基于HBV preS1新表位制备HBV特异性全人中和抗体及其关键技术研究

Although Hepatitis B virus (HBV) vaccine is effective, the prevention of HBV infection under some special conditions, such as blocking mother-to-child transmission, liver transplantation of Hepatitis B patients, and protection from acute HBV exposure, relies more on HBV immunoglobulin (HBIG) applications. HBIG is purified from the plasma of blood donors. The composition of HBIG is complicated and its value is not stable. We also have to consider the biological safety of HBIG application. The antibody with similar activity prepared by antibody engineering is the best alternative of HBIG with great clinical application value. It is well known that HBV preS1 mediated the entrance of HBV into hepatocytes. We previously found that mouse monoclonal antibody against HBV preS1(aa91-107) could block the entrance of HBV into human primary hepatocytes. Non-immune antibody is currently the main method to prepare human antibody, but affinity of the antibodies made in this way is usually not high enough.In this program, we will combine in vitro lymph node cultivation, improved in vitro immunization program, antigen-biased antibody library construction, assembly and expression of full human antibody, to prepare human antibody against preS1(aa91-107), and test its potential application in blockade of HBV infection with in vitro and in vivo models. This program will further explore the key technological points in human antibody preparation, which will promote the progress and application of this important technology, and also, offer new means for prevention of HBV.

疫苗预防HBV感染确实有效，但在阻断母婴传播、HBV患者肝移植及急性病毒暴露防护等情况下多依赖HBV免疫球蛋白(HBIG)。HBIG来源于献血员血浆，成分复杂，效价不稳定，存在安全隐患，利用抗体工程方法制备HBIG替代品具有重要价值。部分HBV preS1特异性抗体能阻断病毒感染，但目前报道的表位有限。本室研究证实HBV preS1(aa91-107)是一免疫原性强的B细胞表位区域，其特异性鼠单克隆抗体能阻止病毒感染肝细胞，提示针对该表位的人抗体可能具有中和HBV的活性，有望用于制备HBIG替代品。本研究拟结合淋巴结体外培养、改良体外免疫程序、抗原偏向性人抗体库构建、全人抗体组装及表达技术，制备抗新表位preS1(aa91-107)全人抗体，应用体内外HBV感染模型验证其中和HBV活性。本项目是对全人抗体制备关键技术的深入探索，将推动该技术的进步与应用，并为HBV的预防提供新的方法和手段。

课题组前期发现HBV preS1(aa91-107)是优势性B细胞表位区域，制备了特异性的鼠单克隆抗体。在此基础上，本课题制备了HBV preS1特异性小鼠单链抗体，建立了基于邻位触及DNA杂交原理的preS1电化学检测免疫传感器，构建超大容量全人源单链抗体库，淘选出HBV preS1(aa91-107)特异性的全人源单链抗体。主要研究结果为：①应用噬菌体展示等技术克隆鼠单抗mAb-D8的轻、重链可变区序列，通过(G4S1)4短肽连接，获得一株亲和力约50 nM的抗preS1 单链抗体（ScFvD8）；在大肠杆菌和CHO细胞中大量表达。②通过同源建模、分子对接等手段构建了preS1(aa91-107)肽段与ScFvD8相互作用的3D模型，通过模型观测到二者的结合关键氨基酸残基；将ScFvD8的L96Tyr和H98Asp分别突变成Ser之后，获得一株亲和力提高10倍的突变体ScFvD8-M。③根据邻位触及DNA杂交原理，设计了preS1检测电化学免疫传感器；优化检测条件，确定DNA杂交时最佳浓度、温度和最适Mg2+浓度分别为；该传感器的线性范围为1pM到50pM，检测下限为0.1pM。④收集了健康成人外周血、骨髓血和脐带血标本，分离淋巴细胞，共提取RNA，逆转录成cDNA，PCR扩增抗体的VH、VL基因，通二者之间添加Loxp连接序列，重叠PCR拼接VH、VL基因，插入到噬菌粒载体pDF，电转大肠杆菌OMNImax构建ScFv形式初级抗体库。分别获得一个库容为5×10^7外周血及脐带血来源的初级抗体库和一个库容为5×10^7骨髓来源的初级抗体库。通过优化重组条件，初级库侵染表达Cre酶的BS1365菌株，经过Cre-Loxp酶介导的轻重链置换重组，获得一个库容为1×10^11的重组抗体库。⑤原核表达preS1抗原及合成preS1(aa91-107)肽段，经过4轮富集筛选，鉴定1000株克隆，获得4株特异性针对preS1的全人源ScFv抗体；经过鉴定，其中1株单链抗体能够用于建立HBV preS1的ELISA检测体系并在体外肝细胞培养中减少HBV的感染。综上所述，本研究建立了一种基于单链抗体和邻位触及反应的传感器检测系统，建立了大容量全人源抗体库，淘选出HBV preS1新表位特异性单链抗体，为HBV的诊断、预防，以及其他有意义靶标特异性抗体的制备奠定了很好的基础。