Fitness costs limit influenza A virus hemagglutinin glycosylation as an immune evasion strategy

Abstract

Here, we address the question of why the influenza A virus hemagglutinin (HA) does not escape immunity by hyperglycosylation. Uniquely among dozens of monoclonal antibodies specific for A/Puerto Rico/8/34, escape from H28-A2 neutralization requires substitutions introducing N-linked glycosylation at residue 131 or 144 in the globular domain. This escape decreases viral binding to cellular receptors, which must be compensated for by additional substitutions in HA or neuraminidase that enable viral replication. Sequence analysis of circulating H1 influenza viruses confirms the in vivo relevance of our findings: natural occurrence of glycosylation at residue 131 is always accompanied by a compensatory mutation known to increase HA receptor avidity. In vaccinated mice challenged with WT vs. H28-A2 escape mutants, the selective advantage conferred by glycan-mediated global reduction in antigenicity is trumped by the costs of diminished receptor avidity. These findings show that, although N-linked glycosylation can broadly diminish HA antigenicity, fitness costs restrict its deployment in immune evasion.

Fig. 1.

Locating amino acid substitutions in H28-A2 escape mutants. 3D model of HA rendered by PyMol software as a solid surface looking at the top and side of the trimeric molecule (using PR8 HA crystal structure 1RVX). Amino acid substitutions in escape mutants are indicated by color and label. In Z1, which zooms in on the receptor binding site (RBS), the two glycosylation sites created by mutations, located at residues 144 (direct introduction of N) and 131 (mutation at residue 133 (red) creates a site for existing N) are shown in pink. Glycosylation at these residues causes global changes in antigenicity and also incurs fitness costs that must be compensated for by epistatic substitutions (at the yellow residues) for virus survival.

Fig. 2.

Biochemical confirmation of N-linked glycosylation at introduced sites. Egg-grown viruses were purified and analyzed by SDS/PAGE. (A) SYPRO Ruby staining or immunoblotting by anti-HA antibody (CM1) shows a shift in HA mobility because of additional glycosylation, which was confirmed by near identical mobilities following PNGase F deglycosylation (B).

Fig. 3.

Glycosylation diminishes receptor binding avidity. (A) Human or (B) turkey RBCs were treated with graded concentrations of receptor-destroying enzymes (RDEs) before the addition of viruses indicated to determine the relative amounts of terminal sialic acid required for agglutination.

Fig. 4.

Receptor binding modulates immune escape in vitro and in vivo. HI assays (A) using pooled mouse primary anti-PR8 antisera show that introduction of a glycosylation site at residue 144 diminishes HI titer. Each experiment was repeated three times with quadruple samples, and one representative experiment is presented here. Similarly, in mouse infections (B), addition of a glycosylation site at residue 144 to the adsorptive mutant S186P abrogates escape from anti-HA Abs induced by vaccination as shown by recovery of infectious virus from lungs. In each group, five mice were used per experiment, and representative data are presented of three separate experiments.

Fig. P1.

Footprint of H28-A2 binding. Alterations in residues that reduce H28-A2 binding (225, 193, and 144) or abrogate it because of glycosylation (144 and 131) are shown in red.