Department of Biotechnology University of Malakand Potency assessment of FMD vaccine using standardized serological assays Syed M. Jamal Department of Biotechnology University of Malakand Pakistan

Potency test for FMD vaccine European Pharmacopoeia Challenge protection studies in cattle Determination of PD50 Limitations of Potency test: Varying outcome Disease security hazards Costly One valency in any given trial Animal welfare

Serological based methods Alternatives to challenge-protection test provided that a correlation is established between antibody titre and protection Advantages: 1. No logistic problems 2. No disease security hazards 3. Relatively in-expensive 4. More than one valency at given time 5. Simultaneous testing of several vaccines

Studies on correlation between antibody titre and protection Mackowiak et al., 1962 van Bekkum, 1970 Sutmoller and Vieira, 1980 Pay and Hingley, 1987 van Maanen and Terpstra, 1989 Amadori et al., 1991 Pay and Hingley, 1992a,b Dus Santos et al., 2000 Barnett et al., 2003

Test results often vary considerably between laboratories Correlation between antibody titre and level of protection accepted worldwide Test results often vary considerably between laboratories difference between route and dose of challenge virus the condition of cattle the interval between vaccination and challenge the type of serological test sensitivity of cells used in neutralization test

Objectives Standardization of antibody titres against O/Manisa for determination of vaccine potency using: A standardized commercial PrioCHECK FMDV type O ELISA Inclusion of a standard 4 week post-vaccination serum from a cow vaccinated with O/Manisa FMD vaccine in both the ELISA and VNT The idea was to evaluate how serology to determine protection can be standardised; by standardising the test (all using the same ELISA kit) or by introducing a standard serum, irrespective of the test used. The latter is more promising, although differences still remain, but that might also be due to differences in vaccines used, vaccination challenge interval (4 weeks in the Netherlands, but 3 weeks in the other potency tests).

Materials and Methods Serum samples from 18 potency tests performed at: Belgium (n=10) The Netherlands (n=6) The United Kingdom (n=2) Standard control serum (4 week post-vaccination serum from O/Manisa vaccinated cow) Serum samples were tested in respective laboratories using: Neutralization test ELISA (PrioCHECK) Unit/standardized antibody titres determined by subtracting the log titre of a standard control serum from that of the test serum Results were analysed using logistic regression Serum samples collected on the day of challenge. Cell used in Pirbright=IBRS-2 Cells used in Ukkal, BHK Cells used in Lelystad=Primary Porcine kidney cells

RESULTS: Antibody titres v/s protection Significant difference in antibody titres among laboratories The slope of the curve steeper in VNT compared to ELISA ---------- Pirbright ---------- Ukkal Lelystad ---------- Pirbright ---------- Ukkal Lelystad

Standardized/Units antibody titres v/s protection Unit antibody titres reduced variation among laboratories BUT Significant difference still present ---------- Pirbright ---------- Ukkal Lelystad ---------- Pirbright ---------- Ukkal Lelystad

Conclusions Inclusion of the standard serum reduced variation between the laboratories. However, significant differences were still present, which could be due to differences in vaccine composition. Inclusion of a standard serum is a good way to make results between laboratories more comparable. Cattle vaccinated with 1/3rd dose of a standard FMD vaccine (containing 3 PD50) should have antibody titre at least as high as that of a standard serum representing 50% protection.

Acknowledgements CVI-lelystad, the Netherlands CODA-CERVA, Belgium Pirbright Institute, United Kingdom Thanks to the EuFMD for supporting my participation in the OS18