Explanation on generic manufacturing process of foot and mouth disease vaccines

1. Generic manufacturing process
of Foot & Mouth Disease
Vaccines
Lim Chloe (0338728)
Tan Poh Hui (0339404)
Wai Kai Wen (0338205)
Woo Xue Rou (0335280)

2. Introduction
● Foot and mouth disease (FMD) is a highly infectious, and often lethal, viral disease
affecting cloven-hound animals, both domestic and wild animals.
● Foot and mouth disease viruses (FMDVs) are a species within the genus Aphthovirus of
the family Picornaviridae.
○ Highly variable - limits the effectiveness of vaccination
○ Develops and mutates continuously - making vaccination difficult
● Seven serotypes of FMDVs: O, A, C, SAT-1, SAT-2, SAT-3, and Asia-1
○ no cross-protection between serotypes
○ Strains from given genotype may have nucleotide sequences that vary by as much
as 30%
○ FMD vaccines must be highly specific to each strain
● First FMD vaccines
○ genetically engineered in the US in 1981
○ killed virus preparations grown in mammalian cell culture

3. FMD vaccine process flow
● Manufacturing is performed in a biosecure environment
● Process for the manufacture of FMD vaccines from raw materials averages 10 weeks,
including all necessary regulatory testing prior to release to the market

4. 3. Clarification
● Remove cell debris and recover virus
● Done at 4 °C.
● Can be divided into:
○ Primary Clarification
■ Zonal centrifugation
■ Normal flow filter
○ Secondary Clarification
■ The Polysep™ II with Durapore® 0.65 μm
■ Cellulosic pad filters
■ Durapore® 0.45 μm (bioburden reduction filter)

5. 4. Virus Inactivation
● Formaldehyde inactivation carries a risk of residual contamination of inactivated
vaccines with live virus.
● 3% binary ethyleneimine (BEI) were used for FMDV inactivation
● binary ethyleneimine (BEI) + formaldehyde (FA) can inactivate FMD rapidly
○ Prepared by binary ethyleneimine, formaldehyde, μ-naphtol violet solution and
NaOH
○ BEI-FA may not enter some parts of the tank (dip tubes)
■ Contamination of inactivated virus antigen due to the inactivated virus
○ Inactivation mixture is transferred into the second inactivation tank that
contain BEI only after 4 hours
● Monitor the inactivation and verification of the correct shape of the regression line
● Inactivation is stopped by the addition of 2% sodium thiosulphate (neutralize BEI)

6. 5. Ultrafiltration
Tangential flow filtration (TFF)
● Removal of inactivating agent by diafiltration &
concentration of virus
● 10-15X concentration and 5-8N diafiltration is
performed.
● Diafiltration buffer:
a. Sodium phosphate buffer [137018 and 137036]
(pH 7.6)
b. glycol buffer (pH 8.7)
● FMD virus antigens in the banks are stored at ultra-
low temperatures (usually -130 °C) to guarantee a
shelf life of at least five years

7. 6. Chromatography (optional)
● FMD antigen bank/FMD marker vaccine:
- differentiate the immune response of a vaccinated animal from a naturally infected
animal
- complete removal of low molecular weight non-structural proteins (NSP) is
required for vaccine purification
● industrial scale chromatography is used in order to produce highly purified, highly
concentrated, inactivated antigens by removing extraneous proteins (including NSPs)
● Antigen will be stored in the gaseous phase of liquid nitrogen until it is needed
7. Sterile Filtration
● Typical final sterilizing grade filter: 0.22 µm Durapore®

8. 8. Formulation
● Typically formulated as quadrivalent inactivated antigens of O, A, C & Asia1
strains
○ Oil or saponin and aluminium hydroxide gel is added to increase the immune
response
● Can be formulated as combination vaccines with other vaccines
○ E.g. haemorrhagic septicaemia & black quarter vaccines.
● Additions: antifoam, phenol red dye (if permitted by the country, lactalbumin
hydrolysate, tryptose phosphate broth, antibiotics, amino acids, vitamins and
buffer salts.
● 2-8 °C is maintained for vaccine distribution
○ To avoid vaccine deterioration
● Emergency FMD vaccines are formulated to a higher potency
○ To create an immune zone and protect the animals within the area
○ To reduce the quantity of virus spread within the suspected infected area

