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

Monoclonal HA stalk-specific antibodies induce the production of ROS by neutrophils, while antibodies recognizing the HA head domain do not.

Broadly neutralizing stalk-specific HA antibodies have been shown to require Fc-Fcγ receptor (FcγR) interaction for optimal protection in vivo (17), yet the full scope of Fc-mediated effector functions elicited by these antibodies has not been fully described. To investigate the interplay between HA stalk-specific antibodies and neutrophils, we utilized an in vitro luminol-based assay to measure the production of ROS by neutrophils when stimulated with immune complexes. The production of various species of oxygen radicals by neutrophils can be induced by soluble and particulate agonists, and these ROS both directly and indirectly aid in pathogen clearance (28). For these studies, we used a panel of mouse monoclonal HA-specific antibodies that have been previously described by our group (29–32). The antibody, 6F12, binds to the stalk domain of H1 viruses, while the antibodies 7B2 and 293E are specific to the head domain of A/California/04/09 (Cal09) virus. Immune complexes were formed by incubating 5 µg/well of purified Cal09 virus with 10 µg of mouse monoclonal antibody. These complexes were used to stimulate murine neutrophils isolated from bone marrow. Finally, to allow for the detection of respiratory burst activity, luminol was added to the system. In this setup, luminol reacts with ROS generated by neutrophils to produce an excited state intermediate that emits light as it relaxes to the ground state (Fig. 1). Furthermore, luminol is capable of crossing biological membranes allowing for the detection of oxygen species originating from the phagosome. When murine neutrophils were incubated with immune complexes formed using the HA stalk-specific antibody 6F12, we observed a potent induction of ROS (Fig. 2A and B). To gauge the relative ability of monoclonal antibodies to induce ROS production, results were indexed according to a baseline activation of neutrophils by purified virus, as described in Materials and Methods. A similar approach has previously been reported (33). Interestingly, antibodies recognizing the head domain of HA did not induce ROS production over baseline levels (Fig. 2A and B). This observation held true even when a high concentration of head-specific antibody was used (Fig. 2C).

To ensure that this phenomenon was not subtype specific, we repeated the experiment using the influenza A virus X-31 (H3N2). These studies used the broadly neutralizing stalk antibody 9H10 as well as the H3 head-specific neutralizing antibody XY102 (34, 35). Consistent with our previous result, we observed that immune complexes formed using the HA stalk-specific antibody 9H10 potently induced ROS production (Fig. 2D and E). Immune complex formation and the induction of ROS occurred in a dose-dependent manner (Fig. 2F). While XY102 induced ROS production to a low level, this induction was fivefold lower than that observed for 9H10 (Fig. 2E).

To confirm whether these findings were relevant in the context of human samples, we utilized chimeric IgG antibodies previously described by our group (36). Briefly, these chimeric antibodies were generated by cloning the variable regions of the H1 stalk-binding antibodies 6F12 and KB2 and the HA head-specific antibody 29E3 into a human IgG1 backbone (h6F12, hKB2, and h29E3). These antibodies were expressed recombinantly in HEK 293T cells. Human neutrophils were isolated from peripheral blood, and the assay was performed using immune complexes formed with chimeric antibodies. Consistent with our findings using a murine system, we observed that only HA stalk-specific antibodies led to the generation of ROS (Fig. 2G and H). The chimeric head-specific antibody h29E3 failed to induce ROS by human neutrophils (Fig. 2G and H). Therefore, only immune complexes formed by HA stalk-specific antibodies were capable of potently stimulating respiratory burst activity by neutrophils.

The production of ROS by monoclonal IgG antibodies is dependent on Fc-FcγR interactions.

As a critical innate effector cell population, neutrophils express both activating and inhibitory Fcγ receptors. Murine neutrophils express all FcγRs (25); however, expression of the activating receptor FcγRI has been shown to be low at steady state (37). On the other hand, human neutrophils constitutively express only three of the six FcγRs, FcγRIIA, FcγRIIB, and FcγRIIIB (26, 27). Of these three FcγRs, only FcγRIIA is an activating receptor. We assessed the dependence of ROS production on Fc-FcγR interactions in two ways. First, we blocked FcγRs on the surfaces of neutrophils by incubating cells with commercially available antibodies prior to stimulation with immune complexes. Incubation of murine neutrophils with anti-CD32/CD16 abrogated the induction of ROS by 6F12 (Fig. 3A) and led to a significant decrease in indexed relative light unit (RLU) values (Fig. 3B). Similarly, incubation of human neutrophils with Fc block (BD Pharmingen) ablated the ability of the human 6F12 (h6F12) to elicit ROS production (Fig. 3C and D). In addition to blocking FcγRs on the surfaces of neutrophils, we engineered a variant of 9H10 that lacks the ability to bind FcγRs. Replacement of aspartic acid with an alanine at position 265 (D265A) in the constant heavy chain has been shown to result in a complete loss of binding of IgG2a and IgG2b to all four classes of FcγRs (38). Compared to the wild-type (WT) 9H10 antibody, the D265A mutant failed to induce ROS by murine neutrophils (Fig. 3E and F). Taken together, these data demonstrate that Fc-FcγR interactions are required for the respiratory burst activity elicited by stalk-specific antibodies.

