HA Antibody-Mediated FcγRIIIa Activity Is Both Dependent on FcR Engagement and Interactions between HA and Sialic Acids

Abstract

Interactions with receptors for the Fc region of IgG (FcγRs) have been shown to contribute to the in vivo protection against influenza A viruses provided by broadly neutralizing antibodies (bnAbs) that bind to the viral hemagglutinin (HA) stem. In particular, Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) has been shown to contribute to protection by stem-binding bnAbs. Fc-mediated effector functions appear not to contribute to protection provided by strain-specific HA head-binding antibodies. We used a panel of anti-stem and anti-head influenza A and B monoclonal antibodies with identical human IgG1 Fc domains and investigated their ability to mediate ADCC-associated FcγRIIIa activation. Antibodies which do not interfere with sialic acid binding of HA can mediate FcγRIIIa activation. However, the FcγRIIIa activation was inhibited when a mutant HA, unable to bind sialic acids, was used. Antibodies which block sialic acid receptor interactions of HA interfered with FcγRIIIa activation. The inhibition of FcγRIIIa activation by HA head-binding and sialic acid receptor-blocking antibodies was confirmed in plasma samples of H5N1 vaccinated human subjects. Together, these results suggest that in addition to Fc-FcγR binding, interactions between HA and sialic acids on immune cells are required for optimal Fc-mediated effector functions by anti-HA antibodies.

Keywords: ADCC; CR6261; CR8033; CR9114; Fc-receptor; head-binding antibody; hemagglutinin; stem-binding antibody.

Figure 1

HAI⁺ antibodies are unable to induce robust FcγRIIIa activation. Left panel indicates approximate epitopes of 2D1 (in red), CR9114, and CR6261 (in blue) superimposed onto A/California/07/09 HA (H1/California) and approximate epitopes of CR8033 (in red), CR8071 (in purple), and CR9114 (in blue) superimposed onto B/Florida/4/2006 HA (B/Florida). Right panel indicates HAI and FcγRIIIa activation of antibodies 2D1, CR8033, CR8071, CR6261, and CR9114. HAI titers were measured for A/California/07/09 (2D1 and CR6261) or B/Florida/4/2006 (CR8033, CR8071, and CR9114). FcγRIIIa activation was measured using A/California/07/09 (2D1 and CR6261) or B/Florida/4/2006 (CR8033, CR8071, and CR9114) HA-transfected target human lung-derived A549 cells at an antibody concentration of 5 μg/ml. The FcγRIIIa activity was normalized against the response obtained with 5 μg/ml of CR9114. All antibodies contain identical human IgG1 Fc domains, making them particularly suitable to compare their ability to mediate FcγRIIIa activation. HAI and FcγRIIIa activity data are representative examples of three independent experiments.

Figure 2

Sialic acid interactions are required for optimal FcγRIIIa activation. (A,B) depict FcγRIIIa activation against target cells transfected with a wild-type HA (blue) or non-SA binding mutant HA (black) of A/California/07/09 (A) or B/Florida/4/2006 (B). (C,D) depict 2D1 or CR8033 (black curves) inhibiting FcγRIIIa activation against HA of A/California/07/09 (C) and B/Florida/04/06 (D), respectively, induced by a fixed concentration (0.25 μg/ml) of CR9114. Background was determined by titrating in CR8033 or 2D1 to a non-binding antibody control (red dashed lines). As a control, a non-binding antibody was titrated in to the fixed concentrations of CR9114 (blue dashed line). RLU is the abbreviation for relative light units. Data presented are representative examples of at least three independent experiments.

Figure 3

Proposed model of differential effects of anti-stem (in blue), anti-head non-receptor blocking (HAI⁻) (in purple), and anti-head receptor blocking (HAI⁺) (in red) HA antibodies on target cell and immune cell interactions, using wild-type and non-SA-binding mutant HA, and the resultant effect on FcγRIIIa activation.

Figure 4

HAI titers strongly correlate with inhibition of FcγRIIIa activity by human plasma. (A,D) depict HAI responses of individual samples against pH1N1 (A) and H5N1 (D). (B,E) depict pH1N1 HA-specific (B) and H5N1-specific (E) FcγRIIIa activation by individual samples. Black lines indicate the group median. (G) depicts the HAI response, and (H) depicts the pH1N1-specific FcγRIIIa activation inhibition of human plasma pools complemented with four different concentrations of mAb 2D1. Plasma samples taken before vaccination are indicated in gray (pre), 21 days after the prime in red (post-prime), and 21 days after the boost in blue (post-boost). The correlations between HAI responses and FcγRIIIa activation inhibition as determined by the Kendall’s tau-b method are depicted in (C,F,I). The correlation is considered significant when p < 0.05. ABC indicates area between curves.