基于冷冻电镜的猪圆环病毒2型中和构象表位结构解析及其抗体介导的中和作用机制研究

Neutralizing antibody is the main factor of anti-virus immunity, and it is also one of the important detection indicators of vaccine protective immunity. PCV2 commercial inactivation and subunit vaccines can induce high levels of neutralizing antibodies, which can effectively prevent and control related diseases caused by PCV2 infection. The Cap protein of PCV2 is the only structural protein, which is associated with its infection and immunity. Previous studies showed that all epitopes of ours PCV2 neutralizing mAbs against Cap protein are conformational epitopes which is difficult to complete solve using conventional methods. Furthermore, the antibody-mediated neutralization mechanism of PCV2 is also unclear. Based on the above scientific issues, we proposed the following four aspects of research: 1) Cryo-electron microscopy was used to solve the structures of different kind of PCV2 neutralizing epitopes; 2) Identification of key amino acids which take part in the antigen-antibody interaction from two aspects of virus and antibody，respectively; 3) Processes analysis of PCV2 attachment, internalization and uncoating using neutralizing mAbs and further clarify its mechanism; 4) The neutralization mechanism of these mAbs will be also evaluated in pigs. The results will reveal and update the molecular basis of neutralizing antibodies production and illuminate the antibody-mediated neutralization mechanism of PCV2 infection which has potential application value.

中和抗体是抗病毒免疫的主要因素，也是疫苗免疫效果重要检测指标之一。PCV2商品化灭活和亚单位疫苗均可以诱导产生高水平中和抗体，能够有效防控PCV2感染所引发的相关疾病。Cap蛋白是PCV2的唯一结构蛋白，与病毒感染和免疫密切相关。前期研究发现，我们所制备的大量PCV2 Cap蛋白中和性单克隆抗体的表位均为构象表位，采用常规方法不能完全解析其表位，且这些抗体所介导的中和作用机制尚不清楚。基于上述科学问题，我们拟进行以下四方面研究: 1)采用冷冻电镜技术解析PCV2不同类型中和性单克隆抗体的构象表位；2）利用点突变技术从病毒和抗体两方面鉴定参与抗原抗体反应的关键氨基酸；3）运用这些中和性抗体对病毒吸附、内化和脱衣壳过程进行分析，阐明其中和作用机制；4）以猪为模型，体内验证上述中和性抗体的中和作用机制。本项目将揭示和更新PCV2中和抗体产生的分子基础及其介导的中和作用机制，具有潜在应用价值。

猪圆环病毒2型（PCV2）是一种免疫抑制性病毒，其在猪群中的感染给全球养猪业造成严重的经济损失。PCV2的衣壳蛋白（Cap蛋白）是该病毒唯一结构蛋白，与病毒的感染和免疫息息相关。本研究报告了一种中和性单克隆抗体3A5，该抗体可以与PCV2a，PCV2b和PCV2d基因型的病毒结合，通过阻断病毒与PK15细胞的吸附来中和PCV2。为了进一步探索其中和作用机制，我们通过冷冻电镜（cryo-EM）单颗粒分析技术，解析了与该单抗Fab片段相结合的PCV2复合物的结构。Fab结合位点定位于病毒五倍体对称轴的顶端边缘，每个表位覆盖两个相邻Cap蛋白的18个氨基酸。通过分析Genebank中2,273条PCV2的ORF2序列，我们发现83.33%的表位氨基酸（15/18）是保守的。通过定点突变和病毒拯救技术发现表位中某些氨基酸的突变对单抗3A5的中和活性具有重要影响。该研究揭示了这种广谱性PCV2中和抗体的分子和结构基础，并为针对PCV2感染的疫苗设计和治疗性抗体开发提供了新的重要信息。