Inhibition of phagocytosis ablates ROS induction by monoclonal IgG antibodies.

We next sought to determine the role of phagocytosis on respiratory burst induction. Phagocytosis represents a well-established effector mechanism utilized by neutrophils to eliminate pathogens (20). To test whether phagocytosis was required for ROS induction, we relied on cytochalasin D, a potent inhibitor of actin polymerization. Murine and human neutrophils were incubated with 10 µg/ml of cytochalasin D prior to stimulation with immune complexes. Inhibition of phagocytosis ablated the induction of ROS by the murine HA stalk-specific antibody 6F12 (Fig. 4A) and the chimeric hKB2 antibody (Fig. 4C), again leading to a significant decrease in indexed RLU values (Fig. 4B and D, respectively). We additionally probed whether the induction of ROS required phagocytosis by coating purified virus on the wells of 96-well plates, instead of allowing immune complexes to form in suspension. We were unable to observe ROS production when the in vitro assay was performed in this manner (data not shown). Chronic granulomatous disease is a disorder characterized by defective phagocytic respiratory burst activity. Our data thus far suggest that HA stalk-specific antibodies stimulate NADPH oxidase activity in an Fc-dependent manner. To test this hypothesis, we utilized mice with a null allele for the 91-kDa subunit of the oxidase cytochrome B. These mice lack phagocytic respiratory burst activity and are used as a model for chronic granulomatous disease. As expected, neutrophils isolated from B6.129S-Cybb^tm1din/J mice did not generate ROS when stimulated with immune complexes formed with either 6F12 or 29E3 antibody (Fig. 4E and F). These data confirm that the ROS species signature elicited by HA stalk-specific antibodies represents activation of NADPH oxidase and phagocytic respiratory burst activity.

Chimeric human stalk-specific IgAs induce ROS production through engagement of FcαR1.

IgA is the most prevalent antibody at mucosal sites, and as such, it plays a critical role in protection against respiratory pathogens such as influenza viruses (39). Our group has previously reported that B cells producing broadly neutralizing antibodies of the IgA isotype are widespread (36). Having observed that HA stalk-specific IgG antibodies induce ROS by neutrophils, we went on to examine whether the same phenomenon could be observed using both HA stalk-specific and head-binding monomeric IgA (mIgA) antibodies. 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. Consistent with our results for chimeric IgG antibodies, only h6F12 was able to induce the production of ROS by human neutrophils (Fig. 5A and B). The IgA-triggering Fc receptor, FcαRI (CD89) is constitutively expressed on neutrophils; therefore, we turned our attention to investigate whether the generation of ROS by mIgA requires FcαRI. To achieve this, human neutrophils were incubated with an anti-CD89 antibody prior to stimulation with immune complexes. In line with our earlier findings, blocking the IgA receptor FcαRI abolished ROS production and led to a significant reduction in indexed RLU values (Fig. 5C and D). These studies were performed using only human neutrophils, as mice lack a FcαRI ortholog. Finally, we went on to probe the role of phagocytosis on respiratory burst activity. As before, neutrophils were incubated with cytochalasin D prior to addition to the assay. While the induction of ROS was delayed when neutrophils were incubated with cytochalasin D, interestingly, ROS production was not entirely ablated (Fig. 5E). This observation held true even when increasing concentrations of cytochalasin D were utilized (data not shown). Therefore, while we observed a decrease in ROS production when phagocytosis was inhibited, no significant difference in indexed RLU values were observed between h6F12 IgA plus vehicle and h6F12 plus cytochalasin D (Fig. 5F). These data suggest that, in addition to phagocytosis, HA stalk-specific IgA antibodies are able to induce ROS by other Fc-related mechanisms. Consequently, our data show that both HA stalk-specific IgG and IgA antibodies elicit respiratory burst activity of human neutrophils.

