Mapping of Antigenic Changes in the Haemagglutinin of Hong Kong Influenza (H3N2) Strains using a Large Panel of Monoclonal Antibodies

H9N2 influenza virus is undergoing extensive genetic and antigenic evolution, warranting detailed antigenic mapping of its hemagglutinin (HA). Through examining antibody escape mutants of an Asian avian H9N2 virus, we identified 9 critical amino acid positions in H9 antigenic sites. Five of these positions, 164, 167, 168, 196, and 207, have not been reported previously and, thus, represent novel molecular markers for monitoring the antigenic change of H9N2 virus. H9N2 influenza virus is circulating in poultry worldwide. H9N2 virus infection is usually mild in nature but may lead to higher mortality if it is associated with secondary infection (1, 2). In Asia, since its introduction into land-based poultry in the late 1980s, H9N2 virus has been spreading to various avian and mammalian species, including pigs (3, 4). Due to the Q226L mutation (change of Gln to Leu at position 226) in the hemagglutinin (HA) (5-7), a significant proportion of H9N2 isolates have acquired human virus-like receptor specificity (5,8). Consistent with this receptor specificity change, multiple human cases of H9N2 virus infection have been reported (9-12). Moreover, H9N2 virus has provided internal genes for the highly pathogenic H5N1 (9, 13, 14) and novel H7N9 (15) viruses. These have put H9N2 virus high on the list of influenza viruses with pandemic potential.Although the crystal structure of H9 has been solved (16), no details for H9 antigenic epitopes have been elucidated. Previous investigations by other groups have identified multiple amino acids in H9 antigenic sites (17, 18). These are nevertheless far from being sufficient for understanding the H9 antigenic structure. To identify more amino acids constituting H9 antigenic sites, we performed an antigenic mapping of the HA of an avian H9N2 virus A/Chicken/Jiangsu/X1/2004 (hereinafter called X1) (GenBank nucleotide sequence accession number KF688983) with monoclonal antibodies (MAbs).H9-specific MAbs were generated through the fusion of myeloma Sp2/0 cells with splenocytes from a BALB/c mouse immunized with X1 virus (19). The immunization included 3 intraperitoneal inoculations at 2-week intervals and a final boost with live X1 virus (on day 3 before the fusion). Hybridomas were screened by indirect immunofluorescence assay using chicken embryo fibroblast cells infected with X1 virus as the antigen, followed by screening with a hemagglutination inhibition (HI) assay using 4 hemagglutination units of X1 virus (20). Ascitic fluid of each selected hybridoma was generated in mice and used directly (e.g., without further purification or treatment with receptor-destroying enzyme) in the characterization of each MAb. All animal experiments were done in accordance with the institutional animal care guidelines, and the protocol (number 06R015) was approved by the Animal Care Committee at Yangzhou University. A microneutralization (MN) assay was performed in Madin-Darby canine kidney (MDCK) cells, following a previous protocol (21), except that 100 median tissue infectious doses (TCID 50 ) o...

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confidence: 67%

Antigenic Mapping of the Hemagglutinin of an H9N2 Avian Influenza Virus Reveals Novel Critical Amino Acid Positions in Antigenic Sites

Wan

et al. 2014

“…Historically, the epitopes recognized by these neutralizing Abs have been mapped by isolation of escape mutants. The epitopes present on the globular head of HA are well characterized, and they are clustered in five sites: A, B, C, D, and E in HA of H3 viruses (2,3) and Sa, Sb, Ca 1 , Ca 2 , and Cb in HA of H1 viruses (4). Because mutations are readily introduced into these sites without losing receptor-binding activity, variant viruses that have acquired resistance to these Abs become dominant and cause annual epidemics.…”

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confidence: 99%

Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses

Iba

Fujii

Ohshima

et al. 2014

Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. IMPORTANCEAntibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines.

“…The molecular mechanisms by which viruses alter their antigenic character are an important subject of study since they ultimately control the epidemics of influenza. From analyses of natural and laboratory-selected antigenic variants, four to five antigenic sites on the HA protein have been identified with H3N2 (29,31). Plotkin et al (19) claimed that multiantigenic regions have changed in recent H3 epidemic strains.…”

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confidence: 99%

Restriction of Amino Acid Change in Influenza A Virus H3HA: Comparison of Amino Acid Changes Observed in Nature and In Vitro

Nakajima¹,

Nobusawa²,

Tonegawa

et al. 2003

We introduced 248 single-point amino acid changes into hemagglutinin (HA) protein of the A/Aichi/2/68 (H3N2) strain by a PCR random mutation method. These changes were classified as positive or negative according to their effect on hemadsorption activity. We observed following results. (i) The percentage of surviving amino acid changes on the HA1 domain that did not abrogate hemadsorption activity was calculated to be ca. 44%. In nature, it is estimated to be ca. 39.6%. This difference in surviving amino acid changes on the HA protein between natural isolates and in vitro mutants might be due to the immune pressure against the former. (ii) A total of 26 amino acid changes in the in vitro mutants matched those at which mainstream amino acid changes had occurred in the H3HA1 polypeptide from 1968 to 2000. Of these, 25 were positive. We suggest that the majority of amino acid changes on the HA protein during evolution might be restricted to those that were positive on the HA of A/Aichi/2/68. (iii) We constructed two-point amino acid changes on the HA protein by using positive mutants. These two-point amino acid changes with a random combination did not inhibit hemadsorption activity. It is possible that an accumulation of amino acid change might occur without order. (iv) From the analysis of amino acids participating in mainstream amino acid change, each antigenic site could be further divided into smaller sites. The amino acid substitutions in the gaps between these smaller sites resulted in mostly hemadsorption-negative changes. These gap positions may play an important role in maintaining the function of the HA protein, and therefore amino acid changes are restricted at these locations.