靶向RSV post-F蛋白的单克隆抗体筛选及其在疫苗构象评估中的应用

Screening of RSV post-F-specific monoclonal antibodies for vaccine conformational evaluation

摘要:

呼吸道合胞病毒(respiratory syncytial virus, RSV)是引起婴幼儿下呼吸道感染的主要病原体之一，其融合蛋白(F)是介导病毒入侵的关键靶点，具有预融合(pre-F)和融合后(post-F)两种构象。其中，对中和抗体最为敏感的抗原表位仅存在于pre-F构象上，因而成为当前疫苗和抗体药物开发的焦点，然而pre-F构象处于亚稳态，在膜融合或自发触发后会不可逆地转变为post-F构象，因此，特异性识别post-F的抗体可用于定量检测疫苗中post-F的含量，从而评估疫苗的稳定性及纯度。基于此，本研究建立了涵盖从免疫到抗体筛选全流程的兔源单B细胞抗体平台，并利用该平台成功获得两株对RSV post-F具有高特异性和亲和力的兔单克隆抗体。表位鉴定结果表明，二者均结合于site I之外的区域。初步应用验证了该抗体在疫苗原液检测中的可用性，提示其在抗原构象稳定性与纯度评估方面具备良好的应用潜力。本研究为RSV及其他呼吸道病毒疫苗的研发提供了重要的平台支持与工具抗体。

Abstract:

Respiratory syncytial virus (respiratory syncytial virus, RSV) is a leading cause of lower respiratory tract infections in infants and young children. The viral fusion (F) protein, which mediates viral entry into host cells, adopts two distinct conformations: the pre-fusion (pre-F) and post-fusion (post-F). The epitopes most sensitive to neutralizing antibodies are exclusively present on the pre-F conformation, making it a major focus of current vaccine and antibody development. However, the pre-F conformation is metastable and undergoes an irreversible transition to the post-F conformation upon membrane fusion or spontaneous triggering. Therefore, antibodies specifically recognize the post-F conformation can be used to quantify the post-F content in vaccine products, thereby assessing the conformational stability and purity. In this study, we established a rabbit-derived single B-cell antibody platform covering the entire workflow from immunization to antibody screening. Using this platform, we successfully obtained two rabbit monoclonal antibodies with high specificity and affinity for RSV post-F protein. Epitope mapping revealed that both antibodies bound to regions outside of site I. Preliminary validation demonstrated the utility of these antibodies in detecting the post-F protein in vaccine drug substance, suggesting their potential for assessing antigen conformational stability and purity. This work provides important platform support and tool antibodies for developing vaccines against RSV and other respiratory viruses.