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.

Wild-type influenza strains were obtained from the Centers for Disease Control and Prevention or purchased from the American Tissue Culture Collection. Cold adapted (ca) live-attenuated influenza vaccine viruses were generated by either classical reassortment or by reverse genetics (Jin et al., 2003) . All viruses were propagated in embryonated chicken eggs, and virus titers were determined by mean 50% tissue culture infective dose (TCID50) per milliliter. Complete viral strain designations are shown in Table S1 . The microneutralization assay was performed as described previously (Benjamin et al., 2014) . Briefly, 60 TCID50 of virus/well was added to three-fold serial dilutions of antibody in a 384-well plate in complete MEM medium containing 0.75ug/ml Trypsin (Worthington) in duplicate wells. After 1 hour incubation at 33°C 5% CO2, 2x10 4 MDCK cells/well were added to the plate. Plates were incubated at 33°C 5% CO2 incubator for approximately 40 hr, and the NA activity was measured by adding a fluorescently-labeled substrate, methylumbelliferyl-N-acetyl neuraminic acid (MU-NANA) (Sigma) to each well and incubated at 37°C for 1 hr. Virus replication represented by NA activity was quantified by reading fluorescence using the following settings: excitation 355 nm, emission 460 nm; 10 flashes per well. The concentration of antibody required for a 50% reduction in viral replication (IC 50 ) was calculated using a non-linear fit algorithm curve fit in Graph Pad Prism. If neutralization curve was not complete, then value was assigned the highest concentration of inhibitor tested.