Session IV Challenge of vaccinated animals for evaluation of foot-and-mouth disease vaccines will always be essential when new strains are implemented in vaccine production. To replace, reduce and refine animal experiments, producers are using alternative tests for batch release of vaccine. Producers either check the antibody response in standardised tests after vaccination or rely on GMP production and the quantification of 146S antigen in the vaccine.

1. Replacement of FMD vaccine potency tests; what
are the options
Aldo Dekker

2.  Background
● Potency test for registration (PD50 and PGP)
 Relation protection for batch release (Replace, Reduce and Refine)
● VNT titre
● 146S content
 Small variation in determination 146S very small difference in
estimated protection level
 QC on formulated vaccines using 12S and or 146S ELISAs
Outline
2

3.  Coda tested 10 times the same vaccine
● 4.6 to 24 PD50/dose (logistic regression)
● Mean 15 PD50/dose (95% CI; 8 – 25)
 100 simulations 2 – 24 PD50/dose
 In PGP only 16/16 95% confidence of 75% prot
 >15 PD50/dose 95% confidence >3 PD50/dose
Inherent variation in potency tests
3
13.93
8.00
18.38
6.06
10.56
13.93
13.93
10.56
18.38
6.06
Min 6.06
Max 18.38
10 simulations

4. Antibody level and protection
 10 times more 146S, 2.5 times
higher VNT titre (0.4 log10)
 Ab predicts protection best
 Type O higher antibody titre
needed
 Titre 0.5 log10 higher to move
from 25% protection to 75%
protection
4
*Pay, T. W. F., Hingley, P. J. 1987. Correlation of 140S antigen dose with
the serum neutralizing antibody response and the level of
protection induced in cattle by foot-and-mouth disease vaccines.
Vaccine 5(1); 60-4.
0.5 1.0 1.5 2.0 2.5 3.0 3.5
0.0
0.2
0.4
0.6
0.8
1.0
VNT titre (log10 )
Protection

5. Amount of 146S and protection
 10 times more 146S, 2.5 times
higher VNT titre (0.4 log10)
 146S predicts protection
 Type O higher 146S needed
 188 times more 146S to move
from 25% protection to 75%
protection
 VNT titre better prediction
5
*Pay, T. W. F., Hingley, P. J. 1987. Correlation of 140S antigen dose with
the serum neutralizing antibody response and the level of
protection induced in cattle by foot-and-mouth disease vaccines.
Vaccine 5(1); 60-4.
0 2 4 6 8
0.0
0.2
0.4
0.6
0.8
1.0
146S dose (ng) (log10 )
Protection

6.  Small difference (0.5 log10) in VNT titre huge difference in protection
 Between laboratory variation testing the same sera is up to 1 log10
 Small differences in 146S content correlate with extreme small
differences in protection
146S determination of vaccine is preferred
 12S and 146S can be determined in formulated vaccine
VNT titre best predictor of protection
6

7.  Extract antigen from vaccine
 Test antigen
● Sucrose gradient analysis combined with (MAb) ELISA
● Quantitative MAb/VHH ELISA
● 12S ELISA for serotypes A, O and Asia1
● 146S ELISA
● Size exclusion chromatography or HPLC
In-vitro quantification in formulated vaccine
7

8.  Potency test for registration (95%
confidence should be >3 PD50/dose
 GMP production system to ensure
reproducible vaccines
 Formulation based on antigen dose
 QC using ELISA based techniques
in formulated vaccine (12S / 146S)
Conclusion
8

9. Semi quantitative comparison 146S/12S
 Isolation using buffer
(lower yield)
 Titration in 12S ELISA
 Black line non-heated
 Red line heated
 Increase due to 146S
 Estimated 36 PD50/dose
homologous
A/Asia/GVII vaccine
 A/IRN05 chal
● 2 PD50/dose
9
Figure 1: Graph of the average OD measurement of the non-heated (black) and the heated
(red) sample
Analysis of the data showed a 146S/12S ratio of 15.