Structural and Antigenic Variation among Diverse Clade 2 H5N1 Viruses

<div><p>Antigenic variation among circulating H5N1 highly pathogenic avian influenza A viruses mandates the continuous production of strain-specific pre-pandemic vaccine candidates and represents a significant challenge for pandemic preparedness. Here we assessed the structural, antigenic and receptor-binding properties of three H5N1 HPAI virus hemagglutinins, which were recently selected by the WHO as vaccine candidates [A/Egypt/N03072/2010 (Egypt10, clade 2.2.1), A/Hubei/1/2010 (Hubei10, clade 2.3.2.1) and A/Anhui/1/2005 (Anhui05, clade 2.3.4)]. These analyses revealed that antigenic diversity among these three isolates was restricted to changes in the size and charge of amino acid side chains at a handful of positions, spatially equivalent to the antigenic sites identified in H1 subtype viruses circulating among humans. All three of the H5N1 viruses analyzed in this study were responsible for fatal human infections, with the most recently-isolated strains, Hubei10 and Egypt10, containing multiple residues in the receptor-binding site of the HA, which were suspected to enhance mammalian transmission. However, glycan-binding analyses demonstrated a lack of binding to human α2-6-linked sialic acid receptor analogs for all three HAs, reinforcing the notion that receptor-binding specificity contributes only partially to transmissibility and pathogenesis of HPAI viruses and suggesting that changes in host specificity must be interpreted in the context of the host and environmental factors, as well as the virus as a whole. Together, our data reveal structural linkages with phylogenetic and antigenic analyses of recently emerged H5N1 virus clades and should assist in interpreting the significance of future changes in antigenic and receptor-binding properties.</p></div

Structural variation among the different H5N1 clades.

<p>(A) Surface representation of the Hubei10 trimeric HA indicating the positions of surface exposed residue substitutions among Clade 1 (Viet04), clade 2.3.4 (Anhui05), clade 2.2.1 (Egypt10) and clade 2.3.2.1 (Hubei10). Positions containing single substitutions are colored cyan and positions containing multiple substitutions are colored magenta. (B) Amino acid consensus sequences of H5N1 HA clades at positions equivalent to the HA antigenic sites, Ca, Cb, Sa and Sb, of human H1N1 viruses <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075209#pone.0075209-Caton1" target="_blank">[39]</a>, are shown. Clade 1 (Viet04), clade 2.3.4 (Anhui05), clade 2.2.1 (Egypt10) and clade 2.3.2.1 (Hubei10) are highlighted in red. Structural positions of these equivalent sites are highlighted on the Hubei10 trimeric structure (Ca; pale yellow, Cb; wheat, Sa; pale green, Sb; pale blue). Asparagine residues that are potentially N-glycosylated are colored orange.</p

Antigenic and amino acid sequence variation among different clades of H5N1 vaccine candidate viruses.

a<p>Hemagglutination inhibition (HI) titers were determined using turkey red blood cells.</p>b<p>Titers for homologous antigen/antisera are shown with values underlined. Titers are presented as the geometric mean titers (GMT) calculated from five independent HI tests.</p>c<p>Differences among strain-specific cross reactivity are quoted as dilutions relative to that of the end-point dilution value for the homologous antigen/sera response. Viruses are considered antigenically diverse if titers are reported as ≥8-fold difference in two-way tests.</p>d<p>Only a reassortant virus for Hubei10 was used in assay. Others were wild type viruses.</p>e<p>Amino acid sequence identities were calculated for the 267 residues of the HA1 structural domain (residues 34–300) of the mature HA, using CLUSTALX <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075209#pone.0075209-Thompson1" target="_blank">[84]</a>.</p>f<p>Surface residue differences were quantified for the HA1 structural domain (residues 34–300) of the mature HA monomer. For this analysis, 166 of the 267 residues were considered surface residues. The number of surface residue substitutions is given in parentheses.</p

Structural comparison between H5 hemagglutinins.

<p>(A) Structural alignment of Anhui05 (green), Egypt10 (blue) and Hubei10 (purple) onto Viet04 (yellow) reveals how structurally related these clades are. (B) Alignment of the receptor-binding site (RBS) reveals conserved structural features and residues. (C) Compared to Viet04, a total of eleven residue differences in and around the RBS are present. Amino acid residues in each structure are numbered consecutively according to the ectodomain fragment of the mature HA1 protein. *Deletion of Leu129 in Egypt10 produces a shift in the numbering of residues 129–324 in Egypt10 relative to structurally equivalent residues in Anhui05 and Hubei10.</p

Receptor specificity of H5 recHAs.

