Shark nanobodies identify pan-coronavirus spike sites of vulnerability fortherapeutic intervention

PROJECT SUMMARY Coronaviruses (CoVs) cause significant disease in humans and have the propensity to shift between zoonotic reservoirs and into human populations, making them a continual global health threat. To date, seven different strains of CoVs have been found to infect humans, of which SARS-CoV-1, SARS-CoV-2, and MERS-CoV (all betacoronaviruses) have previously caused pandemics and still pose severe threats to global health. Worldwide efforts during the COVID-19 pandemic (caused by SARS-CoV-2) led to the rapid development of vaccines and monoclonal antibody therapies (mAbs) that showed great effectiveness in reducing disease severity and preventing death in vulnerable populations. However, viral variants of concern (VOC) have continued to emerge, many of which have escaped the previously approved mAbs, while the pandemic potential of new coronavirus spillover events remains high. Thus, the overall aim of this project is the development of broadly-protective immunotherapeutic molecules that can be easily deployed should a CoV spillover of pandemic potential occur. To achieve this, we will interrogate VNAR nanobody repertoires derived from nurse sharks that have received sequential immunizations with heterologous CoV SpFNs, i.e., primed with SARS-CoV-2 SpFN, then recalled with SARS-CoV-1 SpFN, MERS-CoV, or 229E SpFN immunogens, 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 pseudovirus and live-viral neutralization, and in vivo protection studies with animal models that mimic high-risk human populations. 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 (iii) pan-alphacoronavirus epitopes. Using X-ray crystallography and Cryo-EM imaging we will determine the binding epitope of each VNAR. This atomic-level information will allow a complete understanding of the molecular recognition of antibody specificity and cross-reactivity, as exposed by the unique recognition capabilities of shark VNARs. Using a structure-guided approach the most potent nanobodies will be combined to create multi-valent molecules with prophylactic and therapeutic activity. These will be tested in vivo with administration routes expanded to explore intranasal administration for increased efficacy and ease-of-use in future situations of pandemic potential.