The process of generating and purifying recombinant humanized mAb has been described previously (43, 44). cell-mediated response against influenza virus is achieved and may allow for better vaccine design. Keywords: hemagglutinin, influenza virus, antibody-dependent cell-mediated immunity, broadly reactive antibodies, Fc receptor Abstract Influenza virus strain-specific monoclonal antibodies (mAbs) provide protection impartial of Fc gamma receptor (FcR) engagement. In contrast, optimal in vivo protection achieved by broadly reactive mAbs requires FcCFcR engagement. Most strain-specific mAbs target the head domain name 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 MAIL 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 FcCFcR 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 FcCFcR conversation 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: (and 2 and and and were tested at a starting concentration of 10 g/mL and were serially diluted fourfold, whereas the F(ab)2 in and were coincubated at a constant concentration of 10 g/mL. The F(ab)2 in was added at a starting dilution of 10 g/mL and was diluted fourfold. An H3-specific mAb, XY102, was used as control IgG in and and was generated from a pan-H3 mAb, 9H10. The area under the curve in 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 Sulbutiamine SEM. Results are from one of two impartial experiments. To confirm that this addition of F(ab)2 of PY102 into the system did not compete with 6F12 in binding to its epitope around the HA stalk, we performed a cell-based competition ELISA. In brief, a constant amount of a humanized [murine F(ab)2 and human Fc] 6F12 (10 g/mL) and a variable amount of PY102 F(ab)2 (starting dilution of 10 g/mL; diluted fourfold) were added onto WT HA-transfected HEK 293T cells, and an anti-human IgG-specific secondary antibody conjugated to horseradish peroxidase (HRP) was used to detect binding of the humanized 6F12. The humanized 6F12 alone bound equally well in the presence of either PY102 or control F(ab)2 (Fig. 3might possibly apply to any (viral) pathogen that has suitable receptors on effector cells. Viral Sulbutiamine HAs from Sulbutiamine pathogens such influenza virus and poxvirus (36, 37), as well as the HA-neuraminidase from Sendai virus and Newcastle disease virus, can bind to natural cytotoxic receptors, NKp46 and NKp44, and are able to activate NK cells impartial of antibodies (38C40). In addition, HIV and herpes simplex virus also up-regulate the expression of other natural cytotoxic receptors (41, 42). Although the genetically modified Jurkat T cells used Sulbutiamine in this study might not express pattern recognition receptors, it is plausible that HA can bind to other cell surface receptors around the Jurkat T cells and contribute to antibody-mediated effector activation. Nonetheless, our data help explain the apparent dichotomy between the disparate abilities of head- or stalk-specific antibodies to induce effector cell activity. We suggest that an anti-influenza antibody that does not interfere with the conversation between the receptor-binding site and sialic acid has the ability to induce FcR-mediated effector function, which may include but is not limited to stalk-specific, non-HI active neutralizing head-specific or nonneutralizing antibodies (21, 43, 44). Although our data highlight the importance of this second point of contact, at present it is unclear whether HA binding to sialic acid activates an additional signaling pathway or facilitates stabilization of the immunologic synapse to prolong the conversation between an immune cell Sulbutiamine and the Fc region of an antibody. Our work shed light on the molecular basis of how anti-influenza antibodies can engage the innate immune system to achieve optimal protection against influenza virus contamination. Whereas the isotype of the antibody can be an essential aspect for binding activating FcRs (27C29), our research obviously demonstrate that the positioning of the epitope for the influenza HA is crucial in identifying whether another molecular bridge can be available to completely indulge the antiviral activity of an innate effector cell. We speculate that second discussion required for ideal activation of Fc effector activity with a viral proteins is not.
