Supplementary Figure 1.

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1 F12 IgG2a Isotype Control Supplementary Figure 1. Passive treatment with isotype control mab does not affect viral challenge. Wild type mice were treated with 4 mg/kg of mouse IgG2a 6F12 bnab (filled circles), 4 mg/kg of mouse IgG2a isotype control mab (open circles), or (filled triangles) 2 hours before intranasal infection with 5 mld 5 of PR8 virus. Values represent mean (±SEM) % weight change compared to day (left) or (right). n 6 mice per group. Nature Medicine: doi:1.138/nm.3443

2 c * * * ** ** ** ** ** ** ** 5 5 WT+ 6F12 FcRα-null + 6F12 WT + FcRα-null WT + 6F12 Fcgr4 -/- + 6F12 Fcgr4 -/ WT + 6F12 Fcgr1I -/- + 6F12 Fcgr3 -/- + 6F12 WT Supplementary Figure 2. Activating FcγRs are required for mab-mediated protection from viral infection in vivo. (a) Wild type (WT) mice or FcRα-null mice were treated with 4 mg/kg mouse IgG2a 6F12 mab or 2 hours before intranasal infection with 5 mld 5 of PR8 virus. Values represent mean (±SEM) % weight change compared to day (left panel) or (right panel). n=6-1 mice per group. Significant differences between FcRα-null mice and wild type mice receiving 6F12 mab are shown: **, p<.1. (b) Wild type mice or Fcgr4 -/- mice were treated with 4 mg/kg mouse IgG2a 6F12 mab or 2 hours before intranasal infection with 5 mld 5 of PR8 virus. Values represent mean (±SEM) % weight change compared to day (left panel) or (right panel). n=5-1 mice per group. Significant differences between Fcgr4 -/- mice and wild type mice receiving 6F12 mab are shown: *, p<.5; **, p<.1. (c) Wild type (WT) mice, Fcgr1 -/- mice, or Fcgr3 -/- mice were treated with 4 mg/kg mouse IgG2a 6F12 mab or 2 hours before intranasal infection with 5 mld 5 of PR8 virus. Values represent mean (±SEM) compared to day (left panel) or (right panel). n 6 mice per group. Nature Medicine: doi:1.138/nm.3443

3 1. Relative OD IgG2a IgG1 DA265 Isotype Control. mab concentration ( g/ml) Percent inhibition IgG2a IgG1 DA265 Isotype Control mab concentration ( g/ml) Supplementary Figure 3. In vitro characterization of FI6 mab mutants. (a) Binding of FI6 mab variants to PR8 HA. Mouse IgG2a (filled circles), mouse IgG1 (open circles), and DA265 mutant (filled squares) FI6 mab, and IgG2a isotype control mab (filled triangles), were diluted as indicated and tested for binding to PR8 HA by ELISA. Values represent mean (± SEM) relative OD values from triplicate wells. (b) In vitro plaque reduction neutralization by FI6 mab variants. PR8 virus was pre-incubated with the indicated dilutions of the FI6 mab variants or IgG2a isotype control mab before infecting a monolayer of MDCK cells and applying an agar overlay containing diluted FI6 bnab variants. Plaques were counted after 2 days. Values represent mean % inhibition, which was calculated by comparing plaque numbers in mab-treated wells with wells receiving only. Nature Medicine: doi:1.138/nm.3443

4 ****** ** ** ** 5 WT + FI6 IgG2a Fcer1g -/- + FI6 IgG2a WT + Fcer1g -/ Supplementary Figure 4. Activating FcγRs are required for in vivo protection by FI6 ma (a) Wild type (WT) mice or Fcer1g -/- mice were treated with 4 mg/kg mouse IgG2a 6F12 mab or 2 hours before intranasal infection with 5 mld 5 of PR8 virus. Values represent mean (±SEM) compared to day (left panel) or (right panel). n 4 mice per group. Significant differences between FcγRγ -/- mice and wild type mice receiving 6F12 mab are shown: **, p<.1. Nature Medicine: doi:1.138/nm.3443

