JCI Insight Levy BCH Supplemental Material PDF DD01Dec16 · Dowling et al., JCI Insight, 2016...

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. 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. 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


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



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


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


OD

450

nmDilution

IgG2a


B-cell Response A B

IgG1 IgG2a

9



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


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


OD

450

nm

Dilution

IgG2a


B-cell Response


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


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


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


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


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


IgG total




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


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


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


IgG total




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


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


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


IgG total




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. 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

niza

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

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 6B

DOL

Opso

niza

tion

indi

ces

0 28 56 120101

102

103

104

105Serotype 7F

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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. 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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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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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. 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.

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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

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PCV13 + 3M-052

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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. 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).

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50

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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. 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. 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. 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 )

JCI Insight Levy BCH Supplemental Material PDF DD01Dec16 · Dowling et al., JCI Insight, 2016...

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