Determination of Optimal Antigen Yield and Virus Inactivation Conditions for the Production of the Candidate Foot-and-Mouth Disease Recombinant Vaccine Strain Asia1 Shamir-R in a Bioreactor

Abstract

Since the foot-and-mouth disease (FMD) outbreak in South Korea in 2010-2011, vaccination policies utilizing inactivated FMD vaccines composed of types O and A have been implemented nationwide. However, because type Asia1 occurred in North Korea in 2007 and intermittently in neighboring countries, the risk of type Asia1 introduction cannot be ruled out. This study evaluated the antigen yield and viral inactivation kinetics of the recombinant Asia1 Shamir vaccine strain (Asia1 Shamir-R). When Asia1 Shamir-R was proliferated in shaking flasks (1 L), a 2 L bioreactor (1 L), and a wave bioreactor (25 L), the antigen yields were 7.5 μg/mL, 5.2 μg/mL, and 3.8 μg/mL, respectively. The optimal FMDV inactivation conditions were 2 mM BEI at 26 °C and 1.0 mM BEI at 37 °C. There was no antigen loss due to BEI treatment, and only a decrease in antigen levels was observed during storage. The sera from pigs immunized with antigen derived from a bioreactor exhibited a neutralizing antibody titer of approximately 1/1000 against Asia1 Shamir and Asia1/MOG/05 viruses; therefore, Asia1 Shamir-R is expected to provide sufficient protection against both viruses. If an FMD vaccine production facility is established, this Asia1 Shamir-R can be employed for domestic antigen banks in South Korea.

Keywords: antigen; bioreactor; foot-and-mouth disease; type Asia1; vaccine.

Figure 1

Optimization of the conditions for antigen production on a flask scale. The antigen yield and viral titer were determined according to the virus infection time and concentration. Blue bars indicate the virial titer. Red bars indicate the antigen yield. The results are presented as the mean ± standard deviation. Statistical analysis was conducted using an unpaired t-test (ns, not significant, **** p < 0.0001).

Figure 2

Comparison of antigen yield according to production size. The above conditions were applied to produce the Asia1 Shamir-R antigen from the flask scale to a 2 L bioreactor and a wave bioreactor with an RM bag. Red bars indicate the antigen yield. Blue bars indicate the virial titer. The results are presented as the mean ± standard deviation. Statistical analysis was conducted using an unpaired t-test (ns, not significant, * p < 0.05, ** p < 0.01).

Figure 3

Inactivation kinetics of the Asia1 Shamir-R. The supernatant obtained after the Asia1 Shamir-R was inoculated in the BHK-21 suspension cells was inactivated by each binary ethyleneimine concentration, with samples taken hourly up to 6 and 24 h at 26 °C (A) and at 37 °C (B). The extrapolation of the individual graphs was drawn as a linear line for the analysis of FMDV inactivation kinetics.

Figure 4

Virus neutralization titers post immunization with the Asia1 Shamir-R vaccine. Virus neutralization tests against Asia1 Shamir and Asia1/MOG/05-R viruses were performed using sera collected weekly from pigs immunized twice with the Asia1 Shamir vaccine at 4-week intervals. Data are presented as the mean ± standard deviation. The dotted line indicates the 1.65 log VN titer. Gray bars indicate the VN titer against Asia1 Shamir. Black bars indicate the VN titer against Asia1/MOG/05-R. Statistical analysis was conducted using an unpaired t-test (ns, not significant, * p < 0.05, ** p < 0.01).