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

项目总结 SARS-CoV-2是一种贝塔冠状病毒，是正在进行的新冠肺炎大流行的病原体。作为回应， 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 在普通人群中的受控临床试验和有效性。尽管取得了这些进展，但病毒变种 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 仍然居高不下。因此，我们研究工作的总体目标是指导泛冠状病毒疫苗的研究和 为未来的大流行预防提供具有广泛特异性的免疫治疗分子。为此，我们 将询问从护士鲨克隆的VNAR纳米体库，这些护士鲨已经接受了序列 异种冠状病毒尖峰铁蛋白纳米粒(SpFN)免疫，即用SARS-CoV-2 SpFN启动，以及 与SARS-CoV-1 SpFN或MERS-CoV SpFN一起召回，以识别广泛保守的、中和的 表位。分离的纳米体将以重组形式生产，并对其功能和特异性进行评估 使用结合、亲和力和竞争图谱分析，随后进行病毒中和和体内保护 学习。根据我们的初步研究，我们预计我们将找到能够靶向(I)的VNAR纳米体。 泛肉瘤病毒表位，(Ii)泛肉瘤病毒表位，以及潜在的(Iii)泛肉瘤病毒-斑疹病毒 表位。为了进一步探讨这一点，我们将对我们目前的一套广泛的 肉瘤病毒反应的VNAR以及任何新发现的VNAR。使用X射线结晶学和低温电子显微镜 通过成像，我们将确定每个VNAR的结合表位。该原子级信息将用于映射 交叉中和冠状病毒免疫表位，并提供可用于设计新的 将引发泛肉瘤病毒和泛美白斑病毒保护性免疫反应的免疫原。 拟议的研究具有重要意义，因为它将提供独特的信息，可用于指导 针对SARS-CoV-2 VOC和VOC的新一代免疫原设计和疫苗接种策略 未来的人畜共患病溢出事件。我们项目的创新之处在于：(1) 异源高免疫原性冠状病毒SpFN分子免疫护士鲨可广泛诱导 中和抗体反应；(2)使用结构独特的VNAR纳米体来探测 对肉瘤病毒和肉瘤病毒刺突蛋白的脆弱性；(3)广泛的肉瘤病毒群的利用 和汞病毒RBD分子，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，以及小动物挑战研究。

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.