immunoadjuvant properties of oncolytic schwarz measles virus dr jean-françois fonteneau institut de...
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Immunoadjuvant Properties of Oncolytic Schwarz Measles Virus
Dr Jean-François FonteneauInstitut de Recherche en Santé de
l’Université de NantesINSERM UMR892, CNRS UMR6299
Nantes, France
Anti-tumor Virotherapy using oncolytic virus
Oncolytic virus
Ideal replicating oncolytic virus: (Russell SJ, Nature biotechnol, 2012Vacchelli E et al, Oncoimmunology, 2013)
- Kills efficiently tumor cells (apoptosis)Healthy cells
- No toxicity (genetically stable, minor side effects)
- High viral concentration production capability
- Infects exclusively or preferentially tumor cells
Tumor cells
Infection
apoptosis
- Induction or stimulation of an anti-tumor immune response.
Activation of the immune system
Measles virus vaccine (MV) based anti-tumoral virotherapy
Measles virus vaccine, Schwarz strain (MV): Dr Frédéric Tangy (Viral genomics and Vaccination laboratory, Pasteur Institut, Paris)
- Attenuated replicating vaccine strain of measles virus: Schwarz (MMR vaccine)
- Targets CD46, complement regulatory protein (wt MV targets CD150/SLAM)
- CD46 is expressed at low level by healthy cells
- CD46 is often found overexpressed on tumor cells
- Spontaneously oncolytic:- Lymphoma, glioma, breast, ovary, prostate … (Russell SJ, Mayo Clinic, USA)- MPM, colon and lung adenocarcinoma(Gauvrit A et al, Cancer Research, 2008; Boisgerault N et al, Biomed Res Int, 2013)
Protéine L
Protéine de Fusion (F)
Hémagglutinine (H)
Protéine de Matrice (M)
Double membrane lipidique
Phosphoprotéine (P)
ARN + Nucléoprotéine (N)
- Enveloped, non-segmented, negative-sense, single-stranded RNA (ssRNA) paramyxovirus of the genus Morbillivirus
Guillerme JB et al, Biology, 2013
- antiviral pathway defects are often found in tumor cells
Video : MV infection of meso13 by MV-eGFP (enhanced green fluorescent protein)
MV infection of Malignant Pleural Mesothelioma tumor cells
Limited MV infection of healthy mesothelial cells
Video : healthy mesothelial cells exposed to MV-eGFP
Cross presentation to CD8+ T Lymphocytes (CTL)?
Tumor cells
Immature DC
Apoptosis
MV
Activation of the immune system by MV infected tumor cells ?
Danger signalsDAMPs ? PAMPs ?
Mature DC ? Myeloid DC? Plasmacytoid DC?
20
40
60
80
% C
D83
+ cells
5000
10000
IL-12
p70
(pg/
ml)
10
20
Med
ian
Fluo
resc
ence
Inte
nsity
1000
2000
3000
IL-10
(pg/
ml)
CD83
CD86 IL-10
IL-12p70
Mo-DC activation
Activation of monocyte derived DC (Mo-DC) and tumor antigen cross-priming by MV infected tumor cells
Gauvrit et al, Cancer Research, 2008
Tumor antigen cross-priming
• Expression of TLR7 et TLR9 => specialized in recognition of viral nucleic acids
MVARNsb
IFN-Production and tumor antigen cross-presentation by pDC exposed to MV infected tumor cells?
Plasmacytoid Dendritic Cells (pDC)
•Produce huge quantities of Type I IFN (- and –) in response to virus
•Antigen cross presentation in human:•HIV (Hoeffel, G,.Immunity, 2007) (Crozat, K., J Exp Med, 2010)•Influenza (Lui, G., PLoS One, 2009)
•Antigen cross presentation in mice: •Cross-tolérance (Goubier, A., Dubois, B., Immunity, 2008)•OVA (Mouries, J., Blood, 2008)
• One of the target of imiquimod (R837, TLR7 ligand) in the treatment of basal cell carcinoma with Aldara.
