Conformational and glycan heterogeneity associated with furin cleavage of Spike as a cause of incomplete neutralization of SARS-CoV-2

SARS-CoV-2 Spike is the sole target of neutralizing antibodies (nAbs) but highly resilient to the immune pressure driving continuous genetic evolution. While potency and breadth of neutralization are widely studied, the incomplete neutralization - the mechanism of resistance without needing genetic change, remains unexplored. Several monoclonal antibodies, although potent, showed incomplete neutralization of genetically homogeneous pseudovirus suggesting the existence of distinct spike conformations peculiarly resistant to antibody binding. Incomplete neutralization is more commonly seen with NTD-specific antibodies typically exhibiting shallow neutralization curves in which neutralization saturates well below 100%. The residual infectivity in neutralization is thus accounted for the un-neutralized virions due to their intrinsic resistance to the tested antibody. Although the published studies on spike glycosylation, structure, and conformations provide insights on the spike heterogeneity the precise mechanism for the incomplete neutralization has not been established. In this study, we devised a method to separate the un-neutralized virion population, called as persistent fraction of infectivity (PF), and characterized its on-virus spike protein. The neutralization resistance of PF was stable and was unrelated to the conformational equilibrium that exists in the pseudovirus stock. The spike on the PF was highly cleaved between S1 and S2 subunits, adopted the closed conformation, and expressed more mannosidic glycans on RBD than the total virus population. Our study provides a precise explanation for the incomplete neutralization by the potent neutralizing antibodies and delineate the association between furin cleavage of spike and its conformation and glycosylation.