Background/Objectives: The efficacy of inactivated foot-and-mouth disease virus (FMDV) vaccines depends on the structural integrity of the 146S virions. However, instability of 146S antigens during vaccine manufacturing and storage can compromise vaccine quality. Despite its high immunogenicity, the Korean serotype O strain O Jincheon (O JC) exhibits poor physical stability. Methods: To enhance antigenic stability while preserving strain-specific antigenicity, we engineered a VP1-substituted recombinant virus, (R) O1 M-O JC_VP1, by integrating the VP1 coding region of O JC into the O1 Manisa (O1 M) backbone. Results: The resulting chimeric virus exhibited significantly improved capsid stability, as demonstrated by an increased melting temperature and enhanced resistance to thermal stress, chloroform exposure, and long-term storage. Importantly, the recombinant antigen maintained its immunogenicity and induced antibody responses comparable to those induced by the parental O JC strain in vaccinated pigs. Conclusions: These findings demonstrate that VP1-direct chimeric engineering can improve capsid stability without compromising antigenicity and provide a practical approach for developing a stable FMDV vaccine.

Background: Foot-and-mouth disease (FMD) is a highly contagious viral disease causing substantial economic losses in livestock. Conventional intramuscular (IM) vaccination is widely used for FMD control but may be associated with transient reductions in milk production. This study evaluated whether intradermal (ID) vaccination could induce comparable immune responses and protection while minimizing potential impacts on productivity. Methods: In cattle, the immunogenicity of an experimental bivalent ID vaccine was compared with that of a commercially available IM vaccine by measuring antibody responses for up to 140 days post-vaccination (dpv). Milk production was monitored for 7 dpv in a separate cohort of lactating cattle. Protective efficacy was evaluated in goats following ID vaccination and challenge with a 2025 Republic of Korea serotype O field isolate (O/ME-SA/Ind-2001). Results: ID vaccination induced humoral immune responses comparable to those elicited by IM vaccination throughout the 140-day observation period. Although no statistically significant difference in milk production was observed, the ID group showed a smaller numerical reduction in milk yield. Following viral challenge, ID-vaccinated goats developed rapid neutralizing antibody responses, exhibited no clinical signs, and showed lower viral RNA levels than unvaccinated controls. Conclusions: Intradermal vaccination induced long-lasting immune responses comparable to conventional IM vaccination, was associated with a smaller numerical reduction in milk yield, and protected goats against challenge with a contemporary FMDV field isolate. These findings provide preliminary evidence supporting the potential of ID vaccination as an alternative strategy for FMD control in ruminants and warrant further comparative studies with conventional IM vaccination.

Background: In the Republic of Korea, a bivalent foot-and-mouth disease (FMD) vaccine covering serotypes O and A is administered to livestock, while antigens for the other serotypes are stockpiled in overseas antigen banks. To achieve self-reliance in FMD vaccine production, various vaccine strains have been developed using in-house technology. Although SAT 3 has historically been confined largely to Africa, preparedness against this serotype remains necessary, as the possibility of its introduction into Korea cannot be completely excluded. Methods: In this regard, we evaluated the commercial potential of the SAT 3 ZIM-R vaccine strain by assessing antigen productivity, scalability, inactivation kinetics, and immunogenicity. Results: Supplementation with 3 mM Ca2+ markedly increased antigen yield compared with that obtained in the absence of calcium. Further optimization showed that antigen yield was highest at pH 8.0–8.5. During scale-up, antigen yield was maintained at 9.2–9.8 μg/mL in flask cultures and remained high at approximately 7.8 μg/mL in a bioreactor, demonstrating robust scalability. Treatment with 2 mM binary ethylenimine at 26 °C achieved complete inactivation within 24 h. Vaccines formulated with the SAT 3 ZIM-R antigen produced either in flasks or in a bioreactor induced comparable neutralizing antibody responses in pigs following both the primary and booster immunizations. Conclusions: Collectively, these findings indicate that SAT 3 ZIM-R is a promising vaccine candidate for large-scale vaccine antigen production and the future establishment of a domestic FMD antigen bank in Korea.

