Evaluation of Recombinant Foot-and-Mouth Disease SAT2 Vaccine Strain in Terms of Antigen Productivity, Virus Inactivation Kinetics, and Immunogenicity in Pigs for Domestic Antigen Bank

Abstract

Background: Since the massive outbreak of foot-and-mouth disease (FMD) in South Korea in 2010-2011, cloven-hoofed livestock have been immunized with serotype O and A vaccines across the country. Other serotypes of FMD vaccines were stockpiled in overseas FMD vaccine factories as antigen banks. Once a manufacturing facility has been established in South Korea, the overseas antigen banks will be replaced by domestic one. Therefore, this study aimed to evaluate the commercial potential of the previously developed SAT2 vaccine candidate (SAT2 ZIM-R).

Methods: The optimal condition was determined at various virus concentrations, infection times, and pH levels, resulting in 0.01 MOI for SAT2 ZIM-R for 24 h infection at a pH of 7.5.

Results: When the SAT2 ZIM-R virus was produced in flasks from 40 to 1000 mL in fivefold increments, all scales of production yielded > 7.0 µg/mL of antigens. Using a bioreactor, 5.6 µg/mL of antigens was recovered from a 1 L viral culture. The optimal conditions of viral inactivation kinetics were determined to be 1 mM of binary ethyleneimine (BEI) treatment at 26 °C for 24 h, with approximately 91% of the antigen being retained after virus inactivation. When the SAT2 ZIM-R experimental vaccine was administered twice to pigs, the neutralizing antibody titer increased approximately 500-fold after booster immunization.

Conclusions: To the best of our knowledge, this is the first study to evaluate the antigen productivity, viral inactivation kinetics, and immunogenicity of the SAT vaccine strain in pigs. In the future, the SAT2 ZIM-R vaccine may be a useful candidate vaccine for a domestic antigen bank.

Keywords: SAT 2; antigen bank; antigen productivity; foot-and-mouth disease; inactivation kinetics.

Figure 1

Optimization of antigen production conditions at flask scale. Antigen productivity and viral titer were evaluated based on virus concentration and infection duration. Viral titer is represented by blue bars, and red bars correspond to antigen yield. Data are shown as means ± standard deviations. Statistical comparisons were performed using unpaired Student’s t-test (**** p < 0.0001).

Figure 2

Determination of optimal pH for SAT2 ZIM-R antigen production. Antigen productivity was evaluated based on the medium pH during the virus inoculation phase. Data are shown as means ± standard deviations. Statistical comparisons were performed using unpaired Student’s t-test (**** p < 0.0001).

Figure 3

Evaluation of antigen productivity across different production scales. SAT2 ZIM-R antigen was produced by sequentially increasing the culture volume in 5-fold increments with antigen productivity and titer measured at each stage. Viral titer is represented by blue bars, and red bars correspond to antigen productivity. Data are shown as means ± standard deviations. Statistical comparisons were performed using unpaired Student’s t-test (ns = not significant; *** p < 0.001).

Figure 4

Inactivation kinetics of SAT2 ZIM-R. After inoculating SAT2 ZIM-R into BHK-21 cells in a suspension, the supernatant was treated with various binary ethyleneimine (BEI) concentrations. Samples were collected hourly up to 6 h at 26 °C (A) and 37 °C (B). Linear extrapolation of individual graphs was performed to analyze the time to decrease viral titers to −7 log10 TCID₅₀/mL, which is indicated by a horizontal dotted line.

Figure 5

Virus neutralization (VN) titers (A) and SAT2 SP ELISA results (percent inhibition (PI%)) (B) in pigs following immunization with SAT2 ZIM-R vaccine. VN tests were performed using sera collected weekly from pigs that received two doses of SAT2 ZIM-R vaccine at 4 week intervals. Vaccine antigens were produced using both flask and bioreactor systems. Data are presented as means ± standard deviations. PI values more than 70% (dotted line) were considered positive for sP antibodies. VN titers greater than 1.65 log (dotted line) were considered positive. Gray circles represent titers for antigens produced in a shaking culture flask, and black circles represent titers for antigens produced in a bioreactor. Statistical comparisons were performed using unpaired Student’s t-test (ns = not significant; * p < 0.05; ** p < 0.01).

Figure 5

Virus neutralization (VN) titers (A) and SAT2 SP ELISA results (percent inhibition (PI%)) (B) in pigs following immunization with SAT2 ZIM-R vaccine. VN tests were performed using sera collected weekly from pigs that received two doses of SAT2 ZIM-R vaccine at 4 week intervals. Vaccine antigens were produced using both flask and bioreactor systems. Data are presented as means ± standard deviations. PI values more than 70% (dotted line) were considered positive for sP antibodies. VN titers greater than 1.65 log (dotted line) were considered positive. Gray circles represent titers for antigens produced in a shaking culture flask, and black circles represent titers for antigens produced in a bioreactor. Statistical comparisons were performed using unpaired Student’s t-test (ns = not significant; * p < 0.05; ** p < 0.01).