Abstract
Neutralizing antibodies against influenza viruses have traditionally been thought to provide protection exclusively through their variable region; the contributions of mechanisms conferred by the Fc domain remain controversial. We investigated the in vivo contributions of Fc interactions with their cognate receptors for a collection of neutralizing anti-influenza antibodies. Whereas five broadly neutralizing monoclonal antibodies (bNAbs) targeting the conserved stalk region of hemagglutinin (HA) required interactions between the antibody Fc and Fc receptors for IgG (FcγRs) to confer protection from lethal H1N1 challenge, three strain-specific monoclonal Abs (mAbs) against the variable head domain of HA were equally protective in the presence or absence of FcγR interactions. Although all antibodies blocked infection, only anti-stalk bNAbs were capable of mediating cytotoxicity of infected cells, which accounts for their FcγR dependence. Immune complexes generated with anti–HA stalk mAb efficiently interacted with FcγRs, but anti–HA head immune complexes did not. These results suggest that FcγR binding capacity by anti-HA antibodies was dependent on the interaction of the cognate Fab with antigen. We exploited these disparate mechanisms of mAb-mediated protection to reengineer an anti-stalk bNAb to selectively enhance FcγR engagement to augment its protective activity. These findings reveal a previously uncharacterized property of bNAbs and guide an approach toward enhancing mAb-mediated antiviral therapeutics.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
World Health Organization. Influenza (Seasonal) Fact sheet 211 http://www.who.int/mediacentre/factsheets/fs211/en/ (2009).
Ahmed, R., Oldstone, M.B. & Palese, P. Protective immunity and susceptibility to infectious diseases: lessons from the 1918 influenza pandemic. Nat. Immunol. 8, 1188–1193 (2007).
Martinez, O., Tsibane, T. & Basler, C.F. Neutralizing anti-influenza virus monoclonal antibodies: therapeutics and tools for discovery. Int. Rev. Immunol. 28, 69–92 (2009).
Couch, R.B. & Kasel, J.A. Immunity to influenza in man. Annu. Rev. Microbiol. 37, 529–549 (1983).
Wang, T.T. & Palese, P. Biochemistry. Catching a moving target. Science 333, 834–835 (2011).
Skehel, J.J. & Wiley, D.C. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu. Rev. Biochem. 69, 531–569 (2000).
Knossow, M. et al. Mechanism of neutralization of influenza virus infectivity by antibodies. Virology 302, 294–298 (2002).
Wiley, D.C., Wilson, I.A. & Skehel, J.J. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature 289, 373–378 (1981).
Dreyfus, C. et al. Highly conserved protective epitopes on influenza B viruses. Science 337, 1343–1348 (2012).
Barbey-Martin, C. et al. An antibody that prevents the hemagglutinin low pH fusogenic transition. Virology 294, 70–74 (2002).
Ekiert, D.C. et al. Antibody recognition of a highly conserved influenza virus epitope. Science 324, 246–251 (2009).
Sui, J. et al. Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat. Struct. Mol. Biol. 16, 265–273 (2009).
Corti, D. et al. A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science 333, 850–856 (2011).
Abboud, N. et al. A requirement for FcgγR in antibody-mediated bacterial toxin neutralization. J. Exp. Med. 207, 2395–2405 (2010).
Bournazos, S., Chow, S.K., Abboud, N., Casadevall, A. & Ravetch, J.V. Human IgG Fc domain engineering enhances anti-toxin neutralizing antibody activity. J. Clin. Invest. (in the press).
Schmitz, N. et al. Universal vaccine against influenza virus: linking TLR signaling to anti-viral protection. Eur. J. Immunol. 42, 863–869 (2012).
Huber, V.C., Lynch, J.M., Bucher, D.J., Le, J. & Metzger, D.W. Fc receptor–mediated phagocytosis makes a significant contribution to clearance of influenza virus infections. J. Immunol. 166, 7381–7388 (2001).
Nimmerjahn, F. & Ravetch, J.V. Fcγ receptors as regulators of immune responses. Nat. Rev. Immunol. 8, 34–47 (2008).
Nimmerjahn, F. & Ravetch, J.V. Divergent immunoglobulin g subclass activity through selective Fc receptor binding. Science 310, 1510–1512 (2005).
Tan, G.S. et al. A pan-H1 anti-hemagglutinin monoclonal antibody with potent broad-spectrum efficacy in vivo. J. Virol. 86, 6179–6188 (2012).
