A. DEKKER, N. GORIS, S.M. JAMAL, Y. LI

The Global control of FMD - Tools, ideas and ideals – Erice, Italy 14-17 October 2008

Appendix 75

THE RELATION ANTIBODY AND PROTECTION AFTER FOOT-AND-MOUTH DISEASE VACCINATION CANNOT BE STANDARDISED

A. Dekker1*, N. Goris2, S.M. Jamal

3, Y. Li

4

1Central Veterinary Institute of Wageningen UR, P.O. Box 65, 8200 AB Lelystad, The Netherlands

2Epizootic Diseases Section, Virology Department, Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180 Brussels, Belgium.

3National Veterinary Laboratory, Park Road, 45500, Islamabad, Pakistan

4Institute for Animal Health (IAH), Ash Road, Pirbright, Surrey GU24 0NF, UK

1. INTRODUCTION

Many studies have been performed into the relation antibody response and protection after footand-mouth disease (FMD) vaccination (Black et al. 1984; Pay et al. 1987; Pay et al. 1992). In every study a correlation was observed with the antibodies induced by the vaccine used in the study and protection observed after challenge. In the studies the methodology was never the same, e.g. the type of cells used in the virus neutralisation test (VNT), some studies were using ELISA and the statistical analysis. For countries not in the position to perform challenge experiments a standardised relation between antibody response after FMD vaccination and protection would be very useful. In the current study we try to standardise antibody level detection for FMD type O Manisa in two different ways, first by using a standardised commercial type PrioCHECK® FMDV Type O ELISA and secondly by inclusion of a standard 4 week post vaccination serum from a cow vaccinated with Cedivac® O Manisa FMD vaccine in both the ELISA and the VNT.

2. MATERIALS AND METHODS

2.1 Data cattle potency tests Sera were available from 6 O Manisa potency tests performed in Lelystad (The Netherlands). One of these potency tests were performed with Al(OH)3 adjuvanted vaccine in which the cattle were challenged 3 weeks after vaccination as described in the European Pharmacopoeia. In the other 5 potency tests double oil adjuvanted vaccines were tested, and because the antibody response to oil emulsion vaccines is a bit slower it was decided to challenge the cattle 4 weeks after vaccination. Sera were available from 10 O Manisa potency performed by the Belgium national laboratory (Brussels) at the animal facilities of FGI-ARRIAH using the same batch of a double oil emulsion O Manisa vaccine (Goris et al. 2007). One set of sera from a potency test performed in Pirbright. Serological tests: Sera were titrated for this study in the national reference laboratory that had performed the potency tests, both in the PrioCHECK® FMDV Type O ELISA and the VNT. In each test a titration of a standard serum was included. The units of antibody were calculated by subtracting the 10log titre of the positive control from the observed titre. Statistical analysis: Titres of the control serum were compared using ANOVA followed by a pairwise comparison using Tukey honest significant difference. Serological responses were fitted by logistic regression. In each analysis the contribution of the laboratory performing the tests was included if this resulted in a better fitting model. The latter was tested by the likelihood ratio test. All statistical analyses were performed in R (www.r-project.org).

3. RESULTS

Significant differences were found in the titre of the control serum obtained in the various laboratories, in both the ELISA and the VNT. In the ELISA significant (p<0.01) lower titres in Belgium (titre = 1.72 10log) were found in comparison with the titre found in the Netherlands (titre

The Global control of FMD- Tools, ideas and ideals – Erice, Italy 14-17 October 2008 = 1.82 10log). In the VNT the titre found in Pirbright was significantly (p<0.01) different from the result in both other laboratories. The observed mean titres are given in Table 1. Only a small difference in the mean titre found in protected and non-protected cattle, this is especially true for the ELISA titres found in Brussels. In both the ELISA and the VNT always a very significant (p<0.01) influence of the location was found on the relation between antibodies and protection, but the slope of the relation between antibodies and protection was the same in each laboratory. In the ELISA the slope of the relation between antibody concentration and protection was lower when the titres of the sera collected at the day of challenge were analysed in comparison with the titres found at the day of challenge (for experiments performed by Brussels and Pirbright cattle were challenged at three weeks, so the titre at three weeks and the day of challenge were the same). The slope of the relation between antibodies and protection was steeper when analysing the results obtained in the VNT in comparison with the results obtained in the ELISA. Table 1: Results obtained in various laboratories

Number of PD50 exp Number of vaccinated cattle Percentage protected cattle All cattle Brussels Lelystad Pirbright 10 6 1 150 90 15 72 60 67

