Applied Microbiology and Biotechnology

, Volume 102, Issue 13, pp 5645–5656 | Cite as

Monoclonal antibody against the universal M2 epitope of influenza A virus

  • Mingfang Feng
  • Zhuangchuan Yuan
  • Wenjun Xia
  • Xiaozhi Huang
  • XingBo Wang
  • Yan Yan
  • Min LiaoEmail author
  • Jiyong Zhou
Applied genetics and molecular biotechnology


M2 protein, a highly conserved protein of influenza A virus (IAV), plays an important role in virus particle uncoating, assembly, and budding. In the present study, eight monoclonal antibodies (mAbs) against the M2 protein of the H3N2 IAV strain were generated with recombinant truncated M2 protein or BSA-coupled M2 peptides as immunogens. The linear epitopes recognized by the mAbs were defined by IFA and peptide ELISA. The results showed that mAb 10F4 recognized an epitope located in the N-terminal 6–12 amino acids of the M2 peptide, and the mAbs 10D9, 1E2, 4B5, and 5G10 recognized the epitopes located in the C-terminal 62–77 amino acids of the M2 peptide. Importantly, mAb 10D9 recognized the M2 protein of H1-H13 IAV subtypes, which stained M2 protein located on the membrane of host cells and could be applied in immunoprecipitation and immunohistochemistry assays. The mAb 10D9 which recognizes the universal M2 epitope of IAVs will be a useful tool for studies on the function of IAV M2 protein and for the development of vaccines or detection methods for IAV infection.


Influenza A virus (IAV) Subtypes M2 protein Monoclonal antibody Universal epitope 


Funding information

This work was funded through grants from the National Key R & D Program of China (Grant No. 2015BAD12B01) and the China Agriculture Research System (CARS-40-K13).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical statement

The animal experiment was approved by the Committee on the Ethics of Animal of Zhejiang University (ZJU20170667) and implemented in accordance with the animal care and ethics guidelines. This article does not contain any studies with human participants performed by any of the authors.

Supplementary material

253_2018_9019_MOESM1_ESM.pdf (209 kb)
ESM 1 (PDF 208 kb)