5 G2 IgG2a 2G2 DA B6 migg2a 2B6 DA c F2 migg2a 1F2 DA Supplementary Figure 5. Anti-HA stalk mabs 2G2, 2B6, and 1F2 require FcγR interactions for in vivo protection during lethal viral challenge. (a) Wild type mice were treated with 4 mg/kg of mouse IgG2a 2G2 bnab (filled circles), DA265 mutant 2G2 bnab (filled squares), or (filled triangles) 2 hours before intranasal infection with 5 mld 5 of Neth9 virus. (b) Wild type mice were treated with 4 mg/kg of mouse IgG2a 2B6 bnab (filled circles), DA265 mutant 2G2 bnab (filled squares), or (filled triangles) 2 hours before intranasal infection with 5 mld 5 of Neth9 virus. (c) Wild type mice were treated with 2 mg/kg of mouse IgG2a 1F2 bnab (filled circles), DA265 mutant 1F2 bnab (filled squares), or (filled triangles) 2 hours before intranasal infection with 5 mld 5 of Neth9 virus. Values (a-c) represent mean (±SEM) % weight change compared to day or. n 4 mice per group in all experiments. Nature Medicine: doi:1.138/nm.3443

6 6F12 IgG2a F12 DA265 1mg/kg 2mg/kg 4mg/kg 8mg/kg 16mg/kg c Fcer1g / Mice; 6F12 IgG2a 1mg/kg 2mg/kg 4mg/kg 8mg/kg 16mg/kg mg/kg 8mg/kg 16mg/kg 32mg/kg Supplementary Figure 6. Dose-dependence of the FcγR requirement of bnab during in vivo protection. Wild type mice were treated with the indicated doses of (a) mouse IgG2a or (b) DA265 mutant 6F12 bnab, or, 2 hours before intranasal infection with 5 mld 5 of PR8 virus. (c) Fcer1g -/- mice were treated with the indicated dose of mouse IgG2a 6F12 bnab or before infection as in (a-b). Values represent mean (±SEM) compared to day (left panels) or (right panels). n 5 mice per group. Nature Medicine: doi:1.138/nm.3443

7 Relative OD IgG2a IgG1 DA265 Isotype control. mab concentration (μg/ml) Percent inhibition IgG2a IgG1 DA265 Isotype control mab concentration (μg/ml) Supplementary Figure 7. In vitro characterization of PY12 mab mutants. (a) Binding of PY12 mab variants to PR8 HA. Mouse IgG2a (filled circles), mouse IgG1 (open circles), and DA265 mutant (filled squares) PY12 mab, and IgG2a isotype control mab (filled triangles), were diluted as indicated and tested for binding to PR8 HA by ELISA. Values represent mean (± SEM) relative OD values from triplicate wells. (b) In vitro plaque reduction neutralization by PY12 mab variants. PR8 virus was preincubated with the indicated dilutions of the PY12 mab variants or IgG2a isotype control mab before infecting a monolayer of MDCK cells and applying an agar overlay containing diluted PY12 bnab variants. Plaques were counted after 2 days. Values represent mean % inhibition, which was calculated by comparing plaque numbers in mab-treated wells with wells receiving only. Nature Medicine: doi:1.138/nm.3443

8 9 8 WT + PY12 (IgG2a) Fcer1g -/- + PY12 (IgG2a) WT + Fcer1g -/- + Supplementary Figure 8. PR8 strain-specific anti-h1 head mab PY12 does not require FcγR contributions during protection from viral infection in vivo. Wild type or Fcer1g -/- mice were treated with the.5 mg/kg of mouse IgG2a PY12 mab, or, 2 hours before intranasal infection with 5 mld 5 of PR8 virus. Values represent mean (±SEM) compared to day from n 5 mice per group. Nature Medicine: doi:1.138/nm.3443

