Use of Modified Vaccinia Ankara (MVA) for FMDV Vaccination Javier Castillo-Olivares , David Paton, Bryan Charleston, Satya Parida Institute for Animal Health

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) -The gene of interest is inserted downstream of P7.5

p p 11 7.5 - Expression cassette (also SmaI encoding LacZ) flanked by

TK R MVA DNA sequences lacZ

-Insertion in MVA genome by homologous recombination pSC11 7883 bp - MVA infected cells transfected with pSC11-GOI

pUC9 - Recombinant MVA are TK TK L negative and lacZ positive (blue plaques – plaque assay)

TK L LacZ P11 P7.5 GOI TK R clones Standard Plaque assay of semipure MVA-P1 clones

31b3

1.1

2.1

-1 -2 -3 -3.3 10 10 dilutions 10 10 XGal sensitive staining: High titre stocks of recombinant virus – approx 2x10 9/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 2 3 MWM

1: RNA MVA infected cells 1: RNA MVAP1 infected cells + DNAse 2: RNA MVAP1 infected cells 2: pSC11-P1 + DNAse 3: RNA Mock infected cells 3: pSC11-P1 no DNAse 4: DNA pSC11-P1 5: water Expression of P1 in MVAP1 infected chicken fibroblasts at 48 h p.i.

Neg 0.001 0.01

0.1 1 10 Expression of P1 in MVAP1 infected Bovine skin fibroblasts at 48 h p.i.

Neg m.o.i. 1 m.o.i. 1

m.o.i. 10 m.o.i. 10 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 1

moi 0.1 moi 0.01 Expression of P1 from MVAP1 infected cells – Western blot of cell lysates

24 h p.i. 48 h p.i. MWM BK cells CEF BK cells CEF MVAP1 MVA MVAP1 MVA MVAP1 MVA MVAP1 MVA -

P1 P1

38 31

12 Effect of pSC11-3CD transfection on expression of P1 from MVAP1 infected cells Western blot of cell lysates

MVAP1 MVA No virus

pSC113CDNo plasmid pSC113CD No plasmid pSC113CD No plasmid

76 P1

31 VP0

24 VP1 VP3

12 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