以FcR为基础并以外膜蛋白主要中和位点为靶点的新型RSV粘膜疫苗研究

Respiratory syncytial virus (RSV) is the leading cause of respiratory disease, particularly pneumonia and bronchiolitis, in infants, children and adults, resulting in significant morbidity and mortality worldwide. The failure of the clinical trial of a formalin-inactivated RSV (FI-RSV) vaccine during the 1960's indicates that the development of RSV vaccine is a long-term and tough journey. RSV is mainly transmitted through mucosal sites; however there are no currently available safe and efficient mucosal vaccines capable of inducing strong mucosal immunity cross-protecting against RSV virus challenge. Presented on the viral membrane, G and F proteins are the only proteins of RSVs that induce neutralizing antibodies, thus playing significant roles in protecting against virus infection. As such, these two proteins are considered to be important vaccine targets. The native F protein forms trimeric structures at the viral surface, and several important neutralizing epitopes were identified in the F protein in addition to the G protein. Based on (i) the ability of Fc to induce mucosal immune responses as well as cross-protection against mucosal pathogens via the natural infection pathway, and (ii) the ability of foldon (Fd) trimeric motif to promote recombinant proteins form native trimeric structure to increase immunogenicity, we thus hypothesized that mucosal delivery of a recombinant protein containing the main neutralizing epitopes of G and F proteins of RSV fused with Fc of IgG and Fd trimeric motif will induce broad mucosal and system immunity, including cross-neutralizing protective immunity, against a broad spectrum of RSV strains. The specific aims of the proposed study include: 1) the rational design of mucosal RSV vaccines based on the major neutralizing epitopes of G and F proteins fused to Fc immunoenhancer plus Fd trimeric motif; 2) the comparison, optimization, and confirmation of immune responses and neutralizing activity of designed novel mucosal RSV vaccines in mouse and non-human primate (NMP) animal models; and 3) preclinical evaluation of immune efficacy and cross-protection of optimized mucosal RSV vaccines. The long-term goal of the proposed study is to develop a novel and effective non-live mucosal RSV vaccine capable of inducing broad mucosal and systemic immune responses that cross-protect against divergent RSV strains, without causing immune-enhanced diseases. The proposed mucosal RSV vaccine will be very safe, thus is suitable for rapid production with cost-effective and scalable manufacture.

发展有效的疫苗是防治呼吸道合胞病毒（RSV）感染的关键。迄今，尚无有效的 RSV 疫苗。如何诱导机体产生有效的粘膜保护性中和抗体是 RSV 疫苗发展的关键。RSV 外膜 G 和 F 蛋白是诱导中和抗体产生的主要抗原，是发展 RSV 疫苗的主要靶点。FcR介导的粘膜免疫对呼吸道传播病毒有很强的免疫保护作用，是粘膜病毒疫苗发展的方向。天然 RSV 病毒 F 蛋白以三聚体形式存在，因此融合促三聚体形成分子 Foldon (Fd) 的 RSV 疫苗可形成 F 蛋白的天然空间结构以增强免疫反应。本课题拟发展以 FcR 为基础、融合 G 和 F 蛋白主要中和表位及 Fd 三聚体模式分子的 RSV 新型粘膜疫苗；优化免疫条件和最佳免疫方案；并系统研究候选疫苗免疫机制和功能。长期目标是发展不引起疫苗病理反应的、安全有效的新型 RSV 粘膜疫苗。该研究将为发展以增强粘膜免疫保护作用的疫苗提供理论基础和数据。

呼吸道合胞病毒是导致婴儿、儿童和成年人呼吸道感染，尤其是肺炎和支气管炎的重要病原之一。迄今，国内外仍然没有临床使用的安全有效的疫苗。本研究按主要进行了RSV病毒新型疫苗的探索性研究。按照实验设计，首先将目的蛋白与能介导形成二聚体蛋白形成的分子Fc和介导形成三聚体蛋白的Fd分子进行融合表达。利用上述蛋白探索了RSV病毒G164-186-F85-438重组表达的蛋白诱导的免疫反应性及中和抗体产生情况。试验结果表明，上述重组蛋白能引起良好的免疫反应，产生较高的体液免疫反应，但没有诱导产生中和抗体。其后，根据生物信息技术分析和结构特分析及对RSV 蛋白可能的中和表位功能区蛋白分析，又设计出表达RSV-F151-522蛋白的中和表位功能区蛋白的技术方案。通过小鼠免疫实验发现，该重组蛋白具有良好的免疫原性，可诱导产生高滴度的抗体，同时抗体具有中和活性。采用小鼠和非人灵长类猕猴免疫实验发现重组蛋白可诱导产生良好的免疫反应。这为发展RSV新型疫苗奠定了基础。