  1. Chiarella FC, Daoud Z, Fuentes-Ferrer ME, Amador JTR, Picazo JJ, Culebras E (2017) Characterization and circulation of seasonal influenza viruses in Madrid, 2010-2016. J Med Virol 89(10):1726–1733. CrossRefPubMedGoogle Scholar
  2. Chou JJ, Schnell JR (2010) Structure and mechanism of the M2 channel. In: Wang Q, Tao YJ (eds) Influenza: molecular Virology, pp 109–124Google Scholar
  3. van de Sandt CE, Kreijtz JHCM, Rimmelzwaan GF (2012) Evasion of influenza A viruses from innate and adaptive immune responses. Viruses-Basel 4(9):1438–1476. CrossRefGoogle Scholar
  4. Deng L, Cho KJ, Fiers W, Saelens X (2015) M2e-based universal influenza A vaccines. Vaccine 3(1):105–136. CrossRefGoogle Scholar
  5. FernandezSesma A, Schulman JL, Moran TM (1996) A bispecific antibody recognizing influenza A virus M2 protein redirects effector cells to inhibit virus replication in vitro. J Virol 70(7):4800–4804Google Scholar
  6. Fouchier RAM, Schneeberger PM, Rozendaal FW, Broekman JM, Kemink SAG, Munstert V, Kuiken T, Rimmelzwaan GF, Schutten M, van Doornum GJJ, Koch G, Bosman A, Koopmans M, Osterhaus A (2004) Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome. Proc Natl Acad Sci USA 101(5):1356–1361. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Friguet B, Chaffotte AF, Djavadi-Ohaniance L, Goldberg ME (1985) Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods 77(2):305–319. CrossRefPubMedGoogle Scholar
  8. Fu TM, Freed DC, Horton MS, Fan J, Citron MP, Joyce JG, Garsky VM, Casimiro DR, Zhao Q, Shiver JW, Liang X (2009) Characterizations of four monoclonal antibodies against M2 protein ectodomain of influenza A virus. Virology 385(1):218–226. CrossRefPubMedGoogle Scholar
  9. Gao HN (2013) Clinical findings in 111 cases of influenza A (H7N9) virus infection. N Engl J Med 369(19):1869–1869. CrossRefGoogle Scholar
  10. Holsinger LJ, Shaughnessy MA, Micko A, Pinto LH, Lamb RA (1995) Analysis of the posttranslational modifications of the influenza-virus M(2) protein. J Virol 69(2):1219–1225PubMedPubMedCentralGoogle Scholar
  11. Husain M (2014) Avian influenza A (H7N9) virus infection in humans: epidemiology, evolution, and pathogenesis. Infect Genet Evol 28:304–312. CrossRefPubMedGoogle Scholar
  12. Joyce JG, Hurni WM, Bogusky MJ, Garsky VM, Liang XP, Citron MP, Danzeisen RC, Miller MD, Shiver JW, Keller PM (2002) Enhancement of alpha-helicity in the HIV-1 inhibitory peptide DP178 leads to an increased affinity for human monoclonal antibody 2F5 but does not elicit neutralizing responses in vitro—implications for vaccine design. J Biol Chem 277(48):45811–45820. CrossRefPubMedGoogle Scholar
  13. Kageyama T, Fujisaki S, Takashita E, Xu H, Yamada S, Uchida Y, Neumann G, Saito T, Kawaoka Y, Tashiro M (2013) Genetic analysis of novel avian A (H7N9) influenza viruses isolated from patients in China, February to April 2013. Eur Secur 18(15):20453Google Scholar
  14. Koopmans M, Wilbrink B, Conyn M, Natrop G, van der Nat H, Vennema H, Meijer A, van Steenbergen J, Fouchier R, Osterhaus A, Bosman A (2004) Transmission of H7N7 avian influenza A virus to human beings during a large outbreak in commercial poultry farms in the Netherlands. Lancet 363(9409):587–593. CrossRefPubMedGoogle Scholar
  15. Liu WL, Li H, Chen YH (2003) N-terminus of M2 protein could induce antibodies with inhibitory activity against influenza virus replication. Fems Immunol Med Microbiol 35(2):141–146. CrossRefPubMedGoogle Scholar
  16. Liu WL, Zou P, Chen YH (2004) Monoclonal antibodies recognizing EVETPIRN epitope of influenza A virus M2 protein could protect mice from lethal influenza A virus challenge. Immunol Lett 93(2–3):131–136. CrossRefPubMedGoogle Scholar
  17. Ma C, Polishchuk AL, Ohigashi Y, Stouffer AL, Schoen A, Magavern E, Jing X, Lear JD, Freire E, Lamb RA, DeGrado WF, Pinto LH (2009) Identification of the functional core of the influenza A virus A/M2 proton-selective ion channel. Proc Natl Acad Sci USA 106(30):12283–12288. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Meng Q, Liu G, Liu Y, Deng X, Wang W, Xu K, Zheng X, Zhang D, Pang H, Chen H (2015) A broad protection provided by matrix protein 2 (M2) of avian influenza virus. Vaccine 33(31):3758–3765. CrossRefPubMedGoogle Scholar
  19. Pinto LH, Holsinger LJ, Lamb RA (1992) Influenza-virus M2 protein has ion channel activity. Cell 69(3):517–528. CrossRefPubMedGoogle Scholar
  20. Pissawong T, Maneewatch S, Thueng-in K, Srimanote P, Dong-din-on F, Thanongsaksrikul J, Songserm T, Tongtawe P, Bangphoomi K, Chaicumpa W (2013) Human monoclonal ScFv that bind to different functional domains of M2 and inhibit H5N1 influenza virus replication. Virol J 10:148. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Qi W, Zhou X, Shi W, Huang L, Xia W, Liu D, Li H, Chen S, Lei F, Cao L, Wu J, He F, Song W, Li Q, Li H, Liao M, Liu M (2014) Genesis of the novel human-infecting influenza A(H10N8) virus and potential genetic diversity of the virus in poultry, China. Eur Secur 19(25) doi:
  22. Rossman JS, Lamb RA (2011) Influenza virus assembly and budding. Virology 411(2):229–236. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Shu B, Garten R, Emery S, Balish A, Cooper L, Sessions W, Deyde V, Smith C, Berman L, Klimov A, Lindstrom S, Xu X (2012) Genetic analysis and antigenic characterization of swine origin influenza viruses isolated from humans in the United States, 1990-2010. Virology 422(1):151–160. CrossRefPubMedGoogle Scholar
  24. Tweed SA, Skowronski DM, David ST, Larder A, Petric M, Lees W, Li Y, Katz J, Krajden M, Tellier R, Halpert C, Hirst M, Astell C, Lawrence D, Mak A (2004) Human illness from avian influenza H7N3, British Columbia. Emerg Infect Dis 10(12):2196–2199. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Wei SH, Yang JR, Wu HS, Chang MC, Lin JS, Lin CY, Liu YL, Lo YC, Yang CH, Chuang JH, Lin MC, Chung WC, Liao CH, Lee MS, Huang WT, Chen PJ, Liu MT, Chang FY (2013) Human infection with avian influenza A H6N1 virus: an epidemiological analysis. Lancet Respir Med 1(10):771–778. CrossRefPubMedGoogle Scholar
  26. Zebedee SL, Lamb RA (1988) Influenza A virus M2-protein-monoclonal-antibody restriction of virus growth and detection of M2 in virions. J Virol 62(8):2762–2772PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Mingfang Feng
    • 1
  • Zhuangchuan Yuan
    • 1
  • Wenjun Xia
    • 1
  • Xiaozhi Huang
    • 1
  • XingBo Wang
    • 1
  • Yan Yan
    • 1
  • Min Liao
    • 1
    Email author
  • Jiyong Zhou
    • 1
  1. 1.Key Laboratory of Animal Virology, Ministry of AgricultureZhejiang UniversityHangzhouPeople’s Republic of China

Personalised recommendations