1. Julian Seago Improving quality assurance of FMD vaccine production

2. Intact capsids (146S) are required to generate neutralising antibodies FMDV is both pH and temperature labile. Serotype differences: A and Asia1 are relatively more stable than O and SAT serotypes. Chemical inactivation renders the FMDV capsid less stable WHY MONITOR FMDV CAPSID STABILITY?

3. Thermofluor assay: a simple, fast method to analyse capsid stability Minimal requirements: qPCR machine Fluorescent dye that binds RNA genome – SYBR green II Virus sample Methodology: As temperature increases the FMDV capsid dissociates and releases the viral genome (T R = release temperature), which is bound by the Fluorescent dye. THERMOFLUOR ANALYSIS

4. -1052 = 53.5 o C 53.5 o C T R = Inflection point of slope T R = Most negative Raw fluorescence signal Neg. derivative fluorescence signal THERMOFLUOR ANALYSIS

5. POTENTIAL APPLICATIONS FOR THERMOFLUOR Analysis of stabilising mutations Screening solutions for their ability to stabilise capsids Comparing stability of different strains Monitoring stability during vaccine production Can all be used to produce better vaccines ……….

6. O1M Stabilised FMDV capsids

7. QUALITY CONTROL DURING FMD VACCINE PRODUCTION 1 2 3

8. Purified FMDV VP1 VP2 & 3 VP4 SIMPLIFY SAMPLE PREPARATION Unpurified FMDV

9. THERMOFLUOR ANALYSIS USING LIVE VIRUS

10. THERMOFLUOR ANALYSIS

11. THERMOFLUOR ANALYSIS USING INACTIVATED VIRUS

12. THERMOFLUOR ANALYSIS USING FORMULATED VACCINE ISA 206 produces a double emulsion vaccine

13. THERMOFLUOR ANALYSIS OF STORED VACCINE

14. STORAGE OF UNFORMULATED VACCINE (O K 77/78)

15. STORAGE OF ISA 206 FORMULATED VACCINE (O K 77/78)

16. STORAGE OF ISA 201 FORMULATED VACCINE (O K 77/78)

17. SUMMARY The thermofluor assay can be used to assess stability of non- purified infectious virus, as well as unformulated and formulated inactivated virus (vaccine). The thermofluor assay offers a new tool for quality assurance at different stages of FMD vaccine production.

18. The Pirbright Institute Bryan Charleston Eva Perez Valérie Mioulet Nick Lyons Veronica Carr ACKNOWLEDGEMENTS