Calcium Chloride as a Novel Stabilizer for Foot-and-Mouth Disease Virus and Its Application in the Vaccine Formulation

Abstract

The thermal stability of the in-house-developed foot-and-mouth disease (FMD) type O and A viruses was evaluated, and the O Jincheon virus was found to exhibit the lowest thermal stability. To overcome this instability, we proposed a novel stabilizer, calcium chloride. The thermal stability of FMDVs increased up to a CaCl₂ concentration of 10 mM, and it had a decreasing trend at >30 mM. The O Jincheon virus showed a significant decrease in the amount of antigen over time at 4 °C. In contrast, the samples treated with CaCl₂ showed stable preservation of the virus without significant antigen loss. After the CaCl₂-formulated vaccine was administered twice to pigs, the virus neutralization titer reached approximately 1:1000, suggesting that the vaccine could protect pigs against the FMDV challenge. In summary, the O Jincheon virus is difficult to utilize as a vaccine given its low stability during storage after antigen production. However, following its treatment with CaCl₂, it can be easily utilized as a vaccine. This study evaluated CaCl₂ as a novel stabilizer in FMD vaccines and may contribute to the development of stable vaccine formulations, especially for inherently unstable FMDV strains.

Keywords: calcium chloride; foot-and-mouth disease virus; stabilizer; storage; vaccine.

Figure 1

Analysis of thermal stability of FMD vaccine antigens. The thermal stability of eight strains of FMDVs was monitored by particle stability thermal release assay (PaSTRy). The viral RNA genome released from the capsid was detected by SYBR green II dye. The first derivative of fluorescence was plotted against an increase in temperature from 15 to 95 °C. The single melt peak indicates that RNA is being released, and the corresponding temperature was denoted as T_m.

Figure 2

Effect of excipients for cold chain storage of FMDV. Unformulated, SLA, and calcium-formulated FMDV O Jincheon 146S antigens were stored at 4 °C for 6 months. The amount of 146S particles was measured by high-performance liquid chromatography, and the recovery rate was expressed as a percentage (%). ****, p < 0.0001; ns, not significant. Abbreviations: NC, negative control (no excipients); SLA, 10% sucrose with 5% lactalbumin hydrolysate; Ca, 10 mM calcium chloride.

Figure 3

Immunogenicity of FMD vaccines containing stabilizing excipients. Antibodies elicited by immunization with FMD O Jincheon vaccines prepared by adding SLA (10% sucrose with 5% lactalbumin hydrolysate) and calcium (10 mM). (a) Schematic representation of the animal experiment. Six pigs per group were vaccinated twice at 4-week intervals, and blood samples were collected weekly. (b) Enzyme-linked immunosorbent assay (ELISA) was used to measure the antibody titers of the structural protein (SP). The antibody values were expressed as percent inhibition (PI) values. A PI value of ≥50% (dotted line) was considered to indicate a positive result. (c) The homologous virus-neutralizing (VN) antibody titers were measured using the virus neutralization test. A titer ≥1:45 (1.65 log₁₀) (dotted line) was considered to indicate a positive result. Abbreviations: O JC, O Jincheon virus; SLA, 10% sucrose with 5% lactalbumin hydrolysate; Ca, 10 mM calcium chloride; ns, not significant.