Structure-based design of coronavirus subunit vaccines

项目摘要 病毒亚单位疫苗是安全和方便的，但通常具有低效力。我们的总体假设是 一种内在的限制与亚单位疫苗设计有关， 疫苗含有不利于疫苗效力的表位。受体结合结构域（RBD）的 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， 结合受体并含有中和表位的结合基序（RBM）。RBD是质数 用于亚单位疫苗设计的候选物。在初步的研究中，我们鉴定了在核心亚结构域上的表位， MERS冠状病毒（MERS-CoV）RBD被埋在全长刺突蛋白中，但被人工合成为 暴露在重组RBD中。我们进一步表明，这些表位严重降低疫苗的效力， 诱导强烈的非中和性免疫应答并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 疫苗领域还没有意识到的在本提案中，我们旨在描述这种内在限制， 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。该提案包含三个主要内容。 冠状病毒RBD疫苗的设计方法。首先，我们将人工识别和表征 暴露冠状病毒RBD核心亚结构域上的不利表位。为此，我们介绍一本小说 概念，中和免疫原性指数（NII），以评估每个表位对总体免疫原性的贡献。 疫苗效力我们将通过聚糖屏蔽来掩盖核心亚结构域上的阴性表位， 重铺路面这种设计增强了单独优化的RBD疫苗的效力。二是 构建以冠状病毒RBD核心亚结构域为骨架的嵌合RBD 以及来自另一种冠状病毒RBD的RBM作为免疫原性位点。核心上的不利表位 子域应该从第一个设计方法中被静音。核心子域的接口， 将优化RBM以最大化嵌合RBD疫苗的稳定性。这个设计为我们准备了 高致病性冠状病毒的出现。第三，我们将构建纳米颗粒携带的 冠状病毒RBD疫苗的方式，人工暴露的核心亚结构域上的不利表位， 重新埋在分子界面，以提高RBD疫苗的效力。我们将使用小鼠来评估 本发明的目的是提供上述工程化RBD疫苗的免疫原性，并将使用动物模型（包括hDPP 4敲入 (KI))小鼠和非人灵长类动物），以评估所选RBD疫苗对抗活冠状病毒攻击的能力。 总之，本研究确立了人工暴露的不利表位是病毒的内在限制， 亚单位疫苗，并找到新的方法来克服它。因此，这项研究有希望， 使亚单位疫苗成为对抗病毒感染的更成功和广泛使用的策略。

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.