多聚化抗原诱导高效体液免疫应答的机制研究

Recombinant protein vaccines have distinct advantages in safety and potential indications as compared to traditional attenuated or inactivated vaccines. Most approved vaccines are based on virus-like particles with arrayed epitopes. How such polymeric antigens induce humoral immune responses of high potency and specificity remains poorly understood. It is still a matter of empirical exploration from the discovery of functional protein domains to the design of effective vaccines. During the development of the first hepatitis E virus vaccine and human papillomavirus (HPV) vaccine, we found that multimerization significantly improved the immunogenicity of recombinant protein. Based on the immunogenicity of the particulate and polymeric antigen p239 is 240-fold higher than the dimeric antigen E2, in this proposal, we will perform a panoramic analysis of immune responses induced by p239 and E2, employing state of the art technologies such as Time of Flight Mass Cytometry (CyTOF), Flow Cytometry, Luminex, and antibody repertoire sequencing, to understand at the cellular and molecular levels how particulate and polymeric antigens induce humoral immune responses of high potency and specificity. We will identify the key cell populations and molecules that mediate the effective immune responses induced by particulate and polymeric antigens, and recapitulate the evolution history of functional neutralizing antibodies, with the ultimate aim of understanding the immunological mechanisms underlying the effectiveness of particulate and polymeric antigens. The knowledge obtained from this study will facilitate the design of new and efficacious vaccines.

重组蛋白疫苗在安全性和潜在适应症范围上具有突出优势。已上市的几种重组蛋白疫苗均以多聚化颗粒抗原为主要活性成分。申请人此前在戊型肝炎疫苗研究过程中，亦发现多聚化颗粒抗原的免疫原性较非多聚化抗原高出2个数量级以上，多聚化颗粒的形成是决定表位活性片段能否成为合格疫苗抗原的关键。但是多聚化抗原诱导高效体液免疫应答的细胞和分子机制仍不清楚，从发现关键中和表位到赋予强免疫原性而成为有效疫苗的过程仍停留在经验性探索阶段，缺乏理论指导。本项目拟对这一现象背后的免疫学机理进行深入探索，通过CyTOF、Flow Cytometry、抗体组深度测序等技术对多聚化与非多聚化重组蛋白在刺激抗原递呈、BCR簇集、TFH细胞活化、浆细胞的持久应答或产生抗体的质量等方面进行全面、系统、动态的全景式对比解析，鉴别出多聚化抗原引发高效体液免疫应答的关键细胞亚群及效应因子，揭示其作用机制，为新型疫苗设计提供理论依据。

疫苗是防控传染病最为经济有效的手段，业内普遍认为以病毒样颗粒为代表的多聚体抗原具有更强的免疫原性，但对其中的免疫机制尚缺乏系统研究。..立项以来，我们主要以课题组研发上市的戊型肝炎疫苗抗原作为多聚体抗体模式，探索了多聚体与寡聚体抗原诱导体液免疫应答的各关键环节，发现多聚体抗原相比寡聚体抗原具有以下优势：（1）可促进抗原注射位置的免疫细胞浸润，提高抗原从接种部位迁移至淋巴结的效率；（2）可增强抗原递呈细胞的激活，显著上调其对于抗原的摄取和递呈；（3）可在接种后快速诱导抗原特异性B细胞激活和增殖；（4）可更强烈地诱导抗原特异性的生发中心与浆细胞的分化。将单个B细胞研究技术与低偏差抗体组测序技术相结合，以小样本的戊肝疫苗接种者为研究对象，实现对戊肝疫苗接种全程中抗体的分子演化特征、质量及数量进行精准分析，发现基于多聚化抗原的戊肝疫苗能快速地诱导机体产生保护性抗体应答，保护性抗体无需长期的演化，从抗体分子层面揭示了戊型肝炎疫苗跨型别广谱保护的分子基础。..本项目的目标之一是通过新型小分子佐剂，使难以多聚化的免疫原具备多聚化抗原的强免疫原性。刺突蛋白是新冠疫苗的主要靶标，但将其制成颗粒化抗原有很大挑战。团队筛选获得利赛膦酸锌铝佐剂FH002C，与铝佐剂比，FH002C可上调抗原递呈相关共刺激受体表达并增强T、B细胞激活，促进Tfh、GC B和浆细胞的分化，可诱导显著优于铝佐剂组的体液免疫和细胞免疫应答，在小鼠、仓鼠、水貂、食蟹猴等动物中均表现出突出的免疫增强效应，候选新冠疫苗已完成临床前研究并申报临床试验。..综上，本项目按计划全景式描绘了多聚体与寡聚体抗原诱导的体液免疫应答差异，揭示了基于多聚体抗原的戊肝疫苗在人体中诱导保护性抗体应答的时相特征和跨型保护的分子基础。基于对体液免疫应答机制理解研发的利赛膦酸锌铝微纳米佐剂已申请专利、完成临床前研究并申报临床试验。代表性成果发表于The Lancet Respiratory Medicine、Cell Host & Microbe、Science Translational Medicine、Gut、Nature Communications等高水平期刊。