Murine and human stalk-specific IgG antibodies activate the antibody-dependent cellular phagocytosis pathway in an epitope-specific manner.

Our previous results demonstrated that murine and human HA stalk-specific IgG antibodies led to respiratory burst activity of neutrophils in a manner that is dependent on both Fc-FcγR engagement and phagocytosis. To confirm that these IgG antibodies are capable of eliciting antibody-dependent cellular phagocytosis (ADCP), we relied on a commercially available ADCP assay whereby A549 cells infected with influenza A viruses express HA on the cell surface. These conditions mimic how HA would be presented in the context of a natural infection. Following the addition of HA-specific antibodies to infected cells, a reporter cell line that stably expresses human FcγRIIA is added into this system. This allows for the quantification of ADCP induction by luciferase expression.

Cells infected with A/Netherlands/602/09 (H1N1) were incubated with the chimeric IgG antibodies h6F12, hKB2, and h29E3 at doses ranging from 10 to 0.013 µg/ml. As expected, h6F12 and hKB2 induced ADCP, whereas h29E3 did not (Fig. 6A). Given that human FcγRIIA has previously been shown to bind murine IgG2a and IgG2b antibodies (40), we used this reporter system to additionally test the murine HA stalk-specific antibody 9H10 and the HA head-specific antibody XY102. Cells were infected with X-31 (H3N2) and incubated with antibody concentrations from 25 to 0.000381 µg/ml. Again, we observed that while XY102 failed to activate the reporter cell line, 9H10 potently induced ADCP (Fig. 6B).

The results from the studies presented here support previous work that broadly neutralizing HA stalk-specific antibodies readily induce FcR-mediated effector functions, while HA head-specific antibodies do not (17). However, thus far, the ability of HA monoclonal antibodies to induce downstream Fc-mediated effector function has been assessed in an epitope-dependent fashion. It is possible that binding of each antibody class to its respective epitope affects the accessibility of the Fc region. To dissect whether the ability of HA stalk-specific antibodies to elicit Fc effector functions is an intrinsic property of this antibody class or in fact dependent on the epitope, we made use of a microsphere phagocytosis assay. Yellow fluorescent carboxylate microspheres were coated first with recombinant protein L, followed by adsorption of either h6F12 or h29E3. Protein L binds immunoglobulins via the light chain, and therefore, coating the microspheres in this way leaves the Fc region of each immunoglobulin accessible (Fig. 6C). Human neutrophils were isolated from peripheral blood and incubated with coated microspheres. We compared the internalization of microspheres coated with h6F12 and h29E3 to microspheres coated with protein L only. While neutrophils internalized microspheres coated with protein L, a significant enhancement of phagocytosis was observed when microspheres were coated with h6F12 and h29E3 (Fig. 6D). No significant differences were observed between h6F12- or h29E3-coated beads. Taken together, these results suggest that the differential ability of HA stalk- and head-specific antibodies to mediate Fc effector functions is epitope dependent.

Cells and viruses.

Adenocarcinoma human alveolar basal epithelial cells (A549) and Madin-Darby canine kidney (MDCK) cells were grown in Dulbecco’s modified Eagle’s medium (Gibco) and supplemented with 10% fetal bovine serum and 100 U/ml of penicillin and streptomycin. X-31 is a reassortant virus with the six internal genes of PR8 virus and the hemagglutinin (HA) and neuraminidase (NA) of A/Hong Kong/1/68 virus (H3N2). Two pandemic H1N1 viruses were used in this study. A/California/04/09 was grown in MDCK cells and purified over a 30% sucrose cushion. Virus pellets were resuspended in phosphate-buffered saline (PBS), and protein content was quantified using the Bradford protein assay. A/Netherlands/602/09 was grown in the allantoic fluid of 10-day-old embryonated chicken eggs.

Antibodies.

XY102, 6F12, KB2, 9H10, and 29E3 have been previously described (29–32, 34). Chimeric IgG and IgA antibodies were generated as previously described (36). Briefly, the variable regions of the group 1 HA stalk-binding monoclonal antibodies 6F12 and KB2 were cloned into human IgG and IgA backbones (pFUSE; InvivoGen). An identical strategy was used for 29E3, a strain-specific antibody that recognizes the head domain of A/California/04/09 HA.

Antibody purification.