pDC are not infected by Schwarz MV
Video : pDC exposed to MV-eGFP
MV infected tumor cells induce maturation of plasmacytoid DC
CD
123
SS
C
CD86 CD40
9 %
92.4 %
20 %
90.6 %
7.42 %
CD83
92.4 %
BDCA4FSC
4,27
130,96
5,13
5,99
12,36
5,33
24
10
60
22
85
167
Meso13 MV
Meso13 UV
IL3
R848
MV
IL-3 + MV
CD83
IL3
MV
MV
+IL
3
R8
48
M18
MV
M18
UV
Mes
o1
3 M
V
Mes
o1
3 U
V
A5
49 M
V
A5
49 U
V
0
20
40
60
80
100
** *****
******
% P
os
itiv
e C
ells
CD40
IL3
MV
MV
+IL
3
R8
48
M18
MV
M18
UV
Mes
o1
3 M
V
Mes
o1
3 U
V
A5
49 M
V
A5
49 U
V
0
50
100
150
200
*
R-M
FI
CD86
IL3
MV
MV
+IL
3
R8
48
M18
MV
M18
UV
Mes
o1
3 M
V
Mes
o1
3 U
V
A5
49 M
V
A5
49 U
V
0
20100
150
200
*
R-M
FI
MV infected tumor cells are internalized by plasmacytoid DC
4°C 37°C
MV
UV
M18
MV
UV
A549
37°C
4°C
PKH
67
BDCA-4
PKH
67
HLA-DR
pDCMo-DC
HLA-DR Alexa568 PKH-67 MERGE
pDC +M18 MV
pDC + A549 MV
MV activates pDC IFN- production by TLR7
IRS661: TLR7inhibitor
IL-3 0 0,1 0,5 1 0 0,1 0,5 1 0 0,1 0,5 1
0
20
40
60
80
200
400
600
800
IL-3 + MV*10 CpG-A CpG-AIF
N- (
ng
/ml)
IL-3 0 0,1 0,5 1 0 0,1 0,5 1 0 0,1 0,5 1
0
20
40
60
80
200
400
600
800
IL-3 + MV*10 CpG-A CpG-A
IF
N-
(n
g/m
l)
IRS661
IL-3 0 0,1 0,5 1 0 0,1 0,5 1 0 0,1 0,5 1
0
20
40
60
80
200
400
600
800
IL-3 + MV*10 CpG-A CpG-A
IFN
- (
ng
/ml)
IL-3 0 0,1 0,5 1 0 0,1 0,5 1 0 0,1 0,5 1
0
20
40
60
80
200
400
600
800
IL-3 + MV*10 CpG-A M18 MV
IFN
- (
ng
/ml)
IRS661 IRS661
(TLR-9)
pDC
IFN
-α (n
g/m
l)
IL3
MV
IL3
+ M
V
R8
48
M1
8 M
V
M1
8 U
V
A5
49
MV
A5
49
UV
0
10
20
30
200
400
600
IFN
- (
ng
/ml)
MV probably activates pDC via TLR7 in early endosome
CpG-A (TLR9)
Kerkmann M et al, J Immunol, 2003
CpG-B (TLR9)
-> High IFN- -> Low IFN-
-> Low inflammatory molecules secretion (IL-8)
-> High inflammatory molecules secretion (IL-8)
-> Low costimulation (CD80, CD86)
-> High costimulation (CD80, CD86)
Early endosome Late endosome Honda K et al, Nature, 2005
Guiducci C et al, J Exp Med, 2006
Sadaka C et al, Blood, 2009
Kubo-Murai M et al, Blood, 2008
IRF7 pathway NF-B pathway
HIV (TLR7) R848 (TLR7/TLR8)CpG-B (TLR9)
O’brien M et al, J Clin Invest, 2011
MV (TLR7) R848 (TLR7/TLR8) Guillerme JB et al, clin cancer Res, 2013
NYESO-1 cross-presentation by pDC exposed to MV infected tumor cells
NYESO-1
M 18 A5490
5000
10000
15000NIUVMV
(NY
ES
O-1
/ R
PL
PO
)*10
00
M18 : HLA-A*0201- , NYESO-1+
A549 : HLA-A*0201- , NYESO-1-
pDC : HLA-A*0201+ , NYESO-1-
M117.167 : HLA-A*0201/NYESO-1(157-165) specific CD8+ T cell clone
NYESO-1 cross-presentation by pDC exposed to MV infected tumor cells
LT only pDC + LT
0.1µM NYESO-1[157-165]
1µM NYESO-1[157-165]
M18
MV UV
M18 MV
A549
R848
M18 UV
10.8%
6.5%
0.8% 0.2%
0.2%
87.5%
0.1% 0.1%
0.3% 0.1%
LT - pDC
CD8
IFN-γ
