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
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