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.

项目概要 SARS-CoV-2 是一种 β 冠状病毒，是当前 COVID-19 大流行的病原体。作为回应， 全球范围内的努力已导致多种候选疫苗的快速开发，这些候选疫苗已在以下方面显示出功效： 对照临床试验及其在普通人群中的有效性。尽管取得了这些进展，病毒变种 关注的问题（VOC）继续出现，而未来冠状病毒人畜共患溢出效应的大流行潜力 仍然处于高位。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治疗分子。为此我们 将询问从已接受序列治疗的护士鲨克隆的 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 引发，以及 与 SARS-CoV-1 SpFN 或 MERS-CoV SpFN 一起回忆，以识别广泛保守的、中和的 表位。分离的纳米抗体将以重组形式生产并评估其功能和特异性 使用结合、亲和力和竞争作图分析，然后进行病毒中和和体内保护 研究。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 纳米抗体 泛 sarbecovirus 表位，(ii) 泛 merbecovirus 表位，以及可能的 (iii) 泛 sarbecovirus-merbecovirus 表位。为了进一步探索这一点，我们将对我们目前广泛的一组进行详细的结构生物学研究 sarbecovirus 反应性 VNAR 以及任何新发现的 VNAR。使用 X 射线晶体学和冷冻电镜 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 CoV 免疫表位，并提供可用于设计新疫苗的信息 将引发泛萨贝克病毒和泛默贝克病毒保护性免疫反应的免疫原。 拟议的研究意义重大，因为它将提供可用于指导的独特信息 用于预防 SARS-CoV-2 VOC 的下一代免疫原设计和疫苗接种策略 未来的 sarbecovirus 和 merbecovirus 人畜共患病溢出事件。我们项目的新颖之处在于（1） 使用异源且高免疫原性的 CoV SpFN 分子对护士鲨进行免疫，以广泛引发 中和抗体反应； (2) 使用结构独特的VNAR纳米抗体来探测 sarbecovirus 和 merbecovirus 刺突蛋白的脆弱性； (3) 利用广泛的 sarbecovirus 组 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 领域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。