猪圆环病毒2b/2d亚型优势中和表位鉴定及嵌合病毒样颗粒免疫原性研究

猪圆环病毒2型（PCV2）是引起猪圆环相关疾病的主要病原，是我国养猪业疫病防控的重点。PCV2现已发现8个亚型，目前上市疫苗主要针对PCV2b，而流行病学调查显示PCV2d检测阳性率逐年上升，在局部地区已占据主导。作为PCV2b疫苗免疫的逃逸毒株，PCV2d的流行给商用疫苗带来了压力。本课题组在前期研究中，成功制备了PCV2b和PCV2d的病毒样颗粒，解析了高分辨率的PCV2b-VLP晶体/冷冻电镜结构；结合筛选的PCV2b中和抗体库，初步分析了多个中和抗原表位区域。本项目拟进一步筛选PCV2b/2d-VLP的优势中和单抗，解析PCV2b/2d-VLP与单抗Fab片段复合物的冷冻电镜结构。鉴定PCV2b/2d-VLP两个亚型差异优势中和表位，构建包含PCV2b/2d中和表位的嵌合VLP，以期获得对PCV2b/2d两种亚型提供免疫保护的嵌合颗粒，为研发预防多亚型PCV2嵌合表位疫苗奠定基础。

Porcine circovirus type 2 (PCV2) was reported as the causative agent of post-weaning multisystemic wasting syndrome (PMWS), which is the critical point of disease prevention and control in swine industry of China. There are 8 sub-genotypes of PCV2, named PCV2a, PCV2b, PCV2c, PCV2d, PCV2e PCV2f, PCV2b, PCV2g and PCV2h. Most of the engineered chimeric PCV2 vaccine in the market, derived from PCV2b capsid protein shows superior prophylactic efficacy. However, the concerns have been raised over the efficacy of current commercial PCV2 vaccines due to that PCV2d sub-genotype identified in PCV2b vaccine failure case investigations has become the most predominant in some regions of China. In our previous study, we expressed and purified the soluble sole capsid protein of PCV2b/PCV2d, which was capable of self-assembly into virus-like-particles (VLPs). Moreover, we reported the crystal structure and cryo-EM structure of PCV2b-VLP at 2.8Å and 4.1 Å resolution, respectively. Furthermore, a large pool of neutralizing monoclonal antibodies (mAbs) against PCV2b-VLPs was established. In this proposal, initial efforts will be focused on determining the high-resolution structure of complex of PCV2b/2d-VLP in complex with Fab fragments of neutralizing monoclonal antibodies to map the specific neutralizing epitope by both X-ray crystallography and Cryo-EM approaches. Based on the PCV2b/2d neutralizing epitopes & structural differences, we are going to develop structure-based chimeric VLPs vaccine. The anticipative results would shed light on next generation broad-spectrum vaccine development.