Project 1: Definition of the structural principles underlying broadly protective humoral immunity to coronaviruses

项目摘要--项目1：界定广泛的基本结构原则 保护性体液免疫 尽管新冠肺炎疫情加速了SARS-CoV-2疫苗的发展 史无前例的速度，没有获得许可的疫苗能够产生广泛的保护，抵御人类的大范围感染 冠状病毒。因此，迫切需要疫苗来产生广泛的保护作用，以防止目前 流行的和远亲的贝塔冠状病毒，用于预防大流行。拟议的项目旨在 识别交叉反应和广泛中和的抗β冠状病毒抗体所针对的表位以获得 贝塔冠状病毒刺突三聚体表面存在的靶点抗原图，以指导我们的疫苗设计 努力。识别刺激性受体结合域的广泛中和肉瘤病毒抗体有 然而，最近发现的它们不会与其他亚属的成员发生交叉反应。以前的研究 已经表明，比S1亚基更保守的棘波融合机制(S2亚基)含有 以交叉反应多克隆抗体为靶标的保守表位。虽然有一些β冠状病毒交叉- 反应性的单抗是已知的，深入了解表位的多样性是广泛的目标 缺乏中和抗体及其对中和的定量贡献，从而阻碍了 合理设计疫苗，激发广泛免疫力。我们将使用三种方法来确定分子 通过揭示冠状病毒的类型、特异性和多样性来确定广泛的抗体介导的冠状病毒免疫的决定因素 针对所有三个主要的贝塔冠状病毒亚属的广谱中和抗体(肉瘤病毒， MerbecVirus，和EmbecVirus)。首先，我们将表征多克隆的结合和中和呼吸 用纳米疫苗免疫的非人灵长类动物血清共显示多种不同的RBD-和 基于刺激物的抗原。其次，我们将确定这些细胞中交叉反应抗体的表位特异性。 用血清学分析和直接显示多克隆抗体与疫苗配对的复合体的血清 和异种抗原的冷冻电子显微镜观察。最后，我们将从 非人灵长类多价纳米疫苗免疫及其结构特征的研究 分辨率以及它们的绑定、中和和保护广度。

PROJECT SUMMARY – PROJECT 1: Definition of the structural principles underlying broadly protective humoral immunity Although the COVID-19 pandemic has accelerated the development of SARS-CoV-2 vaccines at an unprecedented pace, no licensed vaccines elicit broad protection against a large spectrum of human coronaviruses. There is therefore an urgent need for vaccines inducing broad protection against currently circulating and distantly related betacoronaviruses for pandemic preparedness. The proposed Project aims to identify epitopes targeted by cross-reactive and broadly neutralizing anti-betacoronavirus antibodies to obtain an antigenic map of targets present at the surface of betacoronavirus spike trimers to guide our vaccine design efforts. Broadly neutralizing sarbecovirus antibodies recognizing the spike receptor-binding domain have recently been discovered, however, they do not cross-react with members of other subgenera. Previous studies have shown that the spike fusion machinery (S2 subunit), which is more conserved than the S1 subunit, harbors conserved epitopes targeted by cross-reactive polyclonal antibodies. Although a few β-coronavirus cross- reactive monoclonal antibodies are known, a deep understanding of the diversity of epitopes targeted by broadly neutralizing antibodies and their quantitative contribution to neutralization is lacking, thereby hindering the rational design of vaccines eliciting broad immunity. We will use three approaches to determine the molecular determinants of broad antibody-mediated coronavirus immunity by unveiling the types, specificities, and diversity of broadly neutralizing antibodies targeting all three main betacoronavirus subgenera (sarbecovirus, merbecovirus, and embecovirus). First, we will characterize the binding and neutralizing breath of polyclonal sera from nonhuman primates immunized with nanoparticle vaccines co-displaying multiple different RBD- and spike-based antigens. Second, we will determine the epitope specificities of cross-reactive antibodies in these sera using serological assays and by directly visualizing polyclonal antibodies in complex with vaccine-matched and heterologous antigens using cryo-electron microscopy. Finally, we will isolate monoclonal antibodies from nonhuman primates immunized with multivalent nanoparticle vaccines and characterize their structures at high resolution as well as their binding, neutralizing, and protective breadth.