Insertion outside the supersite in the N-terminal domain of the SARS-CoV-2 spike glycoprotein may contribute to evasion of neutralizing antibodies.

Variants of the SARS-CoV-2 virus continue to evolve. Rapidly emerging mutations, particularly in the Spike glycoprotein, may increase transmissibility and infectivity, and also lead to evasion of the immune response. The Spike protein contains two immunodominant epitopes: NTD and RBD. Numerous neutralizing antibodies against NTD are found in patient sera, and most of these target "supersite" epitopes. Consequently, mutations outside this region receive less attention. Our work continues the investigation of the effect of insertions outside the "supersite" on the phenotypic properties of SARS-CoV-2. Previously we characterized two closely related SARS-CoV-2 Delta variants differing by a 12-nucleotide insertion in the N-terminal domain of the Spike glycoprotein. In the present study, we modeled these two Spike glycoproteins and used them to perform spatial alignment analyses of S-protein structures in complex with antibodies against the NTD to assess the potential impact of the insertion on virus binding by neutralizing antibodies. We identified four potential scenarios, how the insertion can impact the binding of neutralizing antibodies targeting the NTD. Our study confirms the importance of investigating rapidly emerging mutations in the NTD, not only within the "supersite" but also beyond it. These mutations may contribute to immune evasion; consequently, their investigation is crucial for assessing the efficacy of commercially available monoclonal antibodies and vaccine preparations.