发热伴血小板减少综合征病毒糖蛋白中和表位的鉴定

In recent years, a novel Bunyavirus called Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) has been detected and circulated in the central and east regions of China, with more evidences indicating its worldwide distribution. Clinically, the patients suffered from severe hemorrhage and multiple organ failure, resulting in death with high fatality. SFTSV can be transmitted by person-to-person contact. Currently, there are no effective prophylactic or therapeutic measurements available in the clinical settings. Due to its ability to mutate rapidly, the development of SFTSV-specific epitope-based vaccine, which is composed of viral functional epitopes, will be of great importance in containing this new emerging disease. As the first step for such vaccine development, this study focuses on the identification of conserved neutralizing epitopes on viral GN and GC glycoproteins. It involves of three steps: 1) single B cell antibody cloning and phage display technology are used, respectively, to develop human antibody with wide spectrum neutralizing activities; 2) a novel computational algorithms called Multiple Copy Simultaneous Search (MCSS) is applied to predict the epitopes based on the antibody variable domain structure; 3) the predicted epitopes will be verified by experimental methods. The applicant’s preliminary work has demonstrated the feasibility of the project as the predicted epitope has been confirmed experimentally. The outcomes of the proposed work will give multiple strains of human broadly neutralizing antibodies with their binding epitopes being predicted and validated. This will serve as the foundation for SFTSV epitope-based vaccine design. In addition, the other objectives of this project will include personnel training, publication, and patent application. More importantly, this study could provide references and new vaccine design strategy for other related diseases’ vaccine research.

发热伴血小板减少综合征病毒是近年来流行于我国中东部地区的一种新病毒，已呈全球性分布。该病毒可引起感染者产生严重的出血和多脏器衰竭，死亡率高，存在人传人现象。临床无特异性治疗、预防措施。基于该病毒易于突变的本质，研发由多个中和表位组成的表位疫苗对于控制该病的流行具有重要意义。作为研发表位疫苗的第一步，在该病毒糖蛋白分子上鉴定保守中和表位是本项目的研究内容。首先应用单B细胞抗体克隆、噬菌体展示技术制备具广谱中和活性的人源抗体；应用多切块分子优化算法、结合抗体可变区结构，预测该抗体的抗原表位，并通过实验对该表位加以验证。申请者前期工作表明预测的表位与实验结果具有良好的符合性。通过本研究，能获得多株具广谱中和活性的人源抗体，预测其抗原表位并验证，为该病毒的表位疫苗设计奠定基础，同时可实现人才培养、文章发表和专利申报之目标。本项目的研究成果也为其它相关疾病的疫苗研究提供借鉴。

发热伴血小板减少综合征（Severe Fever with Thrombocytopenia Syndrome, SFTS）是近年来流行于我国中东部地区一种新发传染病，俗称“蜱虫病”，一度引起罹患地区民众的恐慌和广泛的社会关注，其病原为一类新型出血热病毒，称为发热伴血小板减少综合征病毒（Severe Fever With Thrombocytopenia Syndrom Virus, SFTSV）。SFTS病死率较高（10-30%）。该病多为散发，亦可引起家庭聚集性、医院内感染性暴发；存在严重的人传人现象，目前临床上缺乏有效地预防和治疗手段。本项目利用噬菌体展示技术（phage display）平台筛选人源中和抗体，利用多切块分子优化（Multiple Copy Simultaneous Search, MCSS）计算方法对中和抗体的结合表位进行预测，并通过实验对预测的表位加以验证。.通过项目实施，共获得具有广谱人源中和单抗分子6株（其靶标均为GN糖蛋白），分别命名为MAb4-5、MAb4-6、MAb1F6、MAb1B2、MAbTC3和MAbTB10。这6株单抗在体外微量中和实验中对多个SFTSV毒株（毒株来源为江苏、浙江、安徽、河南）均表现出良好的中和效果（可达70-100%）。完成了对部分单抗分子的动物体内保护性实验，MAb1F6对感染SFTSV的小鼠模型可以达到60%的保护力。利用MCSS，在GN分子上共获得4条多肽分子，分别称为GNP1、GNP2、GNP3和GNP4。在体外中和实验中，它们均能不同程度地抑制病毒颗粒对受体细胞的侵袭（抑制率可达30-60%），说明通过MCSS预测出的多肽均是通过阻碍病毒与受体结合而起到抑制作用的。.本项目的产出包括科研论文发表12篇，其中SCI收录5篇，申报国家发明专利4项，均获得授权。研究成果具有良好的应用前景，研发的人源中和单抗可以作为候选分子进行抗体药物的开发；来自于GN糖蛋白上的衍生多肽可以作为重组表位疫苗的候选成分待进一步研究开发。