9. 9. FMD Vaccine Analytics
● Vaccine efficacy is dependent on FMD virus particles integrity
● Quantification of the active ingredient of vaccines:
○ 146S quantitative sucrose density gradient analysis
○ size-exclusion chromatography & measurement of virus by absorption at 254
nm. (during manufacturing process)

10. Current advances
● Improvement in techniques for purifying viral capsids and removing NSPs from
conventional inactivated virus preparations (Uttenthal et al., 2010)
● Development of infectious copy-based master seed vaccine strains into which novel
capsid genes could be rapidly inserted (Blignaut et al., 2010)
● Adenovirus-vectored vaccines delivering interferons or FMDV capsid proteins, co-
expressed with the viral protease required for their processing, have been shown to
provide rapid-onset protection against FMD in pigs and cattle (Grubman, 2005)
● Baculovirus-derived virus-like particles (VLPs) are also highly immunogenic (Li et al., 2011)
and offer advantages of safe production and freedom from non-structural viral proteins
(NSPs)

11. Potential challenges
● Existence of seven serotypes of FMDV that are not cross-protective, with the possible
exception of animals sequentially infected with multiple serotypes
● Experimental vaccines have requirement for multiple doses
○ repeated vaccination with a viral-vector vaccine leads to immunity against the vector and
reduced protection. (Rodriguez and Gay, 2011)(Grubman et al., 2010)
● imperfect antigenic match between the field virus and vaccine strain
○ disease may not be prevented if the vaccine strain is antigenically distinct from the field virus.
(Kitching et al., 2006)
● Vaccines are unstable outside range of 2-8 ◦C
○ Making their effective application in the tropics difficult (Kitching et al., 2006)

12. References
Blignaut, B., Visser, N., Theron, J., Rieder, E. and Maree, F., 2010. Custom-engineered chimeric foot-and-mouth disease vaccine elicits protective
immune responses in pigs. Journal of General Virology, 92(4), pp.849-859.
Grubman, M., 2005. Development of novel strategies to control foot-and-mouth disease: Marker vaccines and antivirals. Biologicals, 33(4), pp.227-
234.
Grubman, M., Moraes, M., Schutta, C., Barrera, J., Neilan, J., Ettyreddy, D., Butman, B., Brough, D. and Brake, D., 2010. Adenovirus serotype 5-vectored
foot-and-mouth disease subunit vaccines: the first decade. Future Virology, 5(1), pp.51-64.
Kitching, P., Hammond, J., Jeggo, M., Charleston, B., Paton, D., Rodriguez, L. and Heckert, R., 2007. Global FMD control—is it an option?. Vaccine,
25(30), pp.5660-5664.
Li, Z., Yin, X., Yi, Y., Li, X., Li, B., Lan, X., Zhang, Z. and Liu, J., 2011. FMD subunit vaccine produced using a silkworm–baculovirus expression system:
Protective efficacy against two type Asia1 isolates in cattle. Veterinary Microbiology, 149(1-2), pp.99-103.
Panina, G., Ahl, R., Amadori, M., Barteling, S., De Clercq, K., Donaldson, A., Have, P. and Marangon, S., 1999. Strategy for Emergency Vaccination
against Foot and Mouth Disease (FMD). [online] Available at: <https://ec.europa.eu/food/sites/food/files/safety/docs/sci-com_scah_out22_en.pdf>
[Accessed 7 July 2020].
Rodriguez, L. and Gay, C., 2011. Development of vaccines toward the global control and eradication of foot-and-mouth disease. Expert Review of
Vaccines, 10(3), pp.377-387.
Uttenthal, Å., Parida, S., Rasmussen, T., Paton, D., Haas, B. and Dundon, W., 2010. Strategies for differentiating infection in vaccinated animals (DIVA)
for foot-and-mouth disease, classical swine fever and avian influenza. Expert Review of Vaccines, 9(1), pp.73-87.