Insertion in the N-Terminal Domain of the SARS-CoV-2 Spike Glycoprotein Affects Antibody Recognition and Phenotypic Properties

SARS-CoV-2, which causes COVID-19, continues to circulate around the world, making it necessary to study the impact of rapidly emerging mutations on escape from neutralizing antibodies and pathogenesis. While RBD mutations are well characterized, mutations in the N-terminal domain (NTD) of the spike protein remain comparatively understudied despite their relevance to antibody recognition. This study investigates two phenotypically distinct SARS-CoV-2 mutants, which exhibited differences in plaque morphology on Vero cells. Whole-genome sequencing via Illumina identified a novel 12-nucleotide insertion in the spike NTD. This insertion induced a frameshift, introducing five new amino acids potentially altering viral behavior, receptor interactions, and antibody detection in ELISAs. The study further explores the pathogenicity of these variants in a hamster model. These findings underscore the importance of monitoring NTD mutations, which may contribute to immune evasion and influence therapeutic antibody efficacy, highlighting gaps in current research on SARS-CoV-2 evolution.