A local virus isolate, O/SKR/JC/2014 (O JC), has been considered as a candidate vaccine strain in the development of a domestic foot-and-mouth disease (FMD) vaccine in Korea. However, producing and preserving a sufficient quantity of intact vaccine antigens from the O JC strain was difficult owing to its distinctive structural instability compared to other candidate vaccine strains. Based on this feature, the O JC strain was adopted as a model virus for the stabilization study to determine the optimal stabilizer composition, which enables long-term storage of the FMD vaccine antigen in both aqueous and frozen phases. In contrast to O JC vaccine antigens stored in routinely used Tris-buffered or phosphate-buffered saline, those stored in Tris-KCl buffer showed extended shelf-life at both 4 °C and −70 °C. Additionally, the combined application of 10% sucrose and 5% lactalbumin hydrolysate could protect O JC 146S particles from massive structural breakdown in an aqueous state for up to one year. The stabilizer composition was also effective for other FMDV strains, including serotypes A and Asia 1. With this stabilizer composition, FMD vaccine antigens could be flexibly preserved during the general production process, pending status under refrigeration and banking under ultrafreezing.

Following the worst outbreak of foot-and-mouth disease (FMD), a highly contagious disease in cloven-hoofed animals caused by the FMD virus, from November 2010–April 2011, the Korean government enforced a mandatory vaccination policy. A bivalent (FMD type O and A; O + A) vaccine has been recently implemented. Although the FMD outbreak was suppressed by vaccination, the intramuscular (IM) injection presents side effects. Therefore, improving FMD vaccine quality is necessary. Here, we investigated the side effects and immune efficacy of the O + A bivalent vaccine using two different routes of administration: intradermal (ID) and IM. To compare the immune efficacy of the two inoculation routes, virus neutralization titers and structural protein (antigen) levels were measured. The protective efficacy of ID vaccines was confirmed using two viruses (FMDV O/AS/SKR/2019 and A/GP/SKR/2018) isolated in the Republic of Korea. Serological analysis revealed that both animals administered by ID and IM injections exhibited equal immune efficacy. A virus challenge test in the target animal (swine) revealed no (or extremely low) clinical symptoms. Swine in the ID injected group exhibited no side effects. In conclusion, we suggest that the ID route of vaccination is an effective alternative to the existing IM route, which is associated with more frequent side effects.

South Korea has experienced FMD outbreaks almost every year since 2014. Therefore, a novel local vaccine that can cover various topotypes of viruses is required. Two virus strains, O/Boeun/SKR/2017 and A/Yeoncheon/SKR/2017, were cultured up to the pilot scale based on the optimized conditions set up on the flask scale. FMDV particles (146S) of 2 µg/mL or more were obtained from the virus culture supernatant using a 100 L bioreactor. The viruses were fully inactivated using binary ethylenimine within 16 h through two inactivation cycles and mixed with an adjuvant into a bivalent vaccine (types O and A) consisting of 15 µg viruses per strain. The experimental bivalent vaccine showed a broad spectrum of high neutralizing antibody titers against heterologous viruses, including type O Cathay strain and type A Asia topotypes, except for GVII. The 50% protective dose was determined as 12.5 for O/Boeun/SKR/2017 and 15.6 for A/Yeoncheon/SKR/2017. Collectively, we expect that the bivalent vaccine could protect against FMDV types O and A circulating in South Korea and neighboring countries. To our knowledge, this is the first report demonstrating that the vaccine strains could be successfully scaled-up to a 100 L bioreactor, with the determination of its protective efficacy in pigs.

In South Korea, a mandatory nation-wide foot-and-mouth disease (FMD) vaccination policy is in place. However, a major side effect of the current method of intramuscular (IM) administration of oil-adjuvanted FMD vaccines is the formation of granulomas in the muscles of pigs. To address this issue, we assessed the possible application of intradermal (ID) vaccination. Initially, we compared the serological immune response in specific pathogen-free pigs inoculated with FMD vaccines formulated with eight different adjuvants, administered twice at the neck site using a syringe with a needle via the ID route. Among the formulations (water-in-oil-in-water (W/O/W), oil-in-water (O/W), and polymer nanomaterials), ISA 207 of W/O/W was the most effective in inducing immunogenicity followed by ISA 201 of W/O/W. ISA 207 was further tested in formulations of different antigen doses (12 or 1.2 μg) delivered via both IM and ID routes. All four treatments successfully protected the pigs against FMD virus challenges. To assess the feasibility of the field application of the vaccines with ISA 207, we conducted ID vaccination of conventional pigs using a needle-free device, resulting in the detection of significant levels of neutralizing antibodies. ISA 207 was shown to be superior to ISA 201 in inducing immunogenicity via the ID route. In conclusion, ISA 207 could be a suitable adjuvant for ID vaccination in terms of vaccine efficacy for FMD, allowing for alternate use of ID vaccination and subsequent reduction in the incidences of granuloma formation in the field.

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that affects cloven-hoofed animals and causes severe economic losses in the livestock industry. Given that this high-risk pathogen has to be handled in a biosafety level (BSL)-3 facility for safety reasons and the limited availability of BSL-3 laboratories, experiments on FMDV call for more attention. Therefore, we aimed to develop an FMDV experimental model that can be handled in BSL-2 laboratories. The NanoBiT luciferase (Nano-luc) assay is a well-known assay for studying protein–protein interactions. To apply the NanoBiT split luciferase assay to the diagnosis and evaluation of FMD, we developed an inactivated HiBiT-tagged Asia1 Shamir FMDV (AS-HiBiT), a recombinant Asia1 shamir FMDV with HiBiT attached to the VP1 region of Asia1 shamir FMDV. In addition, we established LgBiT-expressing LF-BK cell lines, termed LgBit-LF-BK cells. It was confirmed that inactivated AS-HiBiT infected LgBiT-LF-BK cells and produced a luminescence signal by binding to the intracellular LgBiT of LgBiT-LF-BK cells. In addition, the luminescence signal became stronger as the number of LgBiT-LF-BK cells increased or the concentration of inactivated AS-HiBiT increased. Moreover, we confirmed that inactivated AS-HiBiT can detect seroconversion in sera positive for FMDV-neutralizing antibodies. This NanoBiT split luciferase assay system can be used for the diagnosis and evaluation of FMD and expanded to FMD-like virus models to facilitate the evaluation of FMDV vaccines and antibodies.

Although polyethylene glycol (PEG) application is the most widely used method in removing nonstructural proteins (NSPs) for foot-and-mouth disease (FMD) vaccine production, some NSPs remaining in the antigen could elicit antibodies against these proteins after repeated vaccinations in livestock. Therefore, the purpose of this study was to purify the FMD virus (FMDV) via affinity chromatography using a heparin ligand to remove most proteins, including NSPs. Chromatography showed an intact virus (146S) particle recovery of 70% or more for three different strains of serotype O FMDV (two locally isolated strains and one genetically modified strain). The experimental vaccine made with antigens eluted via heparin affinity chromatography elicited virus-neutralizing antibodies against homologous viruses but did not induce antibodies against NSPs even after five immunizations in goats; this indicated that the NSPs were effectively removed from the vaccine antigen. This method can then be used to produce a higher-quality vaccine compared with PEG application in terms of the purity of the FMD vaccine. Therefore, this result would be an important groundwork for advanced FMD vaccine manufacturing in the near future.