Fc-mediated effector functions appear not to contribute to protection provided by strain-specific HA head-binding antibodies. Antibodies which do not interfere with sialic acid binding of HA can mediate FcRIIIa activation. However, the FcRIIIa 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 FcRIIIa activation. The inhibition of FcRIIIa activation by HA head-binding and sialic acid receptor-blocking antibodies was confirmed in plasma Isocarboxazid samples of H5N1 vaccinated human subjects. Together, these results suggest that in addition to FcCFcR binding, interactions between HA and sialic acids on immune cells are required for optimal Isocarboxazid Fc-mediated effector functions by anti-HA antibodies. neutralizing activity against influenza viruses [broadly neutralizing antibodies (bnAbs)] have been isolated from human memory B cells. In agreement with their activity, passive transfer of broadly neutralizing anti-influenza antibodies has been shown to protect mice and ferrets from lethal challenge with antigenically diverse viruses (5C12). The structural characterization of several of these antibodies (5C7, 10C15) has revealed epitopes in the head and stem regions of the HA, where functional constraints appear to restrict the potential for the virus to mutate. These epitopes are of great interest as vaccine targets, and several strategies are being employed to generate vaccines that induce broadly reactive antibodies (16C18). To be able to effectively design these types of Isocarboxazid vaccines, it is essential to elucidate the underlying molecular mechanisms involved in the cross-protective immunity of these broadly reactive antibodies. Influenza-specific antibodies can block essential steps in the viral life cycle. Depending on their epitope, they can directly interfere with the viral life cycle by blocking the binding of HA to its sialic acid receptors on the host cell, by preventing the low pH-induced conformational changes of HA required for membrane fusion, by inhibiting the cleavage of the HA0 precursor protein, or by inhibiting viral egress (5C7, 11, 13, 19, 20). Antibodies can also exert anti-viral effects through other mechanisms, including effector functions mediated by the Fc part of the antibody molecule, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) (21C24). Involvement of Fc-effector functions, in particular ADCC, has been demonstrated in the protection of mice from H1N1 challenge by bnAb FI6 (5). Recent publications have shown that broadly reactive anti-HA head and stem antibodies require Fc receptor (FcR) engagement for optimal protection, while protection by strain-specific anti-HA head antibodies was independent of FcR interactions (25C28). In addition, it was shown that only stem-specific and broadly reactive anti-head antibodies, and not strain-specific anti-HA head antibodies, were able to engage FcRs to trigger ADCC (25). No molecular mechanism to explain this observation has been proposed to date. Here, we investigate the molecular mechanisms behind the observation that anti-stem antibodies and not anti-head antibodies are able to mediate robust FcRIIIa activation. A panel of influenza A- and B-specific monoclonal antibodies with identical human IgG1 Fc domains, making them particularly suitable to compare their ability to mediate FcRIIIa activation, were used. We demonstrate that in particular, anti-head antibodies that specifically inhibit the interactions between the HA receptor-binding site and sialic acids on immune cells fail to induce strong FcRIIIa activation. The addition of such anti-head antibodies that block receptor binding can interfere with FcRIIIa activation in human plasma. Based on our data, we propose a model that describes that optimal HA antibody-mediated FcRIIIa activity is dependent on the interaction between HA on host cells and sialic acid receptors on immune cells. Results HAI-Positive Antibodies Are Unable to Induce Robust FcRIIIa Activation We have previously described broadly reactive antibodies that were protective against group 1 influenza A viruses (CR6261) (12, 13), antigenically diverse influenza B viruses (CR8033 and CR8071), and both group 1 and group 2 influenza A viruses as well as influenza B Pdpn viruses (CR9114) (20). CR6261 binds the stem region of HA and neutralizes the virus Isocarboxazid by preventing the conformational changes of this protein that are required for the viral fusion process (12). In contrast, CR8033 and CR8071 bind non-overlapping epitopes in the head region of influenza B HA and neutralize neutralizing activity against these viruses (20). To further explore the molecular mechanism by which these broadly reactive antibodies provide protection efficacy of HA stem-binding, but not head-binding, antibodies against influenza A viruses (25, 26). In agreement with the previous observation, we found.