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Supplemental Figures TLR7/8 Adjuvant Overcomes Newborn Hyporesponsiveness to Pneumococcal Conjugate Vaccine at Birth Dowling et al., JCI Insight, 2016
Dowling et al.
Dowling et al. Supplemental Table 1
Supplemental Table 1. 3M-052 oil-in-water emulsion (O/W) size details. Name TLR Ave. Diameter
(nm) PdI Index Endotoxin-LAL
Assay (EU/ml)
O/W (vehicle)
N/A 129 0.23 < 1
3M-052 (0.01 mg/kg) O/W 7/8 134 0.18 < 1
3M-052 (0.1 mg/kg) O/W 7/8 141 0.19 < 1
PdI: Polydispersity; TLR: Toll-like receptor; LAL: Limulus amebocyte lysate; EU: Endotoxin Units; O/W: Oil-in-Water.
0 1 3 6 180
5
10
15
20
NaiveVehicle
3M-052R848TNF
Time (hrs)
0 1 3 6 180
10
20
30
40
50MX1
Time (hrs)0 1 3 6 18
0
10
20
30
40
50MX2
Time (hrs)0 1 3 6 18
0
5
10
15OAS
Time (hrs)0 1 3 6 18
0
5
10
15Tnfs10
Time (hrs)0 1 3 6 18
0
2
4
6
8TLR7
Time (hrs)
0 1 3 6 180
200
400
600IL-6
Time (hrs)0 1 3 6 18
0
5
10
15
20
25IL-12p40
Time (hrs)0 1 3 6 18
0
50
100
150IFNγ
Time (hrs)0 1 3 6 18
0
50
100
150
200CCL4
Time (hrs)
A
0 1 3 6 180
10
20
30
40
NaiveVehicle
3M-052R848TNF
Time (hrs)0 1 3 6 18
0
500
1000
1500
2000IL-6
Time (hrs)0 1 3 6 18
0
50
100
150
200IL-12p40
Time (hrs)0 1 3 6 18
0
10
20
30
40
50IFNγ
Time (hrs)0 1 3 6 18
0
50
100
150
200CCL4
Time (hrs)
B
Lym
ph N
ode
(Fol
d C
hang
e)Sp
leen
(Fol
d C
hang
e)
0 1 3 6 180
50
100
150MX1
Time (hrs)0 1 3 6 18
0
20
40
60
80
100MX2
Time (hrs)0 1 3 6 18
0
20
40
60OAS
Time (hrs)0 1 3 6 18
0
5
10
15
20
25Tnfs10
Time (hrs)0 1 3 6 18
0
2
4
6
8TLR7
Time (hrs)
Dowling et al. Supplemental Figure 1
Supplemental Figure 1. Cytokine and IFN-inducible gene expression following free or lipidated TLR7/8 imidazoquinoline subcutaneous injection. Mouse mRNA expression is depicted in (A) draining lymph nodes (brachial and axillary), and (B) spleen post a single subcutaneous injection of 3M-052 or R848 formulated (both 1 mg/kg, (20 µg/mouse)) in oil-in-water emulsion (O/W) (vehicle) to the scruff of the neck. Data represents relative fold-change gene expression (i.e., treatment relative expression / untreated relative expression) (n = 3).
Supplemental Figure 2
570
100
101
102
103
104 Adult Newborn
N = 10
PCV (1:)
3M-052 (µM)
R848 (µM)
- 0.01 0.1 1 10 100- -- --- -- -
-- -- -
-- -- - -- -- -
-- -- --- -- -
NS
NS
NS
NS NS
NS
NS ***
NSNS
**
NS
NS NS
NS
NS *
NS
NS
NS ++
5.7575,70057,000 5.757057,000 575,700
0.01 0.1 1 10 100
0.01 0.1 1 10 100
IFNγ
(pg
/ml)
+++
101
102
103
104
105Adult Newborn
N = 12
NSNS
NS
***
NS
NSNS
NSNS
NS
NS
NS
NS *
NS
NS
NS
NS*
++ NS
TN
F (p
g/m
l)
A
B
Supplemental Figure 2. Addition of 3M-052 to PCV13 enhances TNF and IFNγ responses in newborn cord blood. Human neonatal and adult blood was cultured for 6 hours with sterile buffer control (RPMI, not shown), PCV13 (1:5.7 – 57,000 v/v), 3M-052 or R848 (both 0.01, 0.1, 1, 10, 100 µM), or (PCV13+3M-052). Supernatants were collected for ELISA and multiplex assay. Mean ± SEM of agonist-induced cytokine production are shown for (A) TNF (n = 12) and (B) IFNγ (n = 10). For comparisons between overall groups (e.g., newborn vs. adult), two-way repeated measures ANOVA for non-parametric sample populations were applied and statistical significance denoted as +p < 0.05, ++p < 0.01. For comparison at individual concentrations, the unpaired Mann-Whitney test was applied and statistical significance denoted as *p < 0.05, **p < 0.01, or NS (not significant). Results represent means ± SEM.
Dowling et al.
Supplemental Figure 3 Dowling et al.
Supplemental Figure 3. Human newborn whole blood cytokine responses to 3M-052, PCV13, and (PCV13 + 3M-052). Human cord blood cultured for 6 hours with O/W vehicle, PCV13 alone (1:5.7 – 57,000 v/v), 3M-052 alone (0.01, 0.1, 1, 10, 100 µM) and concentration dependent combinations of each. Supernatants were collected for ELISA and multiplex assay. Mean ± SEM of agonist-induced cytokine production are shown (n = 8 – 10). For comparisons between overall groups (e.g., PCV13 vs. (PCV13 + 3M-052)), two-way repeated measures ANOVA for non-parametric sample populations were applied and statistical significance denoted as +p < 0.05, ++p < 0.01, +++p < 0.001. For comparison at individual concentrations, the unpaired Mann-Whitney test was applied and statistical significance denoted as *p < 0.05, **p < 0.01, ***p < 0.001. Results represent means ± SEM, with p values indicating significance as compared to that colored group.
