Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact

Abstract

Influenza virus strain-specific monoclonal antibodies (mAbs) provide protection independent of Fc gamma receptor (FcγR) engagement. In contrast, optimal in vivo protection achieved by broadly reactive mAbs requires Fc-FcγR engagement. Most strain-specific mAbs target the head domain of the viral hemagglutinin (HA), whereas broadly reactive mAbs typically recognize epitopes within the HA stalk. This observation has led to questions regarding the mechanism regulating the activation of Fc-dependent effector functions by broadly reactive antibodies. To dissect the molecular mechanism responsible for this dichotomy, we inserted the FLAG epitope into discrete locations on HAs. By characterizing the interactions of several FLAG-tagged HAs with a FLAG-specific antibody, we show that in addition to Fc-FcγR engagement mediated by the FLAG-specific antibody, a second intermolecular bridge between the receptor-binding region of the HA and sialic acid on effector cells is required for optimal activation. Inhibition of this second molecular bridge, through the use of an F(ab')₂ or the mutation of the sialic acid-binding site, renders the Fc-FcγR interaction unable to optimally activate effector cells. Our findings indicate that broadly reactive mAbs require two molecular contacts to possibly stabilize the immunologic synapse and potently induce antibody-dependent cell-mediated antiviral responses: (i) the interaction between the Fc of a mAb bound to HA with the FcγR of the effector cell and (ii) the interaction between the HA and its sialic acid receptor on the effector cell. This concept might be broadly applicable for protective antibody responses to viral pathogens that have suitable receptors on effector cells.

Keywords: Fcγ receptor; antibody-dependent cell-mediated immunity; broadly reactive antibodies; hemagglutinin; influenza virus.

Fig. 1.

Characterization of FLAG-tagged HAs. (A) The FLAG epitope (DYKDDDDK) was inserted in frame into PR/8 (H1N1) HA at site 135, 291, 298, 313, or 366 (residues indicated in red). The receptor-binding region represented by amino acids Y108, W166, H196, and L207 (indicated in green) resides on top of the globular head of the HA. (B) Cell surface expression of FLAG-tagged HAs was assessed via FACS analysis on transfected 293T cells using polyclonal sera, head-specific mAb PY102, an anti-FLAG mAb, or an IgG2a control (mAb XY102). Percent binding for each HA construct was normalized to polylconal sera against each respective HA. (C) Expression of the FLAG epitope of the tagged HAs was further confirmed by Western blot analysis of whole cell lysates from transfected 293T cells using mAb PY102 and an anti-FLAG mAb. GAPDH served as a loading control. (D) Binding of FLAG-tagged HAs to sialic acid motifs was assessed by a modified hemolysis assay. FLAG-tagged HA transfected cells were coincubated with a 0.5% solution of chicken RBCs for 1 h at 4 °C. Heme released from agglutinated transfected cells was quantified by absorbance (405 nm). Error bars represent SEM.

Fig. 2.

A stalk-based FLAG epitope can induce FcγR-mediated effector function. To examine the role of epitope location on the induction of effector function, HEK 293T cells were transfected with WT HA (A), 135 FLAG-HA (B), 291 FLAG-HA (C), 298 FLAG-HA (D), 313 FLAG-HA (E), or 366 FLAG-HA (F). WT or FLAG-tagged HA-expressing HEK 293T cells were used as targets for measuring antibody-mediated effector function with a genetically modified Jurkat cell line expressing the murine FcRγIV with an inducible luciferase reporter gene. The mAb PY102 (IgG1) is a strain-specific antibody that recognizes the globular head of the HA of the PR/8 (H1N1) virus, and the mAb 6F12 (IgG2b) is a pan-H1 stalk-specific mAb. An H3-specific mAb, XY102 (IgG2a), served as a control IgG. The anti-FLAG mAb (IgG2a) is a commercially available antibody (Clontech). All mAbs were tested at a starting concentration of 10 µg/mL and were serially diluted fourfold. A nonlinear regression best-fit curve was generated for each dataset using GraphPad Prism 5. Error bars represent SEM. Results are from one of two independent experiments.

Fig. 3.

Inhibition of HA binding to its sialic acid receptor prevents FcγR-mediated effector function induced by HA stalk mAbs. HEK 293T cells were transfected with WT HA (PR/8) (A–C), 366 FLAG-HA (E), WT HA (Cal/09) (F), or a mutant HA with a point mutation in the receptor binding site. Y108F HA (Cal/09) served as FcγR-mediated effector targets for the indicated mAbs. A genetically modified Jurkat cell line expressing the murine FcyRIV with an inducible luciferase reporter gene was used to determine the induction of antibody-mediated effector activity. (A) Induction of FcγR-mediated effector function by a stalk-specific mAb, 6F12, was inhibited by blocking the sialic acid-binding site of the globular head of HA with coincubation of a constant amount (10 µg/mL) of head-specific PY102 F(ab′)₂. (B) FcγR-mediated effector function by a stalk-specific mAb, 6F12 (10 µg/mL), was inhibited in a dose- dependent manner with the addition of a serial dilution of PY102 F(ab′)₂ (starting concentration of 10 µg/mL, diluted fourfold) (C) Coincubation of a constant amount (10 µg/mL) of humanized [murine F(ab′)₂, human Fc] 6F12 with variable amounts of PY102 or control F(ab′)₂ (starting concentration of 10 µg/mL, fourfold dilution) did not prevent 6F12 from binding to the stalk region in a cell-based ELISA. (D) Purified preparations (100 ng) of full-length and F(ab′)₂ of PY102 were resolved in an SDS/PAGE gel (in nonreducing conditions) and assessed by Western blot analysis. An anti-mouse F(ab′)₂-specific secondary antibody conjugated to HRP was used to visualized the antibody isoforms. (E) Induction of an anti-FLAG mAb against 366 FLAG-HA was also inhibited with a constant amount of PY102 F(ab′)₂ (10 µg/mL). (F) A stalk-specific mAb, 6F12, has reduced ability to induce effector activity against Y108F HA (Cal/09) compared with WT HA (Cal/09). Full-length mAbs in A and E were tested at a starting concentration of 10 µg/mL and were serially diluted fourfold, whereas the F(ab′)₂ in A and E were coincubated at a constant concentration of 10 µg/mL. The F(ab′)₂ in B was added at a starting dilution of 10 µg/mL and was diluted fourfold. An H3-specific mAb, XY102, was used as control IgG in A and F, and the control F(ab′)₂ in B and E was generated from a pan-H3 mAb, 9H10. The area under the curve in C was calculated using GraphPad Prism 5 from ELISA values read at 492 nm. A nonlinear regression best-fit curve was generated for each dataset using GraphPad Prism 5. RLU, relative luminescence units. Error bars represent SEM. Results are from one of two independent experiments.

Fig. 4.

Optimal activation of FcγR-mediated effector function requires two intermolecular contacts. (A) Classical hemagglutination-inhibiting antibodies that target the receptor-binding domain of the HA do not efficiently induce Fc-mediated effector functions because of steric hindrance of the receptor-binding site and subsequent loss of the second point of contact. Only one contact point between Fc and FcyR is established (1). (B) The interaction between the receptor-binding domain of the influenza virus HA and sialic acid motifs on the surface of effector cells (2) provides a second intermolecular bridge in addition to the Fc–FcγR interaction (1) and leads to optimal activation of an effector cell. The scenario described in B might possibly apply to any (viral) pathogen that has suitable receptors on effector cells.