Identifying antigenicity-associated sites in highly pathogenic H5N1 influenza virus hemagglutinin by using sparse learning

Abstract

Since the isolation of A/goose/Guangdong/1/1996 (H5N1) in farmed geese in southern China, highly pathogenic H5N1 avian influenza viruses have posed a continuous threat to both public and animal health. The non-synonymous mutation of the H5 hemagglutinin (HA) gene has resulted in antigenic drift, leading to difficulties in both clinical diagnosis and vaccine strain selection. Characterizing H5N1's antigenic profiles would help resolve these problems. In this study, a novel sparse learning method was developed to identify antigenicity-associated sites in influenza A viruses on the basis of immunologic data sets (i.e., from hemagglutination inhibition and microneutralization assays) and HA protein sequences. Twenty-one potential antigenicity-associated sites were identified. A total of 17 H5N1 mutants were used to validate the effects of 11 of these predicted sites on H5N1's antigenicity, including 7 newly identified sites not located in reported antibody binding sites. The experimental data confirmed that mutations of these tested sites lead to changes in viral antigenicity, validating our method.

FIG. 1

The workflow for the different steps involved in the integrative sparse learning method.

FIG. 2

The antigenic cartography of H5N1 highly pathogenic avian influenza virus (HPAIV) made by using AntigenMap (http://sysbio.cvm.msstate.edu/AntigenMap)^{; ;} . Cartography based on the results from hemagglutination inhibition assays in chicken red blood cells (A) or microneutralization assays in MDCK cells (B). One unit (grid) represents a 2-fold change in HI assay results. The cartography includes 27 influenza viruses (listed in TABLE 1), which includes viruses from 9 clades or subclades (C) ^; . The antigenic clusters are marked with a large circle for visualization purpose.

FIG. 2

The antigenic cartography of H5N1 highly pathogenic avian influenza virus (HPAIV) made by using AntigenMap (http://sysbio.cvm.msstate.edu/AntigenMap)^{; ;} . Cartography based on the results from hemagglutination inhibition assays in chicken red blood cells (A) or microneutralization assays in MDCK cells (B). One unit (grid) represents a 2-fold change in HI assay results. The cartography includes 27 influenza viruses (listed in TABLE 1), which includes viruses from 9 clades or subclades (C) ^; . The antigenic clusters are marked with a large circle for visualization purpose.

FIG. 2

The antigenic cartography of H5N1 highly pathogenic avian influenza virus (HPAIV) made by using AntigenMap (http://sysbio.cvm.msstate.edu/AntigenMap)^{; ;} . Cartography based on the results from hemagglutination inhibition assays in chicken red blood cells (A) or microneutralization assays in MDCK cells (B). One unit (grid) represents a 2-fold change in HI assay results. The cartography includes 27 influenza viruses (listed in TABLE 1), which includes viruses from 9 clades or subclades (C) ^; . The antigenic clusters are marked with a large circle for visualization purpose.

FIG. 3

Ribbon diagram of the trimeric hemagglutinin (HA) molecule with the 21 identified antigenicity associated sites (all sites listed in TABLE 2) shown in red spheres. The mutant positions were numbered based on the corresponding residue number in HA of H5N1 HPAIVs, and the corresponding numbers in H3 HA are listed in parentheses. The antibody binding sites A–E are shown in red, and the receptor-binding site is shown in blue.

FIG. 4

The antigenic cartography of H5N1 HPAIVs and their mutants (listed in TABLE 4) made by using AntigenMap (http://sysbio.cvm.msstate.edu/AntigenMap)^{; ;} . The mutant positions were numbered based on the corresponding residue number in HA of H5N1 HPAIVs. The parental viruses are labeled with heavily colored circles, and the mutants with lightly colored circles. The parental viruses are also encircled (circle = 2-unit diameter). A symbol (p) is appended after the name of each parent strain. One unit (grid) corresponds to a 2-fold change in HI titer.