100
101
102
103
104
105
106Human Newborn WB CCL3
0.15.7575705,70057,000
0.01 101 100PCV (1:)3M-052 (µM) -
-
NS+**
CC
L3 (p
g/m
l)
0
10000
20000
30000
40000Human Newborn WB IL-1β
3M-052 (O/W)
O/W Vehicle
PCV133M-052 (O/W) / PCV
O/W Vehicle / PCV
+++
***
*** NS
IL-1β
(pg/
ml)
0
100
200
300
400Human Newborn WB IL-12p40
+++
NS
*
IL-1
2p40
(pg/
ml)
0
20
40
60Human Newborn WB IL-12p70
+
*
NS
IL-1
2p70
(pg/
ml)
0
10000
20000
30000
40000Human Newborn WB CXCL8
0.15.7575705,70057,000
0.01 101 100PCV (1:)3M-052 (µM) -
-
NS
+++
***
***C
XCL8
(pg/
ml)
0
500
1000
1500Human Newborn WB IL-10
NSNS
IL-1
0 (p
g/m
l)
102
103
104
105
106Human Newborn WB CCL2
PCV (1:)3M-052 (µM) 0.1
5.7575705,70057,0000.01 101 100-
-
NSNS**
CC
L2 (p
g/m
l)
0
10000
20000
30000Human Newborn WB IL-6
++++
*
**
*
IL-6
(pg/
ml)
102
103
104
105Human Newborn WB CXCL10
++
++
** ***
CXC
L10
(pg/
ml)
Supplemental Figure 4
Supplemental Figure 4. Human adult whole blood cytokine responses to 3M-052, PCV13 and (PCV13 + 3M-052). Human adult blood cultured for 6 hours with O/W vehicle, PCV13 alone (1:5.7 - 57,000 v/v), 3M-052 alone (0.01, 0.1, 1, 10, 100 µM) and concentration dependent combinations of each. Supernatants were collected for ELISA and multiplex assay. Mean ± SEM of agonist-induced cytokine production are shown (n = 8 – 10). For comparisons between overall groups (e.g., PCV13 vs. (PCV13 + 3M-052)), two-way repeated measures ANOVA for non-parametric sample populations were applied and statistical significance denoted as +p < 0.05, ++p < 0.01, +++p < 0.001. For comparison at individual concentrations, the unpaired Mann-Whitney test was applied and statistical significance denoted as *p < 0.05, **p < 0.01, ***p < 0.001. Results represent means ± SEM, with p values indicating significance as compared to that colored group.
102
103
104
105
106Human Adult WB CCL2
0.15.7575705,70057,000
0.01 101 100PCV (1:)3M-052 (µM) -
-
+++NS
*
CC
L2 (p
g/m
l)
100
101
102
103
104
105
106Human Adult WB CCL3
0.15.7575705,70057,000
0.01 101 100PCV (1:)3M-052 (µM) -
-
NS
*
NS
CC
L3 (p
g/m
l)
0
50
100
150Human Adult WB IL-10
++IL
-10
(pg/
ml)
0
10000
20000
30000Human Adult WB CXCL8
0.15.7575705,70057,000
0.01 101 100PCV (1:)3M-052 (µM) -
-
+++
+
*
***
***
CXC
L8 (p
g/m
l)
102
103
104
105
106Human Adult WB CXCL10
+++
* +++*** **
CXC
L10
(pg/
ml)
0
100
200
300
400Human Adult WB IL-12p40
+
*
NS
IL-1
2p40
(pg/
ml)
0
20
40
60
80
100Human Adult WB IL-12p70
++
**
NS
IL-1
2p70
(pg/
ml)
0
5000
10000
15000Human Adult WB IL-1β
3M-052 (O/W)
O/W Vehicle
PCV13
3M-052 (O/W) / PCV
O/W Vehicle / PCV
+++
+**
*
IL-1β
(pg/
ml)
0
5000
10000
15000Human Adult WB IL-6
++
*
NS
*
IL-6
(pg/
ml)
Dowling et al.
Supplemental Figure 5
Supplemental Figure 5. 3M-052 enhances antigen-specific IgG levels while also skewing the response towards Th1 (IgG2a induction). Balb/c mice were immunized by subcutaneous injection (scruff of neck) with a 10 µg dose of influenza hemagglutinin (HA) alone, or in combination with 0.01, 0.03, 0.1, 0.3, or 1 mg/kg 3M-052 three times (prime, boost, boost) 14 days apart. Serum was collected at Day 77 (21 days after the final immunization) for measurement of HA-specific serum Ig levels by ELISA. Production of both serum (A) IgG1 and (B) IgG2a, suggest induction of a mixed Th1/Th2-response following immunization (n = 5).
Dowling et al.
