New Supplemental data for manuscript: Fc functional antibodies in … · 2017. 6. 22. · Supplemental data for manuscript: Fc functional antibodies in humans with severe H7N9 and

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


*****

**

-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


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


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


*****


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


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

.)


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


-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.)


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.)


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


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


HI t

itre

0 20 40 60 80 1000.0

0.2

0.4

0.6

0.8

1.0


rsFcγR

IIa d

imer

bin

ding

(nor

mal

ised

O.D

.)

0 20 40 60 80 1001

10

100

1000

10000


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


0 20 40 60 800.0

0.5

1.0

1.5


rsFcγR

IIIa

dim

er b

indi

ng(n

orm

alis

ed O

.D.)

0 20 40 60 801

10

100

1000

10000


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


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


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


NA

b tit

re

p< 0.0001 r = 0.69


0.0 0.2 0.4 0.6 0.8 1.0

20

320

1280

5120

20480

<10

80


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


0.0 0.2 0.4 0.6 0.8

20

40

160

320

10

80

<10


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


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


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


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

Documents

New Supplemental data for manuscript: Fc functional antibodies in … · 2017. 6. 22. · Supplemental data for manuscript: Fc functional antibodies in humans with severe H7N9 and