Antigenic remodeling of the SARS-CoV-2 Spike N-terminal domain links sequence variability to structural reorganization of a neutralizing antibody supersite.

Introduction: The N-terminal domain (NTD) of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike harbors a neutralizing antibody supersite and is a recurrent site of antigenic change. However, how sequence variability across pre- and post-vaccination immune landscapes reshapes antibody recognition remains incompletely characterized. Methods: Clinical-genomic surveillance was combined with structural modeling and molecular dynamics to identify antigenically relevant NTD changes and probe their structural impact on the neutralizing antibody interface. SARS-CoV-2 profiles were compared across two periods reflecting contrasting immune contexts: a pre-vaccination period dominated by B.1.1.33 and a post-vaccination period dominated by the Omicron sublineage BQ.1.1. A local cohort (n=126) supported descriptive characterization. An expanded cohort integrating local and GISAID genomes with harmonized clinical metadata (n=534; 401 pre-vaccination, 133 post-vaccination) was used to prioritize candidate loci by their association with clinical severity. Results: In adjusted models, age remained independently associated with severity in both periods, whereas sex did not. In the pre-vaccination cohort, Spike S in the NTD was associated with greater severity, without phylogenetic segregation by outcome. This association is exploratory and was used to prioritize an antigenically located NTD change for structural analysis. Q23K was regionally concentrated in Espírito Santo (57.4%) relative to other Brazilian states (2.7%) and international genomes (1.4%). In the post-vaccination cohort, an overlapping deletion event (ORF9b -29/N -33) was associated with non-severe disease, but should be interpreted cautiously given metadata heterogeneity, cohort structure, and the nature of the locus. However, in this period clinical outcome and sequencing source were partially confounded. A source-stratified sensitivity analysis restricted to GISAID-only samples showed the association persisted, but could not rule out a source- or pipeline-dependent indel-calling artifact, so the finding is reported as exploratory. Although Q23K itself did not persist, adjacent NTD changes (T19I/A27S/D24 -26) became recurrent post-vaccination, indicating remodeling of the same antigenic neighborhood rather than persistence of a single mutation. Structural modeling and molecular dynamics of the NTD-antibody 1 -87 complex supported reorganization of the modeled antibody-antigen interface, with redistribution of supersite contacts rather than loss of structural compatibility. Discussion: These findings link NTD variability across immune contexts to structural reorganization of the modeled antibody-antigen interface and support integrating genomic surveillance with structural analysis to prioritize antigenically relevant changes for functional testing.