CHIMERIC CD40L/SHIV VLPs ENHANCE DENDRITIC CHIMERIC CD40L/SHIV VLPs ENHANCE DENDRITIC CELL ACTIVATION AND BOOST IMMUNE CELL ACTIVATION AND BOOST IMMUNE

RESPONSES AGAINST HIVRESPONSES AGAINST HIV

Qizhi Cathy Yao, M.D., Ph.D.

Molecular Surgeon Research CenterMichael E. DeBakey Department of Surgery

Baylor College of Medicine

Production of Chimeric CD40L/SHIV VLPsProduction of Chimeric CD40L/SHIV VLPs

rBV-SIVGag

rBV-HIVEnv

rBV-hCD40L

Sf-9 cells CD40L/SHIV VLP

hCD40L

Env.

87.2%

Sf9 + rBV-mCD40L

FL2-H10 10 10 10 10

0 1 2 3 4

01

02

03

04

05

0

Co

un

ts

B.

M1

75.0%

Sf9 + rBV-hCD40L

FL2-H10 10 10 10 10

0 1 2 3 4

01

02

03

04

05

0

Co

un

ts

A.

M1

Surface expression of hCD40L and mCD40LSurface expression of hCD40L and mCD40L

hC

D40

L L

evel

s in

VL

Ps

(pg

)

0

200

400

600

800

1000

1200

1400

1600

1800

SHIV-VLP hCD40L/SHIV-VLP

500 ng VLPs

1000 ng VLPs

C.

incorporation of hCD40L in chimeric hCD40L/SHIV-VLPsincorporation of hCD40L in chimeric hCD40L/SHIV-VLPs

mC

D40

L L

evel

s in

VL

Ps

(pg

)

0

200

400

600

800

1000

1200

1400

1600

1800

SHIV-VLP mCD40L/SHIV-VLP

D.2000 500 ng VLPs

1000 ng VLPs

incorporation of mCD40L in chimeric mCD40L/SHIV-VLPsincorporation of mCD40L in chimeric mCD40L/SHIV-VLPs

A. iDC+SHIV-VLP

10 0 10 1 10 2 10 3 104

FL1-Height

M1

51.2%

4080

120

140

0C

ou

nts

60

20

100

Co

un

ts

40

iDC+hCD40L/SHIV-VLPB.

10 0 10 1 10 2 10 3 104

FL1-Height

M1

62.1%

120

140

020

8060

10

0

Enhanced binding of SHIV-VLPs with DCsEnhanced binding of SHIV-VLPs with DCs

Untreated

LPS

SHIV-VLP

Heated

hCD40L/SHIV-VLP

M1

M1

M1

M1

M1

M1

M1

M1

M1

M1

M1

M1

M1

M1

M1

CD40 CD83 CD86

31.2% 1.6% 91.2%

82.3%

80.3%

87.6%

41.5%

43.8%

42.9%

72.1%

6.9%

92.5%

94.5%

95.8%

93.1%

C.

hCD40L/SHIV-VLP

Enhanced phenotypic activation of DCs by chimeric hCD40L/SHIV-VLPsEnhanced phenotypic activation of DCs by chimeric hCD40L/SHIV-VLPs

Untreated

SHIV-VLP

hCD40L/SHIV-VLP

hCD40L

SHIV-VLP+hCD40L

hCD40L Ab+hCD40L/SHIV-VLP

Irr. Ab+hCD40L/SHIV-VLP

LPS

12

3

4

56

7

8

0

20

40

60

80

100

CD40 CD83

1 2 3 4 5 6 7 8

Po

siti

ve C

ells

(%

) D.

1 2 3 4 5 6 7 8

Blockade of hCD40L/SHIV-VLPs-induced DC activation Blockade of hCD40L/SHIV-VLPs-induced DC activation by hCD40L specific antibodyby hCD40L specific antibody

Untreated

SHIV-VLP

hCD40L/SHIV-VLP

hCD40L

SHIV-VLP+hCD40L

hCD40L Ab+hCD40L/SHIV-VLP

Irr. Ab+hCD40L/SHIV-VLP

LPS

12

3

4

56

7

8C

D86

MF

I

0

50

100

150

200

250

1 32 4 5 6 87

[3 H]

Th

ymid

ine

(CP

M)

0

20000

40000

60000

80000

100000

1. Untreated2. LPS3. SHIV-VLP

4. hCD40L/SHIV-VLP5. Heated hCD40L/SHIV-VLP

*

**

1 2 3 4 5

Elevated allogeneic T-cell-stimulatory activity of Elevated allogeneic T-cell-stimulatory activity of chimeric CD40L/SHIV-VLPs-activated DCs chimeric CD40L/SHIV-VLPs-activated DCs