All mouse antibodies described herein were produced from hybridoma cultures. Supernatants from hybridoma cultures were collected, clarified by centrifugation, and filtered prior to purification. IgG antibodies were purified by gravity flow columns containing G Sepharose 4 Fast Flow (GE Healthcare). Supernatants were run over the column twice followed by a subsequent wash step with PBS. Antibodies were eluted with 45 ml of 0.1 M glycine-HCl (pH 2.7), and the eluent was neutralized with 5 ml of 2 M Tris-HCl buffer (pH 10). IgA antibodies were purified in a similar fashion using peptide M/Sepharose resin (InvivoGen). Antibodies were subsequently concentrated and dialyzed against PBS using Amicon ultracentrifugal filter units (Millipore) with a 30-kDa molecular mass cutoff.

Animals.

All animal protocols were reviewed and approved by the Mount Sinai Institutional Animal Care and Use Committee (IACUC). Six- to 8-week-old female BALB/c mice were purchased from Jackson Laboratory and housed under specific-pathogen-free conditions. Age-matched B6.129S-Cybb^tm1Din/J (also known as gp91 phox−) mice were purchased from Jackson Laboratory. These mice possess a null allele for the 91-kDa subunit of oxidase cytochrome B and are a model for chronic granulomatous disease (CGD).

Murine neutrophil isolation.

Murine neutrophils were isolated from bone marrow extracted from harvested femurs and tibias of uninfected naive mice using density gradient centrifugation. A gradient was created by layering 3 ml of Histopaque 1077 over 3 ml of Histopaque 1119. Bone marrow was resuspended in 1 ml of Hanks buffered saline solution (HBSS), and the cell suspension was overlaid on top of the Histopaque 1077. Samples were centrifuged for 30 min at 2,000 rpm at 25°C. Neutrophils were collected at the interface of the Histopaque 1119 and 1077 layers. Cells were washed twice in HBSS, and viability was assessed by trypan blue exclusion. Neutrophils were resuspended to a final concentration of 1 × 10⁷ cells/ml.

Human neutrophil isolation.

Peripheral blood samples were collected from healthy volunteers who provided informed consent. Studies were approved by the Hamilton Integrated Research Ethics Board. Blood was collected in EDTA collection tubes, and whole blood was layered over a gradient of Histopaque 1077/1119 as described above. Samples were centrifuged for 30 min at 2,000 rpm at 25°C. Neutrophils were collected at the interface of the Histopaque 1119 and 1077 layers. Cells were washed twice in HBSS, and viability was assessed by trypan blue exclusion. Neutrophils were resuspended to a final concentration of 1 × 10⁷ cells/ml.

Luminol-based assay.

A suspension-based assay to detect reactive oxygen species was adapted from a similar method (33). Briefly, immune complexes were formed by incubating 5 µg/well of purified virus with 10 µg of a given antibody for 30 min at room temperature in Nunc opaque MaxiSorp 96-well plates. Species appropriate neutrophils were isolated as described above. Prior to the addition of neutrophils, luminol (Sigma) was added to all wells. Following the final wash step, 50 µl of neutrophils (5 × 10⁵ cells per well) was added. Luminescence (in relative light units [RLU]) resulting from interaction of luminol with reactive oxygen species was assessed using a Filter Max F2 microplate reader. Neutrophils spiked with 3 to 5 µl of phorbol myristate acetate (PMA) at a concentration of 0.1 mg/ml served as a positive control. Kinetic measurements were taken at 1-min intervals for 1 h. To investigate the roles of Fc signaling and phagocytosis, neutrophils were incubated with Fc block or cytochalasin D prior to the addition of cells to the assay. Fc blocking antibodies and cytochalasin D were used at a final concentration of 10 µg/ml.

Data analysis.

To compare the relative ability of antibody groups to induce reactive oxygen species, an indexed RLU was calculated as follows (33): indexed RLU = absolute maximum RLU of test sample/absolute maximum of reference sample.

For all experiments, neutrophils incubated in the presence of virus served as the reference sample. This condition provides a baseline measurement for respiratory burst activity induced by virus.

Microsphere-based phagocytosis assay.

Yellow fluorescent carboxylate microspheres (0.5 µm; Polysciences, Warrington, PA) were coated with protein L, followed by either HA head- or stalk-specific monoclonal antibodies. This was achieved by sequentially incubating microspheres first with 750 µg of protein L for 4 h at room temperature and then overnight with 300 µg of the monoclonal antibody with washing steps in between incubation steps. Following the last incubation, beads were pelleted at 12,000 rpm for 10 min and washed with PBS for 30 min at room temperature with gentle mixing. Supernatants were harvested for protein determination. Beads were resuspended in a final volume of 500 µl. Primary human neutrophils were freshly isolated, and microspheres were added to aliquots of neutrophils at a ratio of 500 beads per cell. The tubes were then incubated at 37°C for 15 min with gentle shaking. At the completion of incubation, cells were centrifuged for 10 min at 2,000 rpm. The supernatant was removed from each tube, and cells were washed two times with PBS to remove all unbound microspheres. Samples were added in triplicate to a black 96-well plate, and fluorescence was measured on a SpectraMax i3 plate reader (Molecular Devices) at an excitation wavelength of 526 nm and emission wavelength of 555 nm (Ex₅₂₆/Em₅₅₅). Data were reported in relative fluorescent units (RFU).

