Antigenic evolution of H3N2 influenza A viruses in swine in the United States from 2012 to 2016

Abstract

Background: Six amino acid positions (145, 155, 156, 158, 159, and 189, referred to as the antigenic motif; H3 numbering) in the globular head region of hemagglutinin (HA1 domain) play an important role in defining the antigenic phenotype of swine Clade IV (C-IV) H3N2 IAV, containing an H3 from a late 1990s human-to-swine introduction. We hypothesized that antigenicity of a swine C-IV H3 virus could be inferred based upon the antigenic motif if it matched a previously characterized antigen with the same motif. An increasing number of C-IV H3 genes encoding antigenic motifs that had not been previously characterized were observed in the U.S. pig population between 2012 and 2016.

Objectives: A broad panel of contemporary H3 viruses with uncharacterized antigenic motifs was selected across multiple clades within C-IV to assess the impact of HA1 genetic diversity on the antigenic phenotype.

Methods: Hemagglutination inhibition (HI) assays were performed with isolates selected based on antigenic motif, tested against a panel of swine antisera, and visualized by antigenic cartography.

Results: A previously uncharacterized motif with low but sustained circulation in the swine population demonstrated a distinct phenotype from those previously characterized. Antigenic variation increased for viruses with similar antigenic motifs, likely due to amino acid substitutions outside the motif.

Conclusions: Although antigenic motifs were largely associated with antigenic distances, substantial diversity among co-circulating viruses poses a significant challenge for effective vaccine development. Continued surveillance and antigenic characterization of circulating strains is critical for improving vaccine efforts to control C-IV H3 IAV in U.S. swine.

Keywords: H3N2; antigenic cartography; antigenic evolution; influenza A virus; swine.

Figure 1

Temporal frequency of H3 antigenic clusters. (A) Temporal frequency of H3 antigenic clusters prior to this study. (B) Temporal frequency of H3 antigenic clusters following this study. Cluster designations and coloring follow the color scheme used previously by Lewis et al.12 Strains denoted “Other” encode outlier antigenic motifs of low prevalence. Strains encoding an antigenic motif not yet phenotypically characterized are denoted as “Uncharacterized”, while “New” strains encode an antigenic motif characterized in this study

Figure 2

Antigenic phenotype of strains encoding a previously uncharacterized motif. (A) Three‐dimensional antigenic map of strains encoding a previously uncharacterized motif. Viruses encoding identical antigenic motifs are grouped (dotted circles) and labeled. Predominant antigenic clusters from 2009 to 2016, with cluster representative viruses denoted by an asterisk (*), are visualized for reference (the dominant antigenic motif is indicated for each colored phenotype). (B) Antigenic distance from Cyan (MN/09), Red (NY/11), and Green (IA/14) cluster representative strains. The 3 antigenic unit (AU) line denotes an 8‐fold loss in HI cross‐reactivity, the cutoff typically used in human H3 IAV antigenic studies to define significant antigenic drift

Figure 3

Antigenic evolution within antigenic clusters. (A) Three‐dimensional antigenic map of strains encoding a Red or Green antigenic motif. Newly characterized viruses encoding KYNNYK (bright green) or NYNNYK (bright red), along with a previously characterized Green virus (pale green) and previously characterized Red (pale red) and Cyan (cyan) cluster viruses. (B) Intra‐cluster antigenic distance from Red (PA/10) and Green (IL/12) cluster predecessors across the study time frame. One antigenic unit (AU) is equal to a twofold loss in cross‐reactivity

Figure 4

Cross‐reactivity of swine sera raised against antigenic cluster representatives. Relative fold reduction in heterologous strains from antisera raised to MN/09 (Cyan), NY/11 (Red), and IA/14 (Green). A ≥ 8‐fold reduction in the heterologous GMT from the homologous reaction is considered a significant loss in cross‐reactivity by the sera. The dominant antigenic motif is indicated for colored phenotypes

Figure 5

Antigenic and genetic relationships of H3 C‐IV IAV‐S. (A) Pairwise correlation of the antigenic distance between two antigens and the number of amino acid differences in the HA1 between the two antigens. (B) Pairwise correlation from A zoomed in to antigen pairs with five or less amino acid differences in the HA1 region. (C) Distribution of antigenic motifs within C‐IV phylogenetic clades A‐F