9 2.5 Relative OD IgG2a IgG1 DA265 Isotype Control mab concentration ( g/ml) Percent inhibition IgG2a IgG1 DA265 Isotype Control mab Concentration ( g/ml) Supplementary Figure 9. In vitro characterization of 7B2 mab mutants. (a) Binding of 7B2 mab variants to A/California/7/29 (Cal9) HA. Mouse IgG2a (filled circles), mouse IgG1 (open circles), and DA265 mutant (filled squares) 7B2 mab, and IgG2a isotype control mab (filled triangles), were diluted as indicated and tested for binding to Cal9 HA by ELISA. Values represent mean (± SEM) relative OD values from triplicate wells. (b) In vitro plaque reduction neutralization by 7B2 mab variants. Neth9 virus was pre-incubated with the indicated dilutions of the 7B2 mab variants or IgG2a isotype control mab before infecting a monolayer of MDCK cells and applying an agar overlay containing diluted 7B2 bnab variants. Plaques were counted after 2 days. Values represent mean % inhibition, which was calculated by comparing plaque numbers in mab-treated wells with wells receiving only. Nature Medicine: doi:1.138/nm.3443

10 WT + 7B2.5 mg/kg FcRα-null + 7B2.5 mg/kg WT + 7B2 1.5 mg/kg FcRα-null + 7B2 1.5 mg/kg WT + FcRα-null + c C4 IgG2a 5 4C4 IgG2a 4C4 DA C4 DA mg/kg.5 mg/kg.25 mg/kg Days Supplementary Figure 1. PR8 strain-specific anti-h1 head mab 7B2 and 29 pandemic-specific anti-h1 head mab 4C4 do not require FcγR contributions during protection from viral infection in vivo. (a) Wild type or FcRα-null mice were treated with the indicated doses of mouse IgG2a PY12 mab, or, 2 hours before intranasal infection with 5 mld 5 of PR8 virus. (b) Wild type mice were treated with.25 mg/kg of mouse IgG2a 4C4 mab (filled circles), DA265 mutant 4C4 mab (open circles), or (filled triangles) 2 hours before intranasal infection with 5 mld 5 of Neth9 virus. (c) Wild type mice were treated with the indicated doses of mouse IgG2a or DA265 mutant PY12 mab, or, 2 hours before intranasal infection with 5 mld 5 of Neth9 virus. (a-c) Values represent mean (±SEM) % weight change compared to day or from n 5 mice per group. Nature Medicine: doi:1.138/nm.3443

11 15 MFI 5 6F12 G1 FI6 G1 FI6 N297A PY12 G1 PY12 GASDALIE Antibody concentration (µg/ml) 25 2 MFI F12 G1 FI6 G1 FI6 N297A 7B2 G1 7B2 GASDALIE Antibody concentration (µg/ml) Supplementary Figure 11. huigg1 mab binding to PR8 and Neth9 virus-infected cells. A549 cells infected with (a) PR8 virus or (b) Neth9 virus were incubated with the indicated concentrations of the indicated huigg1 mabs, washed, stained with anti-huigg, and analyzed by flow cytometry. Values represent mean MFI (mean florescence intensity) values from individual samples. Nature Medicine: doi:1.138/nm.3443

12 Relative RU Anti-head PY12 mab Anti-stalk 6F12 mab 2 nm nm 5nM 25nM 12.5 nm 6.25 nm nm nm nm nm 2 4 Time (s) 2 4 Time (s) K D = 5.61x1 1 M k a = x1 5 1/Ms k d =1.625x1 4 1/s K D = x1 9 M k a = x1 4 1/Ms k d = x1 4 1/s Supplementary Figure 12. SPR analysis of the binding of PR8 HA to anti-head mab PY12 (left) or anti-stalk mab 6F12 (right). Real-time sensorgrams with affinity constants are shown. Technical details are described in the Methods section. Nature Medicine: doi:1.138/nm.3443

