Scale-Up Production of Type O and A Foot-and-Mouth Disease Bivalent Vaccine and Its Protective Efficacy in Pigs

Figures

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  Figure 1. Optimization of the conditions for antigen production in a flask scale. (a) Comparison of cell growth curve during cultivation of BHK-21 suspension cell for 3 days in different volumes. Viable cell concentration (filled symbols) and cell viability (empty symbols) were estimated by Vi-cell RX at 0, 1, 2, and 3 days. Determination of optimal virus concentration and harvest time for (b) O/Boeun/SKR/2017 and (c) A/Yeoncheon/SKR/2017. BHK-21 suspension cells were grown up to 3 × 106 cells/mL for 3 days, inoculated with viruses at different MOIs. The samples were collected at 8 h or 12 h to 24 h post-infection. The data shown are representative of three independent experiments and are presented as the mean ± standard deviations (SD). The asterisks represent the statistical differences: *, p < 0.05; **, p < 0.005.

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  Figure 2. Results of antigen production in a 100 L bioreactor. (a,b) Contents of 146S antigen and virus titer from virus supernatant produced by a 100 L bioreactor. (c,d) Virus inactivation kinetics after binary ethyleneimine (BEI) treatment. The virus culture supernatant was treated with 3 mM BEI twice (at 0 h and 16 h) and incubated until 24 h. Samples were taken every hour from the bioreactor for 6 h to measure virus inactivation kinetics for each strain of the virus. The extrapolation of the individual graph is drawn as a linear line for the analysis of FMDV inactivation. (e) Inactivated 146S antigen was concentrated with PEG, purified using sucrose gradient ultracentrifugation, and observed by transmission electron microscope (TEM).

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  Figure 3. Virus neutralizing antibody titers against homologous and heterologous viruses in guinea pigs after immunization with the bivalent vaccine. Virus neutralization tests for O/Boeun/SKR/2017 (a) and A/Yeoncheon/SKR/2017 (b) were performed with sera collected from guinea pig vaccinated with two different volumes (1 mL and 2 mL) of the bivalent vaccine until 84 days post-vaccination (dpv). (c) Virus neutralization test for the serotype O heterologous viruses was performed with sera collected 28 dpv from guinea pig vaccinated with 1 mL of the bivalent vaccine. (d) Virus neutralization test for the serotype A heterologous virus was performed with sera collected 28 dpv from guinea pig vaccinated with 1 mL of the bivalent vaccine. The dotted line represents 1:45 (1.65 log10) of virus neutralizing antibody titer that is regarded to be positive according to the OIE terrestrial manual. The data shown are representative of three independent experiments and are presented as the mean ± SD values. The asterisks represent the statistical differences: **, p < 0.005.

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  Figure 4. Virus neutralizing antibody titers against serotype O homologous virus in pigs after immunization with the bivalent vaccine. Pigs (n = 18) were divided into four groups (full dose, 1/3 dose, 1/9 dose, control) and blood samples were collected at 0, 7, 14, 21, and 28 days post-vaccination. Pigs were challenged with O/Boeun/SKR/2017 at 28 dpv with 1 × 105 TCID50/0.1 mL in the bulb of the heel. Blood samples were collected until 7 days post-challenge. Virus neutralization test with O/Boeun/SKR/2017 was performed using the sera collected from vaccinated pigs (five heads per group) with full dose (a), 1/3 dose (b), and 1/9 dose (c). VN titers of the sera were collected from three heads of non-vaccinated pigs as the control (d). Black arrows indicate the challenge time at 28 dpv.

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  Figure 5. Clinical scores and virus shedding in pigs immunized with a bivalent vaccine after O/Boeun/SKR/2017 challenge. Oral swab and blood samples were collected daily from the pigs (n = 18) that were vaccinated with the bivalent vaccine in the full dose (a), 1/3 dose (b), 1/9 dose (c), and control (d) groups until 7 days post-challenge. Clinical symptoms were examined daily after the virus challenge. The FMDV RNA was identified by extraction of viral RNA from oral swab samples and quantitative real-time polymerase chain reaction (RT-PCR).

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  Figure 6. Virus neutralizing antibody titers against serotype A homologous virus in pigs after immunization with the bivalent vaccine. Pigs (n = 18) were vaccinated with full dose (a), 1/3 dose (b), 1/9 dose (c), control (d) and blood samples were collected and challenged with A/Yeoncheon/SKR/2017 at 28 dpv with 1 × 105 TCID50/0.1 mL in the bulb of the heel. Moreover, a virus neutralization test with A/Yeoncheon/SKR/2017 was performed as described in Figure 4. Black arrows indicate the challenge time at 28 dpv.

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  Figure 7. Clinical scores and virus shedding in pigs immunized with a bivalent vaccine after the A/Yeoncheon/SKR/2017 challenge. Oral swab and blood samples were collected daily from the pigs (n = 18) that were vaccinated with the bivalent vaccine in the full dose (a), 1/3 dose (b), 1/9 dose (c), and control (d–f) groups until 7 days post-challenge. Clinical symptoms were examined daily after the virus challenge. The foot-and-mouth disease virus (FMDV) RNA was identified by the extraction of viral RNA from oral swab samples and quantitative real-time polymerase chain reaction (RT-PCR).