Self-Assembling Spike-EBR Nanoparticles as a Vaccine Platform Technology Against SARS-CoV-2 and Future Pandemic Coronaviruses.

项目摘要/摘要 新冠肺炎大流行是由人畜共传播贝塔冠状病毒引起的第三次疫情 (贝塔冠状病毒)在过去的20年里。因此，迫切需要新的疫苗策略来控制正在进行的 大流行和预防未来的冠状病毒暴发。信使核糖核酸疫苗已经成为一个理想的平台 快速反应疫苗的开发，但临床研究表明，中和抗体滴度会引起 基因疫苗的滴度比蛋白纳米粒(NP)疫苗低约10倍。这是一个令人担忧的问题 关于出现对疫苗不那么敏感的SARS-CoV-2病毒变异株- 诱导抗体。此外，只有不到25%的世界人口完全接种了疫苗。因此，快速响应 需要一次注射和/或更低剂量就能引起强大抗体反应的疫苗技术， 以确保对VOCs的持久保护，降低成本，并加快全球分销。此外，预防 未来的冠状病毒大流行需要开发一种通用的冠状病毒疫苗，以引起交叉反应免疫 通过集中对保守表位的反应来应对广泛的冠状病毒毒株。的范围 建议的研究是设计和评估新的疫苗策略，以增强基于mRNAs的效力。 快速反应疫苗和促进通用冠状病毒疫苗的开发。该提案以EBR为基础 NP技术，它修饰膜蛋白，如冠状病毒尖峰蛋白(S)，以自组装成病毒- 类似于从细胞表面发芽的NPs。NP组装是通过插入一个短的氨基酸序列来诱导的 进入细胞质尾部，从运输所需的内体分选复合体中招募蛋白质 (ESCRT)途径。在小鼠身上的初步研究表明，低剂量注射呈现SARS的EBR纳米粒- 冠状病毒2型S蛋白诱导的中和抗体效价是可溶性S蛋白和蛋白基纳米粒的10倍 这显示了S蛋白的受体结合结构域。EBR NP技术将应用于 完成三个目标：i)设计编码修饰的SARS-CoV-2 S-EBR结构的杂交基因疫苗 它会在细胞表面表达，并自我组装成类似病毒的NPs，从而诱导出更强大的 抗体反应优于批准的辉瑞/莫德纳疫苗，同时保留了制造特性 基因疫苗对T细胞的激活作用。Ii)设计S-EBR NPs，包装并交付S或S-EBR mRNA 疫苗作为脂肪NPs的替代品。S说，这种递送方式将增强基因疫苗的效力。 S-EBR纳米粒上的蛋白质可诱导强大的抗体反应，促进有效的细胞内递送，以及 将RNA疫苗靶向自然感染SARS-CoV-2的组织，以诱导局部免疫反应。 Iii)设计和评价基于S-EBR NP的马赛克全长通用冠状病毒候选疫苗 多株冠状病毒膜相关S蛋白诱导的交叉免疫应答 广泛的冠状病毒和预防未来的暴发。拟议的疫苗策略可能会直接 加强对新冠肺炎全球卫生危机的影响，推进应对下一次大流行的应急准备。

Project Summary/Abstract The COVID-19 pandemic represents the 3rd outbreak caused by zoonotic transmission of a beta-coronavirus (beta-CoV) in the last 20 years. Hence there is an urgent need for new vaccine strategies to control the ongoing pandemic and prevent future CoV outbreaks. mRNA vaccines have emerged as an ideal platform for the development of rapid-response vaccines, but clinical studies have shown that neutralizing antibody titers elicited by mRNA vaccines are ~10-fold lower than titers elicited by protein nanoparticle (NP) vaccines. This is a concern with regards to the emergence of SARS-CoV-2 variants of concern (VOCs) that are less sensitive to vaccine- induced antibodies. In addition, less than 25% of the world population is fully vaccinated. Thus, rapid-response vaccine technologies are needed that elicit potent antibody responses with a single injection and/or lower doses, to ensure lasting protection against VOCs, reduce costs, and accelerate global distribution. Moreover, prevention of future CoV pandemics requires the development of a universal CoV vaccine that elicits cross-reactive immune responses against a broad spectrum of CoV strains by focusing responses to conserved epitopes. The scope of the proposed research is to design and evaluate new vaccine strategies to enhance the potency of mRNA-based rapid-response vaccines and facilitate universal CoV vaccine development. The proposal is based on the EBR NP technology, which modifies membrane proteins such as CoV spike (S) proteins to self-assemble into virus- resembling NPs that bud from the cell surface. NP assembly is induced by inserting a short amino acid sequence into the cytoplasmic tail designed to recruit proteins from the endosomal sorting complex required for transport (ESCRT) pathway. Initial studies in mice showed that low-dose injections of EBR NPs presenting the SARS- CoV-2 S protein elicited 10-fold higher neutralizing antibody titers than soluble S protein and protein-based NPs that displayed the receptor-binding domain (RBD) of the S protein. The EBR NP technology will be applied to accomplish three goals: i) Design a hybrid mRNA vaccine encoding the modified SARS-CoV-2 S-EBR construct that would be expressed at the cell surface and self-assemble into virus-resembling NPs to elicit more potent antibody responses than the approved Pfizer/Moderna vaccines, while retaining the manufacturing properties and T-cell activation of mRNA vaccines. ii) Engineer S-EBR NPs to package and deliver S or S-EBR mRNA vaccines as an alternative to lipid NPs. This delivery approach would enhance mRNA vaccine potency as S proteins presented on S-EBR NPs induce potent antibody responses, facilitate efficient intracellular delivery, and target mRNA vaccines to tissues that are naturally infected by SARS-CoV-2 to induce local immune responses. iii) Design and evaluate mosaic S-EBR NP-based universal CoV vaccine candidates that present full-length membrane-associated S proteins from multiple CoV strains to elicit cross-reactive immune responses against a broad spectrum of CoVs and protect against future outbreaks. The proposed vaccine strategies could have direct impact on the COVID-19 global health crisis and advance our emergency preparedness for the next pandemic.