Defining influenza A virus hemagglutinin antigenic drift by sequential monoclonal antibody selection

Abstract

Human influenza A virus (IAV) vaccination is limited by "antigenic drift," rapid antibody-driven escape reflecting amino acid substitutions in the globular domain of hemagglutinin (HA), the viral attachment protein. To better understand drift, we used anti-hemagglutinin monoclonal Abs (mAbs) to sequentially select IAV escape mutants. Twelve selection steps, each resulting in a single amino acid substitution in the hemagglutinin globular domain, were required to eliminate antigenicity defined by monoclonal or polyclonal Abs. Sequential mutants grow robustly, showing the structural plasticity of HA, although several hemagglutinin substitutions required an epistatic substitution in the neuraminidase glycoprotein to maximize growth. Selecting escape mutants from parental versus sequential variants with the same mAb revealed distinct escape repertoires, attributed to contextual changes in antigenicity and the mutation landscape. Since each hemagglutinin mutation potentially sculpts future mutation space, drift can follow many stochastic paths, undermining its unpredictability and underscoring the need for drift-insensitive vaccines.

Figure 1. Antigenic Map of Sequential Variants

(A) The antigenicity of SEQ variants was determined by measuring the relative binding affinities of a panel of 60 mAbs via RIA. Black shows equivalent binding to mutant and WT viruses; gray shows reduced affinity (2- to 4-fold); white shows greatly reduced affinity. (B) Three-dimensional model of HA rendered by PyMOL software as a solid surface looking at 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. Green shows same substitutions when selected with PR8, blue shows same amino acid position but a new substitution, and red shows new position. (C) Three-dimensional model of NA rendered by PyMOL software as a solid surface looking at top (top panel) and membrane proximal (bottom panel) aspects of the tetramer molecule (using H5N1 NA crystal structure 2HTY). NA active sites (118, 151, 152, 224, 227, 276, 292, and 371) are labeled in red and substitution G357S (H5N1 NA crystal structure 2HTY numbering) that is present in SEQ-8, SEQ-9, and SEQ-10 and decreases NA activity in green. Also see Figure S1.

Figure 2. Sequential Variants Acquire Epistatic Changes in NA to Optimize Receptor Avidity

(A) NA activities of HAU-normalized viruses ± detergent to dissociate viral glycoproteins were determined using a small fluorogenic substrate. (B) NA activities of PR8, SEQ-4, SEQ-8, and SEQ-12 normalized to the amount of NA protein in virions as determined by western blot. (C) Ratios of NA/HA protein content in virions determined by immunoblotting. (D) Receptor binding avidities of SEQ- variants were measured, determining the amount of RDE required to abrogate agglutination of turkey RBCs. Data are represented as mean ± SEM.

Figure 3. Locating H17-L2 and H17-L10 Escape Substitutions

Three-dimensional model of HA rendered by PyMOL software as a solid surface looking at top and side of the trimeric molecule (using PR8 HA crystal structure 1RVX). Amino acid substitutions in sequential variants are labeled in blue. Escape mutants of H17-L10 and H17-L2 that are present in both PR8 and SEQ-7 repertoire are labeled in red. Green shows substitutions exclusively in PR8 escape repertoire, magenta shows substitutions exclusively in SEQ-7 repertoire, and yellow shows substitutions exclusively in SEQ-8 repertoire. See also Table 3 and Table S3.

Figure 4. Antigenic Evolution Is Context Specific

(A) TCID₅₀ titers of WT and mutant viruses rescued (P₀ passage) after eight-plasmid transfection in 293T cells. (B) TCID₅₀ titers of WT and mutant viruses rescued (P₁-M passage) after amplifying in MDCK cells. (C) TCID₅₀ titers of WT and mutant viruses rescued (P₁-E passage) after amplifying in MDCK cells. All the experiments were performed in quadruplet, and the p values were calculated using Prism software (Student’s t test). (D and E) Binding affinities of H17-L10 and H17-L2 to PR8, PR8-D225Y, SEQ-7, and SEQ-7-D225Y were calculated by ELISA using detergent-disrupted viruses. All the experiments were done in quadruplicate, and dissociation constants were calculated using Prism software. All avidities reported demonstrated excellent fit for one site binding with Hill slope curve fitting (R² values > 0.98). Data are represented as mean ± SEM. See also Table S2.