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

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.

Figure 1

c-FcγR interactions are required for protection from viral infection by an anti–HA stalk bNAb in vivo. (a) Binding of 6F12 bNAb variants to PR8 HA. Mouse IgG2a, mouse IgG1 and DA265 mutant 6F12 bNAb and an IgG2a isotype control mAb diluted as indicated and tested for binding to PR8 HA by ELISA. Values represent mean ± s.e.m. relative optical density (OD) values from triplicate wells. (b) In vitro plaque reduction neutralization by 6F12 bNAb variants. Values represent mean % inhibition, calculated by comparing plaque numbers in mAb-treated wells with wells receiving only PBS. (c) Percentage weight change compared to day 0 (left) or percentage survival (right) in wild-type mice treated with mouse IgG2a 6F12 bNAb, mouse IgG1 6F12 bNAb, DA265 mutant 6F12 bNAb or PBS before infection with PR8 virus. Weight change values represent mean ± s.e.m. n ≥ 5 mice per group. (d) PFU per lung in mice treated as in c, with lungs harvested on day 3 and analyzed for viral titers using a plaque assay. Weight change values represent mean ± s.e.m. (n ≥ 4 mice per group). For c,d, significant differences between the indicated sample and PBS-treated sample are shown: *P < 0.05; **P < 0.001.

Figure 2

ctivating FcγRs are required for bNAb-mediated protection from viral infection in vivo. (a) Percentage weight change compared to day 0 and survival over time in wild-type (WT) mice or Fcer1g−/− mice treated with mouse IgG2a 6F12 bNAb or PBS before infection with PR8 virus. n = 6–10 mice per group. (b) Percentage weight change compared to day 0 and survival over time in wild-type mice or Fcgr2b−/− mice treated with mouse IgG1 6F12 bNAb or PBS before infection with PR8 virus. n = 5–10 mice per group. (c) Percentage weight change compared to day 0 and survival over time in wild-type mice treated with mouse IgG2a 6F12 bNAb, IgG1 6F12 bNAb, DA265 mutant 6F12 bNAb or PBS before infection with Neth09 virus. n ≥ 6 mice per group. (d) Percentage weight change compared to day 0 and survival over time in wild-type mice treated with mouse IgG2a 6F12 bNAb, mouse IgG1 6F12 bNAb, DA265 mutant 6F12 bNAb or PBS before infection with PR8 virus. Weight change values represent mean ± s.e.m. in a–d. n ≥ 5 mice per group. For a–c, significant differences between the indicated sample and PBS-treated sample are shown: **P < 0.001.

Figure 3

Two strain-specific anti–H1 head mAbs do not require FcγR contributions during protection from viral infection in vivo. (a) Percentage weight change compared to day 0 and survival over time in wild-type mice treated with mouse IgG2a, mouse IgG1 or DA265 mutant PY102 mAb or PBS before infection with PR8 virus. (b) Percentage survival over time in wild-type mice treated with the indicated doses of mouse IgG2a or DA265 mutant PY102 mAb, or PBS, before infection with PR8 virus. (c) Percentage weight change compared to day 0 and survival over time in wild-type mice treated with 0.5 mg/kg of mouse IgG2a, mouse IgG1 or DA265 mutant 7B2 mAb or PBS before infection with Neth09 virus. (d) Percentage survival over time in wild-type mice treated with the indicated doses of mouse IgG2a or DA265 mutant PY102 mAb, or PBS, before infection with Neth09 virus. Weight change data in a,c are expressed as mean ± s.e.m. n = 5–8 mice per group.

Figure 4

Anti–HA stalk mAbs function through FcγRs after viral entry and induce superior ADCC compared to anti-head mAb. (a) Antibody-mediated inhibition of viral entry is FcγR independent. Mice were infected with PR8 virus alone, virus preincubated with the indicated concentration of IgG2a or DA265 6F12 mAb, or PBS. Values represent mean ± s.e.m. frequency of NP+ cells in the lungs of individual mice 6 h after receiving the indicated treatment. Horizontal bars indicate mean values. (b) Impaired NK cell activation by anti–HA head mAb compared to anti–HA stalk mAb. Values represent the mean ± s.e.m. frequency of CD107a+ cells among NK cells from cultures with target cells bound by the indicated antibody from 4 individual leukocyte donors. Results are representative of 3 independent experiments. Significant differences between the indicated sample and 6F12 huIgG1 sample are shown: **P < 0.01. (c) Immune complexes were generated between huIgG1 anti-stalk 6F12, anti-stalk FI6 or anti-head PY102 mAbs and PR8 HA and assessed for their ability to engage huFcγRIIIa Phe158. Values represent the mean ± s.e.m. relative OD from duplicate ELISA measurements, with background binding by N297A mutant versions of each antibody subtracted. Results are representative of 2 independent experiments.

Figure 5

Selectively enhancing huFc-huFcγR interactions augments bNAb-mediated protection from viral infection in vivo. (a) Percentage weight change compared to day 0 and survival over time in FcγR-humanized mice treated with the indicated doses of wild-type huIgG1 6F12 bNAb or PBS before infection with PR8 virus. Weight change values represent mean ± s.e.m. n ≥ 4 mice per group. (b) Percentage weight change compared to day 0 and survival over time in FcγR-humanized mice treated with wild-type huIgG1 6F12 bNAb, GASD/ALIE mutant 6F12 bNAb or PBS before infection with PR8 virus. Weight change values represent mean ± s.e.m. n ≥ 12 mice per group. Significant differences between wild-type IgG1 mAb–treated and GASD/ALIE mAb–treated groups are shown: **P < 0.01.