adjuvant dependent innate and adaptive immune signatures ...vaccari et al 1 supplemental online...

Vaccarietal

1

Supplemental Online Material for

Adjuvant dependent innate and adaptive immune signatures of risk of SIVmac251 acquisition

Monica Vaccari, Shari N. Gordon, Slim Fourati, Luca Schifanella, Namal P.M. Liyanage, Mark Cameron, Brandon F. Keele, Xiaoying Shen, Georgia D. Tomaras, Erik Billings, Mangala Rao, Amy W. Chung, Karen G. Dowell, Chris Bailey-Kellogg, Eric P. Brown, Margaret E. Ackerman, Diego A. Vargas-Inchaustegui, Stephen Whitney, Melvin N. Doster, Nicolo Binello, Poonam Pegu, David C. Montefiori, Kathryn Foulds, David S. Quinn, Mitzi Donaldson, Frank Liang, Karin Loré, Mario Roederer, Richard A. Koup, Adrian McDermott, Zhong-Min Ma, Christopher J. Miller, Tran B. Phan, Donald N. Forthal, Matthew Blackburn, Francesca Caccuri, Massimiliano Bissa, Guido Ferrari, Vaniambadi Kalyanaraman, Maria G. Ferrari, DeVon Thompson, Marjorie Robert-Guroff, Silvia Ratto-Kim, Jerome H. Kim, Nelson L. Michael, Sanjay Phogat, Susan W. Barnett, Jim Tartaglia, David Venzon, Donald M. Stablein, Galit Alter, Rafick-Pierre Sekaly & Genoveffa Franchini.

*to whom correspondence should be addressed. E-mail: [email protected]

Nature Medicine: doi:10.1038/nm.4105

Vaccarietal

2

Supplementary Figures

Supplementary Figure 1: Vaccines efficacy and activation markers in blood and in rectal mucosa. (a) Twenty-three historical (dotted line) and 24 concurrent controls (solid line; 6 naïve, 6 alum-adjuvant and 12 MF59-adjuvant) showed no differences in the acquisition rate of infections. (b) Vaccine efficacy was maintained in the vaccinated alum group (n = 27) when compared with the alum only controls (n = 6). ). Of note, the alum 6 concurrent controls acquired SIV significantly faster than the concurrent naïve animals. However the historical 11 alum-only controls didn’t significantly differ from naïve animals, casting doubts on the effect of alum. The small number of animals (6) precludes any conclusion on the effect of the alum alone. (c) Acquisition rate in the in the 27 MF59-vaccinated animals compared to the 12 controls immunized with MF59 alone shows no protection. (d) Vaccine efficacy was not affected by gender in the alum or (e) in the MF59 group and (f) remained significant when all 54 vaccinees were compared to all 47 controls (P = 0.022). Log rank test was used in all the distribution comparisons. Markers of activation were measured in blood and mucosa to assess the role of adjuvant-driven inflammation in vaccine efficacy. (g) Both vaccine regimens elicited equivalent frequency of CCR5+ Ki67+ CD4+ T cells (live CD3+ CD4+ cells, n = 8) in blood collected at one week from boost with gp120 with alum or MF59 (wk13) (h) and their frequency did not correlate with SIVmac251 acquisition. (i) There was no difference in the frequency of CCR5+ CD8– CD3+ cells in the rectal mucosa (n = 6). (j) Correlation between the absolute number of CD4+ Ki67+/mm2 and the time of SIVmac251 acquisition in the rectal mucosa of vaccinated animals in both groups, at week 25, 3 weeks before challenge exposure (alum n = 21; MF59 n = 21 animals). Correlations between (k) the frequency of α4β7+ Ki67+ CD4+ T cells in blood at week 13 (n = 12) and the number of challenges in the alum-vaccinated group, and (l) between

0 1 2 3 4 5 6 7 8 9 10 110

102030405060708090

100

Number of IR challenges

Perc

ent u

ninf

ecte

d

Concurrent Controls (n=24)Historical Controls (n=23)

