pubmed.ncbi.nlm.nih.gov

Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner - PubMed

  • ️Fri Jan 01 2016

Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner

Caitlin E Mullarkey et al. mBio. 2016.

Abstract

Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protection in vivo Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.

Importance: The present study provides evidence that broadly neutralizing HA stalk-specific antibodies induce downstream Fc-mediated neutrophil effector functions. In addition to their ability to neutralize, this class of antibodies has been shown to rely on Fc-Fc receptor interactions for optimal protection in vivo Curiously, neutralizing antibodies that bind the HA head domain do not require such interactions. Our findings build on these previous observations and provide a more complete picture of the relationship between stalk-specific antibodies and cells of the innate immune compartment. Furthermore, our data suggest that the ability of HA stalk-specific antibodies to mediate Fc-Fc receptor engagement is epitope dependent. Overall, this work will inform the rational design of improved influenza virus vaccines and therapeutics.

Copyright © 2016 Mullarkey et al.

PubMed Disclaimer

Figures

FIG 1
FIG 1

Schematic of in vitro luminol-based assay to measure ROS. Immune complexes were formed by incubating 5 µg/well purified influenza A viruses with 10 µg of a given antibody for 30 min at room temperature in Nunc opaque MaxiSorp 96-well plates. Neutrophils were isolated either from peripheral human blood or murine bone marrow. Prior to the addition of neutrophils, 50 µl of luminol was added to all wells. Following isolation, 50 µl of neutrophils (5 × 105 cells per well) was added into the system. Luminescence (in relative light units [RLU]), resulting from the interaction of luminol with ROS, was assessed using a microplate reader. To gauge the relative ability of antibodies to induce ROS, we calculated an indexed RLU value (33), where the reference sample for all experiments was neutrophils (PMNs) in the presence of virus.

FIG 2
FIG 2

Monoclonal HA stalk-specific antibodies induce the production of ROS by neutrophils. Primary murine (A to F) or human (G and H) neutrophils (PMNs) were isolated and assessed for their ability to produce ROS in an in vitro luminol-based assay. Immune complexes were formed by incubating antibodies with purified H1N1 A/California/04/09 virus (A to C, G, and H) or X-31 virus (H3N2) (D to F). (A to C) The broadly neutralizing H1 stalk-specific murine antibody 6F12 and strain-specific antibodies 29E3 and 7B2 were tested for ROS induction. (D to F) The same assay was repeated using the H3 stalk-specific antibody 9H10 and head-specific antibody XY102. (G and H) Chimeric H1 broadly neutralizing stalk-specific antibodies h6F12 and hKB2 were also evaluated for their ability to induce ROS along with the strain-specific head antibody h29E3. Representative time course data for a single experiment (A, D, and G) and indexed RLU values (33) from three independent experiments (B, C, E, F, and H) are shown. Values are means ± standard errors of means (error bars). Values that are significantly different by the Kruskal-Wallis test with Dunn’s multiple comparison (B and F) or by the Mann-Whitney test (C) are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.

FIG 3
FIG 3

The production of ROS by monoclonal IgG antibodies is dependent on Fc-FcγR interactions. The dependence of ROS production on Fc-FcR interactions was probed either by incubating neutrophils with FcR blocking antibodies prior to addition in the assay (A to D) or engineering antibodies with a D265A mutation to ablate binding to FcγRs (E and F). The ability of the murine H1 broadly neutralizing stalk-specific antibody 6F12 to induce ROS (A and B) and the chimeric human antibody h6F12 (C and D) were assessed in the presence of Fc blocking antibodies. (E and F) The murine H3 broadly neutralizing stalk-specific antibody 9H10 engineered with a D265A mutation was examined for its ability to induce ROS. Luminol assays using this mutant were performed in parallel with the experiments shown in Fig. 2C and D, and the controls have been duplicated for ease of comparison. (A, C, and E) Representative time course from a single experiment. (B, D, and F) Indexed RLU values (33) pooled from three independent experiments. Means ± standard errors of means (error bars) are shown. Values that are significantly different by the Kruskal-Wallis test with Dunn’s multiple comparison are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.

