Deep mutagenesis scanning using whole trimeric SARS-CoV-2 spike highlights the importance of NTD-RBD interactions in determining spike phenotype

Author summarySARS-CoV-2 continues to evolve into new variants bearing RBD and NTD mutations in spike that confer immune escape. Phenotypic maps of SARS-CoV-2 spike mutations used in surveillance and forecasting are based largely on data using screens with monomeric RBD, rather than trimeric whole spike. Interactions between the NTD and RBD affect receptor binding affinity and immune escape of the SARS-CoV-2 spike. Using deep mutational scanning with whole trimeric spike, we identified antibody escape mutations that have subsequently emerged in nature. Vaccine responses were found to be focused on one or two epitopes, making immune escape possible with single mutations. Because we used whole spike, we uncovered an unexpected role for the NTD in enhancing antibody escape from RBD directed antibodies. Genomic surveillance should take account of this new role of the NTD when scanning for VOCs. New variants of SARS-CoV-2 are continually emerging with mutations in spike associated with increased transmissibility and immune escape. Phenotypic maps can inform the prediction of concerning mutations from genomic surveillance, however most of these maps currently derive from studies using monomeric RBD, while spike is trimeric, and contains additional domains. These maps may fail to reflect interdomain interactions in the prediction of phenotypes. To try to improve on this, we developed a platform for deep mutational scanning using whole trimeric spike. We confirmed a previously reported epistatic effect within the RBD affecting ACE2 binding, that highlights the importance of updating the base spike sequence for future mutational scanning studies. Using post vaccine sera, we found that the immune response of vaccinated individuals was highly focused on one or two epitopes in the RBD and that single point mutations at these positions can account for most of the immune escape mediated by the Omicron BA.1 RBD. However, unexpectedly we found that the BA.1 RBD alone does not account for the high level of antigenic escape by BA.1 spike. We show that the BA.1 NTD amplifies the immune evasion of its associated RBD. BA.1 NTD reduces neutralistion by RBD directed monoclonal antibodies, and impacts ACE2 interaction. NTD variation is thus an important mechanism of immune evasion by SARS-CoV-2. Such effects are not seen when pre-stabilized spike proteins are used, suggesting the interdomain effects require protein mobility to express their phenotype.