<p>Glycan microarray analysis of recombinant Viet04 HA (A), Anhui05 (B), Egypt10 (C) and Hubei10 (D). Colored bars highlight glycans that contain α2–3 Neu5Ac (blue) and α2–6 Neu5Ac (red), α2–6/α2–3 mixed Neu5Ac (purple), N-glycolyl Neu5Ac (green), α2–8 Neu5Ac (brown), β2–6 and 9-O-acetyl Neu5Ac (yellow), and non-Neu5Ac (grey). Error bars reflect the standard error in the signal for six independent replicates on the array. The structures of each of the numbered glycans are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075209#pone.0075209.s008" target="_blank">Table S5</a>.</p

SDS-PAGE of A/bat/Peru/10 HA0 (lanes 1 to 4) and its mature HA (lanes 5 to 8) in trypsin susceptibility assay.

<p>A/bat/Peru/10 HA with a monobasic cleavage site was expressed in its HA0 form in a baculovirus expression system. Lanes 1 and 2 show A/bat/Peru/10 native HA0 at pH 8.0 and pH 4.9, respectively, while lanes 3 and 4 show the equivalent reducing gel of HA0 treated with trypsin at pH 8.0 and pH 4.9, respectively. Similarly, lanes 5 and 6 show A/bat/Peru/10 mature HA at pH 8.0 and pH 4.9, respectively, while lanes 7 and 8 show the equivalent reducing gel of mature HA treated with trypsin at pH 8.0 and pH 4.9, respectively.</p

Important functional motifs in non-bat influenza A internal proteins are largely conserved in bat influenza proteins.

†<p>Letters written as subscripts denote amino acid substitutions in bat viruses compared to the consensus non-bat amino acid sequence.</p

Crystal structures of A/bat/Peru/10 HA.

<p>(A) Overall structure of A/bat/Peru/10 HA. The H18 HA trimer consists of three identical monomers with one RBS per monomer. HA1 is highlighted in green and HA2 in cyan. N-linked glycans observed in the electron density maps are shown with yellow carbons. (B) The A/bat/Peru/10 HA putative RBS (in crystal 1, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003657#ppat.1003657.s014" target="_blank">Table S6</a>) in ribbon representation with the side chains of key binding residues shown. The three highly conserved residues (W153, H183, and Y195) in HAs are colored with green carbon atoms, whereas nine residues that are conserved in A/bat/Peru/10 H18 and two H17 HAs from bat influenza viruses A/little yellow-shouldered bat/Guatemala/164/2009 (H17N10) (GU09-164) and A/little yellow-shouldered bat/Guatemala/060/2010 (H17N10) (GU10-060) are labeled in red. E190 and G225 are also conserved, especially in avian influenza A viruses. (C) Molecular surface of the putative RBS of A/bat/Peru/10 HA compared to the RBS of 2009 H1 HA from A/California/04/2009 (H1N1) (PDB code 3UBQ). A canonical sialic acid is modeled in the HA for comparison as observed in other HA structures. The RBS of A/bat/Peru/10 HA is shallower and wider than 2009 H1 HA with no space for the glycerol moiety of sialic acid (indicated by the red arrow). For comparison, figures (B) and (C) are generated in the same orientation.</p

Detection of influenza A virus and antibody in bats from Peru (2010).

<p>Detection of influenza A virus and antibody in bats from Peru (2010).</p

Crystal structures of A/bat/Peru/10 NAL.

<p>(A) Overall structure of A/bat/Peru/10 NAL with a conserved calcium binding site. The N11 NAL tetramer is viewed from above the viral surface, and consists of four identical monomers with C4 symmetry. One monomer is colored in six different colors to illustrate the canonical <i>β</i>-propeller shape of six four-stranded, anti-parallel <i>β</i>-sheets. The putative active site is located on the membrane-distal surface (on top of the molecule). The observed N-linked glycosylation sites are shown with attached carbohydrates. A single calcium ion is shown in red spheres. (B) The A/bat/Peru/10 NAL putative active site (crystal form 1, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003657#ppat.1003657.s014" target="_blank">Table S6</a>) with the conserved catalytic and active site residues in other NAs shown as well as other polar and charged residues. The six residues (R118, W178, S179, R224, E276 and E425) conserved in all influenza NAs are colored with green carbon atoms in contrast to other putative active site residues in yellow carbon atoms, whereas eight residues that are conserved in two bat influenza N10 and N11 NAL proteins are labeled in red. (C) Molecular surface of the active site of A/bat/Peru/10 N11 NAL and 1918 N1 NA from A/Brevig Mission/1/18 (H1N1) (PDB code 3BEQ). A canonical sialic acid is modeled in A/bat/Peru/10 NAL as in other NA structures and appears to collide with the NA putative active site around the glycerol moiety (as indicated by the red arrow). The putative active site pocket of A/bat/Peru/10 NAL is much wider than 1918 N1 NA. For comparison, figures (B) and (C) are generated in the same orientation.</p