0.7619P =

0 1 2 3 4 5 6 7 8 9 10 110

102030405060708090

100


Perc

ent u

ninf

ecte

d

0.0061P =

Concurrent Alum CTRL(n=6)ALVAC-SIV/gp120 Alum (n=27)

0 1 2 3 4 5 6 7 8 9 10 110

102030405060708090

100


Perc

ent u

ninf

ecte

d

0.4675P =

ALVAC-SIV/gp120 MF59 (n=27)Concurrent MF59 CTRL(n=12)

0 1 2 3 4 5 6 7 8 9 10 110

102030405060708090

100


Per

cent

uni

nfec

ted

0.5685P =

Male (n=15)Female (n=12)

0 1 2 3 4 5 6 7 8 9 10 110

102030405060708090

100


Per

cent

uni

nfec

ted

0.4579P =

Male (n=13)Female (n=14)

0 1 2 3 4 5 6 7 8 9 10 110

102030405060708090

100


Perc

ent u

ninf

ecte

d

ALL VACC (n=54)ALL controls (n=47)

0.0220P =

0.0

0.2

0.4

0.6

0.8

1.0

% o

f KI6

7+ CC

R5+ C

D4+ T

cel

ls

in b

lood

at w

eek1

3

gp120 MF59ALVAC-SIV

gp120 AlumALVAC-SIV

0

1

2

3

4

% o

f CC

R5+ C

D8– C

D3+ c

ells

in

muc

osa

at w

eek

13

gp120 MF59ALVAC-SIV

gp120 AlumALVAC-SIV

0 1 2 3 4 5 6 7 8 9 10 110.0

0.2

0.4

0.6

0.8

1.0


% o

f KI6

7+ CC

R5+ C

D4+ T

cel

ls

in b

lood

at w

eek

13

ALVAC-SIV/gp120 AlumALVAC-SIV/gp120 MF59

0 1 2 3 4 5 6 7 8 9 10 110.0

0.5

1.0

1.5

2.0

2.5


Num

ber o

f Ki6

7+ CD

4+ cel

ls/m

m2

in re

ctal

muc

osa

at w

eek

25

ALVAC-SIV/gp120 AlumALVAC-SIV/gp120 MF59

a b c d

e f g h

i j k l m

0 1 2 3 4 5 6 7 8 9 10 110

2

4

6

Number of IR Challenges

% o

f α4β

7+ Ki6

7+

CD

4+ T c

ells

R = –0.62P = 0.02

0 1 2 3 4 50

2

4

6

8

10

Number of transmitted founder viruses%

of C

D38

+ Ki6

7+

CD

4+ T c

ells

R = 0.56P = 0.01

0 1 2 3 4 50

1

2

3

4

Number of transmitted founder viruses

% o

f α4β

7+ Ki6

7+

CD

4+ T c

ells

R = 0.46P = 0.05


Vaccarietal

3

CD38+ Ki67+ CD4+ T cells or (m) α4β7+ Ki67+ CD4+ T cells (week 13, n = 18) and the number of transmitted variant in the MF59 group. Spearman's rank correlation coefficients are shown.


Vaccarietal

4

Supplementary Figure 2: Envelope-specific IgG and IgA in serum and T cell responses in blood. All vaccinated animals in the alum group are in red (n = 27) and those from the MF59 (n = 27) and control group (n = 8) are in blue and black respectively. (a) IFN-γ or (b) IL-2 producing CD4+ T cells in blood at one week after the last immunization. (c) IgG titers in serum against the gp130 of SIVmac251 and (d) the gp140 SIVsmE660. (e) Serum IgG titers to the gp70-V1/V2 scaffold of SIVmac251. (g) Serum IgA titers to the SIVmac251 gp130 (f) or SIVsmE660 gp140 The Mann-Whitney-Wilcoxon test was used to compare continuous factors between two groups.

