全分子全人源抗狂犬病病毒中和性抗体的制备、鉴定及中和表位的分析

Rabies is a zoonosis, of which mortality rate is almost 100% and is seriously threatting of public health security in China. According to the categorization of exposure defined by the World Health Organization (WHO), the most severe cases, Category III or Category II of immunodeficiency patient, require wound rabies vaccination, and direct injection of rabies immunoglobulin (RIG). At present, rabies prophylaxis after exposure includes the use of human rabies immunoglobin (HRIG) and equine rabies immunoglobin (ERIG). Nevertheless, the use of these two immunoglobins is constrained by problems such as the diffculty of ensuring the quality of antibodies, potential virus reinfection and causing allergic reactions. The use of fully human monoclonal antibody(mAb), which has neutraling potential, can address those problems. However, the technology to establish human mAbs is immature and similar studies remain blank in domestic and international reports. The purposes of this research, based on previous study of the isolation of human Fabs with rabies-virus-neutralizing activity from a human immunized phage display library, are to explore and prepare mutiple strains of fully human and whole molecular anti-rabies virus neutralizing antibodies against different epitopes of glycoprotein of rabies virus, which including human IgM monoclonal antibody, human IgM V-human IgG Fc whole molecular antibody and human Fab-human IgG1 Fc whole molecular antibody, by applying human immunoglobin gene locus transgenic mice, which are firstly established by our laboratory in China, in combination with FACS-HTS high throughput screening technology. Optimizing different designs of potent neutralizing antibodies against different epitopes will be useful to improve neutralizing protective response significantly in prophylaxis and therapy of different subtypes of rabies infections. The clarification of neutralizing epitopes of rabies virus can provide new targets for the rabies subunit vaccine design.

狂犬病为人兽共患疾病，死亡率几乎是100%，我国是受其危害最为严重的国家之一。目前，用于狂犬病病毒暴露接触后预防的免疫球蛋白是人抗狂犬病病毒免疫球蛋白（HRIG）和马抗狂犬病病毒免疫球蛋白（ERIG），但存在抗体品质难以保证、病毒再传染和引起过敏反应等问题。应用全人源具有中和作用的单克隆抗体可解决这些难题，但其制备技术不成熟，国内外尚未见类似的研究报道。本课题拟用我们建立的我国第一个人免疫球蛋白基因座转基因小鼠和FACS-HTS高通量筛选技术，探索、制备针对狂犬病病毒糖蛋白不同表位的多株全分子全人源抗狂犬病病毒中和性抗体：全人源IgM单克隆抗体，人IgM V区-人IgG Fc全分子抗体，人Fab-人IgG1 Fc全分子抗体，优化针对不同表位的高效中和抗体用于预防和治疗不同亚型狂犬病病毒株的感染，有希望显著提高中和保护作用。狂犬病病毒中和表位的阐明，可为狂犬病亚单位疫苗的研制提供新的靶点。

狂犬病为人兽共患疾病，死亡率几乎是100%。目前，用于狂犬病毒暴露接触后预防的免疫球蛋白是人抗狂犬病毒免疫球蛋白和马抗狂犬病毒免疫球蛋白，但存在抗体品质难以保证、病毒再传染和引起过敏反应等问题。应用全人源具有中和作用的单克隆抗体可解决这些难题，但其制备技术不成熟，国内外尚未见类似的研究报道。本课题研究内容包括六部分：全人源抗狂犬病毒单克隆抗体的制备与特性分析；全人源抗狂犬病毒G蛋白scFv、scFv-Fc融合抗体的制备及活性分析；全人源抗狂犬病毒Fab抗体的制备及中和活性分析；全人源双表位特异性抗狂犬病毒IgG抗体的制备及鉴定；全人源抗狂犬病毒抗体对狂犬病毒暴露后的预防作用和安全性评价；抗狂犬病毒中和性抗体针对的RVG中和表位的序列和空间构象分析。本课题以灭活狂犬病毒CTN株、重组RVG蛋白作为抗原，免疫转人IgM基因小鼠，共获得10株分泌全人源单克隆抗体的杂交瘤细胞株，可特异性结合CTN株或重组RVG蛋白。构建了全人源抗狂犬病毒scFv抗体库，获得1株具有中和活性的抗狂犬病毒RVG scFv抗体，在此基础上制备的RVG scFv-Fc融合抗体，增强了其分子稳定性和中和活性。本课题还构建了全人源抗狂犬病毒Fab抗体库，获得4株核酸序列不同的Fab抗体，其中Fab094可与狂犬病毒特异性结合，具有较好的中和效价，在此基础上制备的纳米偶联抗体Fab094-CPNPs，其中和活性明显提高。还制备了人源双表位特异性抗狂犬病毒IgG抗体，能显著提高与RVG的亲和力，特异性结合不同的RVG抗原表位，产生叠加或协同效应。Fab094及 Fab094-CPNPs对狂犬病毒暴露后具有较好的预防作用。预测中和性抗体Fab094、IgG091针对RVG的结合位点为空间表位构象，可能的结合位点序列为CXXL。本课题研发的全人源抗狂犬病毒中和性抗体对狂犬病毒暴露接触后具有较好的预防作用，为研制狂犬病预防性、治疗性抗体药物提供了候选抗体分子，对狂犬病的防治具有重要意义。