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Julian Seago
Improving quality assurance of
FMD vaccine production
• 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?
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 (TR = release temperature), which is bound by the Fluorescent dye.
THERMOFLUOR ANALYSIS
-1052 = 53.5oC
53.5oC TR = Inflection point of slope
TR = Most negative
Raw fluorescence signal
Neg. derivative fluorescence signal
THERMOFLUOR ANALYSIS
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 ……….
O1M
Stabilised FMDV capsids
QUALITY CONTROL DURING FMDVACCINE PRODUCTION
1 2 3
Purified FMDV
VP1VP2 & 3
VP4
SIMPLIFY SAMPLE PREPARATION
Unpurified FMDV
THERMOFLUOR ANALYSIS USING LIVE VIRUS
THERMOFLUOR ANALYSIS
THERMOFLUOR ANALYSIS USING INACTIVATED VIRUS
THERMOFLUOR ANALYSIS USING FORMULATED VACCINE
ISA 206 produces a double emulsion vaccine
THERMOFLUOR ANALYSIS OF STORED VACCINE
STORAGE OF UNFORMULATED VACCINE (O K 77/78)
STORAGE OF ISA 206 FORMULATED VACCINE (O K 77/78)
STORAGE OF ISA 201 FORMULATED VACCINE (O K 77/78)
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.
The Pirbright Institute
Bryan Charleston
Eva Perez
Valérie Mioulet
Nick Lyons
Veronica Carr
ACKNOWLEDGEMENTS