101

102

103

104

105

gp120 MF59ALVAC-SIV

Controlsgp120 AlumALVAC-SIV

P = 0.0039

Ser

um b

indi

ng Ig

G E

C50

gp13

0 S

IVm

ac25

1

101

102

103

104

gp120 MF59ALVAC-SIV


P = 0.037

Ser

um b

indi

ng Ig

G E

C50

gp70

-V1/

V2

SIV

mac

251

100

101

102

103

gp120 MF59ALVAC-SIV


P = 0.021

Ser

um Ig

Agp

130

SIV

mac

251

b

e f g

101

102

103

104

105

gp120 MF59ALVAC-SIV


P = 0.0013

Ser

um b

indi

ng Ig

G E

C50

gp14

0 S

IVsm

E66

0

100

101

102

103

104

gp120 MF59ALVAC-SIV


P = 0.0012

Ser

um Ig

Agp

140

SIV

smE

660

ALVAC/gp120/MF59 ALVAC/gp120/Alum controls

0.0

0.5

1.0

1.5

gp120 MF59ALVAC-SIV


P = 0.001

% C

D4+

T ce

lls p

rodu

cing

IFNγ

SIV

mac

251

Env

0.0

0.5

1.0

1.5

gp120 MF59ALVAC-SIV


P = 0.0045

% C

D4+

T ce

lls p

rodu

cing

IL-2

SIV

mac

251

Env

a c d


Vaccarietal

5

Supplementary Figure 3: Adjuvant associated differences in neutrophils recruitment in lymph nodes and plasmasmablast homing markers in blood. (a) Correlation in mucosal responses to cyclic V2 obtained in two independent laboratories using the same peptides in the alum group (26 animals, Spearman correlation). (b–e) Serum IgA titers and mucosal IgA response unit to the cV2 of SIVmac251 or SIVsmE660. (f) Percentage of vaccine-induced α4β7+ and (g) CXCR3+ plasmablasts in macaques’ blood (MF59 n = 23; alum n = 22 animals) before and one week after the last immunization. In mice, MF59 induces local inflammation and up-regulates IFN-stimulated genes, resulting in the recruitment of neutrophils1. (h) Neutrophil score in four animals vaccinated with MF59 (blue) and five animals vaccinated with the alum-regimen (red). Each dot represents the average of 5 counts- picture-animal and the lines represent the median value. The p-value was calculated using the repeated measures analysis of variance. (i) Immunohistochemistry on inguinal lymph nodes on one animal vaccinated with the MF59 (lower panel) or alum (upper panel) regimen, 2 weeks after the last immunization (week 26), shows persistence of neutrophils in the MF59, but not in the alum. Pictures are at 40x resolution and Elastase+ cells (neutrophils) are in brown.

pre-vax post-vax pre-vax post-vax0

20

40

60

80

100

%α

4β7+

plas

mab

last

s in

mac

aque

blo

od

P = 0.0021

gp120 MF59ALVAC-SIV

gp120 AlumALVAC-SIV

7 days7 dayspre-vax post-vax pre-vax post-vax

0

20

40

60

80

100

% C

XC

R3+

plas

mab

last

s in

mac

aque

blo

od

gp120 MF59ALVAC-SIV

gp120 AlumALVAC-SIV

P < 0.0001

7 days7 days

a b c

0.0

0.5

1.0

1.5

Rec

tal I

gA c

V2

SIV

E54

3-3

resp

onse

uni

ts

gp120 MF59ALVAC-SIV

gp120 AlumALVAC-SIV

0.0107P =

0.0

0.5

1.0

1.5

Rec

tal I

gA c

V2

SIV

mac

251

resp

onse

uni

ts

gp120 MF59ALVAC-SIV

gp120 AlumALVAC-SIV

0.0117P =

102

103

104

Ser

um Ig

A ti

ter

cV

2 S

IVm

ac25

1gp120 MF59ALVAC-SIV


102

103

104

Ser

um Ig

A ti

ter

cV

2 S

IVsm

E54

3

gp120 MF59ALVAC-SIV


0 1 2 3 4 5 6 7 8 9 10 110

100

200

300

400

500

Mucosa IgG cV2a SIVsmE660 post-vax (RU)

Muc

osa

aRhI

gG c

V2c

S

IVsm

543.

