Production of Foot-and-Mouth Disease Type O and A Vaccine Antigens on a Pilot Scale and Determination of Optimal Amount of Antigen for Monovalent Vaccines

Abstract

Foot-and-mouth disease (FMD) is a highly infectious disease affecting cloven-hoofed animals and causes significant economic losses to the livestock industry. The Type O PanAsia-2 (O PA-2) vaccine strain is protective against a wide range of serotype O FMD virus (FMDV) strains in East Asia, and A22 Iraq/24/64 (A22 IRQ) is the most widely used vaccine strain in FMD vaccine antigen banks. The aim of this study was to produce antigens from O PA-2 and A22 IRQ viruses using a 100 L bioreactor and evaluate the protective efficacy of varying antigen concentrations in pigs. More than 2 μg/mL of the antigen was recovered from the O PA-2 and A22 IRQ virus-infected supernatants. Further, inactivation of O PA-2 and A22 IRQ by binary ethyleneimine revealed that the viral titers decreased below 10^-7 TCID₅₀/mL within 13 h and 9 h, respectively. The O PA-2 and A22 IRQ vaccines, containing 10 μg and 5 μg of antigen, respectively, provided protection against homologous viruses in pigs. This is the first report demonstrating that the antigens obtained from the pilot-scale production of O PA-2 and A22 IRQ are viable candidate vaccines. These results will pave the way for industrial-scale FMD vaccine production in South Korea.

Keywords: FMDV; monovalent; scale-up; vaccine.

Figure 1

Antigen production was carried out in a 100 L bioreactor. (a,b) The quantity of 146S antigen and virus titer in the supernatant resulting from virus infection in the bioreactor. (c,d) The virus inactivation kinetics after treatment with binary ethyleneimine (BEI) was also examined. The supernatant obtained from virus infection was subjected to two treatments with 3 mM BEI (at 0 h and 16 h) and allowed to incubate for a total of 24 h. Samples were collected hourly from 0 to 6 h to determine the kinetics of virus inactivation for each virus strain. The resulting data were extrapolated and plotted as a linear line to analyze the inactivation kinetics of FMDV. (e,f) The inactivated 146S antigen was concentrated through PEG precipitation, purified via sucrose gradient ultracentrifugation, and observed using a transmission electron microscope (TEM).

Figure 2

The titers of virus neutralization in pigs following immunization with the O PA-2 vaccine. VN tests were conducted on sera collected from pigs (5 pigs per group except for negative control with 2 pigs) that received trial vaccines containing four different antigen payloads of 20 µg (a), 15 µg (b), 10 µg (c), and negative control (d) up to 28 days post vaccination (dpv). Blood samples were obtained on days 0, 7, 14, 21, and 28 dpv. At 28 dpv, the pigs were exposed to O PA-2 with a viral dose of 1 × 10⁵ TCID₅₀/0.1 mL in the heel bulb. The red arrows indicate the virus challenge introduced at 28 dpv. The dotted line represents the VN titer of 1:45 (1.65 log₁₀), which is considered positive according to the guidelines in the WOAH terrestrial manual [12].

Figure 3

The virus-neutralizing titers observed in pigs after immunization with the A22 IRQ vaccine. The pigs (n = 17) were divided into four groups (5 pigs per group except for negative control with 2 pigs), receiving doses of 15 µg (a), 10 µg (b), and 5 µg (c) and negative control (d). Blood samples were collected at various time points: 0, 7, 14, 21, and 28 days post vaccination. At 28 days post vaccination, the pigs were challenged with A22 IRQ, administered at a viral dose of 1 × 10⁵ TCID₅₀/0.1 mL in the heel bulb. Blood samples were further collected up to 7 days post challenge. The red arrows indicate the virus challenge introduced at 28 days post vaccination. The dotted line on the graph represents the VN titer threshold of 1:45 (1.65 log₁₀), as deemed positive in accordance with the guidelines presented in the WOAH terrestrial manual [12].