Fine antigenic variation within H5N1 influenza virus hemagglutinin's antigenic sites defined by yeast cell surface display

Abstract

Fifteen strains of mAb specific for HA of the A/Hong Kong/482/97 (H5N1) influenza virus were generated. The HA antigenic sites of the human A/Hong Kong/482/97 (H5N1) influenza virus were defined by using yeast cell surface-displaying system and anti-H5 HA mAb. Evolution analysis of H5 HA identified residues that exhibit diversifying selection in the antigenic sites and demonstrated surprising differences between residue variation of H5 HA and H3 HA. A conserved neutralizing epitope in the H5 HA protein recognized by mAb H5M9 was found using viruses isolated from [1997][1998][1999][2000][2001][2002][2003][2004][2005][2006]. Seven single amino acid substitutions were introduced into the HA antigenic sites, respectively, and the alteration of antigenicity was assessed. The structure obtained by homology-modeling and molecular dynamic methods showed that a subtle substitution at residue 124 propagates throughout its nearby loop (152)(153)(154)(155)(156)(157)(158)(159). We discuss how the structural changes caused by point mutation might explain the altered antigenicity of the HA protein. The results demonstrate the existence of immunodominant positions in the H5 HA protein, alteration of these residues might improve the immunogenicity of vaccine strains.Key words: Antigenic sites . Evolution analysis . HA . Immunodominant positions IntroductionIn 1997, an H5N1 influenza virus crossed the species barrier to directly transmit from birds to humans in Hong Kong and caused 18 cases of illness including six deaths [1]. New genotypes of the H5N1 virus continued to circulate and occasionally caused severe human disease in Asia [2][3][4]. A lack of protective immunity in the human population against the H5 influenza virus subtype presents a significant risk of an influenza pandemic.Antigenic drift of the influenza virus is caused by the accumulation of mutations in the antigenic regions of HA. Research on influenza virus antigenic drift has mainly been based on the analysis of the human H3N2 subtype [5,6]. The HA protein of all influenza viruses consists of two chains, HA1 and HA2. The HA1 3498mutates more frequently than HA2 and undergoes strong Darwinian selection for novel variants [7]. Using natural and laboratory-selected antigenic variants, five antigenic domains (sites A-E) were revealed in the H3 subtype HA molecule [8]. Of the 329 amino acids in HA1, 131 lie in or near to the five overlapping antigenic sites [5]. Codon usage at the H3 HA epitopic residues are biased toward diversification relative to the nonepitopic residues [7]. In other words, codon biases in the epitopic residues of H3 HA indicate that these residues are under strong selection for substitutional change to escape Ab pressure. Antigenic drift has also been shown to occur with avian influenza viruses, especially those circulating in poultry in the field and in antigenic variant viruses emerging from the H5N1 HPAI virus [9,10]. Therefore, the study of antigenic variation and codon usage at the H5 HA antigenic regions is necessary for the selection o...

A Unique and Conserved Neutralization Epitope in H5N1 Influenza Viruses Identified by an Antibody against the A/Goose/Guangdong/1/96 Hemagglutinin

“…The epitope was further refined by selection of escape mutants and flow cytometry analyses of H5M9 binding to HA and by site-directed mutagenesis of key HA binding residues. We previously documented that H5M9 is an inhibitor of hemagglutination, as it directly blocks virus binding to cellular receptors (11). We show here that H5M9 is also an inhibitor of the pH-induced conformational change required for virus fusion activity.…”

“…Among them, one antibody, H5M9, elicited in mice from H5 HA that was isolated and concentrated from GD1 H5N1 virus, had broad neutralizing activity against different clades (effective for all tested clades-0, 1, 2.3.4, and 7) of H5N1 influenza viruses isolated from 1997 to 2008 (listed in Table 1 and in our previous report [11]), indicating the presence of a conserved neutralizing epitope in the H5 HA protein recognized by H5M9. Antibody epitope mapping of H5 HA using yeast cell surface display showed that H5M9 binds to the HA1 region (11).…”

“…Antibody H5M9 was elicited by immunization of mice with GD1 HA as described previously (11). The Fab= fragment of antibody H5M9 (IgG1, chain) was produced by standard protocols (15).…”

“…A panel of 15 monoclonal antibodies specific for H5N1 viruses was generated by immunizing mice with viral or recombinant HA of the GD1 virus (11). Among them, one antibody, H5M9, elicited in mice from H5 HA that was isolated and concentrated from GD1 H5N1 virus, had broad neutralizing activity against different clades (effective for all tested clades-0, 1, 2.3.4, and 7) of H5N1 influenza viruses isolated from 1997 to 2008 (listed in Table 1 and in our previous report [11]), indicating the presence of a conserved neutralizing epitope in the H5 HA protein recognized by H5M9.…”

Antigenic Drift in H5N1 Avian Influenza Virus in Poultry Is Driven by Mutations in Major Antigenic Sites of the Hemagglutinin Molecule Analogous to Those for Human Influenza Virus

“…Additionally, substitution P74S introduced together with R140G, S141P, F144Y, and R162K contributed to the further reduction of the HI titer (e.g., compare mutants 30 and 20, Table 1). It is interesting that 3 out of 5 key amino acid substitutions detected in this study (positions 140, 141, and 162) are located in corresponding H3 antigenic sites A and B (16,23). Mutations in 6 positions of 1709-6/2008, namely, 110, 120, 123, 165, 184, and 226, do not appear critical for escape from antibody binding.…”