Abstract
Recombination has played a crucial role in shaping the evolution of human coronaviruses (hCoVs), resulting in the emergence of novel pathogens like SARS-CoV-2. The coronavirus spike protein is heavily influenced by this mechanism and has been involved in most of the major recombination events throughout hCoV history. Interspecific recombination will continue to occur, and the emergence of novel spikes could instigate future pandemics. In this novel study, 22 chimeric hCoV spike proteins were created using the NEBuilder HiFi DNA Assembly Kit to investigate their impacts on hCoV evolution and to evaluate their potential as putative pan-coronavirus vaccine candidates. The proteins were designed to simulate recombination between the spikes of SARS-CoV-2 and the other six hCoV species by exchanging either their N-Terminal Domains (NTD) or Receptor Binding Domains (RBD) between spike backbones. The plasmids encoding for these spikes were sequenced using the MinION MK1C device, which verified they contained chimeric spike genes. HEK-293T cells were then transfected with each plasmid and their expression was confirmed through qRT-PCR and western blotting. The chimeric spikes were translated through a pseudotyped viral particle system, with their tropisms determined by infecting HEK-293T cell lines overexpressing hCoV protein receptors. Chimeric SARS-CoV-2 spikes were constructed that could bind to AP-N and DPP4, receptors not utilised by the wild type virus. Additionally, inserting the SARS-CoV-2 domains into other hCoV spikes often conferred ACE2-mediated binding exceeding the affinities of the parental viruses. In multiple instances, inserting the NTD resulted in a greater tropism shift than inserting the RBD from the same species, emphasising an underappreciated role for this domain in the binding of protein-based receptors. Finally, the neutralising capabilities of COVID-19 convalescent sera were evaluated against the chimeric spikes, with certain proteins unable to be inhibited by the sera and highlighting the impact of NTD-directed antibodies. Multiple chimeric spikes were identified that, should they form in nature, would cause significant threats to human health; able to bind to different receptors with high affinity and unable to be neutralised by the sera. With plans to test these proteins in animal models, it is hoped that their chimeric nature could induce pan-coronavirus immunity, protecting against a range of hCoVs and helping to shift pandemic control to an anticipatory, rather than a reactive, approach.