猪流行性腹泻病毒(PEDV)高效中和抗体的产生机制

The protective mechanism of most vaccines is primarily mediated through the effective induction of neutralizing antibodies, which is a critical factor for a successful vaccine. Porcine epidemic diarrhea virus (PEDV) is the major pathogen causing diarrhea of neonatal piglets and causes serious economic loss for pork industry in China. By studying PEDV, the project will closely focus the important question how the PEDV potent neutralizing antibody is induced. In this proposal, first, we will isolate the PEDV potent neutralizing monoclonal antibodies from sorted single B cells of PEDV vaccinated pigs using single B cell PCR antibody technology, and we will map the epitopes of these neutralizing antibodies. Second, we will explore and analyze the porcine antibody repertoire prior to or after PEDV inoculation using antibody next-generation sequencing (NGS) technology. The results of porcine antibody repertoire acquired by NGS will substantially enhance our understanding of the porcine B cell immunity. Finally, based on the known genes of PEDV potent neutralizing antibodies and the swine antibody repertoire by NGS, we will establish the phylogenetic trees of PEDV potent neutralizing antibodies, track the production and maturation process of these neutralizing antibodies in pigs following PEDV vaccination with the meta-analysis informatics analysis tools of sequencing data. We will identify the dominant immunoglobulin genes selectively expanded by PEDV antigen and the preferential immunoglobulin genes adopted by PEDV potent neutralizing antibodies. The project will clarify the development mechanism of PEDV potent neutralizing antibodies, which enhances substantially to elucidate swine humoral immune responses, and provides the critical hints to the control of PEDV infection and the design of PEDV new vaccines.

大部分疫苗的保护机制主要是通过诱导机体产生中和抗体，因此诱导产生高水平的中和抗体是免疫成功的关键因素。目前，猪流行性腹泻病毒(PEDV)是引起我国新生仔猪腹泻的主要病原。本项目以PEDV这个重要病原为切入点,围绕机体如何诱导产生高效中和抗体这个科学问题，阐明高效中和抗体的产生机制。首先，本项目利用猪单个B细胞PCR抗体技术，从PEDV免疫猪体内B细胞中分离到猪源的PEDV高效中和单克隆抗体并鉴定其中和抗原表位；其次，通过抗体高通量测序，建立PEDV免疫前后不同时间的抗体谱；最后，根据获得的高效中和抗体基因和猪抗体谱，利用生物信息学方法建立PEDV高效中和抗体基因的进化树，解析中和抗体在机体内的演化成熟过程。本项目可望阐明高效中和抗体的产生机制，有助于加深对猪体液免疫应答的认识，为PED的有效防控和新型疫苗的设计提供重要的理论指导。

猪流行性腹泻病毒(PEDV)是引起我国新生仔猪腹泻的主要病原。抗体是机体获得性免疫的重要组成部分，特别是具有保护性的中和抗体, 在机体抵抗PEDV感染中起着关键作用。因此阐述PEDV感染或疫苗接种后的猪抗体谱和机体如何诱导产生高效中和抗体这个科学问题，为PEDV有效防控和新型疫苗的设计提供重要的理论指导。本项目揭示PEDV感染或接种PEDV疫苗后的整个抗体谱，并分析PEDV病毒抗原诱导的优势基因；研究揭示猪PEDV免疫后，IgHV1S2, IgKV1-11和IgLV3-4分别是抗体谱中重链、κ链、λ链的优势抗体基因，并进一步发现在外周血和肠系膜淋巴结中抗体基因的使用特征不存在明显的组织间差异性；相较于猪PEDV免疫后的整体抗体谱，PEDV S蛋白特异性抗体基因对IgHV1-4、IgHV1-14、IgLV3-3具有使用倾向性。而猪PEDV感染后抗体谱中，抗体重链、κ链、λ链分别以IgHV1-4和IgHV1S2、IgKV1-11和IgKV2-10、IgLV3-3和IgLV3-5为主导基因。IgHV1S2 在PEDV感染和免疫后的抗体谱中为优势基因，说明了PEDV对某些抗体基因的使用偏嗜性。这些研究结果不仅增强了我们对PEDV抗原刺激后猪抗体谱的理解和认识，而且有助于PEDV新型疫苗的设计和研发。此外，构建了抗PEDV中和抗体PC10-IgA 高表达CHO细胞克隆株，并申请专利1项，为进一步研发PEDV治疗性抗体制剂奠定基础。最后，利用干细胞3D培养技术，通过肠隐窝干细胞体外培养成肠道类器官，建立PEDV等猪肠道冠状病毒体外感染模型。用于探索PEDV的致病机制及宿主和评价中和抗体的效果的体外感染模型，解决了PEDV缺少合适的体外感染模型的瓶颈问题。总之，项目执行期间申请专利1项，获得专利授权1项；共发表SCI高水平论文5篇和中文核心期刊2篇，达到或超过项目申报书的各项指标。