Broad Cross-Reactive Epitopes of the H5N1 Influenza Virus Identified by Murine Antibodies against the A/Vietnam/1194/2004 Hemagglutinin

Abstract

There is an urgent need for a rapid diagnostic system to detect the H5 subtype of the influenza A virus. We previously developed monoclonal antibodies (mAbs) against the H5 hemagglutinin (HA) for use in a rapid diagnostic kit. In this study, we determined the epitopes of the anti-H5 HA murine mAbs OM-b, AY-2C2, and YH-1A1. Binding assays of the mAbs to different strains of H5 HAs indicated that OM-b and AY-2C2 cross-reacted with HAs from clades 1, 2.1.3.2, 2.2, and 2.3.4, whereas YH-1A1 failed to bind to those of clades 2.1.3.2 and 2.3.4. HA chimeras revealed that the epitopes for each of the mAbs were in the HA1 region. Analysis of escape mutants revealed that OM-b and AY-2C2 mAbs interacted mainly with amino acid residues D43 and G46, and the YH-1A1 mAb interacted with G139 and K or R140 of H5 HA. Multiple alignments of H5 HA protein sequences showed that D43 and G46 were very conserved among H5N1 HAs, except those in clade 2.2.1 and clade 7 (88.7%). The epitope for YH-1A1 mAb was highly variable in the HAs of H5N1, although it was well conserved in those of H5N2-N9. The OM-b and AY-2C2 mAbs could bind to the HAs of clades 1.1 and 2.3.2.1 that are currently epidemic in Asia, and we conclude that these would be effective for the detection of H5N1 infections in this region.

Development of a sensitive novel diagnostic kit for the highly pathogenic avian influenza A (H5N1) virus

“…During the preparation of this manuscript, Sakurai et al reported a 10–100-fold more sensitive detection of H5 HA using fluorescent beads and an improved IC method [ 21 ]; however, the clinical effectiveness of this method remains to be evaluated. We recently determined the epitopes of our anti-H5HA mAbs and found that OM-b, one of mAbs used for the H5/A kit, was broadly reactive to various clades of H5N1 influenza virus isolates in Asia, indicating that ther kit is quite useful for the diagnosis of H5N1 infection in Asian counties [ 22 ].…”

Epitopes in the HA and NA of H5 and H7 avian influenza viruses that are important for antigenic drift

“…In another study, formation of an N -linked glycosite in H7N9 at amino acid N133 (H5: 128; H7: 123), mediated by T135 substitution (H5: 131; H7: 125) led to epitope masking and immune escape (Alvarado-Facundo et al 2016 ) and naturally occurring H7N9 viruses with 135T are able to escape vaccine induced immunity in poultry (Yin et al 2021 ). Additionally, H5 and/or H7 escape mutants with substitutions at subsites 141 (Schmeisser et al 2015 , Tan et al 2016 ), 143 (Kobayashi-Ishihara et al 2014 , Tan et al 2016 , Thornburg et al 2016 , Gronsang et al 2017 ), 144 (discussed in detail below), 145 (discussed in detail below), or 146 (Henry Dunand et al 2016 ) are frequently reported. These subsites are in the H7 antigenic motif 140 RR-SGSS 146 (H5 equivalent: 140 PYQGKSS 146 ), suggesting that this motif may be a hotspot for antigenic diversity.…”

“…Another study selecting for H5N1 A/goose/Guangdong/1/1996 (gs/Gd)-lineage (A/Vietnam/1203/2004) escape mutants using mAbs described S126Y + I155T, G143E, K144E, and S145F/P/T substitutions in antigenic site A (H5: S121Y + I151T, G139E, K140E, and S141F/P/T; H7: S116Y + I144T, G132E, K133E, and S134F/P/T (Kaverin et al 2007 ). H5 escape mutants with antigenic site A substitutions at subsite 144 (H5: 140; H7: 133) (Kobayashi-Ishihara et al 2014 ) and 144–147 (H5: 140–143; H7: 133–136) (Okuda et al 2019 ) have subsequently been described. Importantly, H5 vaccine mismatches at amino 144 (H5: 140; H7: 133) significantly reduced protective efficacy following challenge of chickens (Criado et al 2020 ) and reverse genetics studies have confirmed a crucial role of subsite 144 (H3 numbering) in mediating H5 antigenicity (Cattoli et al 2011 , Hervé et al 2015 ).…”

“…The footprint of the broadly neutralizing antibody (bnAb) MEDI8852 has been partially mapped to antigenic site C (H3 residues 54, 276, and 278; H5: 44, 273, and 275; H7: 44, 267, and 269) (Kallewaard et al 2016 ), with the bnAb 39.29 light chain also interacting with residue 278 (H5: 275; H7: 269) (Nakamura et al 2013 ). Antigenic escape mutants in epitope C have been described, D53N/Y, D54 A Y, and G55E (amino acids 54 A and 55 flank antigenic site C) (Kobayashi-Ishihara et al 2014 ), D53N + H276N (reported as D47N + H287N) (Ohkawara et al 2017 ), Y274H and N278S (immature protein: Y287H and N291S) (Höper et al 2012 ), and D275G (Lyashko et al 2024 ), although substitutions at site C occur at a lower frequency than A and B. Additionally, substitutions at amino acid 55 and 57, which flanks antigenic site C, have been described (H7N9, G55E, R57K/W, H5N1, G55R) (Henry Dunand et al 2015 , Henry Dunand et al 2016 , Ohkawara et al 2017 ). Recently, a novel conserved epitope in antigenic site C of H5 HPAIV was identified (Zhu et al 2013 ).…”

“…The first antigenic evolution study to experimentally characterize antigenic drift was first described in 1950 (Archetti and Horsfall 1950 ). To examine residues crucial to the antigenicity of H5 and H7 AIVs, experimentally derived HA and NA antigenic variants have been generated by in vitro or in ovo selection of escape mutants in the presence of monoclonal antibodies (mAbs) (Lentz et al 1984 , Air et al 1985 , 1990 , Webster et al 1987 , Philpott et al 1989 , Saito et al 1994 , Kaverin et al 2002 , 2007 , Chen et al 2009 , Krylov et al 2009 , Ferreira et al 2010 , Prabakaran et al 2010 , Rudneva et al 2010 , He and Kwang 2013 , Zhu et al 2013 , Itoh et al 2014 , Kobayashi-Ishihara et al 2014 , Henry Dunand et al 2015 , Kaverin et al 2015 , Tan et al 2015 , Claes et al 2016 , Henry Dunand et al 2016 , Thornburg et al 2016 , Wan et al 2016 , Gronsang et al 2017 , Ohkawara et al 2017 , Ito et al 2019 , Li et al 2019a , Okuda et al 2019 , Timofeeva et al 2020a , Xiong et al 2020 , Strohmeier et al 2022 , Chang et al 2023 , Lyashko et al 2024 ), polyclonal antiserum (Lambkin et al 1994 , Cleveland et al 1997 , Höper et al 2012 , Kalthoff et al 2013 , Sitaras et al 2014 , Chang et al 2019 , Sitaras et al 2020 ), or following infection of vaccinated birds (Hinshaw et al 1990 , Beato et al 2014 , Nguyen et al 2017 ). Antigenic variants that lead to evasion of the neutralizing host immune response may also be associated with changes in functional attributes, such as receptor binding dynamics (Hensley et al 2009 ), pH of fusion, or thermostability.…”