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Broadly protective murine monoclonal antibodies against influenza B virus target highly conserved neuraminidase epitopes

Abstract

A substantial proportion of influenza-related childhood deaths are due to infection with influenza B viruses, which co-circulate in the human population as two antigenically distinct lineages defined by the immunodominant receptor binding protein, haemagglutinin. While broadly cross-reactive, protective monoclonal antibodies against the haemagglutinin of influenza B viruses have been described, none targeting the neuraminidase, the second most abundant viral glycoprotein, have been reported. Here, we analyse a panel of five murine anti-neuraminidase monoclonal antibodies that demonstrate broad binding, neuraminidase inhibition, in vitro antibody-dependent cell-mediated cytotoxicity and in vivo protection against influenza B viruses belonging to both haemagglutinin lineages and spanning over 70 years of antigenic drift. Electron microscopic analysis of two neuraminidase–antibody complexes shows that the conserved neuraminidase epitopes are located on the head of the molecule and that they are distinct from the enzymatic active site. In the mouse model, one therapeutic dose of antibody 1F2 was more protective than the current standard of treatment, oseltamivir, given twice daily for six days.

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References

  1. 1.

    Shaw, M. & Palese, P. Orthomyxoviridae: the viruses and their replication. Fields Virol. 2, 1648–1689 (2013).

  2. 2.

    Chen, J. M. et al. Exploration of the emergence of the Victoria lineage of influenza B virus. Arch. Virol. 152, 415–422 (2007).

  3. 3.

    Molinari, N. A. et al. The annual impact of seasonal influenza in the US: measuring disease burden and costs. Vaccine 25, 5086–5096 (2007).

  4. 4.

    Dijkstra, F., Donker, G. A., Wilbrink, B., Van Gageldonk-Lafeber, A. B. & Van Der Sande, M. A. B. Long time trends in influenza-like illness and associated determinants in The Netherlands. Epidemiol. Infect. 137, 473–479 (2009).

  5. 5.

    Heikkinen, T., Ikonen, N. & Ziegler, T. Impact of influenza B lineage-level mismatch between trivalent seasonal influenza vaccines and circulating viruses, 1999–2012. Clin. Infect. Dis. 59, 1519–1524 (2014).

  6. 6.

    Brottet, E. et al. Influenza season in Réunion dominated by infuenza B virus circulation associated with numerous cases of severe disease, France, 2014. Euro. Surveill.  19, 20916 (2014).

  7. 7.

    Su, S. et al. Comparing clinical characteristics between hospitalized adults with laboratory-confirmed influenza A and B virus infection. Clin. Infect. Dis. 59, 252–255 (2014).

  8. 8.

    Centers for Disease Control and Prevention. Influenza-associated pediatric deaths—United States, September 2010–August 2011. MMWR. Morb. Mortal. Wkly. Rep. 60, 1233–1238 (2011).

  9. 9.

    Fiore, A. E., Fry, A., Shay, D., Gubareva, L., Bresee, J. S. & Uyeki, T. M. Antiviral agents for the treatment and chemoprophylaxis of influenza—recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm. Rep. 60, 1–24 (2011).

  10. 10.

    Kawai, N. et al. A comparison of the effectiveness of oseltamivir for the treatment of influenza A and influenza B: a Japanese multicenter study of the 2003–2004 and 2004–2005 influenza seasons. Clin. Infect. Dis. 43, 439–444 (2006).

  11. 11.

    Kawai, N. et al. Longer virus shedding in influenza B than in influenza A among outpatients treated with oseltamivir. J. Infect. 55, 267–272 (2007).

  12. 12.

    Sugaya, N. et al. Lower clinical effectiveness of oseltamivir against influenza B contrasted with influenza A infection in children. Clin. Infect. Dis. 44, 197–202 (2007).

  13. 13.

    Wang, Q., Cheng, F., Lu, M., Tian, X. & Ma, J. Crystal structure of unliganded influenza B virus hemagglutinin. J. Virol. 82, 3011–3020 (2008).

  14. 14.

    Dreyfus, C. et al. Highly conserved protective epitopes on influenza B viruses. Science 337, 1343–1348 (2012).

  15. 15.

    Yasugi, M. et al. Human monoclonal antibodies broadly neutralizing against influenza B virus. PLoS Pathog. 9, e1003150 (2013).

  16. 16.

    Air, G. M., Laver, W. G., Luo, M., Stray, S. J., Legrone, G. & Webster, R. G. Antigenic, sequence, and crystal variation in influenza B neuraminidase. Virology 177, 578–587 (1990).

  17. 17.

    Laver, W. G. et al. Crystallization and preliminary X-ray analysis of type B influenza virus neuraminidase complexed with antibody Fab fragments. Virology 167, 621–624 (1988).

  18. 18.

    Doyle, T. M. et al. A monoclonal antibody targeting a highly conserved epitope in influenza B neuraminidase provides protection against drug resistant strains. Biochem. Biophys. Res. Commun. 441, 226–229 (2013).

  19. 19.

