A PRIME-BOOST VACCINATION STRATEGY IN CATTLE TO PREVENT SEROTYPE O FMDV INFECTION USING A “SINGLE-CYCLE” ALPHAVIRUS VECTOR AND EMPTY CAPSID PARTICLES Maria Gullberg1, Louise Lohse1, Anette Bøtner1, Gerald M. McInerney2, Alison Burman3, Terry Jackson3, Charlotta Polacek1 and Graham J. Belsham1 1DTU National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark 2Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden. 3The Pirbright Institute, Pirbright, Woking, Surrey, U.K

Summary ●Single cycle alphavirus vector (rSFV-FMDV) expresses FMDV empty capsid particles ● Single vaccination of cattle with rSFV-FMDV induces anti-FMDV antibodies but not protection ● Vaccination with rSFV-FMDV primes an anti-FMDV immune response ● Vaccination with rSFV-FMDV followed by empty capsids induces protection ● No viremia observed post-challenge in cattle

Why recombinant FMDV vaccines? Challenge: Current FMDV vaccines are not optimal Chemically inactivated virus has to be grown under high containment Short duration of immunity Aim: Develop an improved, safe, vaccine based on expression of self-assembling empty capsid particles within cells - No serotype cross protection - Short vaccine protection time - Vaccine stability - Airborne - Some animal species high shedders, poor indicator animals 2 "New FMDV vaccine approach" (aims: ... etc. as in this slide) 3

FMDV particle formation 3B1-3 Lab VP4 2A Lb VP2 VP3 VP1 2B 2C 3A 3C 3D FMDV VP0 (Empty capsid particle proteins) VP0 VP3 VP1 Encapsidation of RNA (Virus particle proteins) VP4 VP2 VP3 VP1 Empty capsid particle Virus particle Belsham & Bøtner, 2015

rSFV-FMDV design FMDV cDNA SFV vector

SFV split helper system Vaccine Expression of foreign protein Smerdou and Liljeström, 1999 6

Expression of FMDV capsid proteins by rSFV-FMDV vectors Antigen ELISA WB Abs 450nm Sucrose gradient Gullberg et al., 2016

Single inoculation of cattle with rSFV-FMDV vectors and FMDV challenge Ab ELISA Temperature Viremia Outcome Control FMD rSFV-P1-2A FMD rSFV-P1-2A-mIRES3C FMD

Anti-FMDV antibody titres (ELISA) Group Animal PVD 14 PVD 21 PVD 27 PVD 30 1 Control C1 - 20 1 C2 2 rSFV-P1-2A C3 320 2 C4 C5 5 >640 3 rSFV-P1-2A-mIRES-3C C6 10 3 C7 C8 Low titre anti-FMDV response induced by rSFV-FMDV alone. Much stronger anti-FMDV response post challenge (on PVD 21) in rSFV-FMDV inoculated animals Gullberg et al., 2016

Prime-boost strategy Use of: 1) rSFV-P1-2A-mIRES-3C 2) Purified ”empty capsid” (EC) particles (expressed from vaccinia virus vectors, Porta et al., 2013). Plan (3 groups) 1) Control (no inoculation) 2) Inoculate with rSFV-P1-2A-mIRES-3C (PVD 0), then boost with ECs (on PVD 14) 3) Inoculate with ECs (PVD 0), then boost with rSFV-P1-2A-mIRES-3C (on PVD 14) Challenge with FMDV on PVD 28

Protection against FMDV challenge FMDV RNA Temperature Control FMD rSFV + ECs No disease ECs + rSFV FMD Gullberg et al., 2016

Anti-FMDV responses in cattle Ab ELISA VNT titre VNT titres Pre-boost Control Pre-challenge rSFV + EC EC + rSFV Post-challenge Gullberg et al., 2016

Conclusions rSFV-FMDV vectors can express FMDV empty capsids in cells Single inoculation with rSFV-FMDVs induces low titre anti-FMDV responses BUT primes antibody induction Prime-boost strategy using rSFV-FMDV vector followed by ECs generates strong anti-FMDV response that blocks FMDV dissemination in cattle following challenge The prime-boost strategy using rSFV-FMDV vectors and ECs has properties at least as good as current vaccine and can be made outside of high containment Duration of immunity, minimum dose required etc. … to be determined