抗病毒广谱中和抗体是在感染性疾病患者中发现的一类稀有抗体，其产生机制对通用疫苗设计具有重要指导意义。超高水平体细胞突变、超长CDR3等特征是这类抗体的共性。目前，尚未有疫苗免疫方法成功诱导广谱中和抗体的产生。本课题拟利用广谱中和抗体前体基因敲入和胚系CDR3敲入的小鼠模型，解析广谱中和抗体的进化规律。我们将免疫小鼠模型，利用杂交瘤技术和特异单B细胞分选等免疫学方法筛选高亲和力B细胞，并克隆表达抗体基因进行病毒中和实验。在此流程上，我们将聚焦于广谱中和抗体进化中的瓶颈环节，研究关键突变位点的产生机制，最终利用小鼠免疫学模型探寻诱导广谱中和抗体的疫苗免疫方法。本课题结合小鼠遗传学模型和免疫学方法，拟阐明广谱中抗体产生中的关键位点突变问题。这一问题的顺利解决可以为HIV、流感、新冠等通用疫苗的设计提供新理论。

Antiviral broadly neutralizing antibodies (bnAbs) are a class of rare antibodies found in HIV-, influenza- and 2019nCoV-infected patients, and their discovery is of great significance for the development of universal vaccines. Anti-HIV bnAbs have some common characteristics, such as ultra-high level of somatic hypermutations, long CDR3 and auto-/polyreactivity. No bnAbs have been elicited in humans or mouse models by vaccination so far. In this project, we will explore the molecular mechanism of bnAbs evolution using mouse models carrying a precursor V gene of bnAb and a putative germline CDR3 gene in all B cells. Upon activation, B cells undergo Somatic Hypermutation processes to diversify the V gene and the CDR3. In contrast to previous mouse models where a matured CDR3 was used, our mouse model carries germline CDR3 which has the ability to undergo diversification including insertions that generates long CDR3, possibly giving rise to bnABs. B cell clones with increased antigen-binding ability will be screened by hybridoma or FACS technology. The antibody gene sequence will be determined by RT-PCR and sequencing technology. The antibodies will then be validated by neutralizing assays and mutations on the V gene and CDR3 will be characterized. We will also test whether improbable mutations in bnAbs may be the bottleneck of bnAb production. By comparing the known bnAbs sequences, we determined key amino acid sites that are improbable in the evolution of antibodies. By mutating these sites in advance in mouse models, we will verify whether overcoming these critical mutation sites could accelerate the production of neutralizing antibodies. The overall goal of this study is to elucidate the evolution of bnAbs including the diversification of the CDR3 during Somatic Hypermutation and determine critical mutations that hinders elicitation broadly neutralizing antibodies. The results of this study will provide new information for universal vaccine design.