3

0.60700.0010

R =P =

d e

f g h

MF59(2weekspost-vax)

Alum(2weekspost-vax)

i

0

100

200

300

400

500

gp120 MF59ALVAC-SIV

gp120 AlumALVAC-SIV

P = 0.023

Neu

troph

ils s

core

in ly

mph

nod

es


Vaccarietal

6

Supplementary Figure 4: Functional differences in serum gp120 specific antibodies elicited by the two adjuvants. Bulk and anti-gp120 antibody glycoforms in vaccinated animals (alum n = 27; MF59 n = 27). The variable addition of 4 sugars generate up to 36 distinct glycan profiles that can be added to the Fc-domain of antibodies. These sugars include the addition/removal of: 1 fucose (F) – when absent induces higher levels of ADCC, 2) 1 bisecting-n-acetyl glucosamine (GlcNAc – or B) - when present induces higher ADCC, 3) up to 2 galactoses (G) which can be added to either arm of the glycan (G0= no galactose, G1= 1 galactose, G2= 2 galactoses) - when absent induces elevated complement activation, 4) up to 2 sialic acids which can be added to either arm at the end of galactose (S1= 1 sialic acid, S2= 2 sialic acids) - when present are associated with anti-inflammatory activity. G0 glycans are thought to have enhanced antibody functionality, G1 are less functional and more abundant in healthy subjects, and G2 are thought to have anti-inflammatory activity as they are likely to be sialated. The removal of fucose or the addition of a b-GlcNAc are associated with enhanced ADCC. The relative amount of gp120 specific IgG Fc glycoforms were determined for all immunized animals following the last immunization. Nearly all the glycoforms abundance levels differed significantly between the Alum and MF59 groups. MF59 drives enhanced mono- and di-galactosylated glycan structures on bulk circulating antibodies. Conversely, Alum vaccinated animals generated lower levels of anti-inflammatory di-galactosylated (G2) structures on gp120-specific antibodies, but higher levels of G1F structures (particularly G1_F_1 previously linked to higher ADCC) and strikingly higher levels of G0 glycosylated antibodies (G0F) previously associated with higher ADCVI activity.