Li, G.M. et al. Pandemic H1N1 influenza vaccine induces a recall response in humans that favors broadly cross-reactive memory B cells. Proc. Natl. Acad. Sci. USA 109, 9047–9052 (2012).
Reale, M.A. et al. Characterization of monoclonal antibodies specific for sequential influenza A/PR/8/34 virus variants. J. Immunol. 137, 1352–1358 (1986).
Wrammert, J. et al. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. J. Exp. Med. 208, 181–193 (2011).
Smith, P., DiLillo, D.J., Bournazos, S., Li, F. & Ravetch, J.V. Mouse model recapitulating human Fcγ receptor structural and functional diversity. Proc. Natl. Acad. Sci. USA 109, 6181–6186 (2012).
Manicassamy, B. et al. Protection of mice against lethal challenge with 2009 H1N1 influenza A virus by 1918-like and classical swine H1N1 based vaccines. PLoS Pathog. 6, e1000745 (2010).
Jegaskanda, S. et al. Cross-reactive influenza-specific antibody-dependent cellular cytotoxicity antibodies in the absence of neutralizing antibodies. J. Immunol. 190, 1837–1848 (2013).
Jegaskanda, S., Weinfurter, J.T., Friedrich, T.C. & Kent, S.J. Antibody-dependent cellular cytotoxicity (ADCC) is associated with control of pandemic H1N1 influenza virus infection of macaques. J. Virol. 87, 5512–5522 (2013).
Srivastava, V. et al. Identification of dominant ADCC epitopes on hemagglutinin antigen of pandemic H1N1 influenza virus. J. Virol. 87, 5831–5840 (2013).
Alter, G., Malenfant, J.M. & Altfeld, M. CD107a as a functional marker for the identification of natural killer cell activity. J. Immunol. Methods 294, 15–22 (2004).
Nimmerjahn, F. & Ravetch, J.V. Analyzing antibody–Fc-receptor interactions. Methods Mol. Biol. 415, 151–162 (2008).
El Bakkouri, K. et al. Universal vaccine based on ectodomain of matrix protein 2 of influenza A: Fc receptors and alveolar macrophages mediate protection. J. Immunol. 186, 1022–1031 (2011).
Jegerlehner, A., Schmitz, N., Storni, T. & Bachmann, M.F. Influenza A vaccine based on the extracellular domain of M2: weak protection mediated via antibody-dependent NK cell activity. J. Immunol. 172, 5598–5605 (2004).
Tedder, T.F., Baras, A. & Xiu, Y. Fcγ receptor–dependent effector mechanisms regulate CD19 and CD20 antibody immunotherapies for B lymphocyte malignancies and autoimmunity. Springer Semin. Immunopathol. 28, 351–364 (2006).
Nordstrom, J.L. et al. Anti-tumor activity and toxicokinetics analysis of MGAH22, an anti-HER2 monoclonal antibody with enhanced Fcγ receptor binding properties. Breast Cancer Res. 13, R123 (2011).
Clynes, R.A., Towers, T.L., Presta, L.G. & Ravetch, J.V. Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat. Med. 6, 443–446 (2000).
Yang, X. et al. Cetuximab-mediated tumor regression depends on innate and adaptive immune responses. Mol. Ther. 21, 91–100 (2013).
Belser, J.A., Katz, J.M. & Tumpey, T.M. The ferret as a model organism to study influenza A virus infection. Dis. Model. Mech. 4, 575–579 (2011).
Nimmerjahn, F. & Ravetch, J.V. Antibodies, Fc receptors and cancer. Curr. Opin. Immunol. 19, 239–245 (2007).
Nimmerjahn, F. & Ravetch, J.V. FcγRs in health and disease. Curr. Top. Microbiol. Immunol. 350, 105–125 (2011).
Barkas, T. & Watson, C.M. Induction of an Fc conformational change by binding of antigen: the generation of protein A–reactive sites in chicken immunoglobulin. Immunology 36, 557–561 (1979).
Kilàr, F. & Zavodszky, P. Non-covalent interactions between Fab and Fc regions in immunoglobulin G molecules. Hydrogen-deuterium exchange studies. Eur. J. Biochem. 162, 57–61 (1987).
Torres, M., Fernandez-Fuentes, N., Fiser, A. & Casadevall, A. The immunoglobulin heavy chain constant region affects kinetic and thermodynamic parameters of antibody variable region interactions with antigen. J. Biol. Chem. 282, 13917–13927 (2007).
Oda, M., Kozono, H., Morii, H. & Azuma, T. Evidence of allosteric conformational changes in the antibody constant region upon antigen binding. Int. Immunol. 15, 417–426 (2003).