9
3M Drug Delivery Systems 3M-052 is an Effective Vaccine Adjuvant
3M-052 enhances total IgG levels & skews response towards a Th1
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
OD
450
nm
Dilution
PBS HA HA + 3M052/PC 1 MPK HA + 3M052/PC .3 MPK HA + 3M052/PC .1 MPK HA + 3M052/PC .03 MPK HA + 3M052/PC .01 MPK
IgG1Vehicle HA
1 mg/kg 3M-052 0.3 0.1 0.03 0.01
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
PBS HA HA + 3M052/PC 1 MPK HA + 3M052/PC .3 MPK HA + 3M052/PC .1 MPK HA + 3M052/PC .03 MPK HA + 3M052/PC .01 MPK
OD
450
nm
Dilution
IgG2a
Mice: Balb/c, M, 5/group Route of administration: S.C. Regimen: q14d x 3 vaccinations Dose: 10 mg HA plus 0.01, 0.03, 0.1, 0.3, & 1 mg/kg 3M-052 Endpoint: HA-specific serum Ig; Serum collected 21 days post final immunization
B-cell Response
9
3M Drug Delivery Systems 3M-052 is an Effective Vaccine Adjuvant
3M-052 enhances total IgG levels & skews response towards a Th1
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
OD
450
nm
Dilution
PBS HA HA + 3M052/PC 1 MPK HA + 3M052/PC .3 MPK HA + 3M052/PC .1 MPK HA + 3M052/PC .03 MPK HA + 3M052/PC .01 MPK
IgG1Vehicle HA
1 mg/kg 3M-052 0.3 0.1 0.03 0.01
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
PBS HA HA + 3M052/PC 1 MPK HA + 3M052/PC .3 MPK HA + 3M052/PC .1 MPK HA + 3M052/PC .03 MPK HA + 3M052/PC .01 MPK
OD
450
nmDilution
IgG2a
Mice: Balb/c, M, 5/group Route of administration: S.C. Regimen: q14d x 3 vaccinations Dose: 10 mg HA plus 0.01, 0.03, 0.1, 0.3, & 1 mg/kg 3M-052 Endpoint: HA-specific serum Ig; Serum collected 21 days post final immunization
B-cell Response A B
IgG1 IgG2a
9
3M Drug Delivery Systems 3M-052 is an Effective Vaccine Adjuvant
3M-052 enhances total IgG levels & skews response towards a Th1
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
OD
450
nm
Dilution
PBS HA HA + 3M052/PC 1 MPK HA + 3M052/PC .3 MPK HA + 3M052/PC .1 MPK HA + 3M052/PC .03 MPK HA + 3M052/PC .01 MPK
IgG1Vehicle HA
1 mg/kg 3M-052 0.3 0.1 0.03 0.01
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
PBS HA HA + 3M052/PC 1 MPK HA + 3M052/PC .3 MPK HA + 3M052/PC .1 MPK HA + 3M052/PC .03 MPK HA + 3M052/PC .01 MPK
OD
450
nm
Dilution
IgG2a
Mice: Balb/c, M, 5/group Route of administration: S.C. Regimen: q14d x 3 vaccinations Dose: 10 mg HA plus 0.01, 0.03, 0.1, 0.3, & 1 mg/kg 3M-052 Endpoint: HA-specific serum Ig; Serum collected 21 days post final immunization
B-cell Response
Supplemental Figure 6
Supplemental Figure 6. Addition of 3M-052 augments Th1-responses to alum adjuvanted influenza hemagglutinin antigen. Addition of 3M-052 to Alum-adjuvanted HA antigen markedly enhances IgG2a Ab production. Balb/c mice were immunized by subcutaneous injection (scruff of neck) with a 10 µg dose of influenza hemagglutinin (HA) alone or in combination with Alum or 0.1 mg/kg 3M-052 three times (prime, boost, boost) 14 days apart. The results depict median HA-specific serum (A) IgG, (B) IgG1, and (C) IgG2a levels measured by ELISA on Day 77, which was 21 days after the final immunization (n = 5).
Dowling et al.
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
OD
450n
m
Dilution
PBS HA HA + Alum HA + 3M052 HA + Alum + 3M052
IgG1
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
OD
450n
m
Dilution
PBS HA HA + Alum HA + 3M052 HA + Alum + 3M052
IgG2a
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
OD
450n
m
Dilution
PBS HA HA + Alum HA + 3M052 HA + Alum + 3M052
IgG total
3M-052 Augments Alum Adjuvanticity
10 mg HA, 0.1 mg/kg 3M-052 (0.25 ug 3M-052/mouse) Bled 2 weeks following boost
Mice: Balb/c Regimen: Prime/Boost (q14d x 2 vaccinations) Endpoint: Serum Ig
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
OD
450n
m
Dilution
PBS HA HA + Alum HA + 3M052 HA + Alum + 3M052
IgG1
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
OD
450n
m
Dilution
PBS HA HA + Alum HA + 3M052 HA + Alum + 3M052
IgG2a
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
OD
450n
m
Dilution
PBS HA HA + Alum HA + 3M052 HA + Alum + 3M052
IgG total
3M-052 Augments Alum Adjuvanticity
10 mg HA, 0.1 mg/kg 3M-052 (0.25 ug 3M-052/mouse) Bled 2 weeks following boost
Mice: Balb/c Regimen: Prime/Boost (q14d x 2 vaccinations) Endpoint: Serum Ig
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
OD
450n
m
Dilution
PBS HA HA + Alum HA + 3M052 HA + Alum + 3M052
IgG1
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
OD
450n
m
Dilution
PBS HA HA + Alum HA + 3M052 HA + Alum + 3M052
IgG2a
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
OD
450n
m
Dilution
