Shark nanobodies enable identification of pan-sarbecovirus and pan-merbecovirus spike RBD sites of vulnerability

项目摘要 SARS-CoV-2是一种β冠状病毒，是正在进行的COVID-19大流行的病原体。在答复中， 世界范围内的努力导致了多种候选疫苗的快速开发，这些候选疫苗在以下方面显示出功效： 对照临床试验和一般人群的有效性。尽管取得了这些进展， 关注（VOC）继续出现，而未来冠状病毒人畜共患病溢出的大流行潜力 仍然很高。因此，我们研究工作的总体目标是指导泛冠状病毒疫苗研究和 为未来的大流行预防提供具有广泛特异性的免疫分子。为此我们 将询问从护士鲨克隆的VNAR纳米抗体库， 异源CoV刺突铁蛋白纳米颗粒（SpFN）免疫，即，用SARS-CoV-2 SpFN致敏， 用SARS-CoV-1 SpFN或MERS-CoV SpFN进行召回，以鉴定广泛保守的、中和的 表位分离的纳米抗体将以重组形式产生，并评估其功能和特异性 使用结合、亲和性和竞争作图测定，随后进行病毒中和和体内保护 问题研究基于我们的初步研究，我们预计我们将发现VNAR纳米抗体，可以靶向（i） 泛-肉瘤病毒表位，（ii）泛-梅贝科病毒表位，和潜在的（iii）泛-肉瘤病毒-梅贝科病毒 表位为了进一步探索这一点，我们将对我们目前广泛的一系列结构进行详细的结构生物学研究 肉瘤病毒反应性VNAR以及任何新鉴定的VNAR。利用X射线晶体学和冷冻电镜 通过成像，我们将确定每个VNAR的结合表位。这些原子级信息将用于映射 交叉中和CoV免疫表位，并提供可用于设计新的 免疫原，其将引发泛肉瘤病毒和泛梅贝科病毒保护性免疫应答。 这项拟议中的研究意义重大，因为它将提供独特的信息，可用于指导 下一代免疫原设计和疫苗接种策略，用于预防SARS-CoV-2 VOC， 未来的人畜共患传染病传染事件。我们的项目的新奇之处是（1） 用异源和高免疫原性CoV SpFN分子免疫保育鲨鱼， 中和抗体应答;（2）使用结构独特的VNAR纳米抗体来探测 对Sarbecovirus和merbecovirus刺突蛋白的脆弱性;（3）利用广泛的Sarbecovirus 和merbecovirus RBD分子，以评估结合和结构研究的识别广度; (4)鲨鱼免疫学和VNAR方面的跨职能和长期专业知识的结合 鉴定、疫苗设计和结构生物学以及小动物攻击研究。

PROJECT SUMMARY SARS-CoV-2, a betacoronavirus, is the etiologic agent of the ongoing COVID-19 pandemic. In response, worldwide efforts have led to the rapid development of multiple vaccine candidates that have shown efficacy in controlled clinical trials and effectiveness in the general population. Despite these advances, viral variants of concern (VOC) continue to emerge, while the pandemic potential of future coronavirus zoonotic spillovers remains high. Thus, the overall aim of our research efforts is to guide pan-coronavirus vaccine research and the provision of immunotherapeutic molecules with broad specificity for future pandemic prevention. To this end we will interrogate VNAR nanobody repertoires cloned from nurse sharks that have received sequential heterologous CoV spike ferritin nanoparticle (SpFN) immunizations, i.e., primed with SARS-CoV-2 SpFN, and recalled with either SARS-CoV-1 SpFN or MERS-CoV SpFN, to identify broadly conserved, neutralizing epitopes. Isolated nanobodies will be produced in recombinant form and evaluated for function and specificity using binding, affinity, and competition mapping assays, followed by viral neutralization, and in vivo protection studies. Based upon our preliminary studies we anticipate that we will find VNAR nanobodies that can target (i) pan-sarbecovirus epitopes, (ii) pan-merbecovirus epitopes, and potentially (iii) pan-sarbecovirus-merbecovirus epitopes. To explore this further we will undertake detailed structural biology studies of our current set of broadly sarbecovirus-reactive VNARs as well as any newly identified VNARs. Using X-ray crystallography and Cryo-EM imaging we will determine the binding epitope of each VNAR. This atomic level information will be used to map cross-neutralizing CoV immune epitopes and provide information that can be used in the design of new immunogens that will elicit pan-sarbecovirus and pan-merbecovirus protective immune responses. The proposed research is significant because it will provide unique information that can be used to guide next generation immunogen design and vaccination strategies for protection against SARS-CoV-2 VOC and future sarbecovirus and merbecovirus zoonotic spillover events. The novelties of our project are (1) the immunization of nurse sharks with heterologous and highly immunogenic CoV SpFN molecules to elicit broadly neutralizing antibody responses; (2) the use of structurally unique VNAR nanobodies to probe sites of vulnerability on sarbecovirus and merbecovirus spike proteins; (3) utilization of a broad panel of sarbecovirus and merbecovirus RBD molecules to assess the breadth of recognition for both binding and structural studies; (4) the combination of cross-functional and long-standing expertise in shark immunology and VNAR identification, vaccine design and structural biology, and small animal challenge studies.