Supplemental data for manuscript:
Fc functional antibodies in humans with severe H7N9 and seasonal influenza
Hillary A Vanderven1, Lu Liu2, Fernanda Ana-Sosa-Batiz1, Thi HO Nguyen1, Yanmin Wan2,
Bruce Wines3, P Mark Hogarth3, Danielle Tilmanis4, Arnold Reynaldi5, Matthew S Parsons1,
Aeron C Hurt4, Miles P Davenport5, Tom Kotsimbos6, Allen C Cheng7, Katherine Kedzierska1,
Xiaoyan Zhang2*, Jianqing Xu2* and Stephen J Kent1,8,9*
Contents:
1. Supplementary Table 1…………………………………………………………. page 2
2. Supplementary Figures 1-9 ……………………………………………........pages 3-11
1
Supplementary Table 1 – Infecting virus subtype/strain, HA proteins and influenza viruses studied for H7N9 and seasonal influenza
infected cohorts
Cohort Infecting
virus
subtype
Infecting virus strain HA protein studied
(FcγR dimer ELISA
and NK cell activation
assay)
Influenza virus studied
(HI, FRA and LDH ADCC
assay)
H7N9
(n=18)
H7N9
(n=18)
A/Shanghai/1/2013-like (n=18) A/Shanghai/1/2013 HI: A/Shanghai/4664T/2013
Pseudovirus Neutralisation
assay: non-replicative HIV
expressing H7 and N9
Seasonal
(n=16)
H1N1
(n=4)
A/California/07/2009-like (n=4) A/California/04/2009 HI: A/California/07/2009
FRA: A/California/07/2009
H3N2
(n=8)
A/Newcastle/22/2014-like (n=4) clade 3C.3
A/Switzerland/9715293/2013-like (n=2) clade 3C.3
A/South Australia/91/2014-like (n=1) clade 3C.3
A/New Caledonia/71/2014-like (n=1) clade 3C.2
A/Switzerland/9715293/
2013
HI:
A/Switzerland/9715293/2013
FRA:
A/Switzerland/9715293/2013
LDH ADCC assay: X-31
B (n=4) B/Phuket/3073/2013-like (n=2) Yamagata lineage
B/Brisbane/46/2015-like (n=1) Victoria lineage
unknown (n=1)
B/Phuket/3073/2013 HI: B/Phuket/3073/2013
FRA: B/Phuket/3073/2013
2
Supplementary Figure 1
H7N9 infection Seasonal infection
-0.5
0.0
0.5
1.0
1.5
2.0
rsFcγR
IIIa
dim
er b
indi
ng
(nor
mal
ised
O.D
.)
AH7
R/DH7
Agp140
R/Dgp140
H7 gp140
influenza + healthy controls
******
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
rsFcγR
IIa d
imer
bin
ding
(n
orm
alis
ed O
.D.)
AH7
R/DH7
Agp140
R/Dgp140
H7 gp140
influenza + healthy controls
*****
**
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
rsFcγR
IIIa
dim
er b
indi
ng
(nor
mal
ised
O.D
.)
AHA
R/DHA
D30HA
Agp140
R/Dgp140
D30gp140
influenza +
AHA
R/DHA
D30HA
Agp140
R/Dgp140
D30gp140
influenza -
***ns
***
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
rsFcγR
IIa d
imer
bin
ding
(n
orm
alis
ed O
.D.)
AHA
R/DHA
D30HA
Agp140
R/Dgp140
D30gp140
influenza +
AHA
R/DHA
D30HA
Agp140
R/Dgp140
D30gp140
influenza -
***ns
***
Figure S1. Increased HA-specific FcγR binding Abs after severe influenza infection. Data is
shown as in Figure 1 with the addition of FcγR binding responses to a negative control HIV-1
gp140 protein for all time points. All subjects were HIV negative.
3
Supplementary Figure 2
Lymphocytes69.3
0 50K 100K 150K 200K 250K
FSC-A
0
50K
100K
150K
200K
250K
SS
C-A
Q11.12
Q20.83
Q33.07
Q495.0
0-103
103
104
105
CD107a
0
-103
103
104
105
IFN
γQ10.024
Q26.55E-3
Q30.28
Q499.7
0-103
103
104
105
CD107a
0
-103
103
104
105
IFN
γ
CD3, CD56 subset17.0
0-103
103
104
105
CD3
0
-103
103
104
105
CD
56
Single Cells97.4
0 50K 100K 150K 200K 250K
FSC-A
0
50K
100K
150K
200K
250K
FSC
-H
Healthy Chinese controlH7N9 influenza-infected subject at
hospital release to HA of H7N9
Lymphocytes NK cellsSingle cells
Q10.040
Q20
Q30.34
Q499.6
0-103
103
104
105
CD107a
0
-103
103
104
105
IFN
γ
H7N9 influenza-infected subject at hospital release to gp140 of HIV
Figure S2. Gating strategy for primary NK cell activation assay. Freshly isolated PBMCs were
gated for lymphocytes by size (FSC-A) and granularity (SSC-A) ensuring single cells (FSC-A vs.