Antibody-dependent cellular phagocytosis (ADCP) cell-based assay.

A549 cells were seeded on a 96-well white bottom plate, and 24 h later, cells were infected with influenza A viruses at a multiplicity of infection (MOI) of 3 without trypsin to avoid multicycle replication. At 12 to 16 h postinfection, the inoculum was removed and replaced with assay buffer (RPMI [Gibco] with 4% low IgG serum) followed by the addition of serial dilutions of monoclonal antibodies. Antibodies and infected cells were incubated at 37°C for 30 min. At the completion of incubation, Jurkat effector cells (Promega) stably expressing FcγRIIA were resuspended in assay buffer and added to plates at a concentration of 1.5 × 10⁴ cells per well. Plates were incubated for 6 h at 37°C before the addition of Bio-Glo luciferase assay reagent (Promega). Luminescence was quantified using a Synergy 4 (Bio-Tek) plate reader. Fold induction was calculated as follows: (RLU_induced − RLU_background)/(RLU_uninduced − RLU_background). Curve fitting was performed with GraphPad Prism v6 software using a “log(agonist) versus normalized response variable slope (four-parameter) analysis.”

Statistics.

Statistical analyses were performed using GraphPad Prism v6 (GraphPad Software, San Diego, CA). Data were assessed for normality using a Shapiro-Wilk test, and the appropriate statistical test was applied as indicated in figure legends. A P value of <0.05 was considered statistically significant throughout.

