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