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

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.