Rational design and evaluation of novel mRNA vaccines against MERS-CoV

摘要 传统的疫苗开发策略受到长期和昂贵的制造的影响，因此， 对新出现和再次出现的传染病往往不能迅速作出反应。相比之下，信使 RNA（mRNA）正在崛起为一种新的技术平台，用于开发针对病毒病原体的“按需”疫苗， 提供了吸引人的优点，例如无细胞生产、非病毒递送，以及简单、快速和低成本， 有效制造。进一步提高mRNA的稳定性和翻译效率， 它们的免疫机制，以及它们的保护效力的评估将有助于下一代的发展， 针对不同病毒病原体的mRNA疫苗技术。中东呼吸综合征 （MERS）冠状病毒（MERS-CoV）是一种高致病性、新出现的传染性病毒，构成持续威胁 全世界的公共卫生。目前还没有MERS疫苗被批准用于人类。MERS-CoV 刺突（S）蛋白，特别是其受体结合结构域（RBD），是重要的疫苗靶点。我们有 先前表明MERS-CoV RBD含有一个关键的中和结构域， 交叉中和抗体和保护人二肽基肽酶4转基因（hDPP 4-Tg）小鼠免受 MERS-CoV感染，疗效显著。然而，亚单位疫苗和其他传统疫苗的生产 疫苗具有局限性，例如低表达和复杂的纯化。为了解决这些未得到满足的 挑战，我们建议合理设计和评估新的mRNA疫苗，使用MERS-CoV作为模型 病原体和MERS-CoV S蛋白作为靶抗原。我们假设，通过适当的修改， 和优化，基于MERS-CoV S蛋白RBD的mRNA疫苗将表现出改善的稳定性， 在小鼠和非人灵长类动物（NHP）中提高翻译效率和增强免疫原性 模型，具有与基于RBD的亚单位疫苗相当的保护效力。具体目标是：（1） 合理设计MERS-CoV mRNA疫苗，提高稳定性和翻译效率，（2）仔细 优化mRNA制剂和免疫方案，以在体内评价其免疫原性 以及在野生型小鼠中的作用模式，以及（3）综合评估MERS-CoV的保护效力 mRNA疫苗，并阐明其在hDPP 4-Tg小鼠和NHP中的保护机制。值得注意的是，我们还将 研究微流控和下一代测序（NGS）分析等新技术的实用性 mRNA疫苗开发和评估中的B细胞反应。长期目标是发展一个安全的 和有效的mRNA疫苗，其能够（1）保持足够的数量和质量适合于工业- 规模化生产，以及（2）符合世卫组织的目标产品概况，以便在疫情爆发时迅速产生免疫力 环境和长期保护处于MERS CoV高持续风险的人。共同提出的项目 将阐明mRNA疫苗的保护机制，并提供急需的信息， 开发针对具有大流行潜力的各种病毒病原体的mRNA疫苗的指导方针。

Abstract Traditional strategies of vaccine development suffer from long-term and costly manufacture, and as a result, often fail to respond rapidly to newly emerging and reemerging infectious diseases. By contrast, messenger RNA (mRNA) is rising as a new technology platform to develop vaccines “on demand” against viral pathogens, offering attractive advantages such as cell-free production, non-viral delivery, as well as simple, fast and cost- effective manufacture. Further improvement upon mRNA's stability and translation efficiency, understanding of their immune mechanisms, and evaluation of their protective efficacy will facilitate the development of next- generation mRNA vaccine technologies against diverse viral pathogens. Middle-East respiratory syndrome (MERS) coronavirus (MERS-CoV) is a highly pathogenic, emerging infectious virus posing a continuous threat to public health worldwide. There are currently no MERS vaccines approved for use in humans. MERS-CoV spike (S) protein, particularly its receptor-binding domain (RBD), is an important vaccine target. We have previously shown that MERS-CoV RBD contains a critical neutralizing domain capable of inducing strong cross-neutralizing antibodies and protecting human dipeptidyl peptidase 4-transgenic (hDPP4-Tg) mice against MERS-CoV infection with outstanding efficacy. However, production of subunit vaccines and other traditional vaccines has limitations, such as low expression and complex purification. To address these unmet challenges, we propose to rationally design and evaluate novel mRNA vaccines, using MERS-CoV as a model pathogen and MERS-CoV S protein as a target antigen. We hypothesize that with appropriate modification and optimization, MERS-CoV S protein RBD-based mRNA vaccines will demonstrate improved stability, increased translation efficiency, and enhanced immunogenicity in both mouse and non-human primates (NHP) models, with protective efficacy on par with the RBD-based subunit vaccine. The specific aims are to (1) rationally design MERS-CoV mRNA vaccines with improved stability and translation efficiency, (2) carefully optimize mRNA formulations and immunization regimens towards in-vivo evaluation of their immunogenicity and mode of action in wild-type mice, and (3) comprehensively evaluate protective efficacy of MERS-CoV mRNA vaccines and elucidate their protective mechanisms in hDPP4-Tg mice and NHPs. Of note, we will also examine the utility of new technologies such as microfluidics and next-generation sequencing (NGS) analysis of B-cell response in mRNA vaccine development and evaluation. The long-term goal is to develop a safe and effective mRNA vaccine that is able to (1) maintain sufficient quantity and quality suitable for industrial- scale production, and (2) meet the WHO Target Product Profiles for rapid onset of immunity in outbreak settings and long-term protection of people at high ongoing risk of MERS-CoV. Together, the proposed project will shed light on protective mechanisms of mRNA vaccines, and provide much-needed information and guidelines for developing mRNA vaccines against diverse viral pathogens with pandemic potential.