Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes

Kallewaard, Nicole L.; Corti, Davide; Collins, Patrick J.; Neu, Ursula; McAuliffe, Josephine M.; Benjamin, Ebony; Wachter-Rosati, Leslie; Palmer-Hill, Frances J.; Yuan, Andy Q.; Walker, Philip A.; Vorlaender, Matthias K.; Bianchi, Siro; Guarino, Barbara; De Marco, Anna; Vanzetta, Fabrizia; Agatic, Gloria; Foglierini, Mathilde; Pinna, Debora; Fernandez-Rodriguez, Blanca; Fruehwirth, Alexander; Silacci, Chiara; Ogrodowicz, Roksana W.; Martin, Stephen R.; Sallusto, Federica; Suzich, JoAnn A.; Lanzavecchia, Antonio; Zhu, Qing; Gamblin, Steven J.; Skehel, John J.

ADCC assays were preformed using primary human NK cells as effector cells isolated from the PBMCs of healthy donors and target cells of A/Puerto Rico/8/34 H1N1 or A/Hong Kong/8/68 H3N2 influenza virus infected A549 cells at a target to effector ratio of 6:1. Antibody dependent cell killing was measured using LDH release assay after 4 hours of incubation. For ADCP activity, human monocytes were isolated from PBMCs of healthy donors and incubated with M-CSF for 6-7 days to differentiate into macrophages. Macrophages fluorescently labeled violet were incubated with MDCK target cells stably expressing either H1 or H3 HA proteins from A/South Dakota/06/2007 H1N1 or A/Hong Kong/8/68 H3N2 and fluorescently labeled with CFSE at an effector to target ratio of 6:1. Antibody-mediated phagocytosis was determined after 2 hours incubation by flow cytometry, measuring the total macrophage percentage that contained target cells (i.e., the double positive violet and CFSE stained cells). CDC activity was measured using rabbit low-tox complement that had been pre-adsorbed to infected MDCK cells. Complement was mixed with influenza A/Puerto Rico/8/34 H1N1 infected MDCK cells, and MEDI8852 dependent cell killing was measured using LDH release assay after 2 hours of incubation.