FSC-H). CD3-CD56+dim NK cells were selected for analysis using IFNg and/or CD107a
(Q1+Q2+Q3) as activation markers.
4
Supplementary Figure 3
0
2
4
6
8
10
% N
K c
ells
exp
ress
ing
IFNγ
and/
or C
D10
7a
AHA
R/DHA
D30HA
Agp140
R/Dgp140
D30gp140
influenza +
AHA
R/DHA
D30HA
Agp140
R/Dgp140
D30gp140
influenza -
**ns
0
1
2
3
4
5
6
% N
K c
ells
exp
ress
ing
IFNγ
and/
or C
D10
7a
AH7
R/DH7
Agp140
R/Dgp140
H7 gp140
influenza + healthy controls
*****
H7N9 infection Seasonal infection
Figure S3. Increased NK cell activating HA-specific Abs after severe influenza infection. Data
is shown as in Figure 2A and 2B with the addition of Ab-dependent NK cell activation to a
negative control HIV-1 gp140 protein for all time points. All subjects were HIV negative.
5
Supplementary Figure 4
Seasonal infectionH7N9 infection
Survived
Died
Influenza infected
Influenza negative
0
1
2
3
4
5
rsFcγR
IIIa
dim
er b
indi
ng
(nor
mal
ised
O.D
.)
ControlsAdmissionRelease
H7Shanghai
H1Cali /09
H2Japan
H3Perth
H4Ontario
H5Vietnam
0
1
2
3
4
5
rsFcγR
IIIa
dim
er b
indi
ng
(nor
mal
ised
O.D
.)
ControlsAdmissionDeath
H7Shanghai
H1Cali /09
H2Japan
H3Perth
H4Ontario
H5Vietnam
-0.5
0.0
0.5
1.0
1.5
2.0
rsFcγR
IIIa
dim
er b
indi
ng
(nor
mal
ised
O.D
.)
Admission Release/DeathDay 30
H1Cali /09
H2Japan
H3Switz
H4Ontario
H5Vietnam
H3X-31
H7Shang-
hai
-0.5
0.0
0.5
1.0
1.5
2.0
rsFcγR
IIIa
dim
er b
indi
ng
(nor
mal
ised
O.D
.)
Admission Release/DeathDay 30
H1Cali /09
H2Japan
H3Switz
H4Ontario
H5Vietnam
H3X-31
H7Shang-
hai
Figure S4. Breadth of rsFcγRIIIa binding Abs generated during severe influenza infection.
Individual data points are shown for Figure 4A and 4B for each HA tested at the time points
measured.
6
Supplementary Figure 5
-0.5
0.0
0.5
1.0
1.5
2.0
rsFcγR
IIa d
imer
bin
ding
(n
orm
alis
ed O
.D.)
ControlsAdmissionRelease
H7Shanghai
H1Cali /09
H2Japan
H3Perth
H4Ontario
H5Vietnam
-0.5
0.0
0.5
1.0
1.5
2.0
rsFcγR
IIa d
imer
bin
ding
(n
orm
alis
ed O
.D.)
ControlsAdmissionDeath
H7Shanghai
H1Cali /09
H2Japan
H3Perth
H4Ontario
H5Vietnam
-0.5
0.0
0.5
1.0
1.5
2.0
rsFcγR
IIa d
imer
bin
ding
(n
orm
alis
ed O
.D.)
Admission Release/DeathDay 30
H1Cali /09
H2Japan
H3Switz
H4Ontario
H5Vietnam
H3X-31
H7Shang-
hai
-0.5
0.0
0.5
1.0
1.5
2.0
rsFcγR
IIa d
imer
bin
ding
(n
orm
alis
ed O
.D.)
Admission Release/DeathDay 30
H1Cali /09
H2Japan
H3Switz
H4Ontario
H5Vietnam
H3X-31
H7Shang-
hai
Figure S5. Breadth of rsFcγRIIa binding Abs generated during severe influenza infection.
Individual data points are shown for Figure 4C and 4D for each HA tested at the time points
measured.