    Schulman, J. L., Khakpour, M. & Kilbourne, E. Protective effects of specific immunity to viral neuraminidase on influenza virus infection in mice. J. Virol. 2, 778–776 (1968).

  20. 20.

    Dowdle, W. R., Coleman, M. T., Mostow, S. R., Kaye, H. S. & Schoenbaum, S. C. Inactivated influenza vaccines. 2. Laboratory indices of protection. Postgrad. Med. J. 49, 159–163 (1973).

  21. 21.

    Couch, R. B. et al. Induction of partial immunity to influenza by a neuraminidase-specific vaccine. J. Infect. Dis. 129, 411–420 (1974).

  22. 22.

    Johansson, B. E. & Kilbourne, E. D. Immunization with purified N1 and N2 influenza virus neuraminidases demonstrates cross-reactivity without antigenic competition. Proc. Natl Acad. Sci. USA 91, 2358–2361 (1994).

  23. 23.

    Rockman, S. et al. Neuraminidase-inhibiting antibody is a correlate of cross-protection against lethal H5N1 influenza virus in ferrets immunized with seasonal influenza vaccine. J. Virol. 87, 3053–3061 (2013).

  24. 24.

    Easterbrook, J. D. et al. Protection against a lethal H5N1 influenza challenge by intranasal immunization with virus-like particles containing 2009 pandemic H1N1 neuraminidase in mice. Virology 432, 39–44 (2012).

  25. 25.

    Wan, H. et al. Molecular basis for broad neuraminidase immunity: conserved epitopes in seasonal and pandemic H1N1 as well as H5N1 influenza viruses. J. Virol. 87, 9290–9300 (2013).

  26. 26.

    Wohlbold, T. J. et al. Vaccination with adjuvanted recombinant neuraminidase induces broad heterologous, but not heterosubtypic, cross-protection against influenza virus infection in mice. mBio 6, e02556-14 (2015).

  27. 27.

    Wohlbold, T. J. et al. Hemagglutinin stalk- and neuraminidase-specific monoclonal antibodies protect against lethal H10N8 influenza virus infection in mice. J. Virol. 90, 851–861 (2015).

  28. 28.

    Memoli, M. J. et al. Evaluation of antihemagglutinin and antineuraminidase antibodies as correlates of protection in an influenza A/H1N1 virus healthy human challenge model. mB io 7, e00417-16 (2016).

  29. 29.

    Palese, P., Tobita, K., Ueda, M. & Compans, R. W. Characterization of temperature sensitive influenza virus mutants defective in neuraminidase. Virology 61, 397–410 (1974).

  30. 30.

    Matrosovich, M. N., Matrosovich, T. Y., Roberts, N. A., Klenk, H. & Gray, T. Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium. J. Virol. 78, 12665–12667 (2004).

  31. 31.

    Cohen, M. et al. Influenza A penetrates host mucus by cleaving sialic acids with neuraminidase. Virol. J. 10, 321 (2013).

  32. 32.

    Wan, H. et al. Structural characterization of a protective epitope spanning A(H1N1)pdm09 influenza virus neuraminidase monomers. Nat. Commun. 6, 6114 (2015).

  33. 33.

    Kilbourne, E. D. Comparative efficacy of neuraminidase-specific and conventional influenza virus vaccines in induction of antibody to neuraminidase in humans. J. Infect. Dis. 134, 384–394 (1976).

  34. 34.

    Webster, R. G., Laver, W. G. & Kilbourne, E. D. Reactions of antibodies with surface antigens of influenza virus. J. Gen. Virol. 3, 315–326 (1968).

  35. 35.

    DiLillo, D. J., Tan, G. S., Palese, P. & Ravetch, J. V. Broadly neutralizing hemagglutinin stalk-specific antibodies require FcγR interactions for protection against influenza virus in vivo. Nat. Med. 20, 143–151 (2014).

  36. 36.

    DiLillo, D. J., Palese, P., Wilson, P. C. & Ravetch, J. V. Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection. J. Clin. Invest 126, 605–610 (2016).

  37. 37.

    Henry Dunand, C. J. et al. Both neutralizing and non-neutralizing human H7N9 influenza vaccine-induced monoclonal antibodies confer protection. Cell Host Microbe 19, 800–813 (2016).

  38. 38.

    Webster, R. G., Brown, L. E. & Laver, W. G. Antigenic and biological characterization of influenza virus neuraminidase (N2) with monoclonal antibodies. Virology 135, 30–42 (1984).

  39. 39.

    Air, G. M., Els, M. C., Brown, L. E., Laver, W. G. & Webster, R. G. Location of antigenic sites on the three-dimensional structure of the influenza N2 virus neuraminidase. Virology 145, 337–248 (1985).

  40. 40.

    Gulati, U. et al. Antibody epitopes on the neuraminidase of a recent H3N2 influenza virus (A/Memphis/31/98). J. Virol. 76, 12274–12280 (2002).

  41. 41.

    Nicholson, K. G. et al. Efficacy and safety of oseltamivir in treatment of acute influenza: a randomised controlled trial. Lancet 355, 1845–1850 (2000).