0

1

2

3

G2FB

Fra

ctio

n o

f g

p12

0 sp

ecifi

c g

lyca

ns p< 000.1

MF59 Alum0

5

10

15

20

25

G2F

Fra

ctio

n o

f g

p12

0 sp

ecifi

c g

lyca

ns p< 000.1

MF59 Alum0

20

40

60

G2S1F

Fra

ctio

n o

f g

p12

0 sp

ecifi

c g

lyca

ns p< 000.1

MF59 Alum

0

5

10

15

G1F_1

Fra

ctio

n o

f g

p12

0 sp

ecifi

c g

lyca

ns p= 000.1

MF59 Alum0

5

10

15

20

G1F_2

Fra

ctio

n o

f g

p12

0 sp

ecifi

c g

lyca

ns p=0.028

MF59 Alum0

10

20

30

40

G0F

Fra

ctio

n o

f g

p12

0 sp

ecifi

c g

lyca

ns

p< 000.1

MF59 Alum

Gp120specificIgG

TotalIgG

0.0

0.5

1.0

1.52.0

2.5

Frac

tion

of

bulk

ant

ibod

y gl

ycan

s

G1Fp = 0.0022

MF59 Alum0

2

4

6

8

Fra

ctio

n of

bul

k an

tibod

y gl

ycan

s

G2S1F

p = 0.0066

MF59 Alum


Vaccarietal

7

Supplementary Figure 5: Glycan modifications, cells and mucosal humoral markers are associated with protection from SIV acquisition. The ability of individual sets of aggregate data to predict protection in MF59 (blue) and alum (red) was compared among animals that were infected in 4 or fewer challenges (≤ 4), or after 5 or more challenges (≥ 5), using logistic regression and leave-one-out cross-validation. A full list of immune assessments included in each set of aggregate data is described in Supplementary Table 1. A jitter plot presenting the predicted odd of being protected by the vaccine (odd-ratio, y-axis) of each individual macaque is presented as function of the observed number of SIVmac251 challenge to infected the macaque (x-axis). The accuracy of each logistic regression model is given above each jitter plot.

Number of challenges


Vaccarietal

8

Supplementary Figure 6: Different pathways are modulated by MF59 and alum 24 hours after immunization. Enrichment maps showing the different pathways enriched between immunization by the MF59 vaccine or the alum vaccine. Gene Set Enrichment analysis (GSEA) was used to annotate genes and rank canonical pathways2. In brief, genes were ranked by the absolute differential expression between pre-vax and post-3rd (i.e. absolute moderated t-statistic), separately for the MF59 and alum group. Given a defined set of genes (here Ingenuity canonical pathways), the goal was to determine whether the members of that GeneSet (GS) are found at the top of the list, implying they are not randomly distributed across the ranked list. An Enrichment Score was calculated to quantify the degree to which the GS is over-represented at the top of the entire ranked list. A gene-based permutation test procedure was used to estimate a false discovery rate for a given enrichment score (-log10_pval). Significantly enriched pathways (GSEA: FDR q-value ≤ 0.05) were organized in networks using the enrichment maps strategy3. This was accomplished by linking GS by the number of genes differently expressed overlapping between GS. Overlap between significant GS is computed according to the Jaccard index. A Jaccard index of 0.5 was used to generate the enrichment maps. This technique allows the identification of the major enriched functional themes and interprets the enrichment results of GSEA. The functional themes are given different colors.


Vaccarietal

9

Supplementary Figure 7: Pre-vaccination markers of alum-conferred protection from SIV acquisition are significantly association to protection post-vaccination. The heatmap presenting the evaluation of predictors of protection on each combination vaccine × immunization step. The Pearson correlation between the predicted protection (see material and method section microarray analysis) and the observed protection is proportional to the size of the dots. The p-values of a Pearson t-test are presented using a blue-white-red color scale.


Vaccarietal

10

Supplementary Table 1: Full list of evaluated immune assessments.

Note: DN (double negative for NKG2a and NKp44).


Vaccarietal

11

Supplementary Table 2: List of individual vaccine-induced immune assessments differently expressed between MF59 and alum.



Vaccarietal

12

Supplementary Table 3: List of individual vaccine-induced immunological assessments associated with risk of SIV acquisition in the MF59 and alum group.



Vaccarietal

13

Supplementary Table 4: Contributions of individual vaccine-induced immunological assessments to logistic regression models trained to classify animals according to the number of challenges required to achieve infection within alum or MF59 study arms.


Vaccarietal

14

Supplementary Table 5: Genes significantly up-regulated in vaccine-specific CD4+ T cellsassessment to logistic regression models trained to classify animals according to the number of challenges required to achieve infection within alum or MF59 study arms.Gene Type Elevated in p valueGABPA Transcription factor MF59 0.03IL-2 Cytokine MF59 0.03NFATc1 Transcription factor MF59 0.02RORA Orphan receptor MF59 0.04CD154+ (responding) CD4+ TEM were sorted from NHP PBMC stimulated with matched Env peptides. Gene expression was quantified by Fluidigm “nano-array”. Animals analyzed =10 per group. The four (of 96) genes differentially expressed with a p-value ≤ 0.05.


Vaccarietal

15

Supplementary Table 6: Number of transcripts differentially expressed between immunization steps and vaccines.

