1.
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

2.
Open Session of the EuFMD - Cascais –Portugal 26-28 October 2016
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

3.
OS16
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

4.
VP2 VP3 VP1 2B 2C 3A 3C 3D
VP4 2A
P1-2A
VP0 VP3 VP1
VP4 VP2 VP3 VP1
Virus particle
Empty capsid particle
Encapsidation of RNA
(Empty capsid particle proteins)
(Virus particle proteins)
VP0
Lab
Lb
3B1-3
FMDV particle formation
Belsham & Bøtner, 2015
FMDV

5.
OS16
rSFV-FMDV design
FMDV cDNA SFV vector

6.
OS16
SFV split helper system
Expression of
foreign protein
Smerdou and Liljeström, 1999
Vaccine

7.
OS16
Expression of FMDV capsid proteins by rSFV-FMDV vectors
Abs
450nm
Antigen ELISA
WB
Sucrose
gradient
Gullberg et al., 2016

8.
OS16
Group 1 (control)
0 5 10 15 20 25 30
0
25
50
75
100
C1
C2
PVD
ODP(%)
0 5 10 15 20 25 30
37
38
39
40
41
42 C1
C2
Group 1 (control)
PVD
Temp
(
o
C)
Group 1 (control)
5 10 15 20 25 30
100
101
102
103
104
105
106
107
108
109 C1
C2
PVD
RNA
copies/ml
Group 2 (rSFV-P1-2A)
0 5 10 15 20 25 30
0
25
50
75
100 C3
C4
C5
PVD
ODP(%)
Group 2 (rSFV-FMDV-P1-2A)
0 5 10 15 20 25 30
37
38
39
40
41
42
C3
C4
C5
PVD
Temp
(
o
C)
Group 2 (rSFV-P1-2A)
5 10 15 20 25 30
100
101
102
103
104
105
106
107
108
109
C3
C4
C5
PVD
RNA
copies/ml
Group 3 (rSFV-P1-2A-mIRES-3C)
0 5 10 15 20 25 30
0
25
50
75
100 C6
C7
C8
PVD
ODP(%)
Group 3 (rSFV-FMDV-P1-2A-mIRES-3C)
0 5 10 15 20 25 30
37
38
39
40
41
42
C6
C7
C8
PVD
Temp
(
o
C)
Group 3 (rSFV-P1-2A-mIRES-3C)
0 5 10 15 20 25 30
100
101
102
103
104
105
106
107
108
109
C6
C7
C8
PVD
RNA
copies/ml
A B C
Single inoculation of cattle with rSFV-FMDV vectors and FMDV challenge
FMD
FMD
FMD
Control
rSFV-P1-2A
rSFV-P1-2A-mIRES3C
Outcome
Ab ELISA Temperature Viremia

9.
OS16
Group Animal PVD 14 PVD 21 PVD 27 PVD 30
1 Control C1 - - - 20
1 C2 - - - 20
2 rSFV-P1-2A C3 - - 320 320
2 C4 - - 320 320
2 C5 - 5 >640 320
3 rSFV-P1-2A-mIRES-3C C6 5 10 320 320
3 C7 - - 320 320
3 C8 - - 320 320
Anti-FMDV antibody titres (ELISA)
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

10.
OS16
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
Prime-boost strategy

11.
OS16
Group 1
0 5 10 15 20 25 30 35 40
10- 1
100
101
102
103
104
105
106
107
108
109
101 0
C1
C2
C3
PVD
RNA
copies/ml
Group 1
0 5 10 15 20 25 30 35 40
37
38
39
40
41
42
C1
C2
C3
PVD
Temp
(
o
C)
Group 2
0 5 10 15 20 25 30 35 40
10- 1
100
101
102
103
104
105
106
107
108
109
101 0 C4
C5
C6
PVD
RNA
copies/ml
Group 2
0 5 10 15 20 25 30 35 40
37
38
39
40
41
42 C4
C5
C6
PVD
Temp
(
o
C)
Group 3
0 5 10 15 20 25 30 35 40
10- 1
100
101
102
103
104
105
106
107
108
109
101 0
C7
C8
C9
PVD
RNA
copies/ml
Group 3
0 5 10 15 20 25 30 35 40
37
38
39
40
41
42
C7
C8
C9
PVD
Temp
(
o
C)
Temperature
Gullberg et al., 2016
Control
rSFV + ECs
ECs + rSFV
FMDV RNA
No disease
FMD
FMD
Protection against FMDV challenge

12.
OS16 Gullberg et al., 2016
Control
rSFV + EC
EC + rSFV
Ab ELISA
VNT titres
Anti-FMDV responses in cattle
Group 1
0 10 20 30 40
0
25
50
75
100
C1
C2
C3
PVD
ODP(%)
PVD 14
C
1
C
2
C
3
C
4
C
5
C
6
C
7
C
8
C
9
0
1
2
3
4
Calf
Log
10
titre
Group 2
0 10 20 30 40
0
25
50
75
100 C4
C5
C6
PVD
ODP(%)
PVD 28
C
1
C
2
C
3
C
4
C
5
C
6
C
7
C
8
C
9
0
1
2
3
4
Calf
Log10
titre
Group 3
0 10 20 30 40
0
25
50
75
100 C7
C8
C9
PVD
ODP(%)
PVD 36
C
1
C
2
C
3
C
4
C
5
C
6
C
7
C
8
C
9
0
1
2
3
4
Calf
Log10
titre
A
B
C
D
E
F
Pre-challenge
Post-challenge
Pre-boost
VNT titre

13.
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