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. 2024 Feb 23;12(3):231.
doi: 10.3390/vaccines12030231.

Calcium Chloride Treatment Enhances Antigen Production in Foot-and-Mouth Disease Vaccines for Serotypes SAT1 and SAT3

Dohyun Kim  1 , Sun Young Park  1 , Gyeongmin Lee  1 , Eun-Sol Kim  1 , Jong-Sook Jin  1 , Jae Young Kim  1 , SooAh Lee  1 , Jong-Hyeon Park  1 , Young-Joon Ko  1
Affiliations

Calcium Chloride Treatment Enhances Antigen Production in Foot-and-Mouth Disease Vaccines for Serotypes SAT1 and SAT3

Dohyun Kim et al. Vaccines (Basel). .

Abstract

Foot-and-mouth disease (FMD) is a highly contagious viral infection causing acute and severe vesicular lesions in cattle and pigs, which has prompted global vaccination policies. This study presents a technique for enhancing antigen yield in SAT1 BOT and SAT3 ZIM by treatment with calcium chloride (CaCl2). We tested changes in cell viability in BHK-21 suspension cells treated with varying concentrations of CaCl2. The optimal CaCl2 concentration was determined based on antigen yield. The timing of CaCl2 supplementation relative to FMD virus inoculation was tested. Finally, the optimal medium for antigen production was identified. We observed a concentration-dependent decrease in BHK-21 cell viability at >7.5 mM CaCl2. A CaCl2 concentration of 3 mM yielded the most antigens. CaCl2 supplementation relative to FMD virus infection was optimal 2 h before or with viral inoculation. CD-BHK 21 medium supplemented with CaCl2 was the most productive medium. Specifically, SAT1 BOT and SAT3 ZIM showed improved antigen production in CD-BHK 21 medium with 3 mM CaCl2, while Provero-1 and Cellvento BHK-200 media showed no significant enhancement. Overall, CaCl2 supplementation enhanced FMD antigen productivity. This study provides a useful framework for enhancing antigen production efficiently in the FMD vaccine industry.

Keywords: SAT; antigen; calcium; foot-and-mouth disease virus; medium.

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Conflict of interest statement

The authors declare no conflicts of interests.

Figures

Figure 1
Figure 1
Cytotoxicity of CaCl2 in BHK-21 suspension cell line for FMD vaccine antigen production. BHK-21 cells were treated with 60, 30, 15, 7.5, 3.75, 1.8, and 0.7 mM calcium chloride and cultured for 24 h. Cell viability was observed using Vi-CELL XR Cell viability analyzer system. Each experiment was conducted three times, and the error bars indicate standard deviations (SDs) from the mean. n.s., non-significant; **** p < 0.001; unpaired t-test.
Figure 2
Figure 2
Comparison of SAT1 BOT virial titer and antigen productivity at various CaCl2 concentrations. BHK-21 suspension cells were treated with 30, 10, 3, and 1 mM CaCl2 simultaneously (MOI: 0.01) and cultured for 24 h at 37 °C. The virial titer (A) and antigen productivity (B) were measured by virus titration and size-exclusion high-performance liquid chromatography (SE-HPLC). Each experiment was conducted three times, and the error bars indicate SDs from the mean. n.s., non-significant; * p < 0.05, *** p < 0.005, **** p < 0.001; unpaired t-test.
Figure 3
Figure 3
Comparison of SAT3 ZIM virial titer and antigen productivity at various CaCl2 concentrations. BHK-21 suspension cells were treated with 30, 10, 3, and 1 mM CaCl2 simultaneously (MOI: 0.01) and cultured for 24 h at 37 °C. The virial titer (A) and antigen productivity (B) were measured by virus titration and SE-HPLC. Each experiment was conducted three times, and the error bars indicate SDs from the mean. n.s., non-significant; * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001; unpaired t-test.
Figure 4
Figure 4
Antigen production in SAT1 BOT according to the timing of CaCl2 addition. The BHK-21 suspension cells were treated with 3 mM calcium chloride at 2 h before FMDV infection (MOI: 0.01), at FMDV infection, 2 hpi, and 24 hpi. The virial titer (A) and antigen productivity (B) were determined in the culture supernatant after 24 h. Each experiment was conducted three times, and the error bars indicate SDs from the mean. n.s., not significant; ** p < 0.01, **** p < 0.001; unpaired t-test.
Figure 5
Figure 5
Antigen production in SAT3 ZIM according to the timing of CaCl2 addition. The BHK-21 suspension cells were treated with 3 mM calcium chloride at 2 h before FMDV infection (MOI: 0.01), at FMDV infection, 2 hpi, and 24 hpi. The virial titer (A) and antigen productivity (B) were determined in the culture supernatant after 24 h. Each experiment was conducted three times, and the error bars indicate SDs from the mean. n.s., non-significant; * p < 0.05, **** p < 0.001; unpaired t-test.
Figure 6
Figure 6
Effect of CaCl2 addition on SAT1 BOT virus titer and antigen production in various media. Three mM of CaCl2 was added at 0 hpi with SAT1 BOT in 30 mL of CD-BHK 21, Provero-1, or Cellvento medium. White bars indicate viral titer. Black bars indicate FMD vaccine antigen content measured using SE-HPLC. Each experiment was conducted three times, and the error bars indicate SDs from the mean. ND, not detected, * p < 0.05, **** p < 0.001; unpaired t-test.
Figure 7
Figure 7
Effect of CaCl2 addition on SAT3 ZIM virus titer and antigen production in various media. Three mM of CaCl2 was added at 0 hpi with SAT3 ZIM in 30 mL of CD-BHK 21, Provero-1, or Cellvento medium. White bars indicate viral titer. Black bars indicate FMD vaccine antigen content measured using SE-HPLC. Each experiment was conducted three times, and the error bars indicate SDs from the mean. ND, not detected, ** p < 0.01, **** p < 0.001; unpaired t-test.
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