Identification of two novel B-cell epitopes targeting the hemagglutinin protein of H9N2 avian influenza virus

Abstract

H9N2 avian influenza virus (AIV) remains a global threat to poultry health and has zoonotic potential. Antigenic drift in the hemagglutinin (HA) protein complicates vaccine efficacy and diagnostic accuracy, highlighting the need for precise epitope characterization. In this study, the HA protein of H9N2 AIV was expressed in a eukaryotic system, and two monoclonal antibodies (mAbs), 9C12 and 9F4, were generated. Both mAbs specifically bound HA, as shown by ELISA, Western blot, and immunofluorescence, but lacked hemagglutination inhibition activity. Epitope mapping revealed two minimal linear epitopes: ¹²³FSSSRSYQ¹³⁰ within the vestigial esterase domain and ²⁰¹NLYTRTDTT²⁰⁹ within the receptor-binding domain. Alanine scanning revealed key residues required for antibody binding, whereas structural modeling confirmed that both epitopes are surface exposed. Sequence analysis demonstrated strong conservation across H9N2 strains, with the 9F4 epitope showing near-complete invariance, whereas both epitopes exhibited low conservation among other influenza A virus subtypes. These findings define two novel, nonneutralizing epitopes on H9N2 HA that expand the antigenic map and represent promising targets for subtype-specific diagnostic assays.

Keywords

H9N2 avian influenza virus / Hemagglutinin / Linear B-cell epitope / Monoclonal antibody / Diagnostic marker

References

[1] Alexander DJ. An overview of the epidemiology of avian influenza. Vaccine, 2007, 25: 5637-5644

[2] Altman MO, Angeletti D, Yewdell JW. Antibody immunodominance: The key to understanding influenza virus antigenic drift. Viral Immunology, 2018, 31: 142-149

[3] Arafat N, Eladl AH, Marghani BH, Saif MA, El-Shafei RA. Enhanced infection of avian influenza virus H9N2 with infectious laryngeotracheitis vaccination in chickens. Veterinary Microbiology, 2018, 219: 8-16

[4] Arafat N, Abd El Rahman S, Naguib D, El-Shafei RA, Abdo W, Eladl AH. Coinfection of Salmonella enteritidis with H9N2 avian influenza virus in chickens. Avian Pathology, 2020, 49: 496-506

[5] Azeem S, Yoon K-J. Diagnostic Assays for Avian Influenza Virus Surveillance and Monitoring in Poultry. Viruses, 2025, 17: 228

[6] Bin Aslam, H., Häsler, B., Iqbal, M., Yaqub, T., Alarcon, P., 2024. Financial impact of low pathogenic avian influenza virus subtype H9N2 on commercial chicken production systems in Pakistan. Barbara and Iqbal, Munir and Yaqub, Tahir and Alarcon, Pablo, Financial Impact of Low Pathogenic Avian Influenza Virus Subtype H9N2 on Commercial Chicken Production Systems in Pakistan. https://doi.org/10.2139/ssrn.4569419.

[7] Byrd-Leotis L, Cummings RD, Steinhauer DA. The interplay between the host receptor and influenza virus hemagglutinin and neuraminidase. International Journal of Molecular Sciences, 2017, 18 1541

[8] Chen H, Yuan H, Gao R, Zhang J, Wang D, Xiong Y, Fan G, Yang F, Li X, Zhou J. Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: A descriptive study. Lancet, 2014, 383: 714-721

[9] Cong YL, Pu J, Liu QF, Wang S, Zhang GZ, Zhang XL, Fan WX, Brown EG, Liu JH. Antigenic and genetic characterization of H9N2 swine influenza viruses in China. Journal of General Virology, 2007, 88: 2035-2041

[10] Cross KJ, Wharton SA, Skehel JJ, Wiley DC, Steinhauer DA. Studies on influenza hemagglutinin fusion peptide mutants generated by reverse genetics. The EMBO Journal, 2001, 20(16): 4432-4442

