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

鲨鱼纳米抗体能够识别泛萨贝克病毒和泛默贝克病毒的 RBD 漏洞位点

• 批准号: 10644226
• 负责人: Helen M. Dooley
• 金额: $ 26.63万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-17 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10644226
• 关键词: 2019-nCoV; Affinity; Animals; Antibody Response; Antibody Specificity; Antigen-Antibody Complex; Antigens; B-Lymphocytes; Baltimore; Binding; Biological Assay; Blood; COVID-19 pandemic; Clinical effectiveness; Collaborations; Controlled Clinical Trials; Coronavirus; Cryoelectron Microscopy; Development; Emerging Communicable Diseases; Epitopes; Etiology; Ferritin; Foundations; Future; General Population; Goals; Human; Humoral Immunities; Image; Immune; Immune response; Immunization; Immunize; Immunology; Immunotherapeutic agent; Maps; Maryland; Merbecovirus; Methods; Middle East Respiratory Syndrome Coronavirus; Molecular; Negative Staining; Nurses; Phage Display; Proteins; Recombinants; Research; Research Institute; Resolution; SARS coronavirus; SARS-CoV-2 variant; Sarbecovirus; Shark; Site; Specificity; Structure; Techniques; Universities; Vaccination; Vaccine Design; Vaccine Research; Viral; X-Ray Crystallography; betacoronavirus; coronavirus vaccination; cross reactivity; design; future pandemic; immunogenic; in vivo; innovation; medical schools; molecular recognition; nanobodies; nanoparticle; neutralizing antibody; next generation; novel; pandemic potential; prevent pandemics; response; spillover event; structural biology; universal coronavirus vaccine; vaccination strategy; vaccine candidate; variants of concern; zoonotic coronavirus; zoonotic spillover

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.

项目摘要 SARS-COV-2是一种Betacoronavirus，是正在进行的COVID-19大流行的病因学药物。作为回应， 全球努力导致了多种候选疫苗的迅速发展，这些疫苗在 对照临床试验和普通人群的有效性。尽管有这些进展，但病毒变体的 关注（VOC）继续出现，而未来冠状病毒的大流行潜力 保持高。因此，我们的研究工作的总体目的是指导泛氧化病毒疫苗研究和 提供具有广泛特异性的免疫治疗分子，以预防未来的大流行。为此，我们 将询问从已接收顺序的护士鲨鱼克隆的vnar纳米机曲目 异源COV尖峰铁蛋白纳米颗粒（SPFN）免疫，即用SARS-COV-2 SPFN启动，并且 用SARS-COV-1 SPFN或MERS-COV SPFN召回，以确定广泛保守的，中和 表位。孤立的纳米结构将以重组形式产生，并评估功能和特异性 使用结合，亲和力和竞争映射测定，然后进行病毒中和和体内保护 研究。基于我们的初步研究 Pan-Sarbecovirus表位，（ii）泛肉片病毒表位，并有可能（iii）pan-sarbecovirus-merbecovirus 表位。为了进一步探讨这一点，我们将对当前一组广泛的结构生物学研究 SARBECOVIRUS反应性VNAR以及任何新确定的VNAR。使用X射线晶体学和冷冻EM 成像我们将确定每个VNAR的结合表位。此原子级信息将用于映射 交叉中和COV免疫表位，并提供可用于新设计的信息 将引起泛核病毒和泛肉毒病毒保护性免疫反应的免疫原子。 拟议的研究很重要，因为它将提供可用于指导的独特信息 下一代免疫原设计和疫苗接种策略，以防止SARS-COV-2 VOC和 未来的SARBECOVIRUS和MERBECOVIRUS人畜共患溢出事件。我们项目的新颖性是（1） 用异源和高度免疫原性的COV SPFN分子免疫鲨鱼的免疫鲨鱼，以广泛引起 中和抗体反应； （2）使用结构唯一的VNAR纳米构造在探测位点 SARBECOVIRUS和MERBECOVIRUS SPIKE蛋白的脆弱性； （3）利用一大堆SARBECOVIRUS Merbecovirus 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型动物挑战研究。