20000

40000

60000

80000

1:40 1:80 1:160

hCD40L/SHIV-VLP

hCD40L+SHIV-VLP

hCD40L Ab+hCD40L/SHIV-VLP

irr. Ab+hCD40L/SHIV-VLP

*

[3 H]

Th

ymid

ine

(CP

M)

DC and T Cell Ratio

Blockade of allogeneic T-cell-stimulatory activity ofBlockade of allogeneic T-cell-stimulatory activity of hCD40L/SHIV-VLPs-induced DC by hCD40L AbhCD40L/SHIV-VLPs-induced DC by hCD40L Ab

Effect of hCD40L antibody on hCD40L/SHIV-VLP-induced secretion of Th1 type of cytokines.

TNF-α (pg/ml)

IFN-γ (pg/ml)

IL-12 (pg/ml)

Untreated 18 81 3

SHIV-VLP 428 105 4

hCD40L/SHIV-VLP 3200 194 49

hCD40L Ab+hCD40L/SHIV-VLP

339 103 6

SV5 Ab+hCD40L/SHIV-VLP

2277 164 47

LPS 2701 157 11

SIV-VLP

SHIV-VLP

mCD40L/SHIV-VLP

0

0.04

0.08

0.12

0.16

20 μg VLPs 100 μg VLPs

An

ti-H

IV E

nv

IgG

(O

.D.

405

nm

)

*

Elevated anti-HIV Env Ab production in chimeric Elevated anti-HIV Env Ab production in chimeric mCD40L/SHIV-VLP immunized micemCD40L/SHIV-VLP immunized mice

0

200

400

600

800

1000

1200

Gag pool 1 Gag pool 2

# o

f sp

ots

/106

sple

no

cyte

s

IFN-γ

*

*

SIV-VLPmCD40L/SIV-VLP

Elevated levels of IFN-Elevated levels of IFN-γγ production in mCD40L/SIV-VLPs immunized production in mCD40L/SIV-VLPs immunized mouse splenocytes upon SIV Gag peptides pulse mouse splenocytes upon SIV Gag peptides pulse

0

50

100

150

200

250

300

350

**

Gag pool 1 Gag pool 2

# o

f sp

ots

/106

sple

no

cyte

s

IL-4

SIV-VLPmCD40L/SIV-VLP

Elevated levels of IL-4 production in mCD40L/SIV-VLPs immunized Elevated levels of IL-4 production in mCD40L/SIV-VLPs immunized mouse splenocytes upon SIV Gag peptides pulse mouse splenocytes upon SIV Gag peptides pulse

0

500

1000

1500

2000

2500

Gag pool 1 Gag pool 2 Env

# o

f S

po

ts/1

06 S

ple

no

cyte

s

*

*

*

*

**

IFN-γ

SHIV-VLP (20 μg)

mCD40L/SHIV-VLP (20 μg)SHIV-VLP (100 μg)

mCD40L/SHIV-VLP (100 μg)

Elevated cytokines production in mCD40L/SHIV-VLPs immunized Elevated cytokines production in mCD40L/SHIV-VLPs immunized mouse splenocytes stimulated with Gag or Env peptides mouse splenocytes stimulated with Gag or Env peptides

0

200

400

600

800

1000

1200

1400

1600

*

**

Gag pool 1 Gag pool 2 Env

# o

f S

po

ts/1

06 S

ple

no

cyte

s

IL-4

SHIV-VLP (20 μg)

mCD40L/SHIV-VLP (20 μg)SHIV-VLP (100 μg)

mCD40L/SHIV-VLP (100 μg)

Group/PBS

SHIV VLP 20 μg

peptides

SHIV VLP 100 μg

mCD40L/SHIV VLP 20 μg

mCD40L/SHIV

VLP 100 μg

peptidesSIV Gag HIV Env.

IFN

-γ

100 101 102 103 104

FL1-H

100 101 102 103 104

FL1-H

FL1-H

FL1-H

101 102 103

FL1-HCD8

1.4

3.1

4.4

4.7

7.41010

04

100

1010

04

1010

04

1010

04

1010

04

FL1-H

FL1-H

FL1-H

FL1-H

100 101 102 103 104

FL1-H

1.6

2.8

4.1

4.5

6.91010

04

1010

04

1010

04

1010

04

1010

04

• Chimeric CD40L/SHIV VLPs can phenotypically and functionally activate DC in vitro.

• Chimeric CD40L/SHIV VLPs induce strong humoral and cellular immune responses in a mouse model.

ConclusionsConclusions

AcknowledgmentsAcknowledgments

Yao’s LabDr. Rongxin ZhangDr. Uddalak Bharadwaj Dr. David Doan

CollaboratorsDr. Min LiDr. Johnny Chen