CD8
IFN-γ
LT + pDCCD
8
IFN-γ
CD8
IFN-γ
CD8
IFN-γ
CD8
IFN-γ
LT
on
ly (
n=
5)
pD
C +
LT
(n
=6
)
pD
C 0
.1 µ
M N
YE
SO
-1 (
n=
3)
pD
C +
M1
8 M
V (
n=
6)
pD
C +
M1
8 U
V(n
=5
)0
10
20
% o
f IF
N +
T C
D8+
cel
ls
NYESO-1 cross-presentation by pDC and Mo-DC exposed to MV infected tumor cells
HLA-A*0201neg/NY-ESO-1pos
HLA-A*0201neg/NY-ESO-1pos
Mo-DC pDC
Ø
NYESO-1 [157-165](10µM)
M18MV
M18 UV
IFN-y IFN-y
0.6%
11.7%
68%
0.16%
0.3%
8.15%
79.6%
0.4%
CD8CD8
LT
on
ly
Mo
-DC
i
Mo
-DC
m +
0
,1µ
M N
Y-E
SO
-1
Mo
-DC
+ M
18M
V
Mo
-DC
+ M
18U
V0
5
10
15
20
% o
f IF
N +
T C
D8
+ c
ells
Mo- DC Cross- presentation
LT
on
ly (
n=
5)
pD
C +
LT
(n
=6
)
pD
C 0
.1 µ
M N
YE
SO
-1 (
n=
3)
pD
C +
M1
8 M
V (
n=
6)
pD
C +
M1
8 U
V(n
=5
)0
10
20
% o
f IF
N +
T C
D8+
cel
ls
pDC cross-presentation
0,5
Mo-DC
95,1
Mo-DC + pep 1µM
M18 MV
M18 UV
Mo-DC Mo-DC + IFN-γ + TNF-α
Mo-DC + R848
10,0 22,7 7,0
0,5 2,2 0,9
M18 MV
M18 UV
1,2 4,9 1,3
0,5 2,3 0,7
(-) IFN-γ + TNF-α R848
0
10
20
30
% C
D8+ /
IFN
γ+ cel
ls
Mo-
DC (-)
R848
IFN
-γ T
NF-
(-)
R848 (-)
R848 (-
)
R848
M18 MV M18 UV M18 MV M18 UV
(+) Mo-DC (-) Mo-DC
IFN
-γ T
NF-
IFN
-γ T
NF-
IFN
-γ T
NF-
Deauvieau F et al, Int J Cancer, 2014
NYESO-1 cross-presentation by Mo-DC is increased by TNF-/IFN-
Conclusions
PreferedInfection
Limited infection
MV
Tumor cells(CD46high)
Healthy cells(CD46low)
Apoptosis
Immature Mo-DC
Immature plasmacytoid DC
MV-infectedTumor cells
Mature Mo-DC
Mature plasmacytoid DC
TAAPAMP?DAMP(HSP70, gp96)
TAAPAMP(MV Single-strand RNA)DAMP?
Phagocytosis
Phagocytosis
Maturation
Maturation
->Cross-priming of TAA-specific CD8+ T cell response
->Cross-presentation of TAA to specific CD8+ T cells
Cross-priming ?
->Production of a large quantity of IFN-
Gauvrit A et al, Cancer Research, 2008
Guillerme JB et al, Clinical Cancer Research, 2013
Donnelly OG et al, Gene Ther, 2013
(IFN-, IFN-,HMGB1, IL-6IL-8)
Blood myeloid DC ? CD1c+ DC ? CD141+ DC ?
Plateforme de dévelopement et de transfert à la clinique (CHU Nantes) :Delphine Coulais, ingénieurClarisse Panterne, ingénieur
Service de pneumologie du CHU de NantesLaurent Cellerin, Christine SaganAntoine Magnan
Service de pneumologie du CHU de LilleArnaud Scherpereel
Institut Pasteur, Viral genomics and Vaccination laboratory Frédéric TangyMariana Mesel-Lemoine
Collaborations
Centre de Recherche en cancérologie de LyonJenny ValladeauChristophe Caux
Marc Grégoire, INSERM Christophe Blanquart, CNRSDaniel Pouliquen, INSERMJean-François Fonteneau, INSERMNicolas BoisgeraultJean-Baptiste GuillermeCarole Achard, Ferdaous AllaguiIza DenisClarisse PanterneSophie Deshayes
Team (Inserm U892,Nantes)
Funding
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