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.

A/Vietnam/1194/2004 recombinant virus containing substitution Asn186Lys and H7 A/Turkey/Italy/214845/2002, Group 1 and Group 2 viruses respectively, Figure 2 . The viruses were grown in hens eggs and purified by sucrose density gradient centrifugation, according to standard protocols (Skehel and Schild, 1971 ). H5 and H7 HAs were purified from the virus membrane by detergent extraction followed by trypsin digestion (H5) or bromelain digestion (H7). The digestion was then further purified by ion exchange chromatography using a Q FF Sepharose column and finally by gel filtration chromatography, on a superdex-200 16/60 column (GE) in 20mM Tris/HCl pH 8.0, 150 mM NaCl. The Fab fragments from MEDI8852 IgG were prepared by Endoproteinase Lys-C digestion. The Fab fragments were purified using Protein A sepharose affinity chromatography (HiTrap Protein A, 1 ml column) followed by gel filtration chromatography using a superdex-200 16/60 column (GE) in 20 mM Tris/HCl pH 8.0, 150 mM NaCl. Excess MEDI8852 Fab was added to purified H5 and H7 HA and incubated overnight at 4⁰C for complex formation in 20 mM Tris/HCl pH 8.0, 150 mM NaCl. The complex was then loaded onto a superdex-200 gel filtration column to purify the predicted 3Fab-HA complex from any excess Fab. Peak fractions corresponding to the Fab-HA complex were pooled and concentrated for crystallization. MEDI8855_H5 complex crystals were obtained from 16% PEG 3350, 0.1 M Pipes pH 7.0, cryoprotected with 25% ethylene glycol prior to freezing. MEDI8852_H7 complex crystals were obtained from 45% pentaerythritol propoxylate (PEP) 15/4, 0.1 M Tris, pH 8.5. MEDI8852 Fab crystals were obtained from 20% PEG 3350, 0.2 M Ammonium Dihydrogen Phosphate after seeding with crystals obtained from 18% PEG8000, 0.1 M Sodium Cacodylate pH 6.5 and 0.2 M Calcium Acetate Hydrate. Crystals were cryoprotected with 25% ethylene glycol prior to freezing.