Background: In South Korea, foot-and-mouth disease (FMD), a highly contagious viral infection that affects cloven-hoofed animals, has led to the implementation of a bivalent FMD vaccination program. The current FMD vaccination strategy involves intramuscular (IM) administration to the shoulder region of the swine. However, this method is associated with adverse reactions at injection sites. Our previous studies have demonstrated that intradermal (ID) vaccination eliminates these side effects while maintaining immunogenicity comparable to that of IM vaccination. This study aimed to assess the early immune response induced by ID vaccination and compare its protective ability against FMDV serotype O with that of a commercial IM vaccine recently used in South Korea. Methods: An ID FMD vaccine was evaluated using two adjuvants, ISA 207 (50%) and EMULSIGEN-D (15%). Virus neutralization (VN) titers and structural protein levels were measured to compare efficacy across groups. To assess the early protective efficacy of ID vaccination, viral challenge experiments were conducted at 7 and 14 days post-vaccination (dpv). Results: Swine vaccinated via the ID route exhibited no clinical symptoms at 14 dpv, indicating effective early protection against FMD (O/AS/SKR/2019). In addition, no side effects of FMD ID vaccination were observed. Conclusions: These results suggest that ID vaccination could serve as a viable alternative to conventional IM vaccination, which is frequently associated with adverse effects. Importantly, this study demonstrates that ID vaccination can provide effective early protection within 7-14 days post-vaccination, highlighting its potential utility for emergency outbreak control.

The Republic of Korea has implemented routine vaccination against foot-and-mouth disease virus (FMDV) in livestock using a bivalent vaccine comprising serotypes O and A following the massive FMD outbreak in 2010, while antigens for the remaining serotypes are maintained in overseas antigen banks. The recent geographic expansion of FMDV Southern African Territories 1 (SAT1) beyond Africa underscores the need for enhanced preparedness in previously unaffected regions. In this study, we evaluated the SAT1 BOT-R strain as a candidate vaccine seed for potential domestic vaccine production by optimizing antigen production conditions, assessing scalability, determining virus inactivation parameters, and examining immunogenicity in pigs. Optimal antigen yield was achieved at 20 h−24 h post infection with a multiplicity of infection of 0.005−0.01, with production remaining stable under mildly alkaline conditions. Antigen productivity was consistently maintained during scale-up from shake flasks to a bioreactor, yielding up to 9.5 μg/mL. Complete virus inactivation was achieved using binary ethylenimine at 2 mM for 24 h at 26 °C. Vaccines formulated from both flask- and bioreactor-derived antigens elicited comparable neutralizing antibody responses in pigs, reaching a median titer of 1:500 following booster immunization. Collectively, these findings demonstrate that the SAT1 BOT-R strain is a viable and scalable candidate for SAT1 antigen banking and future domestic vaccine production, providing a practical framework for strengthening national preparedness against potential incursions of FMDV SAT1.

Background/Objectives: Foot-and-mouth disease (FMD) is a highly contagious class 1 animal disease that affects cloven-hoofed animals, such as cattle, pigs, and goats. Diagnosis and research on live FMD virus (FMDV) typically require biosafety level 3 facilities, which are challenging to maintain due to strict protocols and high costs. The development of NanoBiT-based assays has accelerated in response to the coronavirus disease pandemic, providing safer alternatives for viral research, and is now applicable for general laboratories. This study aimed to develop a NanoBiT-based virus-like particle (VLP) assay for the rapid and safe screening of neutralizing antibodies against FMDV Asia1 Shamir (AS). Methods: We developed an AS VLP with an inserted HiBiT tag that enabled the detection of entry into LgBiT cells through luminescence signals. Results: HiBiT-tagged AS VLPs mixed with anti-serum and introduced into LgBiT-expressing cells led to a reduction in luciferase activity. Therefore, we established a NanoBiT-based viral neutralizing antibody test (VNT) that demonstrated a high correlation (R2 = 0.881) with the traditional gold standard VNT. Conclusions: The assay demonstrated high sensitivity and could be performed in BL-2 facilities, offering a safer and more efficient alternative to traditional assays while reducing the need to handle live viruses in high-containment facilities. This method provides a valuable tool for rapid screening of neutralizing antibodies and can be adapted for broader applications in FMDV research.

