Bile and capsid variation coregulate histo-blood group antigen recognition and serum neutralization potency in GII.12 human norovirus

Human norovirus (HuNoV) is a leading cause of acute gastroenteritis, with young children and the elderly bearing the greatest disease burden. GII strains cause the majority of HuNoV outbreaks globally, with host susceptibility influenced by genetic polymorphisms that regulate mucosal expression of histo-blood group antigen (HBGA) binding ligands. HuNoV viral capsid (VP1) diversity in residues adjacent to the conserved HBGA binding pocket selectively stabilizes ligand interactions. Additionally, select HuNoV capsids demonstrate bile-influenced conformational plasticity, further enabling HBGA engagement. Combined, these factors have the potential to enhance the breadth of binding of HuNoV to HBGAs, driving population susceptibility. Here we characterized two GII.12 outbreak strains that co-circulated cotemporaneously and were divergent at five key VP1 sites. Mutational analysis demonstrated amino acids at VP1 positions 373 and 392 coregulate HBGA ligand binding. Serological analysis demonstrated minimal antigenic impact at these positions. Broader GII comparisons demonstrated variable impacts of bile. Bile improved GII.2 and GII.12 binding to HBGA, in contrast to GII.4, GII.3, and GII.6. Further, bile reduced surrogate neutralizing antibody potency for GII.2 and GII.12 but not GII.4 and GII.6 strains. Collectively, these studies suggest that HuNoV bile-dependence is not a general feature of GII norovirus, but instead can be genotype or strain specific. These data inform how select GII HuNoV strains can modulate capsid residues linked to host-ligand and immune recognition, providing potential mechanistic evidence for continual viral circulation. IMPORTANCE: Human norovirus-host interactions are complex, influenced by viral diversity, host-ligand expression profiles, and the gastrointestinal environment. Periodically, outbreaks of endemic human noroviruses, like GII.12, demonstrate genetic diversity between viral strains of a genotype. Exploring this, two GII.12 outbreak strains were used to model the effects of genetic changes on the viral capsid ligand and antibody binding. Residue changes between the two strains modulated affinity for histo-blood group antigens that could be compensated for by addition of bile. Although bile improved HBGA-binding, it reduced serum and monoclonal antibody neutralization potency to GII.12. These observations demonstrate the balanced impact of bile-mediated viral capsid conformational plasticity in ligand recognition and potential immune evasion for select GII genotypes. Bile had similar effects for GII.2 HuNoV, but did not impact GII.6 or GII.4 HuNoV HBGA-binding and serum-ligand blocking potency, indicating that GII HuNoV strains potentially utilize multiple mechanisms to ensure host breadth and antigenic diversity.