• 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] [Google Scholar]
• 2.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 free article] [PubMed] [Google Scholar]
• 3.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 free article] [PubMed] [Google Scholar]
• 4.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 free article] [PubMed] [Google Scholar]
• 5.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 free article] [PubMed] [Google Scholar]
• 6.Nachbagauer R, Miller MS, Hai R, Ryder AB, Rose JK, Palese P, García-Sastre A, Krammer F, Albrecht RA. 2016. Hemagglutinin stalk immunity reduces influenza virus replication and transmission in ferrets. J Virol 90:3268–3273. doi: 10.1128/JVI.02481-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 7.Wohlbold TJ, Nachbagauer R, Margine I, Tan GS, Hirsh A, Krammer F. 2015. Vaccination with soluble headless hemagglutinin protects mice from challenge with divergent influenza viruses. Vaccine 33:3314–3321. doi: 10.1016/j.vaccine.2015.05.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 8.Miller MS, Tsibane T, Krammer F, Hai R, Rahmat S, Basler CF, Palese P. 2013. 1976 and 2009 H1N1 influenza virus vaccines boost anti-hemagglutinin stalk antibodies in humans. J Infect Dis 207:98–105. doi: 10.1093/infdis/jis652. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 9.Ellebedy AH, Krammer F, Li G-M, Miller MS, Chiu C, Wrammert J, Chang CY, Davis CW, McCausland M, Elbein R, Edupuganti S, Spearman P, Andrews SF, Wilson PC, García-Sastre A, Mulligan MJ, Mehta AK, Palese P, Ahmed R. 2014. Induction of broadly cross-reactive antibody responses to the influenza HA stem region following H5N1 vaccination in humans. Proc Natl Acad Sci U S A 111:13133–13138. doi: 10.1073/pnas.1414070111. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 10.Miller MS, Gardner TJ, Krammer F, Aguado LC, Tortorella D, Basler CF, Palese P. 2013. Neutralizing antibodies against previously encountered influenza virus strains increase over time: a longitudinal analysis. Sci Transl Med 5:198ra107. doi: 10.1126/scitranslmed.3006637. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 11.Li G-M, Chiu C, Wrammert J, McCausland M, Andrews SF, Zheng N-Y, Lee J-H, Huang M, Qu X, Edupuganti S, Mulligan M, Das SR, Yewdell JW, Mehta K, Wilson PC, Ahmed R. 2012. 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. doi: 10.1073/pnas.1118979109. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 12.Wrammert J, Koutsonanos D, Li G-M, Edupuganti S, Sui J, Morrissey M, McCausland M, Skountzou I, Hornig M, Lipkin WI, Mehta A, Razavi B, Del Rio C, Zheng N-Y, Lee J-H, Huang M, Ali Z, Kaur K, Andrews S, Amara RR, Wang Y, Das SR, O’Donnell CD, Yewdell JW, Subbarao K, Marasco WA, Mulligan MJ, Compans R, Ahmed R, Wilson PC. 2011. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. J Exp Med 208:181–193. doi: 10.1084/jem.20101352. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 13.Pica N, Hai R, Krammer F, Wang TT, Maamary J, Eggink D, Tan GS, Krause JC, Moran T, Stein CR, Banach D, Wrammert J, Belshe RB, García-Sastre A, Palese P. 2012. Hemagglutinin stalk antibodies elicited by the 2009 pandemic influenza virus as a mechanism for the extinction of seasonal H1N1 viruses. Proc Natl Acad Sci U S A 109:2573–2578. doi: 10.1073/pnas.1200039109. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 14.Halliley JL, Khurana S, Krammer F, Fitzgerald T, Coyle EM, Chung KY, Baker SF, Yang H, Martínez-Sobrido L, Treanor JJ, Subbarao K, Golding H, Topham DJ, Sangster MY. 2015. High-affinity H7 head and stalk domain-specific antibody responses to an inactivated influenza H7N7 vaccine after priming with live attenuated influenza vaccine. J Infect Dis 212:1270–1278. doi: 10.1093/infdis/jiv210. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 15.Nachbagauer R, Wohlbold TJ, Hirsh A, Hai R, Sjursen H, Palese P, Cox RJ, Krammer F. 2014. Induction of broadly reactive anti-hemagglutinin stalk antibodies by an H5N1 vaccine in humans. J Virol 88:13260–13268. doi: 10.1128/JVI.02133-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 16.Brandenburg B, Koudstaal W, Goudsmit J, Klaren V, Tang C, Bujny MV, Korse HJWM, Kwaks T, Otterstrom JJ, Juraszek J, Van Oijen AM, Vogels R, Friesen RHE. 2013. Mechanisms of hemagglutinin targeted influenza virus neutralization. PLoS One 8:e80034. doi: 10.1371/journal.pone.0080034. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 17.DiLillo DJ, Tan GS, Palese P, Ravetch JV. 2014. Broadly neutralizing hemagglutinin stalk-specific antibodies require FcγR interactions for protection against influenza virus in vivo. Nat Med 20:143–151. doi: 10.1038/nm.3443. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 18.Ackerman ME, Moldt B, Wyatt RT, Dugast AS, McAndrew E, Tsoukas S, Jost S, Berger CT, Sciaranghella G, Liu Q, Irvine DJ, Burton DR, Alter G. 2011. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples. J Immunol Methods 366:8–19. doi: 10.1016/j.jim.2010.12.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 19.Huber VC, Lynch JM, Bucher DJ, Le J, Metzger DW. 2001. Fc receptor-mediated phagocytosis makes a significant contribution to clearance of influenza virus infections. J Immunol 166:7381–7388. doi: 10.4049/jimmunol.166.12.7381. [DOI] [PubMed] [Google Scholar]
