Download PPT 21

Vaccinia
FMDV
Pirbright
Compton
Use of Modified Vaccinia Ankara
(MVA) for FMDV vaccination
Javier Castillo-Olivares, David Paton, Bryan Charleston, Satya Parida
Institute for Animal Health
Vaccinia
FMDV
FMDV Vaccines
• Inactivated whole virus vaccine – drawbacks
– Serotype and even sub-type specific immunity – need to match
circulating strains with vaccine strains
– Sterile immunity difficult to achieve probably due to limited
range of effector mechanisms of immunity they provide
– Serological discrimination between vaccinated and infected
animals is still problematic
– Duration and rapid onset of immunity can be improved
Objective
•
Primary goal: To enhance the efficacy of FMDV inactivated vaccines by
increasing the range of effector mechanisms of immunity (i.e. Cytotoxic T
lymphocyte) and / or providing additional T helper epitopes
•
Generate recombinant MVA viruses expressing FMDV antigens (P1 and
3CD) and co-administer with conventional FMDV vaccine.
•
Rationale:
– Poxviruses extensively used as antigen delivery vectors
– MVA is highly safe as does not replicate well in mammalian cells
– Molecular biology very well known (completely sequenced-easy to manipulate)
– Expression of FMDV antigens within host cells via MVA-FMDV infection favour
antigen presentation to T cells through MHC-I potentially increasing capacity to
stimulate cell-mediated immune responses (CTL’s)
Strategy - Generation of FMDV inserts
From viral RNA of FMDV infected cell cultures
•
BTY cells infected with FMDV A22 (Iraq64)
•
RNA extracted from supernatants (Trizol)
•
One-step RT-PCR with P1 or 3CD specific
primers (Sma I)
•
Re-amplify by Hi-Fi PCR
•
Sma I digest and clone in SmaI site pSC11
Strategy – Generation of recombinant MVA
GOI – FMDV (P1, 3CD)
p11
p7.5
SmaI
TKR
lacZ
-The gene of interest is
inserted downstream of P7.5
- Expression cassette (also
encoding LacZ) flanked by
MVA DNA sequences
-Insertion in MVA genome by
homologous recombination
pSC11
7883 bp
- MVA infected cells
transfected with pSC11-GOI
pUC9
TKL
TK L
LacZ
- Recombinant MVA are TK
negative and lacZ positive
(blue plaques – plaque assay)
P11 P7.5 GOI
TK R
Standard Plaque assay of semipure MVA-P1 clones
clones
31b3
1.1
2.1
10-1
10-2
dilutions
10-3
10-3.3
XGal sensitive staining: High titre stocks of recombinant virus – approx 2x109/ml
Characterisation of recombinant MVA P1
• Transcription of P1 by RT-PCR from RNA from
MVAP1 infected cells
• Expression by immunofluorescence and Western
blotting
– Avian cells: CEF, DF-1
– Bovine cells:
• Bovine kidney
• Bovine dermal fibroblasts
• Bovine dendritic cells (PBMC derived IL-4 + GM-CSF)
– P815 (mouse mastocytoma) transfected with bovine
MHC-I
XGal sensitive staining CEF 24 h p.i. MVA-P1 (7b21b-1 p2) - - moi 1
MVA
MVAP1
Detection of P1 RNA in MVAP1 infected cells – 24 hours p.i.
One-step RTPCR: SIII transcriptase + High fidelity Taq DNA polymerase
1
2
3
4
5 MWM
1: RNA MVA infected cells
2: RNA MVAP1 infected cells
3: RNA Mock infected cells
4: DNA pSC11-P1
5: water
1
2
3 MWM
1: RNA MVAP1 infected cells + DNAse
2: pSC11-P1 + DNAse
3: pSC11-P1 no DNAse
Expression of P1 in MVAP1 infected chicken fibroblasts at 48 h p.i.
Neg
0.1
0.001
1
0.01
10
Expression of P1 in MVAP1 infected Bovine skin fibroblasts at 48 h p.i.
Neg
m.o.i. 10
m.o.i. 1
m.o.i. 10
m.o.i. 1
m.o.i. 10
P815 infected with MVA-P1 7B21B-1 p2 – XGal staining 2 hours
P815 infected with MVA-P1 7b21b-1p2 – XGal sensitive stain
moi 10
moi 0.1
moi 1
moi 0.01
Expression of P1 from MVAP1 infected cells – Western blot of cell lysates
24 h p.i.
BK cells
MWM
MVAP1
MVA
48 h p.i.
CEF
MVAP1
BK cells
MVA
MVAP1
MVA
CEF
MVAP1
MVA
P1
76
52
38
31
24
17
12
P1
Effect of pSC11-3CD transfection on expression of P1 from MVAP1 infected cells
Western blot of cell lysates
MVAP1
pSC113CD No plasmid
76
P1
52
38
31
VP0
24
VP1
VP3
17
12
MVA
pSC113CD
No plasmid
No virus
pSC113CD
No plasmid
Conclusions
• Recombinant MVA-FMDV can be produced at high titres
• MVAP1 expresses P1 CEF > P815 > BSF = BDC > BK
• P1 can be cleaved by 3CD co-expressed from P7.5
vaccinia promoter
Short term plans
• Does MVAP1 vaccination provide protection?
• Does MVA3CD vaccination provide protection?
• Can MVA-P1-3C(D) be generated?
– Can we use it for in vitro production of FMDV capsids?
– Can we use it as a vaccine?
• MVA vaccines:
– on their own, combination, prime-boost regimes
Acknowledgements
DEFRA
IAH Pirbright
Satya Parida, David Paton, Bryan Charleston, Aravindh Babu,
Miriam Windsor, Nigel Ferris and Geoff Pero
IAH Compton
Gillian Hill, Efrain Guzman