[11] Dreyfus C, Laursen NS, Kwaks T, Zuijdgeest D, Khayat R, Ekiert DC, Lee JH, Metlagel Z, Bujny MV, Jongeneelen M. Highly conserved protective epitopes on influenza B viruses. Science (New York, n.y.), 1979, 337: 1343-1348

[12] Duan Y, Guo Q, Tu S, Zou J, Li G, Liang C, Cheng Y, Zhou Yijie, Chen L, Zhou Yuanbao. The generation of hemagglutinin monoclonal antibodies against H9N2 influenza virus. Animal Diseases, 2023, 3 38

[13] Ekiert DC, Bhabha G, Elsliger M-A, Friesen RHE, Jongeneelen M, Throsby M, Goudsmit J, Wilson IA. Antibody recognition of a highly conserved influenza virus epitope. Science (New York, n.y.), 1979, 324: 246-251

[14] Gu M, Xu L, Wang X, Liu X. Current situation of H9N2 subtype avian influenza in China. Veterinary Research, 2017, 48 49

[15] Guan Y, Shortridge KF, Krauss S, Webster RG. Molecular characterization of H9N2 influenza viruses: Were they the donors of the “internal” genes of H5N1 viruses in Hong Kong?. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96: 9363-9367

[16] Guo Y, Li J, Cheng X. Discovery of men infected by avian influenza A (H9N2) virus. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1999, 13: 105-108 PMID: 12569771

[17] Han T, Marasco WA. Structural basis of influenza virus neutralization. Annals of the New York Academy of Sciences, 2011, 1217: 178-190

[18] Homme PJ, Easterday BC. Avian influenza virus infections. I. Characteristics of influenza A/Turkey/Wisconsin/1966 virus. Avian Diseases, 1970

[19] Huang Y, Li X, Zhang H, Chen B, Jiang Y, Yang L, Zhu W, Hu S, Zhou S, Tang Y. Human infection with an avian influenza A (H9N2) virus in the middle region of China. Journal of Medical Virology, 2015, 87: 1641-1648

[20] Huang X, Yin G, Cai Y, Hu J, Huang J, Liu Q, Feng X. Identification of unique and conserved neutralizing epitopes of vestigial esterase domain in HA protein of the H9N2 subtype of avian influenza virus. Viruses, 2022, 14 2739

[21] Julkunen I, Pyhälä R, Hovi T. Enzyme immunoassay, complement fixation and hemagglutination inhibition tests in the diagnosis of influenza A and B virus infections. Purified hemagglutinin in subtype-specific diagnosis. Journal of Virological Methods, 1985, 10: 75-84

[22] Lam TT-Y, Wang J, Shen Y, Zhou B, Duan L, Cheung C-L, Ma C, Lycett SJ, Leung CY-H, Chen X. The genesis and source of the H7N9 influenza viruses causing human infections in China. Nature, 2013, 502: 241-244

[23] Li X, Qu B, He G, Cardona CJ, Song Y, Xing Z. Critical role of HAX-1 in promoting avian influenza virus replication in lung epithelial cells. Mediators of Inflammation, 2018, 2018: 3586132

[24] Ming F, Cheng Y, Ren C, Suolang S, Zhou H. Development of a DAS-ELISA for detection of H9N2 avian influenza virus. Journal of Virological Methods, 2019, 263: 38-43

[25] Mostafa A, Abdelwhab EM, Mettenleiter TC, Pleschka S. Zoonotic potential of influenza A viruses: A comprehensive overview. Viruses, 2018, 10: 497

[26] Padilla-Quirarte HO, Lopez-Guerrero DV, Gutierrez-Xicotencatl L, Esquivel-Guadarrama F. Protective antibodies against influenza proteins. Frontiers in Immunology, 2019, 10 1677

[27] Pandey S, Malviya G, Chottova Dvorakova M. Role of peptides in diagnostics. International Journal of Molecular Sciences, 2021, 22 8828

[28] Peacock T, Reddy K, James J, Adamiak B, Barclay W, Shelton H, Iqbal M. Antigenic mapping of an H9N2 avian influenza virus reveals two discrete antigenic sites and a novel mechanism of immune escape. Science and Reports, 2016, 6: 18745

