Fc functional antibodies in humans with severe H7N9 and seasonal influenza

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Clifton Merritt
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1 Supplemental data for manuscript: Fc functional antibodies in humans with severe N9 and seasonal influenza Hillary Vanderven, Lu Liu 2, Fernanda na-sosa-batiz, Thi HO Nguyen, Yanmin Wan 2, Bruce Wines 3, P Mark Hogarth 3, Danielle Tilmanis 4, rnold Reynaldi 5, Matthew S Parsons, eron C Hurt 4, Miles P Davenport 5, Tom Kotsimbos 6, llen C Cheng 7, Katherine Kedzierska, Xiaoyan Zhang 2*, Jianqing Xu 2* and Stephen J Kent,8,9* Contents:. Supplementary Table. page 2 2. Supplementary Figures -9...pages 3-

2 Supplementary Table Infecting virus subtype/strain, H proteins and influenza viruses studied for N9 and seasonal influenza infected cohorts Cohort N9 (n=8) Seasonal (n=6) Infecting virus subtype N9 (n=8) HN (n=4) N2 (n=8) B (n=4) Infecting virus strain H protein studied (FcγR dimer ELIS and NK cell activation assay) Influenza virus studied (HI, FR and LDH DCC assay) ///23-like (n=8) ///23 HI: //4664T/23 Pseudovirus Neutralisation assay: non-replicative HIV expressing and N9 /California/7/29-like (n=4) /California/4/29 HI: /California/7/29 FR: /California/7/29 /Newcastle/22/24-like (n=4) clade 3C.3 /Switzerland/975293/23-like (n=2) clade 3C.3 /South ustralia/9/24-like (n=) clade 3C.3 /New Caledonia/7/24-like (n=) clade 3C.2 B/Phuket/373/23-like (n=2) Yamagata lineage B/Brisbane/46/25-like (n=) Victoria lineage unknown (n=) /Switzerland/975293/ 23 B/Phuket/373/23 HI: /Switzerland/975293/23 FR: /Switzerland/975293/23 LDH DCC assay: X-3 HI: B/Phuket/373/23 FR: B/Phuket/373/23 2

3 Supplementary Figure ns - gp4 gp4 healthy controls gp4 -.2 H H D3 D3 H gp4 gp4 gp4 H H D3 D3 H gp4 gp4 gp4 influenza ** ** ns -.2 gp4 gp4 gp4 healthy controls -.2 H H D3 D3 H gp4 gp4 gp4 H H D3 D3 H gp4 gp4 gp4 influenza - Figure S. Increased H-specific FcγR binding bs after severe influenza infection. Data is shown as in Figure with the addition of FcγR binding responses to a negative control HIV- gp4 protein for all time points. ll subjects were HIV negative. 3

Supplementary Figure 2 Lymphocytes Single cells NK cells 25K 2K 25K 2K 5 4 CD3, CD56 subset 7. SSC- 5K K FSC-H 5K K Single Cells 97.4 CD56 3 5K Lymphocytes 69.

to H of N9 5 Q 24 Q2 6.55E-3 5 Q 4 Q2 5 Q.2 Q2.83 4 4 4 IFNγ 3 IFNγ 3 IFNγ 3-3 Q4 99.7 Q3.28-3 Q4 99.6 Q3.34-3 Q4 95. Q3 3.7-3 3 4 5 CD7a - 3 3 4 5 CD7a - 3 3 4 5 CD7a Figure S2.

4 Supplementary Figure 2 Lymphocytes Single cells NK cells 25K 2K 25K 2K 5 4 CD3, CD56 subset 7. SSC- 5K K FSC-H 5K K Single Cells 97.4 CD56 3 5K Lymphocytes K K 5K 2K 25K FSC- 5K 5K K 5K 2K 25K FSC CD3 Healthy Chinese control N9 influenza-infected subject at hospital release to gp4 of HIV N9 influenza-infected subject at hospital release to H of N9 5 Q 24 Q2 6.55E-3 5 Q 4 Q2 5 Q.2 Q IFNγ 3 IFNγ 3 IFNγ 3-3 Q Q Q Q Q4 95. Q CD7a CD7a CD7a Figure S2. Gating strategy for primary NK cell activation assay. Freshly isolated PBMCs were gated for lymphocytes by size (FSC-) and granularity (SSC-) ensuring single cells (FSC- vs. FSC-H). CD3-CD56+dim NK cells were selected for analysis using IFNg and/or CD7a (Q+Q2+Q3) as activation markers. 4

