Dissecting the Clonal Composition and Determinants of Neutralization Potency Enhancement of Serum Dimeric and Monomeric IgA to Human Norovirus

Protection against human norovirus infection, which is the major cause of acute gastroenteritis, is strongly correlated with humoral immunity, particularly with IgA binding titers in serum and mucosal surfaces. Serum IgA is mostly monomeric (mIgA), with 10-20% present as dimeric (dIgA). Using serum antibody proteomics, we determined the clonal composition of the serum IgG, mIgA, and dIgA repertoires specific to GII.4 virus-like particles (VLPs) following oral vaccination with an adenoviral vectored norovirus vaccine candidate, which phenocopies the route of administration and the higher IgA to IgG serum titers associated with natural infection. We detected a low level of clonal overlap between circulating IgG and IgA at steady state, which, however, increased up to 65% following vaccination. Importantly, > 80% of serum mIgA specific to VLP was also found as dIgA, yet the mIgA:dIgA abundance ratio for different clonotypes varied by up to 250-fold (0.4190). We observed a similar congruence of the influenza hemagglutinin-binding dIgA and mIgA repertoires in serum following intramuscular flu vaccination. For antibodies targeting the apex, but not the lateral cleft, of the protruding (P) domain on human norovirus VLPs, IgA dimerization conferred markedly improved ligand blockade titers, resulting in higher potency towards the neutralization of live virus in the human intestinal enteroid system. Cryo-EM structures of prototypical apical and lateral cleft binding antibodies, coupled with cryo-ET imaging of VLP cross-linking mediated by dIgA, revealed that epitope accessibility, antibody binding orientation, and proximity to the attachment ligand binding site required for virus entry into host cells all influence the epitope-dependent differential potency of dIgA. Our findings elucidate key features of the molecular nature of the IgA serological response and the structural basis for the high neutralization potency of dIgA in an epitope-specific manner.