  42. 42.

    Heinonen, S. et al. Early oseltamivir treatment of influenza in children 1–3 years of age: a randomized controlled trial. Clin. Infect. Dis. 51, 887–894 (2010).

  43. 43.

    Leon, P. E. et al. Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact. Proc. Natl Acad. Sci. USA 113, E5944–E5951 (2016).

  44. 44.

    He, W. et al. Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus. Proc. Natl Acad. Sci. USA 113, 11931–11936 (2016).

  45. 45.

    Krammer, F., Schinko, T., Palmberger, D., Tauer, C., Messner, P. & Grabherr, R. Trichoplusia ni cells (High FiveTM) are highly efficient for the production of influenza A virus-like particles: a comparison of two insect cell lines as production platforms for influenza vaccines. Mol. Biotechnol. 45, 226–234 (2010).

  46. 46.

    Krammer, F., Margine, I., Tan, G. S., Pica, N., Krause, J. C. & Palese, P. A carboxy-terminal trimerization domain stabilizes conformational epitopes on the stalk domain of soluble recombinant hemagglutinin substrates. PLoS ONE 7, e43603 (2012).

  47. 47.

    Margine, I., Palese, P. & Krammer, F. Expression of functional recombinant hemagglutinin and neuraminidase proteins from the novel H7N9 influenza virus using the baculovirus expression system. J. Vis. Exp. 2013, e51112 (2013).

  48. 48.

    Wang, T. T. et al. Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins. PLoS Pathog. 6, e1000796 (2010).

  49. 49.

    Nachbagauer, R. et al. Induction of broadly-reactive anti-hemagglutinin stalk antibodies by an H5N1 vaccine in humans. J. Virol. 88, 13260–13268 (2014).

  50. 50.

    Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

  51. 51.

    Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

  52. 52.

    Wohlbold, T. J., Hirsh, A. & Krammer, F. An H10N8 influenza virus vaccine strain and mouse challenge model based on the human isolate A/Jiangxi-Donghu/346/13. Vaccine 33, 1102–1106 (2015).

  53. 53.

    Klausberger, M. et al. One-shot vaccination with an insect cell-derived low-dose influenza A H7 virus-like particle preparation protects mice against H7N9 challenge. Vaccine 32, 355–362 (2014).

  54. 54.

    Krammer, F. et al. Divergent H7 immunogens offer protection from H7N9 virus challenge. J. Virol. 88, 3976–3985 (2014).

  55. 55.

    Tan, G. S., Krammer, F., Eggink, D., Kongchanagul, A., Moran, T. M. & Palese, P. A pan-H1 anti-hemagglutinin monoclonal antibody with potent broad-spectrum efficacy in vivo. J. Virol. 86, 6179–6188 (2012).

  56. 56.

    Hai, R. et al. Influenza A(H7N9) virus gains neuraminidase inhibitor resistance without loss of in vivo virulence or transmissibility. Nat. Commun. 4, 2854 (2013).

  57. 57.

    Marathe, B. M. et al. Combinations of oseltamivir and T-705 extend the treatment window for highly pathogenic influenza A(H5N1) virus infection in mice. Sci. Rep. 6, 26742 (2016).

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Acknowledgements

The authors thank I. Margine for pilot studies, M. Rajendran Marilyne Panis and R. Nachbagauer for their assistance in the deep sequencing analysis of IBV mutants and competition ELISAs, A. Hirsh for producing recombinant neuraminidase proteins and N. Bouvier for her instructions regarding oral gavaging. We thank A. Hurt (WHO Influenza Collaborating Centre For Reference And Research On Influenza, Melbourne, Australia) and E. Govorkova (St. Jude Children's Hospital, Memphis, TN) for providing NA-inhibitor resistant influenza B virus isolates. This work was funded by NIAID grants R01 AI117287 (to F.K.) and U19 AI109946 (to P.P. and F.K.).

Author information

T.J.W., K.A.P., S.S. and F.K. designed experiments and wrote the manuscript. T.J.W., K.A.P., V.C. and P.M. performed experiments. J.T. and F.A. assisted with experiments. F.A. and G.S.T. generated reagents. T.J.W., K.A.P., V.C., V.F., J.T., E.K., B.R.t., P.P., S.S. and F.K. analysed and interpreted data.

Competing interests

The Icahn School of Medicine at Mount Sinai has filed patents regarding use of the described mAbs as therapeutics (application no. 62/483,262). T.J.W., P.P. and F.K. are named as inventors on the application.

Correspondence to Florian Krammer.

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Fig. 1: In vitro binding of IBV anti-NA mAbs.
Fig. 2: Negative-stain electron microscopy analysis of NA structures reveals binding footprints for 1F2 and 4F11.
Fig. 3: In vivo efficacy of IBV anti-NA mAbs.
Fig. 4: Non-neutralizing IBV anti-NA mAbs reduce viral lung titres in mice, activate ADCC, inhibit activity of a drug-resistant IBV and demonstrate superior effectiveness to oseltamivir.