13 % CD17a+ NK cells ** ** ** FI6 hug1 2B6 hug1 2G2 hug1 IF2 hug1 4C4 hug1 mab concentration (µg/ml) % CD17a+ NK cells ** ** ** ** 6F12 hug1 FI6 hug1 FI6 N297A 7B2 hug1 7B2 GASDALIE Antibody concentration (µg/ml) Supplementary Figure 13. Impaired NK cell activation by anti-ha head mab compared to anti-ha stalk ma A549 lung epithelial cells were infected with Neth9 virus, trypsinized, and coated with the indicated concentrations of ma In (a), anti-stalk bnabs FI6, 2G2, 2B6, and IF2 were compared to the anti-head mab 4C4. In (b), anti-stalk 6F12, anti-stalk FI6, or anti-head 7B2 mabs (huigg1 isotype mabs), a FI6 huigg1 mab mutant (N297A) that does not engage FcγRs, or a 7B2 huigg1 mab mutant (GASDALIE) that preferentially engages hufcγriiia with augmented binding were compared.the cells were washed and incubated at a 1:1 ratio with PBMCs from 4 different leukocyte donors for 3 hours before cell-surface CD17a expression was assessed on CD5 + CD3 - NK cells by immunofluorescence staining with flow cytometry analysis. Values represent the mean frequency of CD17a + cells among NK cells from cultures with the indicated cells from 4 individual leukocyte donors. Results are representative of 2 independent experiments. Significant differences between the indicated sample and the (a) F16 huigg1 sample or (b) 6F12 huigg1 sample are shown. **, p<.1. Nature Medicine: doi:1.138/nm.3443

14 Antibody concentration (ng/ml) Antibody:HA immune complex Supplementary Figure 14. Anti-HA head and anti-ha stalk immune complexes contain similar levels of IgG. Immune complexes were generated with the indicated huigg1 mab and PR8 HA at a 1:1 molar ratio. The anti-ha head and anti-ha stalk immune complexes were diluted and captured on and ELISA plate by plate-bound anti-stalk or anti-head mab, respectively, with IgG concentrations determined by standard curve. Values represent the mean concentration (ng/ml) of IgG in each sample. Nature Medicine: doi:1.138/nm.3443

15 Relative OD Human IgG1 GASD/ALIE Isotype Control % inhibition Human IgG1 GASD/ALIE Isotype Control. mab Concentration (μg/ml) mab Concentration (μg/ml) Supplementary Figure 15. In vitro characterization of huigg1 6F12 mab mutants. (a) Binding of huigg1 6F12 mab variants to PR8 HA. Wild type huigg1 (filled circles), GASD/ALIE mutant (open circles), and huigg1 isotype control mab (filled triangles) were diluted as indicated and tested for binding to PR8 HA by ELISA. Values represent mean (± SEM) relative OD values from triplicate wells. (b) In vitro plaque reduction neutralization by 6F12 mab variants. The assay was performed as described in Fig.1 using the huigg1 6F12 mab variants. Values represent mean percent inhibition, which was calculated by comparing plaque numbers in duplicate mab-treated wells with wells receiving only. Nature Medicine: doi:1.138/nm.3443

16 Supplementary Table 1. Antibody characteristics and half-lives Antibody Reactivity Epitope Isotype/Mutant T 1/2 (days) a 6F12 PY12 Pan-H1 A/Puerto Rico/8/34 (PR8)-specific HA Stalk Domain (HA2) HA Globular Head Domain (HA1) Mouse IgG2a 6.4±.2 Mouse IgG1 8.4±.3 Mouse DA ±.3 Human IgG1 6.3±.6 GASD/ALIE 5.1±.3 Mouse IgG2a 8.7±2.4 Mouse IgG1 8.2±1.7 Mouse DA ±1.5 a Wild type mice received 2 μg of the indicated mab, with serum harvested and HA-specific mab concentrations determined by ELISA. n.d., not determined Nature Medicine: doi:1.138/nm.3443