Structure-guided immunogens to elicit pan-coronavirus B cell responses

项目摘要 由严重急性呼吸道综合征病毒2型（SARS-CoV-2）冠状病毒（CoV）引起的全球大流行 强调了开发下一代病毒疫苗的必要性， 关注的Sarbecoviruses和Merbecoviruses。一种方法是实现基于结构的免疫原 设计策略，引发对表面暴露的刺突（S）病毒上保守位点的体液应答 糖蛋白潜在的位点包括受体结合结构域（RBD）和受体结合基序（RBM） 病毒进入所必需的。S上RBD以外的其他保守位点也可能是中和和抑制的靶点。 和提供广泛CoV保护的非中和反应。项目1将结合联合收割机的计算，结构， 生物物理和单B细胞分析，以设计S和RBD免疫原， 对冠状病毒免疫。基于CoV序列的进化分析，单一抗原不太可能 赋予对整个β冠状病毒属足够广泛的免疫力;因此，我们预计将产生一种 含多价S或RBD的疫苗（项目3）。最初，我们使用CoV优先考虑ACE 2受体作为 概念验证，但设想我们的设计原则可以扩展到其他新兴的冠状病毒，其中ACE 2 不是受体。施密特实验室将实施结构导向的免疫原设计策略， 将免疫应答集中于RBM和RBD上的保守表位。我们将使用异源冠状病毒 RBD作为分子支架，呈现选定的ACE 2结合CoV RBM并使用进一步的免疫焦点 工程化聚糖以掩盖非保守表位。Scheuermann实验室将使用 重建以鉴定现存的CoV作为另外的基于S的免疫原。埃莱贝迪实验室将分离出 从自然感染的，接种疫苗的，或幼稚的人类受试者的单克隆抗体（mAb），以确定冠状病毒交叉 S上的反应性保守表位被中和和非中和mAb识别。我们将定义 mAb交叉反应性、交叉中和和效应器功能的广度（与项目3一起）。弗里蒙特 实验室将通过X射线晶体学和低温电子显微镜（Fremont）进行结构分析 实验室）的分离的单克隆抗体，以确定S上的靶向表位;这一信息将有助于迭代免疫原 优化以引发对这些新定义的交叉反应性表位的应答。项目1的产出将是 优化的RBD和S基因转移到项目3中，用于VSV或ChAd疫苗平台中的疫苗制剂。

Project Summary The global pandemic caused by Severe Acute Respiratory Syndrome virus 2 (SARS-CoV-2) coronavirus (CoV) has highlighted the need to develop next-generation viral vaccines that offer protection against future Sarbecoviruses and Merbecoviruses of concern. One approach is to implement structure-based immunogen design strategies that elicit humoral responses to conserved sites on the surface-exposed spike (S) viral glycoprotein. Potential sites include the receptor binding domain (RBD) and the receptor binding motif (RBM) required for viral entry. Other conserved sites on S, outside the RBD, may also be targets of both neutralizing and non-neutralizing responses offering broad CoV protection. Project 1 will combine computational, structural, biophysical, and single B cell analyses to design S and RBD immunogens that elicit broad-spectrum humoral immunity against CoVs. Based on evolutionary analysis of CoV sequences, it is unlikely that a single antigen will confer broad enough immunity against the entire Betacoronavirus genus; we therefore anticipate generating a polyvalent S or RBD containing vaccine (with Project 3). Initially, we prioritize ACE2-receptor using CoVs as a proof-of-concept but envision that our design principles can be extended to other emerging CoVs where ACE2 is not the receptor. The Schmidt laboratory will implement structure-guided immunogen design strategies to focus immune responses towards conserved epitopes on the RBM and RBD. We will use heterologous CoV RBDs as molecular scaffolds to present selected ACE2-binding CoV RBMs and further immune focus using engineered glycans to mask non-conserved epitopes. The Scheuermann laboratory will use ancestral reconstructions to identify extant CoVs as additional S-based immunogens. The Ellebedy laboratory will isolate monoclonal antibodies (mAbs) from naturally infected, vaccinated, or naïve human subjects to define CoV cross- reactive, conserved epitopes on S recognized by neutralizing and non-neutralizing mAbs. We will define the breadth of mAb cross-reactivity, cross-neutralization, and effector functions (with Project 3). The Fremont laboratory will perform structural analyses by X-ray crystallography and cryo-electron microscopy (Fremont laboratory) of the isolated mAbs to define targeted epitopes on S; this information will aid in iterative immunogen optimization to elicit responses to these newly defined, cross-reactive epitopes. The output of Project 1 will be optimized RBD and S genes to transfer to Project 3 for vaccine formulation in VSV or ChAd vaccine platforms.