Abstract The SARS-CoV-2 spike glycoprotein has 22 potential N-linked glycosylation sites per monomer that are highly conserved among diverse variants, but how individual glycans affect virus entry and neutralization of Omicron variants has not been extensively characterized. Here we compared the effects of specific glycan deletions or modifications in the Omicron BA.1 and D614G spikes on spike expression, processing, and incorporation into pseudoviruses, as well as on virus infectivity and neutralization by therapeutic antibodies. We found that loss of potential glycans at spike residues N717 and N801 each conferred a loss of pseudovirus infectivity for Omicron but not for D614G or Delta variants. This decrease in infectivity correlated with decreased spike processing and incorporation into Omicron pseudoviruses. Oligomannose-enriched Omicron pseudoviruses generated in GnTI - cells or in the presence of kifunensine were non-infectious, whereas D614G or Delta pseudoviruses generated under similar conditions remained infectious. Similarly, authentic SARS-CoV-2 grown in the presence of kifunensine decreased titers more for the BA.1.1 variant than Delta or D614G variants relative to their respective, untreated controls. Finally, we found that loss of some N-glycans, including N343 and N234, increased the maximum percent neutralization by the class 3 S309 monoclonal antibody against D614G but not BA.1 variants, while these glycan deletions altered the neutralization potency of the class 1 COV2-2196 and Etesevimab monoclonal antibodies without affecting maximum percent neutralization. The maximum neutralization by some antibodies also varied with the glycan composition, with oligomannose-enriched pseudoviruses conferring the highest percent neutralization. These results highlight differences in the interactions between spike glycans and residues among SARS-CoV-2 variants that can affect spike expression, virus infectivity, and susceptibility of variants to antibody neutralization. Author summary (non-technical) The SARS-CoV-2 spike surface protein is covered in glycans that may affect its function and ability to evade antibodies. Omicron variants have over 30 mutations compared to the D614G variant, yet all 22 potential N-glycosylation sites are highly conserved. Here we compared the impact of glycan changes in the spikes of the Omicron and D614G variants on virus infectivity and neutralization. We found that loss of specific glycans in the transmembrane subunit of spike greatly reduced Omicron, but not D614G, spike expression and incorporation into pseudoviruses. Changes in the overall glycan composition also reduced the infectivity of Omicron pseudovirus and authentic virus more than D614G pseudoviruses and authentic viruses. We further show that changes in specific glycans directly or indirectly affected susceptibility of pseudoviruses to therapeutic antibodies, but the effects differed among the variants. These findings highlight differences in the interplay between spike glycans and amino acid residues among SARS-CoV-2 variants that can contribute to spike plasticity and modify spike expression, function, and immune evasion properties.

D614G and Omicron SARS-CoV-2 variant spike proteins differ in the effects of N-glycan modifications on spike expression, virus infectivity, and neutralization by some therapeutic antibodies