A. Immunization effect

Number of transcripts diff. expressed p-value < 0.05

Number of transcripts diff. expressed FDR < 0.05

MF59 post-1st v pre-vaxpost-3rd v pre-vaxpost-3rd v post-1st

574187555273

10314481757

alum post-1st v pre-vaxpost-3rd v pre-vaxpost-3rd v post-1st

539868523516

2488100

B. Vaccine effect Number of transcripts diff.

expressed p-value < 0.05Number of transcripts diff. expressed FDR < 0.05

pre-vaxMF59 v alum

2380

0

post-1st MF59 v alum

1920

1

post-3rd MF59 v alum

2516

0


Vaccarietal

16

Supplementary Table 7: 37 genes are associated to protection with alum and SIV-gp96 + 120 vaccines. Comparison Geneset NES FDR q-val LEADING_EDGE Selinger_(post33w5d-pre)

alum_post3rd 1.49 0.00382 CTF1,LOC100429709,LOC100430236,EEF1A2,NOS2,SLC18A3,UBE2C,LOC707320,TM6SF2,BAMBI,CDC27,KIR3DL12,LOC720327,FXYD2,ADAMTS7,LOC697051,NKG7,TBC1D25,CCNE1,CPA5,CLYBL,PTGES2,LOC700041,CCDC80,NONO,LOC697263,ZNF208,LOC711788,LOC699203,TMEM35,LOC694489

Selinger_(post26w-pre)

alum_post3rd 1.44 0.00983 TBC1D25,CAD,PTGES2,CCNE1,KIR3DL17,LOC720327,UBN1,EEF1A2,PLK1S1,ZNF219,UBE2C,LOC707320,LOC711788,LOC716166,HUS1,ZNF208,MBD5,LOC707953,CDC27,LOC714549,PHACTR1,CCDC80,LOC694489,BAMBI,CTF1,BCLAF1,RHOC,PTGR2,LOC719392,LOC100424807,LOC100425576,ARHGDIA,LOC699203,LOC721106,LOC697051,CCNH,LOC100430236,LOC722126,KIR3DL12,ABCA9,CLYBL,LIN28B,DFNB31,TMEM35,NONO,LOC697263,LOC100429709,LOC708613

Selinger_post26w

alum_post3rd 1.34 0.0435 KIR3DL17,LOC721106,MBD5,PHACTR1,PTGES2,CABYR,LOC716166,LOC713561,CCNE1,SNTG2,GPR61,LOC707953,CAD,LIN28B,LOC714549,RHOC,DNCI1,POLD1,ABCA9,UBN1,UBE2C,LOC707320,TMEM160,LOC704106,LOC100430236,LOC720327,DFNB31,CCNH,ARHGDIA,RPS11,PIAS2,UBA1,LOC710713,KIR3DL12,LOC707105,TMEM147,RELN

GSEA assessment of the top 200 genes positively and negatively correlated to number of SIV challenges (ordered by LIMMA t statistic) separately for pre-vaccination, 24 hour post-1st immunization and 24 hour post-3rd immunization after alum. To evaluate the statistical significance of the enrichment, a gene-wise permutation was performed and the calculated false-discovery rate (FDR q-value) is reported. Only significant enrichment corresponding to FDR q-value below 5 % are presented in the table.


Vaccarietal

17

Reference List: 1. Nakajima, C., et al. Induction of the chemokine receptor CXCR3 on TCR-stimulated T cells: dependence on the

release from persistent TCR-triggering and requirement for IFN-gamma stimulation. Eur J Immunol 32, 1792-1801 (2002).

2. A. Subramanian et al., Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proceedings of the National Academy of Sciences of the United States of America 102, 15545 (Oct 25, 2005).

3. D. Merico, R. Isserlin, O. Stueker, A. Emili, G. D. Bader, Enrichment map: a network-based method for gene-set enrichment visualization and interpretation. PloS one 5, e13984 (2010).


adjuvant dependent innate and adaptive immune signatures ...vaccari et al 1 supplemental online...

Documents