单细胞筛选结合深度测序快速获取HCV人源中和抗体及其抗病毒特性研究

Hepatitis C virus (HCV) is one of the main pathogens that leads the global causes of liver disease and liver cancer. Currently, the intervention is limited by the lack of a vaccine and universally successful drug treatments. Human antibodies with broadly neutralization to virus are potential therapeutics, preventing and treating viral infections, to protect the infected person. Rapid capture of HCV broadly neutralizing antibodies (bNAbs) would be significant for vaccine development and individualized virus prevention.. Both the single cell sorting and deep-sequencing-based DNA sequencing of immunoglobulin genes (Ig-seq) are the newest technologies in fast antibody screening. Our group has laid good foundation in both technologies and HCV research. Under these circumstances, we could develop a novel strategy by combining both methods for HCV bNAbs fast screening. Promisingly, we would obtain genes of anthropogenic HCV bNAbs with the following procedure: first off, collect peripheral blood lymphocytes (PBMC) from blood samples with advantage of neutralization activity; secondly, analyze the nucleic acid sequence of the extracts from PBMC with deep sequencing.. Anti-HCV monoclonal antibody (mAb), with verified high affinity and broad neutralization, can be gained by in vitro expression and pan out from few objects. Based on the antigen epitope and antiviral mechanisms analysis, with these potential mAbs, therapeutic antibody drugs and prophylactic HCV vaccine can be developed. Furthermore, our strategy can cover the full set information of antibodymic and significantly reduce the operation procedures. So it is a realistic target to obtain humanized antibody with low cost and high-efficiency, and to create a platform for individualized immunoassay and virus prevention based on this research..

丙型肝炎病毒（HCV）是当前威胁人类健康的主要传染病原之一，尚无能够有效预防其感染的疫苗。中和抗体具有抵抗病毒保护机体的作用，快速获取广谱中和抗体的下一代筛选技术对于HCV病毒的防治具有重要意义。前期研究中我们采用单细胞筛选进行了大量的抗体研究，也通过建立基于深度测序的抗体基因分析技术（Ig-seq）成功获得了HIV广谱中和抗体。本项目拟对两者进行结合优化，初步实现新一代HCV抗体筛选策略，即在对具有中和活性优势的HCV感染血样提取外周血淋巴细胞（PBMC）后，采用深度测序分析直接获取其中的抗HCV中和抗体基因。然后表达优势基因，获取具有高亲和力和广谱中和性的抗HCV人源单抗；对有活性优势的单抗进行抗病毒特性研究，分析其作用靶点与机制。本研究可实现人源中和抗体的低成本高效获取，有望在此基础上建立个体化的免疫分析与病毒防治平台，并为开发有效的HCV治疗性抗体药物和预防性疫苗奠定基础。

丙型肝炎病毒（HCV）在全球约有1.7 亿感染者，其中相当比例的感染者因此罹患肝硬化甚至肝癌。近年直接抗病毒药物迅速发展，数据显示能够有效的实现病毒清除，但是尚无有效的疫苗和抗体成功应用于HCV 的防治。广谱中和抗体具有抵抗病毒保护机体的作用。利用中和抗体的抗病毒特性，分析其具体的抗原抗体结合与病毒阻断机制，有助于更全面地揭示病毒与宿主免疫系统间的相互作用关系，为开发新一代HCV 疫苗奠定基础。本研究利用全人源单抗新技术，通过对单个抗原特异性B 细胞直接进行抗体基因扩增，在此基础上快速制备人源抗HCV单抗，并对其抗病毒的作用机制进行分析。 . 通过利用所建立的HCV中和抗体筛选平台，从HCV抗体阳性患者中筛选获得中和抗体活性优势样本。采用HCV E2探针，对优势样本中抗原特异性记忆性B细胞进行单细胞分选。对细胞裂解后，进行单个B细胞基因扩增获取其免疫球蛋白编码序列。将扩增获得的基因克隆至表达载体，转染CHO或293F细胞后对上清进行抗体活性检测，从中获得了10株具有HCV抗原结合活性的抗体基因和相应的抗体产物。通过中和活性检测和分析，我们发现其中两株抗体HNC5和HNC43具有广谱中和活性。.利用所获得的HCV广谱中和抗体HNC5/43进行抗病毒作用和机制的研究。通过采用丙氨酸扫描，我们已经获得了抗体的抗原结合位点，发现HNC43与对照抗体AR3A的结合位点相似，但HNC5的结合位点则有较大区别。另外，采用胚系抗体分析，我们发现HNC5抗体具有独特的胚系来源，并且成熟的路径较短，是进行靶向疫苗研究的良好工具，并将在此发现基础上进行深入探索。 . 综上所述，本研究建立了完整中和抗体筛选平台，掌握了快速从单个B细胞直接进行抗体制备的技术，并籍此获得了数株具有广谱中和活性的HCV人源广谱中和抗体。通过对所获抗体的抗病毒中和活性与作用机制进行分析和研究，为进一步进行新型HCV疫苗的设计和自主抗体药物研发提供充分的依据，具有重要的理论意义和应用价值。