Broadly neutralizing hemagglutinin stalk–specific antibodies require FcγR interactions for protection against influenza virus in vivo

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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.

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Figure 1: Fc-FcγR interactions are required for protection from viral infection by an anti–HA stalk bNAb in vivo.
Figure 2: Activating FcγRs are required for bNAb-mediated protection from viral infection in vivo.
Figure 3: Two strain-specific anti–H1 head mAbs do not require FcγR contributions during protection from viral infection in vivo.
Figure 4: Anti–HA stalk mAbs function through FcγRs after viral entry and induce superior ADCC compared to anti-head mAb.
Figure 5: Selectively enhancing huFc-huFcγR interactions augments bNAb-mediated protection from viral infection in vivo.

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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.

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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.

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Correspondence to Jeffrey V Ravetch.

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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

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