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.

Antibody mediated fusion inhibition was tested using a low pH induced red blood cell fusion model adaptated from protocol described in (Wang et al., 2010) . In brief, A/Puerto Rico/8/34 virus (10 x 106 TCID50) propagated in the presence of trypsin was incubated with human red blood cells (2% final red cell concentration) on ice for 10 minutes. Antibody dilutions were incubated with virus for 30 minutes at room temperature. Red blood cells were added to the virus-antibody mixture for 30 minutes at 37°C, then sodium acetate buffer (0.5 M pH 5.0) was added for additional 45 minutes at 37°C. Samples were centrifuged for 6 minutes at 400xg and incubated for additional 45 minutes at room temperature and then re-pelleted for 6 minutes at 400xg. Supernatants were transferred to an ELISA plate for determination of absorbance at 540 nm. To evaluate the ability of MEDI8852 to inhibit the low pH activated conformational change in HA, H5 HA (A/Vietnam/1194/2004 carrying substitution N186K) and H5-MEDI8852 Fab complex solutions (0.5 mg/ml) were incubated at various pH values (obtained by adding 0.15 M citrate buffer, pH 3.5), adjusted to neutral pH by using 1M Tris-HCl, pH 8.0, and digested with TPCK treated trypsin at a ratio of 20:1 (wt:wt) for 30 min at 37 ⁰C. The digestion was stopped using an equal amount, to trypsin, of soybean trypsin inhibitor. Tryptic digestion products were analyzed by SDS/PAGE. To assess the ability of antibody to block the cleavage of HA protein, baculovirus expressed recombinant HA from A/New Caledonia/20/99 (H1N1) or A/Hong Kong/8/68 (H3N2) was incubated with antibody at molar ratio of 15:1 (mAb:HA) for 40 min. The antibody-HA mixture was then exposed to 2.5 µg/ml of TPCK-treated trypsin and further incubated for 5, 10, 20, and 40 minutes at 37°C. Samples were separated on a polyacrylamide gel and then transferred to a PVDF membrane for Western blot analysis using a biotinylated human monoclonal antibody (FO32) (Humabs BioMed) that is specific for the HA2 and HA0 of influenza A strains.