PBS HA HA + Alum HA + 3M052 HA + Alum + 3M052
IgG total
3M-052 Augments Alum Adjuvanticity
10 mg HA, 0.1 mg/kg 3M-052 (0.25 ug 3M-052/mouse) Bled 2 weeks following boost
Mice: Balb/c Regimen: Prime/Boost (q14d x 2 vaccinations) Endpoint: Serum Ig
B CIgG1 IgG2a
ATotal IgG
OD
450
nm
OD
450
nm
OD
450
nm
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
OD
450n
m
Dilution
PBS HA HA + Alum HA + 3M052 HA + Alum + 3M052
IgG1
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
OD
450n
m
Dilution
PBS HA HA + Alum HA + 3M052 HA + Alum + 3M052
IgG2a
0.01 1E-3 1E-4 1E-5 1E-6 1E-7 1E-8
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
OD
450n
m
Dilution
PBS HA HA + Alum HA + 3M052 HA + Alum + 3M052
IgG total
3M-052 Augments Alum Adjuvanticity
10 mg HA, 0.1 mg/kg 3M-052 (0.25 ug 3M-052/mouse) Bled 2 weeks following boost
Mice: Balb/c Regimen: Prime/Boost (q14d x 2 vaccinations) Endpoint: Serum Ig
101
102
103
104
105
106Serotype 3
7 63 90 120 1500 28 56
***
*NSNS
Pneu
moc
occa
l spe
cific
IgG
(ng/
ml)
101
102
103
104
105
106Serotype 6B
7 63 90 120 1500 28 56
++NS
***
*
Pneu
moc
occa
l spe
cific
IgG
(ng/
ml)
101
102
103
104
105
106
Serotype 5
7 63 90 120 1500 28 56
+++* *
*** ***
0.07
Pneu
moc
occa
l spe
cific
IgG
(ng/
ml)
101
102
103
104
105
106Serotype 7F
7 63 90 120 1500 28 56
+++NS
**** * * ** ***
*
Pneu
moc
occa
l spe
cific
IgG
(ng/
ml)
+++++
101
102
103
104
105
106Serotype 6A
7 63 90 120 1500 28 56
**** **** *
**
Pneu
moc
occa
l spe
cific
IgG
(ng/
ml)
101
102
103
104
105
106
Serotype 9V
7 63 90 120 1500 28 56
++
****** ** **
Pneu
moc
occa
l spe
cific
IgG
(ng/
ml)
101
102
103
104
105
106Serotype 18C
7 63 90 120 1500 28 56
++NS
0.056 0.056 ** ** *
Pneu
moc
occa
l spe
cific
IgG
(ng/
ml)
101
102
103
104
105
106Serotype 19A
7 63 90 120 1500 28 56
++++
***
* **
**
0.07
Pneu
moc
occa
l spe
cific
IgG
(ng/
ml)
101
102
103
104
105
106Serotype 19F
7 63 90 120 1500 28 56
++
NS
0.056
** ** ***
Pneu
moc
occa
l spe
cific
IgG
(ng/
ml)
101
102
103
104
105
106Serotype 1
7 63 90 120 1500 28 56
+++
** *** **
*
Pneu
moc
occa
l spe
cific
IgG
(ng/
ml)
101
102
103
104
105
106Serotype 4
7 63 90 120 1500 28 56
+++
*** *
Pneu
moc
occa
l spe
cific
IgG
(ng/
ml)
101
102
103
104
105
106Serotype 14
7 63 90 120 1500 28 56
NSNS
0.056
0.056
Pneu
moc
occa
l spe
cific
IgG
(ng/
ml)
101
102
103
104
105
106Serotype 23F
7 63 90 120 1500 28 56
+++
0.056
**** ** ** *** * * * *
Pneu
moc
occa
l spe
cific
IgG
(ng/
ml)
PCV13 PCV13 + 3M-052 (High)Saline PCV13 + 3M-052 (Low) 3M-052 (Low)
3M-052 (High)
Supplemental Figure 7 Dowling et al.
Supplemental Figure 7. Addition of a TLR7/8 agonist accelerates serotype-specific antibody responses to PCV13 in a dose dependent manner. Thirteen PCV13 serotypes are shown. Neonatal and infant rhesus macaques were immunized at DOL0, 28, and 56 with saline control, PCV13 alone, 3M-052 alone (0.01 or 0.1 mg/kg), or (PCV13 + 3M-052 (0.01 or 0.1 mg/kg)). Peripheral blood was collected at the indicated time-points to obtain serum for anti-pneumococcal serotype titers by polysaccharide-IgG binding microarray (n = 3 - 5 per group) run in triplicate. Horizontal broken line indicates 0.35 µg/ml, the WHO recommended reference Ab concentration of IgG used as a correlate of protective levels in humans. For comparisons between overall groups (i.e., PCV13 vs. (PCV13 + 3M-052)), two-way repeated measures ANOVA for non-parametric sample populations were applied and statistical significance denoted as +p < 0.05, ++p < 0.01, or NS (not significant). For comparison at individual time-points (i.e. PCV13 vs. (PCV13 + 3M-052) at DOL28), unpaired Mann-Whitney test was applied at each time-point. Results represent means ± SEM, with statistical significance denoted as *p < 0.05, **p < 0.01, ***p < 0.001.
Supplemental Table 2 Dowling et al.
Supplemental Table 2. Longitudinal non-human primate study design overview DOL 0 2 7 14 28 30 35 42 56 58 63 70 90 120 150 180 360 Vaccination x x x Micro-chipping/Tattooing x Weight monitoring x x x x x x x x x x x x x Physical Exam x x x x x x x x x x x x x x x x x Representative Photography x x x x x x x x x x x x Phlebotomy x x x x x x x x x x x x Muscle Biopsy* x x x x LN Biopsy* x x Neonatal and infant NHP were vaccinated on DOL0 (within 24 hours of birth), DOL28, and DOL56. Phlebotomy, routine physical exams, weight measurements were performed up to DOL360. Representative photography for each animal was performed up to DOL70. * Indicates sub study group of animals (n = 8) that underwent muscle and lymph node biopsies.