• 20.Mócsai A. 2013. Diverse novel functions of neutrophils in immunity, inflammation, and beyond. J Exp Med 210:1283–1299. doi: 10.1084/jem.20122220. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 21.Lim K, Hyun Y-M, Lambert-Emo K, Capece T, Bae S, Miller R, Topham DJ, Kim M. 2015. Neutrophil trails guide influenza-specific CD8+ T cells in the airways. Science 349:aaa4352. doi: 10.1126/science.aaa4352. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 22.Brinkmann V, Zychlinsky A. 2007. Beneficial suicide: why neutrophils die to make NETs. Nat Rev Microbiol 5:577–582. doi: 10.1038/nrmicro1710. [DOI] [PubMed] [Google Scholar]
• 23.Tate MD, Brooks AG, Reading PC. 2008. The role of neutrophils in the upper and lower respiratory tract during influenza virus infection of mice. Respir Res 9:57. doi: 10.1186/1465-9921-9-57. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 24.Tate MD, Ioannidis LJ, Croker B, Brown LE, Brooks AG, Reading PC. 2011. The role of neutrophils during mild and severe influenza virus infections of mice. PLoS One 6:e17618. doi: 10.1371/journal.pone.0017618. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 25.DiLillo DJ, Ravetch JV. 2015. Fc-receptor interactions regulate both cytotoxic and immunomodulatory therapeutic antibody effector functions. Cancer Immunol Res 3:704–713. doi: 10.1158/2326-6066.CIR-15-0120. [DOI] [PubMed] [Google Scholar]
• 26.Nimmerjahn F, Bruhns P, Horiuchi K, Ravetch JV. 2005. FcgammaRIV: a novel FcR with distinct IgG subclass specificity. Immunity 23:41–51. [DOI] [PubMed] [Google Scholar]
• 27.Bruhns P, Iannascoli B, England P, Mancardi DA, Fernandez N, Jorieux S, Daëron M. 2009. Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses. Blood 113:3716–3725. doi: 10.1182/blood-2008-09-179754. [DOI] [PubMed] [Google Scholar]
• 28.Lundqvist H, Dahlgren C. 1996. Isoluminol-enhanced chemiluminescence: a sensitive method to study the release of superoxide anion from human neutrophils. Free Radic Biol Med 20:785–792. doi: 10.1016/0891-5849(95)02189-2. [DOI] [PubMed] [Google Scholar]
• 29.Tan GS, Krammer F, Eggink D, Kongchanagul A, Moran TM, Palese P. 2012. A pan-H1 anti-hemagglutinin monoclonal antibody with potent broad-spectrum efficacy in vivo. J Virol 86:6179–6188. doi: 10.1128/JVI.00469-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 30.Heaton NS, Leyva-Grado VH, Tan GS, Eggink D, Hai R, Palese P. 2013. In vivo bioluminescent imaging of influenza A virus infection and characterization of novel cross-protective monoclonal antibodies. J Virol 87:8272–8281. doi: 10.1128/JVI.00969-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 31.Hai R, Krammer F, Tan GS, Pica N, Eggink D, Maamary J, Margine I, Albrecht RA, Palese P. 2012. Influenza viruses expressing chimeric hemagglutinins: globular head and stalk domains derived from different subtypes. J Virol 86:5774–5781. doi: 10.1128/JVI.00137-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 32.Manicassamy B, Medina RA, Hai R, Tsibane T, Stertz S, Nistal-Villán E, Palese P, Basler CF, García-Sastre A. 2010. 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. doi: 10.1371/journal.ppat.1000745. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 33.Llewellyn D, de Cassan SC, Williams AR, Douglas AD, Forbes EK, Adame-Gallegos JR, Shi J, Pleass RJ, Draper SJ. 2014. Assessment of antibody-dependent respiratory burst activity from mouse neutrophils on Plasmodium yoelii malaria challenge outcome. J Leukoc Biol 95:369–382. doi: 10.1189/jlb.0513274. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 34.Tan GS, Lee PS, Hoffman RMB, Mazel-Sanchez B, Krammer F, Leon PE, Ward AB, Wilson IA, Palese P. 2014. Characterization of a broadly neutralizing monoclonal antibody that targets the fusion domain of group 2 influenza A virus hemagglutinin. J Virol 88:13580–13592. doi: 10.1128/JVI.02289-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 35.Moran TM, Monestier M, Lai AC, Norton G, Reale MA, Thompson MA, Schulman JL, Riblet R, Bona CA. 1987. Characterization of variable-region genes and shared crossreactive idiotypes of antibodies specific for antigens of various influenza viruses. Viral Immunol 1:1–12. doi: 10.1089/vim.1987.1.1. [DOI] [PubMed] [Google Scholar]
