagnosis and potentially threat blood transfusion safety. The study is to clarify the influence and relevant mechanisms of HBV preS/S gene mutations on anti-HBV immune responses, the underlying mechanisms, as well as their clinical implications. Study schedules are as follows: 1) Population-based screening of HBV preS/S gene mutations in patients is performed by direct sequencing to clarify their prevalent characteristics in clinic. Special attention is paid to the impact of the mutations on HBsAg status and clinical presentations. Frequency of epitope-specific T cells is detected using ELISPOT. The mutations in intrahepatic HBV cccDNA from patients receiving biopsy are analyzed by clonal sequencing to verify if the mutations correspondingly occur in cccDNA pool. 2) Long-term follow-up is conducted and clonal sequencing is used to elucidate evolution of the mutations and their relationship with disease progression and therapeutic effcacy in clinic. Frequency of epitope-specific T cells is monitored. 3) Infulence of preS/S gene mutations on B and T cell epitopes is studied using bioinformatics. Replicons of various preS and S gene mutants and their wild-type counterparts are constructed respectively to investigate viral replication capacity, and HBsAg level and immunogenicity. In addition, the impact of preS1 deletion on HBV SP2 promotor activity is assayed. 4) Adeno-associated virus-mediated preS1-deleted HBV persistent infection model is established in mice for monitoring impact of the muntant infection on immune responses in vivo. The applicants have a solid accumulation in relevant studies, with advantage of key technique and abundant sample resources. Results are important for management of HBV infection.

HBV前S/S基因突变可致免疫逃逸，影响疾病进展和疗效，并可造成漏诊、输血安全等问题。本研究旨在阐明HBV前S/S基因突变对抗HBV免疫应答的影响、相关作用机制及临床意义。方案：1)大样本直接测序筛查前S/S基因突变的临床流行特点，重点分析突变对HBsAg等指标及病程进展的影响；ELISPOT检测表位特异性T细胞频率；克隆测序分析肝穿病例肝组织cccDNA是否同步突变。2)长期随访部分患者，克隆测序分析突变株演变规律，监测特异性T细胞频率。3)生物信息学分析突变对B、T细胞表位影响；构建各种前S、S基因突变株和对应野生株复制子，测定病毒复制力、HBsAg水平和反应原性；测定前S1缺失对SP2启动子活性影响。4)建立腺相关病毒介导的前S1缺失HBV持续感染小鼠模型，监测前S1突变株感染对抗体和T细胞应答的影响。申报人与课题组研究基础扎实，具关键技术和样本资源优势。结果对HBV防治具重要意义。

本研究拟在前期工作基础上，通过较大样本病例随访，临床患者研究结合体外细胞与体内动物水平实验研究，深入阐明HBV前S/S基因突变的临床发生特点、发生演变机制、对免疫学应答的影响及其临床意义。.本课题取得的主要成果：1. 对538例不同疾病进程HBV感染患者前S/S区基因测序及缺失突变与疾病进展相关性分析结果显示，慢性HBV感染患者中，随着疾病进展HBV前S基因缺失率呈上升趋势，其中前S1基因缺失突变在肝硬化患者显著增高，前S2基因缺失突变在肝癌患者显著增高，前S基因缺失可能参与驱动HBV慢性感染的疾病进展。2. 对598例HBV感染患者S基因主要亲水区（MHR）新增N-糖基化位点突变与HBsAg/HBsAb双阳性及HCC相关性分析结果显示，在双阳性患者中HBV S基因MHR区新增N-糖基化突变与HCC发生密切相关，提示双阳性叠加MHR新增糖基化突变是HCC发生的高风险因素，可做为慢性HBV感染患者发生HCC的独立预测指标。3. 针对HBV包膜蛋白11个已鉴定的CTL表位，分析显示多个表位变异检出频率与疾病进展相关。进一步进行体外表型、计算机模建、免疫功能和动物实验研究表明，首次明确了sM133L突变可形成与HLA分子亲和力较高的新型CTL表位，是影响某些HBV慢性感染患者炎性反应波动的一个始动因素。4. 我们通过对数例HBsAg阴性HBV DNA阳性的隐匿性HBV感染（OBI）的HCC患者的长期随访，结合HBV前S/S基因序列分析，发现有1例典型的OBI HCC患者携有多种免疫逃逸相关突变的9种病毒株，其中5种突变以往文献未见报道新型免疫逃逸相关突变。通过系统的功能学研究，我们明确了包括新型突变在内的9种突变均可以显著或基本消除HBsAg的抗原性，进而影响机体对HBV感染细胞的免疫监视。5. 动物实验结果显示，某些前S缺失型蛋白与野生型前S蛋白相比较可诱发较强的免疫反应，提示该类突变有可能是驱动疾病进展的因素之一。.本课题共发表论文13篇（8篇为第一标注），包括SCI论文4篇，IF 12.9分；中文核心期刊论文9篇，项目负责人为通讯作者8篇。研究成果在重要的国内外学术会议上交流6次；培养硕士研究生4名（3名已毕业），获国家发明专利授权1项，主编专著1部。总体上完成了课题任务书的研究指标。