0 28 56 120101
102
103Serotype 1
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104Serotype 3
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 4
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104Serotype 5
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 6A
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 7F
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 6B
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 9V
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 14
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104Serotype 18C
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 19A
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 19F
DOL
Opso
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tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 23F
DOL
Opso
niza
tion
indi
ces
PCV13
PCV13 + 0.01 mg/kg 3M-052
PCV13 + 0.1 mg/kg 3M-052
0 28 56 120101
102
103Serotype 1
DOL
Opso
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indi
ces
0 28 56 120101
102
103
104Serotype 3
DOL
Opso
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indi
ces
0 28 56 120101
102
103
104
105Serotype 4
DOL
Opso
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tion
indi
ces
0 28 56 120101
102
103
104Serotype 5
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 6A
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 6B
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 7F
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 9V
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 14
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104Serotype 18C
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 19A
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 19F
DOL
Opso
niza
tion
indi
ces
0 28 56 120101
102
103
104
105Serotype 23F
DOL
Opso
niza
tion
indi
ces
Supplemental Figure 8
Supplemental Figure 8. TLR7/8 adjuvantation markedly accelerates and enhances serotype-specific pneumococcal opsonophagocytic killing capacity in neonatal serum. Neonatal and infant rhesus macaques were immunized at DOL0, 28, and 56 with either PCV13 alone or (PCV13 + 3M-052). Peripheral blood was collected at the indicated time-points to obtain serum for measurement of IgG concentrations and opsonization indicies (OIs) as described in Methods. Geometric mean titers of serotype-specific opsonophagocytic killing activity from n = 3 rhesus macaques per treatment group are shown. Samples with undetectable OIs were assigned an OI of 12. Results represent means ± SEM.
Dowling et al.
Supplemental Figure 9
Supplemental Figure 9. TLR7/8 adjuvantation dramatically accelerates and enhances serotype-specific pneumococcal opsonophagocytic killing capacity in neonatal serum. Neonatal and infant rhesus macaques were immunized at DOL0, 28, and 56 days with either PCV13 alone or PCV13 co-administered with 3M-052. Peripheral blood was collected at the indicated time-points to obtain serum. Average geometric mean titers of serotype-specific opsonophagocytic killing activity from rhesus macaques per treatment group (n = 3). The results are expressed as opsonization indices (Ois), defined as the interpolated dilution of serum that kills 50% of bacteria. Samples identified as negative in the assay (i.e., samples having no functional activity detected) were assigned an OI of 12. Radar plot analysis of all 13 serotypes tested, including raw OI (A) and fold-change analysis (B) at DOL0, 28, 56, and 120. After a single dose of (PCV13 + 3M-052), all immunized infants exceeded this level for all 13 serotypes tested.
Dowling et al.
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Opso
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OIs)
OI -
Fold
Cha
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To P
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A
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PCV13 PCV13 + 0.01 mg/kg 3M-052 PCV13 + 0.1 mg/kg 3M-052
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Pneumococcal specific IgG (ng/ml)
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Serotype 23F
PCV13
PCV13 + 0.01 mg/kg 3M-052
PCV13 + 0.1 mg/kg 3M-052
Supplemental Figure 10 Dowling et al.
Supplemental Figure 10. Day 28 opsonophagocytic killing activity corresponds with accelerated serotype-specific antibody responses to TLR7/8 agonist-adjuvanted pneumococcal conjugate vaccine. Neonatal and infant rhesus macaques were immunized at DOL0 with either PCV13 alone or (PCV13 + 3M-052). Peripheral blood was collected at the indicated time-points to obtain serum for measurement of IgG concentrations and opsonization indicies (OIs) as described in Methods. Day 28 post-first immunization OIs (y-axis) are plotted as a function of IgG concentrations (x-axis) depicted as geometric mean titers (n = 3 per treatment group). Samples with undetectable OIs were assigned an OI of 12. Results represent means ± SEM.
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PCV13 + 0.01 mg/kg 3M-052
PCV13 + 0.1 mg/kg 3M-052
Supplemental Figure 11 Dowling et al.
Supplemental Figure 11. TLR7/8 agonist-adjuvantation of PCV13 enhances Day 56 opsonophagocytic killing activity. Neonatal and infant rhesus macaques were immunized at DOL0 and 28 with either PCV13 alone or (PCV13 + 3M-052). Peripheral blood was collected at the indicated time-points to obtain serum for measurement of IgG concentrations and opsonization indicies (OIs) as described in Methods. Day 56 post-first immunization OIs (y-axis) are plotted as a function of IgG concentrations (x-axis) depicted as geometric mean titers (n = 3 per treatment group). Samples with undetectable OIs were assigned an OI of 12. Results represent means ± SEM.
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Supplemental Figure 12 Dowling et al.
Supplemental Figure 12. TLR7/8 agonist-adjuvantation of PCV13 enhances Day 120 opsonophagocytic killing activity. Neonatal and infant rhesus macaques were immunized at DOL0, 28, and 56 with either PCV13 alone or (PCV13 + 3M-052). Peripheral blood was collected at the indicated time-points to obtain serum. Day 120 post-first immunization OIs (y-axis) are plotted as a function of IgG concentrations (x-axis) depicted as geometric mean titers (n = 3 per treatment group). Samples with undetectable OIs were assigned an OI of 12. Results represent means ± SEM.
0 2 0 2 0 2 0 232
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Saline PCV13 Adj PCV13+AdjDOL
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SalinePCV133M-052 (0.1 mg/kg)3M-052 (0.01 mg/kg)PCV + 3M-052 (0.1 mg/kg)PCV + 3M-052 (0.01 mg/kg)
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56 58 56 58 56 58 56 5832
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38.6*
Saline PCV13 Adj PCV13+Adj
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NSNS
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(C)
Supplemental Figure 13 Dowling et al.
Supplemental Figure 13. Weight and body temperature of immunized neonatal and infant rhesus macaques. (A) Weight, a sensitive indicator of neonatal well-being, was measured regularly to DOL150 and are depicted as normalized values relative to birth weight (100%) for each treatment group. Red dotted lines indicate normal age-matched norms with standard deviations. (B) Body temperature was measured by rectal thermometer at regular intervals up to DOL150. (C) Body temperatures pre- and post- each immunization at DOL0, 28, and 56. For comparison at individual time-points, the unpaired Mann-Whitney test was applied, with statistical significance denoted as *p < 0.05. Results represent means ± SEM of 3-5 animal's per group.
Supplemental Table 3 Dowling et al.
