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Hemagglutination titer value was used to confirm virus egress from cell surface to supernatant. (C) Egress inhibition of FluA-20 was tested using H3 Texas virus.

HA was also incubated with (4) PBS at pH 8.0 and treated with trypsin at pH 8.4 as a control. Non-reducing SDS-PAGE of recombinant HA (H3 Perth) pre-incubated with either (1) PBS or (2) mAb FluA-20 or (3) mAb CR8020 for 1 h at pH 5.0 were neutralized to pH 8.4 and further treated with PBS or TPCK-trypsin for 12 hours. (A) SDS-PAGE of 4 μg of recombinant HA0 protein (from A/Perth/16/2009 (H3N2) that was premixed with either PBS or 40 μg of mAb FluA-20 or mAb CR8020 was either not treated (NT) or treated with TPCK-trypsin for 5, 20 or 40 minutes at 37 ☌. FluA-20 was tested for (A) HA cleavage inhibition, (B) pH-induced HA conformational change inhibition, and (C) egress inhibition. Functional characterization of FluA-20 IgG. ( C) Difference map from deuterium exchange of cleaved HA trimer compared to HA0 trimer from A/Netherlands/219/2003(H7N7) by HDX-MS.Ĩ: Figure S7, related to Figure 7. The dotted line indicates the baseline signal from the noninfectious HA0 virus (untreated). Following incubation, the cells were fixed and the presence of influenza nucleoprotein in the cells was determined by ELISA using a mouse anti-NP Ab. As a control, HA0 virus untreated with trypsin (and therefore inactive) was also added to MDCK cell monolayers.

The samples were trypsin-inactivated with 10% FBS before adding to MDCK cell monolayers.

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(B) HA0 virus (V) produced in the absence of trypsin was incubated with serial dilutions of mAbs (FluA-20 or CR8020) either before or after treatment with 1 μg/mL of trypsin (Trp) at 37 ☌ for 45 minutes. The Ab used for binding to H1 HA (A/California/04/2009) was 5J8 and for H7 HA (A/Shanghai/02/2013) was H7.137. (A) Binding traces of HA0 trimer or cleaved HA trimer (HA1/HA2) to receptor-binding site Abs. Comparison of Ab binding to HA0 versus cleaved HA trimers. Lungs were collected for virus titration at 5 days post-inoculation.ħ: Figure S6, related to Figure 6. (B) Groups of mice (n=3) were lethally challenged with 1.2 LD 50 of H3N2 A/X-31 or H5N1 A/barn swallow/Hong Kong/D10–1161/2010 or H7N9 A/Shanghai/1/2013 on PR8 backbone and were treated therapeutically with 10 mg/kg of FluA-20 or an irrelevant Ab (MRSA) via the intraperitoneal route on days 1, 2 and 4 post-challenge. The graph shows pulmonary virus titers in FluA-20 and control treated mice. Lung samples were collected from mice for each Ab treated group at 6 days post-inoculation.

(A) Groups of mice (n=3) were treated prophylactically with 10 mg/mL of either FluA-20 or a similarly prepared control Ab to an unrelated target (MRSA) and challenged 24 h later with a sublethal dose of 0.1 LD 50 with either H1N1 A/Netherlands/602/2009 or H3N2 A/X-31 (6:2 PR8 backbone) or H5N1 A/barn swallow/Hong Kong/D10–1161/2010 (7:1 PR8 backbone) or H7N9 A/Shanghai/1/2013 (6:2 PR8 backbone). Lung titers of mice treated with FluA-20 in prophylactic or therapeutic settings.