7
Seasonal infectionH7N9 infection
Survived
Died
Influenza infected
Influenza negative
Supplementary Figure 6
0 20 40 60 800.0
0.5
1.0
1.5
2.0
2.5
Days after disease onset
rsFcγR
IIIa
dim
er b
indi
ng(n
orm
alis
ed O
.D.)
0 10 20 30 40 501
10
100
1000
Days after disease onset
HI t
itre
0 20 40 60 80 1000.0
0.2
0.4
0.6
0.8
1.0
Days after disease onset
rsFcγR
IIa d
imer
bin
ding
(nor
mal
ised
O.D
.)
0 20 40 60 80 1001
10
100
1000
10000
Days after disease onset
Neu
tral
isin
g an
tibod
y tit
re
Predicted
a150a90a118a22a131a33a11a12a79a130a9a20a78a134a49a10a107a73
A
C
B
D
Figure S6. Kinetics of Fc functional and neutralising Ab responses in H7N9 influenza.
Individual subjects and data points are shown for the data that was modelled in Figure 5A 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
Supplementary Figure 7
0 20 40 60 800.0
0.5
1.0
1.5
Days after disease onset
rsFcγR
IIIa
dim
er b
indi
ng(n
orm
alis
ed O
.D.)
0 20 40 60 801
10
100
1000
10000
Days after disease onset
HI t
itre
0 20 40 60 800.0
0.2
0.4
0.6
0.8
1.0
Days after disease onsetrs
FcγR
IIa d
imer
bin
ding
(nor
mal
ised
O.D
.)
0 20 40 60 801
10
100
1000
10000
100000
Days after disease onset
Neu
tral
isin
g an
tibod
y tit
re
AH0001AH0005AH0006AH0009AH0011AH0012AH0013AH0014AH0016AH0019AH0029AH0030AH0033AH0036AH0037AH0040Predicted
A
C
B
D
Figure S7. Kinetics of Fc functional and neutralising Ab 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
Supplementary Figure 8
0.0 0.2 0.4 0.6 0.8 1.0
20
320
1280
5120
80
<10
rsFcγRIIa dimer binding (normalised O.D.)
NA
b tit
re
p= 0.006 r = 0.46
0.0 0.5 1.0 1.5 2.0 2.5
20
320
1280
5120
80
<10
rsFcγRIIIa dimer binding (normalised O.D.)
NA
b tit
re
p= 0.02 r = 0.41
0.0 0.2 0.4 0.6 0.8 1.0
20
320
1280
5120
20480
<10
80
rsFcγRIIIa dimer binding (normalised O.D.)
NA
b tit
re
p< 0.0001 r = 0.69
H7N9 infection Seasonal infection
0.0 0.2 0.4 0.6 0.8 1.0
20
320
1280
5120
20480
<10
80
rsFcγRIIa dimer binding (normalised O.D.)
NA
b tit
re
p< 0.0001 r = 0.71
BA
C D
Figure S8. Correlation between neutralising and Fc functional Ab generation. NAb responses
were plotted against rsFcγRIIIa binding (A, B) and rsFcγRIIa binding (C, D) Abs in severe H7N9
influenza using data from 18 subjects over multiple time points (A, C) and in severe seasonal
influenza using data from 16 subjects over multiple time points (B, D). Viruses and HA proteins
used for each cohort are described in the Methods and Table S1.
10
Supplementary Figure 9
0.0 0.2 0.4 0.6 0.8
20
40
160
320
10
80
<10
rsFcγRIIa dimer binding (normalised O.D.)
HI ti
tre
p= 0.09 r = 0.47
0.0 0.5 1.0 1.5 2.0 2.5
20
40
160
320
10
80
<10
rsFcγRIIIa dimer binding (normalised O.D.)
HI ti
tre
p= 0.40 r = 0.25
0.0 0.2 0.4 0.6 0.8 1.0
20
320
1280
5120
<10
80
rsFcγRIIIa dimer binding (normalised O.D.)
HI ti
tre
p< 0.0001 r = 0.76
Seasonal infection
0.0 0.2 0.4 0.6 0.8 1.0
20
320
1280
5120
<10
80
rsFcγRIIIa dimer binding (normalised O.D.)
HI ti
tre
p< 0.0001 r = 0.76
H7N9 infection BA
C D
Figure S9. Correlation between HI and Fc functional Ab generation. HI Ab responses were
plotted against rsFcγRIIIa binding (A, B) and rsFcγRIIa binding (C, D) Abs in severe H7N9
influenza using data from 18 subjects over multiple time points (A, C) and in severe seasonal
influenza using data from 16 subjects over multiple time points (B, D). Viruses and HA proteins
used for each cohort are described in the Methods and Table S1.
11