Measurement Unit Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SDWhite Blood Cell Count per μl 8.42 1.89 11.59 2.37 9.05 0.78 11.60 4.24 8.65 1.77 10.11 2.96 10.27 1.34 12.12 5.92Red Blood Cell Count per μl 5.22 0.40 5.11 0.37 5.19 0.21 5.27 0.60 5.50 0.19 5.73 0.55 5.36 0.33 5.25 0.76
Hemoglobin g/dl 11.48 1.04 9.18 2.86 10.60 1.21 10.70 0.88 11.45 1.33 8.38 3.06 10.80 1.25 9.98 2.04Hematocrit % 35.38 2.68 29.73 6.93 33.96 3.25 34.53 3.61 36.10 2.84 29.30 7.85 34.93 2.07 33.04 6.03
Corpusular Volume fl 67.93 2.89 57.80 10.06 65.28 4.17 65.63 1.33 65.60 4.87 51.12 13.00 65.40 6.41 62.78 3.97Corpuscular Hemoglobin pg 22.03 1.40 17.75 4.48 20.36 1.70 20.35 1.03 20.80 2.38 14.62 5.26 20.25 3.16 18.88 1.61
Corpuscular Hemoglobin Conc. g/dl 32.40 0.96 30.43 2.56 31.14 0.90 31.03 1.19 31.65 1.53 28.18 3.24 30.80 2.22 30.08 0.90Red Blood Cell Distribution Width % 12.93 0.43 14.75 2.06 13.72 0.68 13.70 1.28 13.23 1.47 23.70 7.54 13.57 6.80 14.46 1.83
Platelet Count μl 550.50 128.28 732.00 428.78 703.80 93.89 465.75 256.20 661.75 119.75 739.60 219.59 532.25 295.28 657.20 220.97Mean platelet volume fl 10.63 2.11 12.43 4.45 10.38 1.13 11.25 0.90 10.33 1.20 9.00 5.03 11.45 2.68 10.40 1.14
% Reticuloyctes % 0.00 0.00 1.38 1.63 0.26 0.58 0.00 0.00 0.00 0.00 1.60 1.37 0.00 0.00 1.12 1.55% Neutrophils % 22.05 5.74 26.13 15.28 18.98 3.79 22.83 3.01 19.50 5.53 24.38 8.07 25.03 4.82 35.00 5.58
% Lymphocytes % 71.65 3.50 68.68 15.04 74.84 5.44 70.28 4.12 75.80 4.87 70.74 8.23 69.30 3.54 58.78 5.54% Monocytes % 2.78 0.45 3.20 0.55 3.02 0.65 4.05 2.25 2.88 1.06 3.22 1.22 3.50 0.63 4.30 1.25% Eosinophils % 2.80 1.97 1.35 1.34 2.42 1.66 2.00 1.33 1.28 0.39 1.04 0.84 1.80 1.89 1.40 0.70% Basophils % 0.73 0.48 0.65 0.13 0.74 0.34 0.88 0.35 0.55 0.13 0.62 0.50 0.40 0.12 0.52 0.13# Neutrophils per μl 1.91 0.86 2.90 1.40 1.71 0.36 2.56 0.69 1.69 0.60 2.33 0.56 2.56 0.57 4.40 2.72
# Lymphocytes per μl 6.01 1.18 8.10 2.98 6.78 0.83 8.25 3.36 6.56 1.40 7.26 2.61 7.13 1.05 7.01 3.09# Monocytes per μl 0.23 0.04 0.36 0.02 0.27 0.07 0.50 0.32 0.24 0.08 0.32 0.17 0.36 0.08 0.50 0.18# Eosinophils per μl 0.22 0.15 0.15 0.13 0.21 0.14 0.20 0.06 0.11 0.03 0.11 0.11 0.19 0.21 0.15 0.06# Basophils per μl 0.06 0.04 0.07 0.01 0.07 0.03 0.10 0.05 0.05 0.02 0.07 0.07 0.04 0.02 0.06 0.01
Measurement Unit Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SDWhite Blood Cell Count per μl 9.06 2.23 9.91 0.79 12.40 3.20 12.80 1.49 9.23 3.10 11.85 4.19 10.38 1.92 10.95 1.98Red Blood Cell Count per μl 6.05 0.62 5.53 1.01 5.47 0.33 5.05 1.09 6.07 0.69 5.78 0.69 5.72 0.67 5.23 1.36
Hemoglobin g/dl 12.27 0.74 8.63 4.20 10.45 1.77 9.10 3.78 12.60 0.87 10.13 4.89 9.63 0.90 8.57 4.84Hematocrit % 39.10 1.61 29.48 11.10 34.05 3.71 29.83 11.21 39.30 2.62 34.50 11.44 32.83 0.93 28.20 13.98
Corpusular Volume fl 65.07 6.84 52.20 13.94 62.75 9.50 57.70 10.21 64.97 3.79 58.90 15.65 57.97 6.98 52.23 15.48Corpuscular Hemoglobin pg 20.47 2.82 15.28 6.21 19.28 4.06 17.48 3.65 20.87 1.75 17.13 7.46 17.07 3.23 15.77 5.84
Corpuscular Hemoglobin Conc. g/dl 31.37 1.10 28.60 4.83 30.48 2.20 30.18 1.23 32.07 0.83 28.07 6.06 29.30 2.14 29.87 2.14Red Blood Cell Distribution Width % 16.27 5.74 24.48 9.96 12.87 6.45 17.60 3.29 17.07 7.30 19.30 13.21 15.23 3.70 19.23 5.58
Platelet Count μl 611.33 88.56 650.75 316.08 567.50 400.01 1256.75 1615.86 487.33 141.64 659.67 258.02 852.00 109.78 1364.67 1253.15Mean platelet volume fl 10.00 1.04 8.15 4.71 10.80 1.90 11.40 1.64 9.50 5.51 8.15 4.78 10.20 1.73 11.85 7.14