5 Supplementary Figure 3 % NK cells expressing IFNγ and/or CD7a gp4 ** gp4 healthy controls gp4 % NK cells expressing IFNγ and/or CD7a H H ns ** D3 D3 H gp4 gp4 gp4 H H D3 D3 H gp4 gp4 gp4 influenza - Figure S3. Increased NK cell activating H-specific bs after severe influenza infection. Data is shown as in Figure 2 and 2B with the addition of b-dependent NK cell activation to a negative control HIV- gp4 protein for all time points. ll subjects were HIV negative. 5

6 Supplementary Figure 4 Survived Influenza infected Controls dmission Release.5. dmission Release/ Day 3 H Cali /9 Perth H4 Ontario H5 Vietnam - H Cali /9 Switz X-3 H4 H5 Ontario Vietnam Died Influenza negative H Cali /9 Perth H4 Ontario Controls dmission H5 Vietnam.5. - H Cali /9 Switz X-3 H4 H5 Ontario Vietnam dmission Release/ Day 3 Figure S4. Breadth of rsfcγriiia binding bs generated during severe influenza infection. Individual data points are shown for Figure 4 and 4B for each H tested at the time points measured. 6

7 Supplementary Figure 5 Survived Influenza infected.5. Controls dmission Release.5. dmission Release/ Day 3 - H Cali /9 Perth H4 Ontario H5 Vietnam - H Cali /9 Switz X-3 H4 H5 Ontario Vietnam Died Influenza negative.5. Controls dmission.5. dmission Release/ Day 3 - H Cali /9 Perth H4 Ontario H5 Vietnam - H Cali /9 Switz X-3 H4 H5 Ontario Vietnam Figure S5. Breadth of rsfcγriia binding bs generated during severe influenza infection. Individual data points are shown for Figure 4C and 4D for each H tested at the time points measured. 7

8 Supplementary Figure 6 B C D Neutralising antibody titre a5 a9 a8 a22 a3 a33 a a2 a79 a3 a9 a2 a78 a34 a49 a a7 a73 Predicted Figure S6. Kinetics of Fc functional and neutralising b responses in N9 influenza. Individual subjects and data points are shown for the data that was modelled in Figure 5 in the main text. Solid black line represents the population level model prediction, and dashed line indicates 95%CI for the time to peak. 8

9 Supplementary Figure 7 B C Neutralising antibody titre D H H5 H6 H9 H H4 H6 H H4 Predicted Figure S7. Kinetics of Fc functional and neutralising b responses in severe seasonal influenza. Individual subjects and data points are shown for the data that was modelled in Figure 5B in the main text. Solid black line represents the population level model prediction, and dashed line indicates 95%CI for the time to peak. 9

10 Supplementary Figure 8 B Nb titre Nb titre p< r =.69 2 < p= 2 r =.4 2 < C D Nb titre Nb titre p< r =.7 2 < p= 6 r =.46 2 < Figure S8. Correlation between neutralising and Fc functional b generation. Nb responses were plotted against rsfcγriiia binding (, B) and rsfcγriia binding (C, D) bs in severe N9 influenza using data from 8 subjects over multiple time points (, C) and in severe seasonal influenza using data from 6 subjects over multiple time points (B, D). Viruses and H proteins used for each cohort are described in the Methods and Table S.

11 Supplementary Figure 9 B p< r =.76 < p=.4 r =.25 2 < C D p< r =.76 < p= 9 r =.47 2 < Figure S9. Correlation between HI and Fc functional b generation. HI b responses were plotted against rsfcγriiia binding (, B) and rsfcγriia binding (C, D) bs in severe N9 influenza using data from 8 subjects over multiple time points (, C) and in severe seasonal influenza using data from 6 subjects over multiple time points (B, D). Viruses and H proteins used for each cohort are described in the Methods and Table S.

Fc functional antibodies in humans with severe H7N9 and seasonal influenza

Fc functional antibodies in humans with severe H7N9 and seasonal influenza Hillary A. Vanderven, 1 Lu Liu, 2 Fernanda Ana-Sosa-Batiz, 1 Thi H.O. Nguyen, 1 Yanmin Wan, 2 Bruce Wines, 3 P. Mark Hogarth,