Mean ELISA titre 3 weeks post vaccination 1.6 1.9 1.6 Mean Units of antibodies by ELISA 3 weeks post-vaccination -0.1 0.1 -0.2 Mean ELISA titre at challenge 1.6 2.1 1.6 Mean Units of antibodies by ELISA at challenge -0.1 0.3 -0.2 Mean VNT titre 3 weeks post vaccination 1.7 1.7 1.3 Mean Units of antibodies by VNT 3 weeks post-vaccination 0.4 0.3 -1.2 Mean VNT titre at challenge 1.7 1.9 1.3 Mean Units of antibodies by VNT at challenge 0.4 0.5 -1.2 Non-Protected cattle Mean ELISA titre 3 weeks post vaccination 1.4 1.6 1.2 Mean Units of antibodies by ELISA 3 weeks post-vaccination -0.3 -0.3 -0.5 Mean ELISA titre at challenge 1.4 1.8 1.2 Mean Units of antibodies by ELISA at challenge -0.3 0 -0.5 Mean VNT titre 3 weeks post vaccination 1.4 1.4 1.0 Mean Units of antibodies by VNT 3 weeks post-vaccination 0.1 0 -1.5 Mean VNT titre at challenge 1.4 1.5 1.0 Mean Units of antibodies by VNT at challenge 0.1 0.1 -1.5 Protected cattle Mean ELISA titre 3 weeks post vaccination 1.6 2.1 1.6 Mean Units of antibodies by ELISA 3 weeks post-vaccination 0 0.3 -0.1 Mean ELISA titre at challenge 1.6 2.1 1.6 Mean Units of antibodies by ELISA at challenge 0 0.4 -0.1 Mean VNT titre 3 weeks post vaccination 1.7 1.9 1.5 Mean Units of antibodies by VNT 3 weeks post-vaccination 0.4 0.6 -1.1 Mean VNT titre at challenge 1.7 2.0 1.5 Mean Units of antibodies by VNT at challenge 0.4 0.6 -1.1

Figure 1 shows the relation found between antibody titre in the VNT at three weeks post vaccination and protection. The maximum difference between the curves at the 50% point is 0.55, so close to a 4 fold difference in titre. When correcting the titre by using the titre of the control serum we get the curves shown in Figure 2. The distance between the laboratory in Brussels and Lelystad becomes smaller (0.17), but the difference between Pirbright and the other two laboratories becomes much bigger. A similar result is obtained when using the antibody titres and

The Global control of FMD - Tools, ideas and ideals – Erice, Italy 14-17 October 2008 units obtained by ELISA, although the difference between the laboratories is then smaller the maximum difference at the 50% point is still 0.6.

4. DISCUSSION

The current study was set up to standardise the serology for FMD type O Manisa and the relation with protection, either by a standard test (PrioCHECK® FMDV Type O ELISA), by using a standard control serum to calculate units of antibody or by using both. The analysis shows a significant difference between laboratories. There are several explanations possible for this phenomenon, differences in challenge dose in the different laboratories, differences in match between challenge virus and vaccine virus, the difference in time of challenge, differences in adjuvant used and the difference in 140/12 S composition of the vaccine. In a recent comparison between pigs vaccinated intramuscularly and intradermally there was also a significant difference between both application methods and the relation between neutralising antibody titre and protection against virus shedding detected by mouth swabbing (Eblé non published data). The relation between antibody response and protection was valid within a laboratory. In each laboratory only vaccines from one producer had been tested, so producers can determine the relation between antibodies and protection for their own vaccine but that it is not possible to produce a more general estimate of this relation.

5. CONCLUSIONS

Standardisation of serological tests relating protection to antibody response is not yet possible Producers should set-up their own relation between antibody response and protection

6. RECOMMENDATIONS

More data from more sources should be analysed to finalise the conclusion Mathematical optimisation studies should be performed to provide guidance on how producers most effectively can relate antibody response to protection

Protection 0.0 0.2 0.4 0.6 0.8 1.0

0.0 0.5 1.0 1.5 2.0 2.5

Log VNT antibody titre

Figure 1: Comparison of protection and the VNT titre of sera collected 3 weeks post vaccination. In solid the results of the laboratory in the Netherlands, dashed the results obtained in Brussels and dotted the results obtained in Pirbright.

The Global control of FMD- Tools, ideas and ideals – Erice, Italy 14-17 October 2008

Protection 0.0 0.2 0.4 0.6 0.8 1.0

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0

Log VNT units

Figure 2: Comparison of protection and the VNT units of sera collected 3 weeks post vaccination. In solid the results of the laboratory in the Netherlands, dashed the results obtained in Brussels and dotted the results obtained in Pirbright. It would be interesting to analyse more data from more laboratories performing O Manisa vaccination and challenge experiments.

7. REFERENCES

[1] Black, L., Francis, M. J., et al. (1984). "The relationship between serum antibody titres and protection from foot-and-mouth disease in pigs after oil emulsion vaccination." J Biol Stand 12(4): 379-89. [2] Goris, N., Merkelbach-Peters, P., et al. (2007). "European Pharmacopoeia foot-and-mouth disease vaccine potency testing in cattle: between test variability and its consequences." Vaccine 25(17): 3373-9. [3] Pay, T. W. F. and 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. [4] Pay, T. W. F. and Hingley, P. J. (1992). "Foot-and-mouth disease vaccine potency tests in cattle: the interrelationship of antigen dose, serum neutralizing antibody response and protection from challenge." Vaccine 10 (10): 699-706.