FIG 4
FIG 4

Inhibition of phagocytosis ablates ROS induction by monoclonal IgG antibodies. In vitro luminol-based assays were performed using murine (A and B) or human (C and D) neutrophils incubated with cytochalasin D prior to addition to the assay. (E and F) To confirm activation of NADPH oxidase by murine monoclonal antibodies, neutrophils were isolated from B6.129S-Cybbtm1din/J mice which are a model for chronic granulomatous disease (CGD). (A, C, and E) Representative time course from a single experiment. (B, D, and F) Indexed RLU values (33) pooled from three independent experiments. Means ± standard errors of means are shown. Values that are significantly different by the Mann-Whitney test are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.01.

FIG 5
FIG 5

Chimeric human stalk-specific IgAs induce ROS production through engagement of FcαR1. Chimeric IgA antibodies were generated by cloning the variable regions of the H1 broadly neutralizing stalk-specific antibody 6F12 and strain-specific head antibody 29E3 into a human IgA backbone. (A and B) Chimeric IgA antibodies were assessed for ROS induction in a luminol-based assay using human neutrophils and immune complexes formed with Cal09. (C and D) Neutrophils were incubated with anti-CD89 (FcαR1) prior to the addition to the assay. (E and F) Luminol assays were repeated using neutrophils incubated with cytochalasin D prior to stimulation with immune complexes. (A, C, and E) Representative time course from a single experiment. (B, D, and F) Indexed RLU values (33) pooled from three independent experiments. Means ± standard errors of means are shown. Values that are significantly different by Student’s t test (B) and Kruskal-Wallis test with Dunn’s multiple comparison (D) are indicated by bars and asterisks as follows: *, P < 0.05; ****, P < 0.0001. Values that are not significant (ns) are also indicated.

FIG 6
FIG 6

Murine and human stalk-specific IgG antibodies activate the antibody-dependent cellular phagocytosis (ADCP) pathway in an epitope-specific manner. (A and B) ADCP reporter assays were performed on Cal09-infected (A) or X-31-infected (B) A549 cells using a panel of human (A) or murine (B) monoclonal antibodies. (A) The broadly neutralizing stalk-specific antibodies h6F12 and hKB2 were assessed for their ability to activate FcγRIIA. (B) The same assay was repeated using the broadly neutralizing HA stalk binding antibody 9H10 and head binding antibody XY102. The means and standard errors of means are shown for three independent experiments. (C) Bead-based phagocytosis assays were performed using fluorescent microspheres sequentially coated with protein L, followed by either the HA stalk-specific antibody h6F12 or the strain-specific head antibody h29E3. Preparation of the beads in this way leaves the antibody Fc region accessible. (D) Freshly isolated human neutrophils were incubated with microspheres. Following incubation, unbound microspheres were removed by centrifugation and washing; uptake was measured by fluorescence. Means and standard errors of means are shown. Data are representative of three independent experiments (n = 8 donors in total). Values that are significantly different (P < 0.0001) by Kruskal-Wallis test with Dunn’s multiple comparison are indicated by the bar and four asterisks. Values that are not significant (ns) are also indicated.

Similar articles

Cited by

References

    1. Krammer F, Palese P. 2015. Advances in the development of influenza virus vaccines. Nat Rev Drug Discov 14:167–182. doi:10.1038/nrd4529. - DOI - PubMed
    1. Krammer F, Palese P. 2013. Influenza virus hemagglutinin stalk-based antibodies and vaccines. Curr Opin Virol 3:521–530. doi:10.1016/j.coviro.2013.07.007. - DOI - PMC - PubMed
    1. Krammer F, Margine I, Hai R, Flood A, Hirsh A, Tsvetnitsky V, Chen D, Palese P. 2014. H3 stalk-based chimeric hemagglutinin influenza virus constructs protect mice from H7N9 challenge. J Virol 88:2340–2343. doi:10.1128/JVI.03183-13. - DOI - PMC - PubMed
    1. Krammer F, Pica N, Hai R, Margine I, Palese P. 2013. Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies. J Virol 87:6542–6550. doi:10.1128/JVI.00641-13. - DOI - PMC - PubMed
    1. Krammer F, Hai R, Yondola M, Tan GS, Leyva-Grado VH, Ryder AB, Miller MS, Rose JK, Palese P, García-Sastre A, Albrecht RA. 2014. Assessment of influenza virus hemagglutinin stalk-based immunity in ferrets. J Virol 88:3432–3442. doi:10.1128/JVI.03004-13. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances