新型抗体对慢性乙型肝炎病毒感染的治疗作用与机制研究

Chronic hepatitis B virus infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The approved anti-HBV drugs can only induce disease remission, but not effectively eradicate the virus. In our previous studies, we found a novel mAb E6F6, which targets a unique epitope on hepatitis B surface antigen (HBsAg), could profoundly suppress the levels of HBsAg and HBV DNA via Fcγ receptor-dependent phagocytosis. E6F6 regimen efficiently prevented initial HBV infection, and reduced viral dissemination from infected hepatocytes in human-liver chimeric mice. E6F6-based immunotherapy also facilitated the restoration of anti-HBV T-cell response in mice. Based on these findings, this proposal aim to evaluate the therapeutic effects of E6F6 mediated immunotherapy alone or in combination with interferon in various HBV mouse models, including HBV transgenic mice, hydrodynamic injection (HDI)-based HBV carrier mice and FRG human-liver chimeric mice. We also would like to systematically investigate the molecular mechanism and structural basis to explain how and why the binding epitope difference impacts the in vivo viral clearance potency. The expected results of our proposal may provide more better understanding of the effects and the underlying mechanism of antibody based immunotherapy against persistent viral infection, as well as facilitate the development of therapeutic antibody against chronic HBV infection.

慢性乙型肝炎病毒（HBV）感染可导致慢性肝炎、肝硬化和肝癌等严重疾病，现有药物仅缓解疾病进展，无法实现有效治愈。我们在前期研究中发现识别HBV外膜蛋白（HBsAg）上1个特定表位的抗体（E6F6）能在HBV小鼠模型中通过Fcγ受体介导的phagocytosis持久抑制HBV和HBsAg，阻止HBV肝内感染的扩散并部分恢复耐受宿主对HBV的T细胞应答功能，可能成为慢性HBV感染的治疗新手段。在上述基础上，本项目拟进一步在HBV携带/感染动物模型（HBV转基因小鼠、基于尾静脉高压注射的HBV携带小鼠、FRG人肝嵌合小鼠等）中全面评估E6F6抗体单独或与干扰素（IFN）联合的治疗作用，系统研究识别不同表位的抗体Anti-HBV治疗作用差异的分子机制及与之相关的结构基础。预期研究成果将为阐释抗体对持续性病毒感染的治疗作用和机制提供科学依据，也将为发展针对慢性HBV感染的治疗性抗体奠定基础。

本项目对识别HBV表面蛋白HBsAg上的aa119-aa125表位的E6F6抗体在动物模型中的治疗作用和机制进行了系统研究。通过在DEN/HBV-Tg小鼠模型中的治疗评估，我们发现多剂E6F6治疗能在小鼠中降低HBV相关的HCC发生风险。在AAV-HBV和HDI-HBV小鼠模型中探索了治疗性抗体和干扰素及Poly IC联合用于抗HBV治疗的策略和效果，发现抗体联合Poly IC治疗HDI-HBV持续携带小鼠，具有显著的协同治疗作用，有助于实现HBsAg的持续阴转和Anti-HBs的产生。在冷冻电镜结构解析中，我们发现E6F6抗体的两个抗原结合臂(Fab)倾向于结合在HBsAg颗粒上相邻的两个蛋白单体上，而不造成跨颗粒间的交联的大聚合物。我们采用重组抗体技术改变E6F6人源化抗体两个Fab臂之间的距离，可导致抗体诱导形成的免疫复合物变大，也会同时造成抗体介导吞噬效应的明显减弱。基于本研究，我们推测对于颗粒状病毒样抗原而言，抗体识别表位与抗体两Fab臂之间的空间距离和相对位置关系决定抗体是否诱导形成颗粒间交联的大的免疫复合物，相比之下，未交联的、小而分散的抗原抗体复合物对吞噬作用可能更为有利。本项目利用动物模型揭示了抗体对持续性HBV感染的治疗作用和机制，为发展针对慢性乙肝的治疗新技术奠定了基础。

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