1. Volume 19, Issue 6, Pages 800-813 (June 2016)
Both Neutralizing and Non-Neutralizing Human H7N9 Influenza Vaccine-Induced Monoclonal Antibodies Confer Protection 
Carole J. Henry Dunand, Paul E. Leon, Min Huang, Angela Choi, Veronika Chromikova, Irvin Y. Ho, Gene S. Tan, John Cruz, Ariana Hirsh, Nai-Ying Zheng, Caitlin E. Mullarkey, Francis A. Ennis, Masanori Terajima, John J. Treanor, David J. Topham, Kanta Subbarao, Peter Palese, Florian Krammer, Patrick C. Wilson 
Cell Host & Microbe 
Volume 19, Issue 6, Pages (June 2016)
DOI: /j.chom
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2. Cell Host & Microbe 2016 19, 800-813DOI: (10.1016/j.chom.2016.05.014)
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3. Figure 1 Characterization of Human H7-Reactive Antibodies
(A) Human monoclonal antibodies (mAbs) were cloned from plasmablasts isolated at day 7 after the inactivated H7N9 vaccine.
(B) Twenty H7-reactive mAbs were screened for HAI, neutralization, and cross-reactivity to diverse influenza A HAs (group 1 and 2).
(C) Binding to recombinant HA proteins was tested by ELISA. Representative minimum positive concentrations (μg/ml) from three independent experiments are plotted as a heatmap. The different HAs were clustered by amino acid sequence phylogeny, and multiple alignments were performed using the CLUSTALW algorithm. A rooted tree was constructed using the neighbor-joining method and was visualized using FigTree v1.4.0 software. mAbs are grouped by individuals (22, 07, 24, and 41), and HAI+ Neut+, HAI− Neut+ and HAI− Neut− categories are mentioned. Affinity measurements (KD) determined by biolayer interferometry for A/Shanghai/1/2013 (H7N9) HA are displayed in purple. See also Table S1 and Figure S1.
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4. Figure 2
Human H7-Reactive Antibodies Exhibit Various In Vitro Functional Characteristics
(A–D) Minimum positive concentrations in μg/ml (mean ± SEM) are reported for HAI; IC50 in μg/ml (mean ± SEM) are reported for neutralization. Experiments were done in duplicate three times.
(A) HAI and neutralization assays were performed with A/Shanghai/1/2013 (H7N9) virus.
(B) Neutralization of various group 1 and 2 viruses based on previous ELISA results; n.d., not determined. In color are reported previous neutralization results from (A) with H7N9.
(C) HAI was performed with various H7 viruses from the Eurasian (H7N7) and North American (H7N3 and H7N1) lineages. Only the HAI+ Neut+ mAbs were tested.
(D) HAI was performed with wild-type A/Anhui/1/2013 (H7N9) virus and with an escape mutant virus that displays mutations in the H7 HA antigenic site A (R149G). Only the HAI+ Neut+ mAbs were tested. No HAI activity was observed for 07-4D05 with the escape mutant (R149G). See also Figure S1.
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5. Figure 3 Human H7-Reactive Antibodies Confer Protection In Vivo
6- to 8-week-old female BALB/c mice were injected with 5, 1, or 0.3 mg/kg of each mAb and then infected with 7.5 LD50 of A/Shanghai/1/2013 (H7N9) virus. Values represent mean ± SEM (n = 5 mice per group).
(A–J) Percent of initial weight and percent survival are plotted for each mAb: (A) 07-5D03, (B) 07-5F01, (C) 07-5G01, (D) 07-4B03, (E) 22-3E05, (F) 07-5B05, (G) 41-5E04, (H), 41-5D06, (I) 07-5E01, and (J) 24-4C01. Mice that received an H3-reactive but not H7-reactive mAb (011-2C01) died at day 6 after the challenge. The same control group of mice was used for all panels. See also Figure S2.
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6. Figure 4
Human H7-Neutralizing Antibodies Bind to Various Epitopes on the HA Stalk and Head Domains
Escape mutant variants with A/Shanghai/1/2013 (H7N9) virus were generated for the neutralizing mAbs. Sequences for each mutant were compared to the wild-type virus, and unique mutations were reported.
(A) Critical residues for binding of Neut+ mAbs are reported. Nomenclature for substitutions follows the rule of original residue, position of the amino acid, new residue (H3 numbering).
(B and C) Modeling of A/Shanghai/1/2013 H7 HA was done using PyMOL (PDB: 4LN3). HAI+ Neut+ critical residues are shown in red and HAI− Neut+ residues are shown in yellow. The Arg65 residue (HAI+ and HAI−) is shown in orange. See also Figure S3.
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7. Figure 5
Neutralizing and Non-Neutralizing H7-Reactive Antibodies Bind to Different Epitopes on Hemagglutinin
(A) Biotinylated mAbs 41-5E04 (HAI− Neut+) and 41-5D06 (HAI− Neut−) were tested for binding to A/Shanghai/1/2013 HA by ELISA with or without the presence of a competitor mAb. The experiment was done in duplicate three times. The percentage of competition between the 41-5E04 and 41-5D06 (in red) and the non-neutralizing mAbs or CR9114 is shown. Absorbance value of each mAb against itself is scored at 100% inhibition, and comparison of different mAbs was done as a percentage of this 100% inhibition.
(B) Purified virus preparations were treated with various pH-buffered solutions (from pH 7.0 to 4.4), and binding (at pH 7.4) to HA was measured by ELISA. Antibody binding was tested in duplicate and in two independent experiments. Values represented here are mean ± SEM. See also Figure S4.
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8. Figure 6
Fc-FcγR Interactions Are Important for In Vivo Protection by Non-Neutralizing Antibodies
The HAI− Neut− mAbs and one HAI+ Neut+ antibody were cloned in an IgG2a mouse construct with or without the D265A mutation (DiLillo et al., 2014).
(A) Binding of the mouse mAbs to A/Shanghai/1/2013 (H7N9) viruses by ELISA. Values represent mean ± SEM from triplicate wells.
(B) In vitro MN with A/Shanghai/1/2013 (H7N9) viruses. Values represent mean from duplicate wells and shown here is one representative of two independent experiments.
(C) 6- to 8-week-old female BALB/c mice were injected with one dose of each mouse mAb and then infected with a sublethal dose of A/Shanghai/1/2013 (H7N9) virus. Values represent mean ± SEM (n = 4 mice per group). Percent of initial weight are plotted for each mAb: 3 mg/kg for 07-5E01, 4 mg/kg for 41-5D06, 3 mg/kg for 24-4C01, and 4 mg/kg 07-5G01. Mice that received an irrelevant H6-reactive IgG2a mAb were used as a control. The same control group of mice was used for all panels. See also Figures S5 and S6.
Cell Host & Microbe  , DOI: ( /j.chom )
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