1.
VACCINE EFFICACY OF FMD VIRUS-LIKE PARTICLES
PRODUCED BY THE BACULOVIRUS EXPRESSION SYSTEM
Erwin van den Born

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2
Acknowledgements
Pirbright Institute, UK
Bryan Charleston
Claudine Porta
Eva Perez
University of Reading, UK
Ian Jones
Silvia Loureiro
MSD Animal Health, Netherlands
Ruud Segers
Amaya Serrano García
Alexandra Jiménez-Melsió
Holger Hönemann
University of Oxford, UK
Dave Stuart
Abhay Kotecha
Elizabeth Fry
This work was supported by the Wellcome Trust grant number 101122/Z/13/Z

3.
Background

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There is a need for novel innovative FMD vaccines
• Vaccination is the only method available to control FMD in low
and middle income countries.
• Availability of vaccines is poor, most strikingly in Africa.
• Current ‘killed virus’ vaccines:
- High potency vaccines (6PD50) provide sufficient protection
- Live virus production in high containment facilities
- Require expensive cold chains to deliver effective products
Decivac FMD DOE

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There is a need for novel innovative FMD vaccines
Kotecha et al. (2015) Nat Struct Mol Biol

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Virus-like particles are promising alternative antigens
Virus-like particles (VLPs):
- If vaccines contain 75S particles, sufficient protection is anticipated
- Vaccine production in standard facilities (baculovirus expression)
- Stability/antigenicity can be improved (e.g. stabilizing mutations)
Jamal and Belsham (2013) Vet Res

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VLP production in baculovirus expression system
Porta et al. (2013) J Virol Met
A22 75S particles

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Capsid stabilization through mutations in VP2
93His 93Cys
O1Manisa VP2-S93Y O1Manisa VP2-S93F
Kotecha et al. (2015) Nat Struct Mol Biol
Stabilization of the
VP2-VP2 interface

10.
Production & characterization
of VLPs

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Optimization of VLP production
• Baculovirus expression system
• High yield of VLPs important
• Optimization parameters:
- Type of insect cells
- Type of baculovirus vector
- Time of harvest after infection
- Translation initiation site of expression cassette
- Codon optimization of expression cassette
- Harvest method
- Downstream process
• Yield of FMDV protein increased up to 102 fold

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14 20 27 32.5 36.5 40 42 cells A/IRN
Classic
1:20 dil
A/IRN
Classic
1:40 dil
Sucr%:
25
20
37
15
25
20
37
15
> <
VP0
VP1,2,3
kD
Analysis of VLPs by several techniques (A/IRN H93C)
25 kD
33 kD
p
p
p
pp
p
p
pp
p
p
∅AVG 27 nm
0.0
0.5
1.0
1.5
20 60 180 540 1620 48601458043740
OD(450nm)
Dilution
Harvest
0.0
0.5
1.0
1.5
20 60 180 540 1620 48601458043740
OD(450nm)
Dilution
Harvest & sucr cushion
ELISA
EM
Western blotting with cattle serum
Sucrose cushion centrifugation
VLP Classic
VP0
VP1,2,3

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Immunogenicity: virus neutralizing titres in guinea pigs
0.0
0.5
1.0
1.5
2.0
2.5
classic VLP
S93F
classic VLP
H93F
VLP
H93C
classic VLP
S93Y
classic VLP
K93Y
O A Asia1 SAT2
VNT (log10) Study design
- Vaccines contain fixed
volume of antigen
- 4 serotypes included
- 5 animals/group
- Blood sample at 4 wpv
- VN assay on serum
Cattle vaccination/challenge trial planned
Note: strain in VN assay not always
optimal for classic antigen and/or VLP

14.
Conclusions & Summary

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• Virus-like particles (i.e. 75S capsids) can be produced in the
baculovirus expression system
• Yield and quality of capsids were significantly improved
• Amino acid position 93 of VP2 can be mutated to stabilize the
capsids
• Virus neutralizing titres induced in guinea pigs by VLPs are
comparable to that of classic antigen
àVirus-like particles have the potential to be a commercially viable
alternative to conventional killed vaccines