Spike conformational and glycan heterogeneity associated with furin cleavage causes incomplete neutralization of SARS-CoV-2

TL;DR

This study explores how SARS-CoV-2 evades antibodies through spike protein conformation and glycosylation, without needing genetic changes.

Contribution

The study identifies a persistent fraction of infectivity with cleaved, closed spike conformations and increased mannosidic glycans as a novel mechanism of antibody resistance.

Findings

The persistent fraction of infectivity (PF) has a cleaved, closed spike conformation and more mannosidic glycans.

Incomplete neutralization is linked to furin cleavage and glycosylation, not genetic evolution.

The PF's resistance is stable and unrelated to conformational equilibrium in pseudovirus stocks.

Abstract

SARS-CoV-2 Spike - the sole neutralization target, is highly resilient to the immune pressure driving 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. The residual infectivity at high antibody concentration indicates a viral fraction with intrinsic resistance to the antibody. Although the published studies on spike glycosylation, structure, and conformations provide evidence of spike heterogeneity the precise mechanism for the incomplete neutralization has not been established. In this study, we devise a method to separate the un-neutralized virion population, called as persistent fraction of infectivity (PF), and characterize the viral spike protein. The neutralization resistance of PF is stable and unrelated to the conformational equilibrium that exists in the pseudovirus stock. The spike on the PF is highly cleaved between S1 and S2, adopts the closed conformation, and express more mannosidic glycans on RBD than the total virus population. Our study provides possible explanations for the incomplete neutralization by antibodies and delineates the association between furin cleavage of spike, its conformation and glycosylation.

The tightly closed conformation of the Spike helps SARS-CoV-2 evade the immune system. This cryptic conformation is shaped by increased mannosidic glycosylation and furin cleavage shielding conformational epitopes enable virus to persist in the presence of neutralizing antibodies.

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