% Reticuloyctes % 0.00 0.00 1.13 1.33 5.03 10.05 1.90 2.20 0.00 0.00 0.00 0.00 6.87 11.89 0.73 1.27% Neutrophils % 19.00 5.60 26.15 9.37 25.23 15.43 28.33 8.69 20.07 9.34 36.57 18.90 26.90 2.23 27.37 9.12
% Lymphocytes % 73.37 5.13 68.95 9.76 69.73 14.04 65.55 7.42 73.60 11.84 57.77 18.25 67.17 2.76 68.63 10.34% Monocytes % 5.27 1.19 2.90 0.90 3.33 1.71 4.35 1.09 4.67 3.31 4.00 0.95 2.90 0.79 2.53 1.07% Eosinophils % 1.37 0.55 1.58 0.80 1.30 0.39 1.18 0.78 1.07 0.99 1.13 0.76 2.47 2.80 1.10 0.36% Basophils % 1.03 0.15 0.43 0.17 0.45 0.10 0.60 0.22 0.60 0.20 0.50 0.36 0.50 0.17 0.40 0.20# Neutrophils per μl 1.66 0.29 2.58 0.91 2.96 1.70 3.69 1.46 1.74 0.65 4.07 1.92 2.77 0.32 2.92 0.71
# Lymphocytes per μl 6.70 1.96 6.84 1.22 8.76 3.28 8.35 1.04 6.89 3.13 7.10 4.36 7.01 1.59 7.58 2.25# Monocytes per μl 0.49 0.20 0.28 0.08 0.44 0.30 0.55 0.14 0.43 0.28 0.45 0.05 0.31 0.13 0.27 0.11# Eosinophils per μl 0.12 0.03 0.17 0.09 0.17 0.07 0.14 0.07 0.08 0.06 0.15 0.15 0.24 0.26 0.12 0.03# Basophils per μl 0.09 0.02 0.04 0.01 0.06 0.02 0.08 0.01 0.06 0.04 0.07 0.07 0.05 0.01 0.04 0.02
Supplemental Table 3. Average complete blood count per study treatment group. Day Of Life 90 Day Of Life 120
Saline PCV13 3M-052 PCV13 + 3M-052 Saline PCV13 3M-052
PCV13 + 3M-052
SD: standard deviation. Statistial difference as compared to saline treatment alone denoted by enbolded/italics. n = 3 - 5.
PCV13 + 3M-052
Day Of Life 150 Day Of Life 180Saline PCV13 3M-052 PCV13 + 3M-052 Saline PCV13 3M-052
Supplemental Figure 14 Dowling et al.
Supplemental Figure 14. Intramuscular injection of 3M-052 induces injection site erythema post-second immunization. Neonatal and infant rhesus macaques were immunized at DOL0, 28, and 56 with either PCV13 alone or (PCV13 + 3M-052). (A) Significant injection site erythema (diameter of redness in mm as measured using calipers), as compared to saline (n = 5), was only observed after the second of three immunizations with PCV13 co-administered with 3M-052 (n = 8, combining both 0.01 and 0.1 mg/kg treatment groups). (B) Photographs are labeled 1 – 12 in the top left corner and each individual animal study identification code is indicated in the top right corner of each image. Left thigh ventral photography of representative infant rhesus macaques on DOL30 (images 1 – 4), 35 (images 5 – 8), and 42 (images 9 – 12). While there was a trend towards increased erythema for some individual animals treated with 3M-052 or (PCV13 + 3M-052), no significant erythema at the site of injection was observed pre- or post-first or third immunization. As the maculopapular rash was only observed in a) the second of two birthing/enrollment seasons, b) co-housed animals and c) 3 of the total of 16 3M-052- (or (PCV13 + 3M-052))-treated infant animals, it was unclear whether it was adjuvant-related. For comparison at individual time-points, the unpaired Mann-Whitney test was applied, with with statistical significance denoted as *p < 0.05 or NS (not significant) as compared to saline treatment group. Results represent means ± SEM.
Supplemental Figure 15 Dowling et al.
Supplemental Figure 15. 3M-052 administration with or without PCV13 does not induce systemic cytokines in neonatal/infant rhesus macaques. (A) Evaluation of rhesus plasma cytokine kinetics post- each dose of IM PCV13 or (PCV13 + 0.1 mg/kg 3M-052) formulated in O/W emulsion (vehicle) or Saline (n = 3 - 5). (B – D) Evaluation of rhesus plasma cytokine pre- (DOL28, gray) and post- (DOL30, black, blue and red) single dose of (PCV13 + 0.1 mg/kg 3M-052) (n = 3 - 4).
