Structure-based design of coronavirus subunit vaccines

项目摘要 病毒亚单位疫苗安全、方便，但效果普遍较低。我们的总体假设是 一个固有的限制与亚基疫苗设计有关，在这种设计中，亚基表面被人工暴露 疫苗含有不利于疫苗效力的表位。人血清白蛋白受体结合区(RBD) 冠状病毒刺突蛋白由作为结构支架的核心亚结构域和受体组成。 与受体结合的结合基序(RBM)，包含中和表位。Rbd是质数。 亚基疫苗设计的候选者。在初步研究中，我们确定了核心亚区上的表位 MERS冠状病毒(MERS-CoV)RBD被埋在全长刺突蛋白中，但人工变成 暴露在重组RBDS中。我们进一步表明，这些表位通过以下方式严重降低疫苗效力 诱导强烈的非中和免疫反应，分散宿主免疫系统对 RBM上的中和表位。这一新发现揭示了病毒亚单位疫苗的内在局限性。 疫苗领域一直没有意识到的事情。在这项提案中，我们的目标是描述这种内在的局限性和 建立新的方法来克服它。我们使用来自高致病性冠状病毒的rbd，包括 MERS-CoV和SARS冠状病毒(SARS-CoV)作为模型系统。这份提案包含三个主要内容 冠状病毒RBD疫苗的设计方法。首先，我们将识别和表征人工 暴露了冠状病毒rbds核心亚区上的不利表位。为此，我们介绍了一部小说 概念，中和免疫原性指数(NII)，以评估每个表位对整体的贡献 疫苗的效力。我们将通过葡聚糖屏蔽或屏蔽核心亚区上的负性表位 重新浮出水面。这一设计增强了单独优化的RBD疫苗的效力。第二，我们将 构建以冠状病毒RBD核心区为结构支架的嵌合RBDS 另一种冠状病毒RBD的RBM作为免疫原点。核心上的不利表位 从第一个设计方法开始，子域就应该是静默的。核心子域接口和 将对RBM进行优化，以最大限度地提高嵌合RBD疫苗的稳定性。这一设计为我们准备迎接 未来高致病性冠状病毒的出现。第三，我们将构建纳米颗粒载体 冠状病毒RBD疫苗以一种人为暴露核心亚区上不利表位的方式是 重新埋藏在分子界面上，以提高RBD疫苗的效力。我们将使用老鼠来评估 上述工程RBD疫苗的免疫原性，并将使用动物模型(包括hDPP4-敲入 (Ki))小鼠和非人灵长类动物)，以评估选定的RBD疫苗应对活冠状病毒挑战。 总之，这项研究确立了人为暴露的不利表位为病毒的内在限制。 亚单位疫苗，并找到新的方法来克服它。因此，这项研究有望实现 使亚单位疫苗成为抗击病毒感染的更成功和更广泛使用的策略。

Project Summary Viral subunit vaccines are safe and convenient, but generally suffer low efficacy. Our overall hypothesis is that an intrinsic limitation is associated with subunit vaccine designs in which artificially exposed surfaces of subunit vaccines contain epitopes unfavorable for vaccine efficacy. The receptor-binding domain (RBD) of a coronavirus spike protein consists of a core subdomain that serves as the structural scaffold and a receptor- binding motif (RBM) that binds the receptor and contains neutralizing epitopes. The RBDs are prime candidates for subunit vaccine designs. In preliminary studies, we identified epitopes on the core subdomain of MERS coronavirus (MERS-CoV) RBD that were buried in the full-length spike protein but became artificially exposed in recombinant RBDs. We further showed that these epitopes severely reduce vaccine efficacy by inducing strong non-neutralizing immune responses and distracting the host immune system from reacting to the neutralizing epitopes on the RBM. This novel finding reveals an intrinsic limitation of viral subunit vaccines that the vaccine field had been unaware of. In this proposal, we aim to characterize this intrinsic limitation and establish novel approaches to overcome it. We use the RBDs from highly pathogenic coronaviruses, including MERS-CoV and SARS coronavirus (SARS-CoV), as the model system. This proposal contains three major design approaches for coronavirus RBD vaccines. First, we will identify and characterize the artificially exposed unfavorable epitopes on the core subdomain of coronavirus RBDs. To this end, we introduce a novel concept, neutralizing immunogenicity index (NII), to evaluate the contribution of each epitope to the overall vaccine efficacy. We will mask the negative epitopes on the core subdomain through glycan shielding or resurfacing. This design enhances the efficacy of the individually optimized RBD vaccines. Second, we will construct chimeric RBDs containing the core subdomain from one coronavirus RBD as the structural scaffold and the RBM from another coronavirus RBD as the immunogenic sites. The unfavorable epitopes on the core subdomain should have been silenced from the first design approach. The interface of the core subdomain and RBM will be optimized to maximize the stability of the chimeric RBD vaccines. This design prepares us for the emergence of highly pathogenic coronaviruses in the future. Third, we will construct nanoparticle-carried coronavirus RBD vaccines in a way that artificially exposed unfavorable epitopes on the core subdomain are re-buried at the molecular interfaces to enhance the RBD vaccine's efficacy. We will use mice to evaluate the immunogenicity of the above engineered RBD vaccines and will use animal models (including hDPP4-knock-in (KI)) mice and non-human primates) to assess the selected RBD vaccines against live coronavirus challenge. Overall, this research establishes the artificially exposed unfavorable epitopes as the intrinsic limitation of viral subunit vaccines and finds novel approaches to overcome it. Therefore, this research holds the promise of making subunit vaccines a more successful and widely used strategy in combating virus infections.