The Molecular Determinants of Antibody Recognition and Antigenic Drift in the H3 Hemagglutinin of Swine Influenza A Virus

Abstract

Influenza A virus (IAV) of the H3 subtype is an important respiratory pathogen that affects both humans and swine. Vaccination to induce neutralizing antibodies against the surface glycoprotein hemagglutinin (HA) is the primary method used to control disease. However, due to antigenic drift, vaccine strains must be periodically updated. Six of the 7 positions previously identified in human seasonal H3 (positions 145, 155, 156, 158, 159, 189, and 193) were also indicated in swine H3 antigenic evolution. To experimentally test the effect on virus antigenicity of these 7 positions, substitutions were introduced into the HA of an isogenic swine lineage virus. We tested the antigenic effect of these introduced substitutions by using hemagglutination inhibition (HI) data with monovalent swine antisera and antigenic cartography to evaluate the antigenic phenotype of the mutant viruses. Combinations of substitutions within the antigenic motif caused significant changes in antigenicity. One virus mutant that varied at only two positions relative to the wild type had a >4-fold reduction in HI titers compared to homologous antisera. Potential changes in pathogenesis and transmission of the double mutant were evaluated in pigs. Although the double mutant had virus shedding titers and transmissibility comparable to those of the wild type, it caused a significantly lower percentage of lung lesions. Elucidating the antigenic effects of specific amino acid substitutions at these sites in swine H3 IAV has important implications for understanding IAV evolution within pigs as well as for improved vaccine development and control strategies in swine.

Importance: A key component of influenza virus evolution is antigenic drift mediated by the accumulation of amino acid substitutions in the hemagglutinin (HA) protein, resulting in escape from prior immunity generated by natural infection or vaccination. Understanding which amino acid positions of the HA contribute to the ability of the virus to avoid prior immunity is important for understanding antigenic evolution and informs vaccine efficacy predictions based on the genetic sequence data from currently circulating strains. Following our previous work characterizing antigenic phenotypes of contemporary wild-type swine H3 influenza viruses, we experimentally validated that substitutions at 6 amino acid positions in the HA protein have major effects on antigenicity. An improved understanding of the antigenic diversity of swine influenza will facilitate a rational approach for selecting more effective vaccine components to control the circulation of influenza in pigs and reduce the potential for zoonotic viruses to emerge.

FIG 1

Three-dimensional (3D) antigenic maps of OH/04 mutants and reference viruses. (A) Relative positions of wild-type OH/04 and reference viruses; (B) OH/04_H155Y+R189K and a representative red antigenic virus, A/Swine/Pennsylvania/A01076777/2010 (PA/10); (C) OH/04_N145K+N156K+R189E+N193S and the orange outlier virus, A/Swine/Iowa/A01203196/2012 (IA/12); (D) OH/04_N145K+H155Y+R189K and the light green outlier virus, A/Swine/Wyoming/A01444562/2013 (WY/13); (E) OH/04_N145K+H155Y+N156K+R189E and the dark green outlier virus, A/Swine/North Carolina/A01432566/2013 (NC/13); (F) OH/04_N145K+R189K_N193S and the blue outlier virus, A/Swine/Michigan/A01203498/2012 (MI/12); (G) OH/04_N145K+R189S, the double mutant used to test for pathogenesis and transmission, and A/Swine/Nebraska/A01271549/2013 (NE/12). Wild-type OH/04 and OH/04 mutants are shown as larger spheres, and the corresponding antigenic motifs are labeled. Reference viruses corresponding to antigenic clusters and outliers are color coded in accordance with the color scheme used previously by Lewis et al. (8). The scale bar in each map represents 2 antigenic units (1 antigenic unit corresponds to a 2-fold dilution of antiserum in the HI assay).

FIG 2

Pathogenesis and transmissibility of wild-type OH/04 and OH/04_N145K+R189S. (A) Percentages of macroscopic lung pathology in the principal pigs. (B to E) Virus titers in the bronchoalveolar lavage fluid (BALF) (B) and nasal swabs (C to E) of principal pigs. (F) Virus titers in nasal swabs of contact pigs. In panels A to E, different lowercase letters within the same sampling day indicate statistically significant differences (P ≤ 0.05). In panel F, an asterisk indicates statistically significant differences. NC, nonchallenged.

FIG 3

Temporal and phylogenetic distribution of antigenic motifs. (A) All IAV-S H3 HA sequences available from the Influenza Research Database as of 9 July 2015 were obtained and analyzed to determine their antigenic motifs (amino acids at positions 145, 155, 156, 158, 159, and 189). Shown are the percentages of virus strains that encode antigenic motifs matching the red, light green, or cyan antigenic cluster or none of those (“other”) over time. (B) Percentages of “other” virus strains that encode antigenic motifs matching outlier antigen motifs. Virus strains encoding an antigenic motif that has not been previously characterized are designated “unknown.” (C) Maximum-likelihood phylogeny of the HA of 1,341 swine influenza viruses, 3 turkey viruses, and 12 human viruses. H3N2 genetic clades are colored: clade IV is brown, clade IV-A is red, clade IV-B is blue, clade IV-C is green, clade IV-D is yellow, clade IV-E is gray, clade IV-F is dark yellow, and human and recent human-like swine viruses are pink. The tree is midpoint rooted for clarity, and all branch lengths are drawn to scale; the scale bar indicates nucleotide substitutions per site. Pie charts to the right of the phylogeny show distribution of predicted antigenic phenotypes for each clade. The number of HA sequences corresponding to each clade is indicated adjacent to the pie chart.