0 7 28 30 35 56 630
2000
4000
6000
8000CCL2
DOLC
CL2
(pg/
ml)
0.1
1
10 IFNγ%
IL'10%
sCD40L%
IL'1β%
IL'6%
CCL2%
TNF%
IL'12p40%
0.1
1
10 IFNγ%
IL'10%
sCD40L%
IL'1β%
IL'6%
CCL2%
TNF%
IL'12p40%
0.1
1
10 IFNγ%
IL'10%
sCD40L%
IL'1β%
IL'6%
CCL2%
TNF%
IL'12p40%
0 7 28 30 35 56 630
50
100
150
200
250IL-6
DOL
IL-6
(pg/
ml)
0 7 28 30 35 56 630
50
100
150
200
250
PCV13
TNFPCV13 + 3M-052
Saline Treatment
DOL
TNF
(pg/
ml)
0 7 28 30 35 56 630
50
100
150
200
250IL-10
DOL
IL-1
0 (p
g/m
l)
0 7 28 30 35 56 630
50
100
150
200
250IFNγ
DOL
IFNγ
(pg/
ml)
B
A
C D
0 7 28 30 35 56 630
5000
10000
15000
20000sCD40 ligand
DOL
sCD
40 (p
g/m
l)
0 7 28 30 35 56 630
50
100
150
200
250IL-1β
DOL
IL-1β
(pg/
ml)
0 7 28 30 35 56 630
50
100
150
200
250IL-12p40
DOL
IL-1
2p40
(pg/
ml)
Saline - DOL28 vs DOL30 PCV13 - DOL28 vs DOL30 PCV13 + 3M-052 - DOL28 vs DOL30
Supplemental Figure 16
Supplemental Figure 16. Experimental approach used for mononuclear cell sorting and ex vivo assessment of vaccine-specific B and T cells in infant rhesus macaques. (A) Sorted leukocytes were incubated as depicted. B cell subsets (left) were non-specifically stimulated with R848/IL-2/IFNγ to induce differentiation to Ab-secreting plasma cells. Plasma cells were subsequently plated on ELISpot plates for detection of pneumococcal polysaccharide (PnPS)-specific B cells. Monocytes were differentiated to monocyte-derived dendritic cells (MoDCs) by the addition of GM-CSF and IL-4. After treatment of MoDCs with CRM197 (the protein component of PCV13), cells were co-cultured with CD4+ and CD8+ T cells and activation of vaccine-specific T cells was measured. (B) Frozen PBMCs were thawed and stained with CD20-Pacific Blue, CD27-PE.Cy7, CD14-PE, CD4-FITC, and CD8-APC.Cy7. Cells were subsequently sorted according to the gating strategy depicted on a FACSAria II cytometer.
Dowling et al.
15
~5*106 cryopreserved PBMCs
~2*105 Naïve B cells (CD20+CD27-)
~2*105 Memory B cells(CD20+CD27+)
~2*105
Monocytes~1*106
CD4+ T cells
Non-specific stimulation (R848/IL-2/IFNγ) to generate antibody-secreting plasma
cells (ASCs) (5 days)
ELISpot (total IgG/IgM, PnPS)
MoDC generation (5 days)
Endocytosis of CRM-197
Co-culture MoDCs with CD4+ T cells (1:10) (7 days)
100 ng/ml Concanavalin A (5 days)
T-cell cytokine secretion
0
5
6
Day
A B
Supplemental Figure 17 Dowling et al.
Supplemental Figure 17. 3M-052 accelerated and enhanced the magnitude of neonatal and infant anti-PnPS antibody (IgG) responses and may enhance antibody avidity. Ab titer to pneumococcal conjugate vaccine serotypes 4, 6B, 14, and 23F were compared and confirmed using (A) WHO recommended ELISA total (n = 3), (B) 96-well electrochemiluminescence (ECL) multiplex assay (n = 5). Ab titer to all 13 pneumococcal conjugate vaccine serotypes were compared and confirmed using (C) ELISA and (D) avidity assay (n = 3). For comparisons between overall groups (i.e., PCV13 vs. (PCV13 + 3M-052)), two-way repeated measures ANOVA for non-parametric sample populations were applied and statistical significance denoted as +p < 0.05, or NS (not significant). For comparison at individual time-points (i.e. PCV13 vs. (PCV13 + 3M-052) at DOL56), unpaired Mann-Whitney test was applied at each time-point. Results represent means ± SEM, with statistical significance denoted as *p < 0.05.
0 28 56 90 1202-2
2-1
20
21
22
PCVPCV + 3M-052
Avidity
DOL
NS*Av
idity
Inde
x (M
NaS
CN)
0 28 56 90 120102
103
104
105
106
107
PCVPCV + 3M-052
Total 13 Serotypes
DOL
+*
Imm
unog
lobu
lin (n
g/m
l)
0 28 56 90 120101
102
103
104
105
106
PCV13PCV13 + 3M-052
ECL - Serotype 4
Day of Life
Imm
unog
lobu
lin (n
g/m
l)
0 28 56 90 120101
102
103
104
105
106
PCV13PCV13 + 3M-052
ELISA - Serotype 4
Day of Life
Imm
unog
lobu
lin (n
g/m
l)
0 28 56 90 120100
101
102
103
104
105
106ELISA - Serotype 6B
Day of LifeIm
mun
oglo
bulin
(ng/
ml)
0 28 56 90 120100
101
102
103
104
105
106ELISA - Serotype 14
Day of Life
Imm
unog
lobu
lin (n
g/m
l)
0 28 56 90 120101
102
103
104
105
106ELISA - Serotype 23F
Day of Life
Imm
unog
lobu
lin (n
g/m
l)
A
B
C
0 28 56 90 120100
101
102
103
104
105
106ECL - Serotype 6B
Day of Life
Imm
unog
lobu
lin (n
g/m
l)
0 28 56 90 120100
101
102
103
104
105
106ECL - Serotype 14
Day of Life
Imm
unog
lobu
lin (n
g/m
l)
0 28 56 90 120101
102
103
104
105
106ECL - Serotype 23F
Day of Life
Imm
unog
lobu
lin (n
g/m
l)
D
Supplemental Figure 18
Supplemental Figure 18. Co-administration of 3M-052 with PCV13 increased infiltration of CD68+ cells at the vaccine injection site. Immunization with (PCV13 + 3M-052) accelerates injection site infiltration by monocytes/macrophages. 2 mm cube muscle biopsies were obtained from the injection site (quadriceps muscle) prior to and 48 hours after each immunization (obtained in an alternating pattern (e.g. DOL0 left leg, DOL2 right leg)). Frequencies of CD68+ cells in muscle were determined by immunofluorescence. For comparison at individual time-points, the unpaired Mann-Whitney test was applied, with statistical significance denoted as *p < 0.05, **p < 0.01, or NS (not significant). Data are representative of two animals per treatment group.
Dowling et al.
NS
0 2 0 2 0 2 0 2 0 2 0 20
1000
2000
3000
4000CD68 - Imm 1
DOL
PCV13PCV13 + 3M-052
Saline
NS NS
***
**NS
CD68
+ ce
lls (m
m2 )