• 36.He W, Mullarkey CE, Duty JA, Moran TM, Palese P, Miller MS. 2015. Broadly neutralizing anti-influenza virus antibodies: enhancement of neutralizing potency in polyclonal mixtures and IgA backbones. J Virol 89:3610–3618. doi: 10.1128/JVI.03099-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 37.Tan PS, Gavin AL, Barnes N, Sears DW, Vremec D, Shortman K, Amigorena S, Mottram PL, Hogarth PM. 2003. Unique monoclonal antibodies define expression of Fc RI on macrophages and mast cell lines and demonstrate heterogeneity among subcutaneous and other dendritic cells. J Immunol 170:2549–2556. doi: 10.4049/jimmunol.170.5.2549. [DOI] [PubMed] [Google Scholar]
• 38.Baudino L, Shinohara Y, Nimmerjahn F, Furukawa J, Nakata M, Martínez-Soria E, Petry F, Ravetch JV, Nishimura S, Izui S. 2008. Crucial role of aspartic acid at position 265 in the CH2 domain for murine IgG2a and IgG2b Fc-associated effector functions. J Immunol 181:6664–6669. doi: 10.4049/jimmunol.181.9.6664. [DOI] [PubMed] [Google Scholar]
• 39.Aleyd E, Heineke MH, van Egmond M. 2015. The era of the immunoglobulin A Fc receptor FcαRI; its function and potential as target in disease. Immunol Rev 268:123–138. doi: 10.1111/imr.12337. [DOI] [PubMed] [Google Scholar]
• 40.Jönsson F, Mancardi DA, Kita Y, Karasuyama H, Iannascoli B, Van Rooijen N, Shimizu T, Daëron M, Bruhns P. 2011. Mouse and human neutrophils induce anaphylaxis. J Clin Invest 121:1484–1496. doi: 10.1172/JCI45232. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 41.Steel J, Lowen AC, Wang TT, Yondola M, Gao Q, Haye K, García-Sastre A, Palese P. 2010. Influenza virus vaccine based on the conserved hemagglutinin stalk domain. mBio 1(1):1–9. doi: 10.1128/mBio.00018-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 42.Yassine HM, Boyington JC, McTamney PM, Wei C-J, Kanekiyo M, Kong W-P, Gallagher JR, Wang L, Zhang Y, Joyce MG, Lingwood D, Moin SM, Andersen H, Okuno Y, Rao SS, Harris AK, Kwong PD, Mascola JR, Nabel GJ, Graham BS. 2015. Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection. Nat Med 21:1065–1070. doi: 10.1038/nm.3927. [DOI] [PubMed] [Google Scholar]
• 43.Impagliazzo A, Milder F, Kuipers H, Wagner M, Zhu X, Hoffman RMB, van Meersbergen R, Huizingh J, Wanningen P, Verspuij J, de Man M, Ding Z, Apetri A, Kukrer B, Sneekes-Vriese E, Tomkiewicz D, Laursen NS, Lee PS, Zakrzewska A, Dekking L, Tolboom J, Tettero L, van Meerten S, Yu W, Koudstaal W, Goudsmit J, Ward AB, Meijberg W, Wilson IA, Radosevic K. 2015. A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen. Science 349:1301–1306. doi: 10.1126/science.aac7263. [DOI] [PubMed] [Google Scholar]
• 44.Kanekiyo M, Wei C-J, Yassine HM, McTamney PM, Boyington JC, Whittle JRR, Rao SS, Kong W-P, Wang L, Nabel GJ. 2013. Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature 499:102–106. doi: 10.1038/nature12202. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 45.DiLillo DJ, Palese P, Wilson PC, Ravetch JV. 2016. Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection. J Clin Invest 126:605–610. doi: 10.1172/JCI84428. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 46.Ana-Sosa-Batiz F, Vanderven H, Jegaskanda S, Johnston A, Rockman S, Laurie K, Barr I, Reading P, Lichtfuss M, Kent SJ. 2016. Influenza-specific antibody-dependent phagocytosis. PLoS One 11:e0154461. doi: 10.1371/journal.pone.0154461. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 47.Wang TT, Maamary J, Tan GS, Bournazos S, Davis CW, Krammer F, Schlesinger SJ, Palese P, Ahmed R, Ravetch JV. 2015. Anti-HA glycoforms drive B cell affinity selection and determine influenza vaccine efficacy. Cell 162:160–169. doi: 10.1016/j.cell.2015.06.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 48.Kramski M, Stratov I, Kent SJ. 2015. The role of HIV-specific antibody-dependent cellular cytotoxicity in HIV prevention and the influence of the HIV-1 Vpu protein. AIDS 29:137–144. doi: 10.1097/QAD.0000000000000523. [DOI] [PubMed] [Google Scholar]
• 49.DiLillo DJ, Ravetch JV. 2015. Differential Fc-receptor engagement drives an anti-tumor vaccinal effect. Cell 161:1035–1045. doi: 10.1016/j.cell.2015.04.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 50.Taylor RP, Lindorfer MA. 2008. Immunotherapeutic mechanisms of anti-CD20 monoclonal antibodies. Curr Opin Immunol 20:444–449. doi: 10.1016/j.coi.2008.05.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 51.Uchida J, Hamaguchi Y, Oliver JA, Ravetch JV, Poe JC, Haas KM, Tedder TF. 2004. The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody immunotherapy. J Exp Med 199:1659–1669. doi: 10.1084/jem.20040119. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 52.Clynes RA, Towers TL, Presta LG, Ravetch JV. 2000. Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat Med 6:443–446. doi: 10.1038/74704. [DOI] [PubMed] [Google Scholar]