[29] Peacock T(homas)P, James J, Sealy JE, Iqbal M. A global perspective on H9N2 avian influenza virus. Viruses, 2019, 11 620

[30] Quan K, Zhang N, Lin M, Liu Y, Li Y, Hu Q, Nie M, Qin T, Chen S, Peng D. Development of a broad-spectrum subunit vaccine against H9N2 avian influenza using HA stem domain scaffold and snoopligase system. NPJ Vaccines, 2025, 10: 136

[31] Shen W, Wang Q, Wang Z, Liu M, Du Y, Yuan L, Han L, Smietanka K, Chen H, Xu S. Specific monoclonal antibodies targeting unique HA epitopes block H7N9 influenza A viral replication. Journal of Virology, 2022, 96 e01238-22

[32] Skehel JJ, Wiley DC. Receptor binding and membrane fusion in virus entry: The influenza hemagglutinin. Annual Review of Biochemistry, 2000, 69: 531-569

[33] Sui J, Hwang WC, Perez S, Wei G, Aird D, Chen L, Santelli E, Stec B, Cadwell G, Ali M. Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nature Structural & Molecular Biology, 2009, 16: 265-273

[34] Sun X, Xu X, Liu Q, Liang D, Li C, He Q, Jiang J, Cui Y, Li J, Zheng L. Evidence of avian-like H9N2 influenza A virus among dogs in Guangxi, China. Infection, Genetics and Evolution, 2013, 20: 471-475

[35] Taubenberger JK, Layne SP. Diagnosis of influenza virus: Coming to grips with the molecular era. Molecular Diagnosis, 2001, 6: 291-305

[36] Van Regenmortel MHV. What is a B-cell epitope?. Epitope mapping protocols: Second edition, 2009Springer: 3-20

[37] Wan Z, Ye J, Xu L, Shao H, Jin W, Qian K, Wan H, Qin A. Antigenic mapping of the hemagglutinin of an H9N2 avian influenza virus reveals novel critical amino acid positions in antigenic sites. Journal of Virology, 2014, 88: 3898-3901

[38] Wang Z, Huang B, Thomas M, Sreenivasan CC, Sheng Z, Yu J, Hause BM, Wang D, Francis DH, Kaushik RS. Detailed mapping of the linear B-cell epitopes of the hemagglutinin (HA) protein of swine influenza virus. Virology, 2018, 522: 131-137

[39] Wang Y, Li X, Xu Q, Niu X, Zhang S, Qu X, Chu H, Chen J, Shi Q, Zhang E. Characterization of neutralizing monoclonal antibodies and identification of a novel conserved C-terminal linear epitope on the hemagglutinin protein of the H9N2 avian influenza virus. Viruses, 2022, 14 2530

[40] Wu NC, Wilson IA. Influenza hemagglutinin structures and antibody recognition. Cold Spring Harbor Perspectives in Medicine, 2020, 10 a038778

[41] Xiao Y, Yang F, Liu F, Yao H, Wu N, Wu H. Antigen-capture ELISA and immunochromatographic test strip to detect the H9N2 subtype avian influenza virus rapidly based on monoclonal antibodies. Virology Journal, 2021, 18: 198

[42] Yang H, Hu M, Wang B, Jin Y, Gong X, Liang L, Yue J, Chen W, Ren H. Characterizing the core internal gene pool of H9N2 responsible for continuous reassortment with other influenza A viruses. Frontiers in Microbiology, 2021, 12 751142

[43] Zhu Y, Yang D, Ren Q, Yang Y, Liu Xin, Xu X, Liu W, Chen S, Peng D, Liu Xiufan. Identification and characterization of a novel antigenic epitope in the hemagglutinin of the escape mutants of H9N2 avian influenza viruses. Veterinary Microbiology, 2015, 178: 144-149

Funding

National Key Research and Development Program of China(2022YFD1801005)

earmarked fund(CARS-41)

RIGHTS & PERMISSIONS

The Author(s)