戊型肝炎病毒中和抗体间协同作用的单向增强机制研究

Neutralizing antibody plays vital roles in infection blockage and immune clearance upon viral life cycle, identified sites of which are major targets in antiviral research, and functional antibody is also an ideal candidate for antiviral drug. In common cases, monoclonal antibody functionally acts alone in terms of single specific epitope recognition. The synergistic phenomenon is rare event in antigen-antibody interaction. Our previous studies found a couple of monoclonal antibodies, 8C11 and 8H3, bound with HEV antigen and authentic virion in unidirectional synergistic manner, i.e. the pre-incubation of 8C11 will significantly enhance the bind affinity of 8H3 against HEV subunit protein with at least 2 log, no synergistic effect in converse turn. The evidences from authentic HEV capture or HEV infection animal model verified this synergistic action. In the present grant application, we have collected a diffraction dataset of the crystal of triple-immune complex, E2s-8C11-8H3, and now try to solve the structure by molecular replacement in lights of our previous high-resolution E2s-8C11 complex structure (PDB no. 3RKD published in PNAS 2011). We will also pursue the cryo-EM structure of the couple mAbs in complex with HEV T=1 virus-like particles. The molecular mechanism of the synergistic effect by all the structures will be verified by mutagenesis assay on both E2 protein and mAbs via robust HEV cell and animal models. Hopefully, the unique enhancement mechanism will be unveiled in atomic level, which might contribute to understand the anti-HEV immune protection and synergistic effect among antibodies, and allow us to think about the potential immune function raised by synergistic antibodies.

中和抗体在阻断病毒感染和行使病毒免疫清除中发挥重要作用，其识别的位点是抗病毒研究的主要对象，抗体本身是良好的药物候选。单克隆抗体一般是单独行使功能的，抗体间协同作用较为罕见，本课题组前期工作发现戊型肝炎病毒的两株中和单抗8C11和8H3在结合实验和中和病毒时存在单向正协同作用，即8C11与抗原的预结合可使8H3的结合亲和力提高2个数量级，反之不然，真病毒捕获实验和体内动物保护实验证实了这种正协同作用。本课题拟在获得三蛋白复合物晶体衍射图的基础上解析E2s-8C11-8H3单抗原结合两个单抗的复合物晶体结构，同时解析结合2种单抗的戊肝病毒样颗粒低温电镜结构，并通过突变实验和细胞-动物模型研究，从分子水平阐明这对协同作用单抗的增强机制，为理解戊肝病毒的免疫保护机制和抗体分子间协同作用现象等提供结构基础和理论依据，并探讨协同抗体分子潜在的免疫学功能。

目前基于p239类病毒颗粒开发的戊型肝炎病毒预防性疫苗HecolinⓇ已经在中国上市，该疫苗具有良好的免疫原性和较高的保护率。本研究针对戊型肝炎病毒抗体盘中与8C11抗体具有协同作用的单克隆抗体，利用抗体间的协同作用制备抗原-双抗体免疫复合物，以X射线衍射晶体学为手段，解析免疫复合物的空间结构，对抗体所识别的中和表位进行定位的同时获得抗体间协同作用的结构基础，并辅以生化方法辅助阐明抗体间协同作用的分子机制。. 本课题按照预定的目标已经全面完成各项指标，通过酶联免疫吸附实验考察了抗体8C11对8H3与HEV抗原二聚体的结合能力的影响，抗体8C11存在时，8H3与抗原E2的结合能力提升了10倍；其次，成功制备高质量的8H3Fab晶体，晶体衍射的分辨率为1.98Å以及E2s-8C11Fab-8H3Fab复合物晶体分辨率3.5 Å。通过分析8H3Fab与E2s的相互作用界面，选择了13个氨基酸位点为8H3所识别表位的关键位点进行验证。复合物中，8H3的作用使得8C11Fab在空间位置发生6.3°的旋转。最后利用丙氨酸扫描的方式对8C11重链上与8H3相互作用的可能的氨基酸位点Lys8C11-H66，Ser8C11-H67，Asp8C11-H88，Arg8C11-H68进行单点突变和Lys8C11-H66，Ser8C11-H67，Asp8C11-H88三点联合突变，验证了突变抗体的活性，发现R68位点为影响8C11抗体活性的关键氨基酸位点；考察了单点及三点联合突变的8C11抗体对8H3的协同作用，得到8C11上影响抗体8C11与8H3相互作用的关键位点为Lys8C11-H66，Ser8C11-H67，Asp8C11-H88，即8C11通过上述三个氨基酸位点与8H3发生相互作用。从分子水平上初步验证了8C11Fab与8H3Fab分子之间的相互作用是导致抗体间协同增强作用的原因。