noël tordo
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Noël TORDO
CANINE ADENOVIRUS-BASED VACCINES AGAINST RABIES
working together to stop the ongoing tragedy of rabies!
Institut Pasteur, Paris, FranceUnit Antiviral Strategies
Corinne JALLETNoël TORDO
INRA-AFSSA-ENVA Maisons-Alfort, France UMR1161 - Virologie
Marion SZELECHOWSKIAnnie FOURNIER Bernard KLONJKOWSKIMarc ELOIT
previous papers…
G PV
E10
E3+E1+
left ITR right ITR
E30E10
E1+ E3+
GFP
Replicative Cav2Cav-G R+
Non Replic. Cav2Cav-G R0
Non Replic. HumanAd5-G R0
Replicative Cav2Cav-GFP R+
Wild-type Cav2
Recombinant canine adenovirus (Cav2)
E3+E1+
G PV
E3+
G PV
Ad5-G R0Cav2-G R+Cav2-G R0 Cav2
Ad5G R0
Cav2G R+
Cav2G R0
Cav2Mpurif.G NI
Data.004
100 101 102 103 104
FL1-Height
M1M2
Data.002
100 101 102 103 104
FL1-Height
M1M2
Data.006
100 101 102 103 104
FL1-Height
M1
M2
Data.008
100 101 102 103 104
FL1-Height
M1
M295.68 %531.80
92.44 %396
2.08 %21.26
99.51 %1162.35
Ad5-G R0Cav-G R+Cav-G R0 Cav2
Expression of recombinant Gpv in CHO-CAR cells (24h; 10 TCID50)
Western blotPolyclonal G4846
IF MAb G D1
99.51 %1162.35Flow
cytometry MAb G D1
0 24 48 72 9610 0
10 1
10 2
10 3
10 4
10 5
10 6
10 7
10 8
10 9
10 10
To
tal I
nfe
ctio
us
Un
its/
ml
Hours
DK
DK-E1
293
CRFK
Multiplication of CaV2-derived viruses in various cell lignes (MOI = 1)
DK
0 24 48 72 9610 0
10 1
10 2
10 3
10 4
10 5
10 6
10 7
10 8
10 9
10 10
Hours
To
tal I
nfe
ctio
us
Un
its/
ml
Cav2-GFP R+
Cav2-G R+
Cav2
Hours
DK-E1
To
tal I
nfe
ctio
us
Un
its/
ml
0 24 48 72 9610 0
10 1
10 2
10 3
10 4
10 5
10 6
10 7
10 8
10 9
10 10
Cav2-GFP R+
Cav2-G R+
Cav2
Cav2
dog cells
other cells
I.M. immunization (tibialis muscle) 107 TCID50 Cav2 in 50 l / mouse 27 day (VNAbs) 48 days (VNAbs + T cell response)
Immunogenicity of recombinant Cav2 in C57BL/6 mice by IM route (107 TCID50 )
0
1
2
3
4
5
Pro
lifer
ativ
e In
dex
1
10
100
1000
UI/m
l
Ad5-G R0
Virus Neutralizing Antibodies (VNA) Proliferative Index (G protein)
Cav-G R0 Cav-G R+ Cav2 Ad5-G R0 Cav-G R0 Cav-G R+ Cav2
Immunogenicity of recombinant Cav2 by IM route (107 TCID50 )
individual results
D27 D48
VNA GMT RangeVNA GMT
Range
Ad5-GR0 316,77 101-546 194,35 76-504
Cav-G R0 58,23 47,6-72,3 46,18 23,1-72,4
Cav-G R+ 56,33 44,2-68,9 68,27 52-78
Cav2 0 0
D48
Proliferative index
Range
Ad5-G0 2,23 1,5-3
Cav-G R0 1,94 1,1-2,7
Cav-G R+ 1,98 1,7-4,6
Cav2 1
Immunogenicity of recombinant Cav2 by IM route (107 TCID50 )
Virus Neutralizing Antibodies (VNA) Proliferative Index (G protein)
21 day (VNAbs) 25 day (challenge)
Protection of recombinant Cav2IM (7.106 TCID50 ) or oral (7.107 TCID50 ) routes
7.106 TCID50
7.107 TCID50
Vaccination RouteVNA GMT
(IU)Range Survivors
Cav-G R+ IM 45,28 25-83 9/9
Cav-G R+ oral 1,64 0,38-7,5 7/9
Cav-GFP R+
IM 0 0/5
Cav-GFP R+
oral 0 0/5
Control 0 0/3
0
20
40
60
80
100
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
days post challenge
% s
urv
ival
Cav-G R+ (I.M)
Cav-G R+ (oral)
Cav-GFP R+ (I.M)
Cav-GFP R+ (oral)
Control
Protection of recombinant Cav2IM (7.106 TCID50 ) or oral (7.107 TCID50 ) routes
IM Cav-G R+
OR Cav-G R+
IM Cav-GFP R+
OR Cav-GFP R+
Control
Virus Neutralizing Antibodies (day 21) Challenge (day 25)
Experiments in dogs in progress…
Collaboration with the AFSSA NancyFlorence CLIQUETJacques BARRAT
CONCLUSIONS
•.
Cav2 grow well in canine cell lines and produce significant quantities of recombinant rabies G protein
•.
Canine adenovirus (Cav2) -derived viruses (replicative and non replicative) expressing the rabies (PV strain) G protein have been produced
•.
Cav2 are immunogenic (humoral and cellular responses) by both IM and oral routes
•. Dog experiments are in progress…
•.
IM and oral vaccination of mice with replicative Cav2 G viruses protect them against a peripheral challenge
Cav2 G viruses are promising tools for oral vaccination of dogs
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