基于噬菌体展示文库的新布尼亚病毒人源治疗性抗体筛选

Severe fever with thrombocytopenia syndrome virus (SFTSV), a novel phlebovirus in the Bunyaviridae family, was first isolated from an SFTS patient and characterized by Chinese researchers in 2011.Since then, SFTSV has been found in 11 provinces of China with case-fatality rates being more than 30%, causing a serious threat to human health. However, neither antivirals nor vaccines are available for SFTSV at the moment. In order to prevent an outbreak it is imperative to recruit therapeutic antibodies as an public－health defense line. Furthermore long serum half-life of therapeutic antibodies provides either neutralization protection to the patients or efficacy against infection to health people who live in high infection risk area. Yet, the development of the SFTSV neutralization antibody (nAb) at unprecedented speed, which is the core part of the antibody therapy. Here we plan to provide an optimized nAb selection and characterization system based on human scFv phage display library. Strategically, we will setup a parallel outline to select Gn and Gc glycoproteins of SFTSV nAbs respectively, then to find the best combination of Gn and Gc proteins with best neutralization activity. We will take two optimized approaches to improve the efficiency of phage display based nAb selection and characterization system. Firstly we will constructe a new helper phage, which could minimize translation-ending ratio of the phage display library. Secondly we will express glycoprotein on two platform to provide to different conformations (soluble/membrane integrated) of glycoprotein, which will enhance the coverage of various glycoprotein conformation. Finally, for safety concern, we will performance mAb neutralization experiment on antigen expression pseudo virus platform.

发热伴血小板减少综合症病毒即新布尼亚病毒SFTSV，于2011年在我国首次从SFTS病人中分离获得并确认为布尼亚病毒科白蛉热病毒属病毒。目前，这种病毒在我国11个省份传播，局部地区死亡率高达30%，给人民健康造成极大危害。然而，迄今仍没有针对该病毒的有效疫苗和特效抗病毒药物的研究报道。本项申请目的就是想解决这一问题。我们拟建立一种优化的人源噬菌体展示文库筛选系统，策略上先用独立寻找亲和抗体，再交叉寻找高效中和抗体组合的策略，针对新布尼亚病毒囊膜蛋白Gn与Gc进行筛选。筛选效率上先对噬菌体展示技术本身效率，构建了新型的辅助噬菌体；再通过不同方式表达新布尼亚病毒的囊膜蛋白，提高了筛选过程中噬菌体文库对靶蛋白各种形态的覆盖率。最后，通过假病毒为载体进行抗体中和实验，提高安全性与效率。相关研究成果将为白蛉热病毒属病毒病的防治提供理论参考。

本项目以非免疫人源scfv噬菌体展示文库对新布尼亚主要抗原进行筛选为出发点，研究非免疫人源抗体库对于新布尼亚主要抗原的可筛选性。完成了通过自主设计的人类抗体库引物序列组合，构建高效的非免疫人源scfv噬菌体展示文库的目标。完成了顺利表达与纯化新布尼亚表面抗原Gn与Gc的目标。通过4轮针对抗原的的筛选，得到了scfv序列富集结果，发现在非免疫人源噬菌体展示库中VH4-39这个基因座被大量富集，而且被富集的抗体多是核酸序列与原始基因座完全一致的未经免疫成熟的基因座型抗体。通过ELISA试验检测所构建到IgG1亚型的抗体与新布尼亚抗原的结合特性，发现基因座抗体亲和力普遍很弱。为了回答VH4-39在非免疫人源库中可以富集，但多以亲和力弱的基因座抗体存在的特点。研究近一步通过高通量免疫库测序技术，对一位新布尼亚感染急性期病人PBMC样本，构建测序用抗体库。经过分析后发现VH4-39在急性期的调动水平可以达到总抗体重链数量的60%。这也从侧面回答了本研究工作中，为何以未感染新布尼亚感染史的健康人构建噬菌体库，可以有效富集抗体，但所获得抗体亲和力较低的特点，当新布尼亚这类未曾大规模流行的病毒感染非免疫人群时，人类体内并无因既往免疫而储备的高亲力抗体，此时天然存在于非免疫人群中的低亲和力的基因座型抗体在急性期大量调动至关重要。有学者认为对于急性致死性感染病，比如埃博拉病毒，高致病性禽流感病毒，这类抗体被称为sos抗体，此类抗体并非直接中和病毒，而是减缓病毒感染速度，为更高活性的低丰度抗体在抗原的刺激下调动与免疫成熟创造时间，在进化中此类抗体的存在意义必不可少。..