Schlessinger, J., Steinberg, I.Z., Givol, D., Hochman, J. & Pecht, I. Antigen-induced conformational changes in antibodies and their Fab fragments studied by circular polarization of fluorescence. Proc. Natl. Acad. Sci. USA 72, 2775–2779 (1975).
Tong, H. et al. Peptide-conjugation induced conformational changes in human IgG1 observed by optimized negative-staining and individual-particle electron tomography. Sci. Rep. 3, 1089 (2013).
Takai, T., Li, M., Sylvestre, D., Clynes, R. & Ravetch, J.V. FcR γ chain deletion results in pleiotrophic effector cell defects. Cell 76, 519–529 (1994).
Takai, T., Ono, M., Hikida, M., Ohmori, H. & Ravetch, J.V. Augmented humoral and anaphylactic responses in FcγRII-deficient mice. Nature 379, 346–349 (1996).
Ioan-Facsinay, A. et al. FcγRI (CD64) contributes substantially to severity of arthritis, hypersensitivity responses, and protection from bacterial infection. Immunity 16, 391–402 (2002).
Hazenbos, W.L. et al. Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc γ RIII (CD16) deficient mice. Immunity 5, 181–188 (1996).
Nimmerjahn, F. et al. FcγRIV deletion reveals its central role for IgG2a and IgG2b activity in vivo. Proc. Natl. Acad. Sci. USA 107, 19396–19401 (2010).
Li, F. & Ravetch, J.V. Inhibitory Fcγ receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies. Science 333, 1030–1034 (2011).
Nimmerjahn, F., Bruhns, P., Horiuchi, K. & Ravetch, J.V. FcγRIV: a novel FcR with distinct IgG subclass specificity. Immunity 23, 41–51 (2005).
Wang, T.T. et al. Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins. PLoS Pathog. 6, e1000796 (2010).
Acknowledgements
We thank P. Wilson (University of Chicago), J. Wrammert (Emory University) and R. Ahmed (Emory University) for providing mAb constructs, F. Krammer (Mt. Sinai Medical Center) for providing recombinant soluble HA protein, S. Bournazos for assistance with surface plasmon resonance studies and J. Carroll and P. Smith for technical assistance. We also thank the Biodefense and Emerging Infections Research Resources Repository for supplying recombinant soluble HA protein. Research reported in this publication was partially supported by the National Institute of Allergy and Infectious Disease of the US National Institutes of Health (NIH) under award numbers P01AI081677, R01AI035875 and U54AI057158 to J.V.R. and AI097092 to P.P. Research support was also provided by the Bill & Melinda Gates Foundation grant OPP1033115 to J.V.R. This work was also supported in part by the US Army Research Laboratory and the US Army Research Office under contract number W911NF-13-2-0036 (to J.V.R.). Partial support for P.P. was provided by the Program for Appropriate Technology in Health and by the NIH-funded Center for Research on Influenza Pathogenesis (HHSN266200700010C). D.J.D. received fellowship support from the Leukemia and Lymphoma Society and received funding support from the New York Community Trust.
Author information
Authors and Affiliations
Contributions
D.J.D., G.S.T., P.P. and J.V.R. designed experiments, D.J.D. and G.S.T. performed the experiments, D.J.D., G.S.T., P.P. and J.V.R. analyzed data and D.J.D. and J.V.R. wrote the paper.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–15 and Supplementary Table 1 (PDF 1290 kb)
Rights and permissions
About this article
Cite this article
DiLillo, D., Tan, G., Palese, P. et al. Broadly neutralizing hemagglutinin stalk–specific antibodies require FcγR interactions for protection against influenza virus in vivo. Nat Med 20, 143–151 (2014). https://doi.org/10.1038/nm.3443
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nm.3443
This article is cited by
-
Modulating the immune response to SARS-CoV-2 by different nanocarriers delivering an mRNA expressing trimeric RBD of the spike protein: COVARNA Consortium
npj Vaccines (2024)
-
Enhancing breadth and durability of humoral immune responses in non-human primates with an adjuvanted group 1 influenza hemagglutinin stem antigen
npj Vaccines (2023)
-
B-cell and antibody responses to SARS-CoV-2: infection, vaccination, and hybrid immunity
Cellular & Molecular Immunology (2023)
-
Antibody-mediated NK cell activation as a correlate of immunity against influenza infection
Nature Communications (2023)
-
Systems serology-based comparison of antibody effector functions induced by adjuvanted vaccines to guide vaccine design
npj Vaccines (2023)