Background: Since the massive outbreak of foot-and-mouth disease (FMD) in South Korea in 2010–2011, cloven-hoofed livestock have been immunized with serotype O and A vaccines across the country. Other serotypes of FMD vaccines were stockpiled in overseas FMD vaccine factories as antigen banks. Once a manufacturing facility has been established in South Korea, the overseas antigen banks will be replaced by domestic one. Therefore, this study aimed to evaluate the commercial potential of the previously developed SAT2 vaccine candidate (SAT2 ZIM-R). Methods: The optimal condition was determined at various virus concentrations, infection times, and pH levels, resulting in 0.01 MOI for SAT2 ZIM-R for 24 h infection at a pH of 7.5. Results: When the SAT2 ZIM-R virus was produced in flasks from 40 to 1000 mL in fivefold increments, all scales of production yielded >7.0 µg/mL of antigens. Using a bioreactor, 5.6 µg/mL of antigens was recovered from a 1 L viral culture. The optimal conditions of viral inactivation kinetics were determined to be 1 mM of binary ethyleneimine (BEI) treatment at 26 °C for 24 h, with approximately 91% of the antigen being retained after virus inactivation. When the SAT2 ZIM-R experimental vaccine was administered twice to pigs, the neutralizing antibody titer increased approximately 500-fold after booster immunization. Conclusions: To the best of our knowledge, this is the first study to evaluate the antigen productivity, viral inactivation kinetics, and immunogenicity of the SAT vaccine strain in pigs. In the future, the SAT2 ZIM-R vaccine may be a useful candidate vaccine for a domestic antigen bank.

Background: Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals such as cattle and pigs, characterized by vesicular lesions in the mouth, nose, teats, and feet. Globally, the most commonly used FMD vaccines are inactivated vaccines produced by chemical inactivation of the infectious FMD virus (FMDV). This study aimed to establish an infectious clone of the O/Boeun/SKR/2017 virus that has demonstrated the highest antigen productivity among the various type O vaccine strains developed in South Korea to date. Methods: An infectious clone was generated from a type O virus isolated during the 2017 FMD outbreak in South Korea. The viral genome was divided into two fragments, each amplified separately, and subsequently ligated to produce a full-length infectious clone. Results: Rescue of infectious FMDV was confirmed using a commercial antigen detection kit and electron microscopy. Under optimized culture conditions, the rescued virus titer reached 2 × 107 TCID50/mL, and the antigen yield was 6.4 µg/mL. Following inactivation, the antigen was formulated into a vaccine and administered to pigs. Four weeks post-vaccination, challenge with the live virus resulted in no clinical symptoms, demonstrating complete protective efficacy. Conclusions: To the best of our knowledge, this is the first report describing the construction of an infectious clone derived from a field FMDV isolate in South Korea and its application in vaccine development. The O/Boeun/SKR/2017 infectious clone may serve as a genetic backbone for the rapid generation of new FMD vaccine candidates with high antigen productivity by substituting epitopes from other FMDV.

Foot-and-mouth disease (FMD), a vesicular disease, causes lesions in the mouth, nose, teats, and feet of cloven-hoofed animals. Vaccination remains the most effective method to prevent FMD outbreaks. Since 2010, South Korea has implemented nationwide vaccination and developed multiple domestic vaccine strains to achieve vaccine self-sufficiency. Here, we aimed to construct an infectious clone using the A/SKR/Yeoncheon/2017 virus, which exhibits the highest antigen productivity among previously developed vaccine strains. An infectious clone was constructed based on the A/Yeoncheon/SKR/2017 virus isolated during an FMD outbreak in Korea in 2017. The viral genome was amplified in two fragments and assembled into a full-length clone, from which infectious recombinant virus was successfully rescued. The rescued virus was confirmed via serotyping and transmission electron microscopy to exhibit canonical 25–30 nm icosahedral morphology. Under optimized culture conditions using suspension-adapted BHK-21 cells (multiplicity of infection 0.001; 12 h post-infection), the recombinant virus achieved titers of 108 TCID50/mL and produced 6.2 μg/mL of 146S antigen, comparable to its parental counterpart. The experimental vaccine formulated with the rescued virus (15 μg/dose), 1% saponin, 1% aluminum hydroxide gel, and ISA 206 VG, induced protective immunity in eight-week-old pigs, with vaccinated animals exhibiting no clinical signs following homologous challenge. To our knowledge, this study represents the first successful construction of an infectious clone derived from a field-isolated serotype A FMDV in South Korea. In the future, this A/SKR/Yeoncheon/2017 infectious clone can serve as a platform backbone for the rapid development of next-generation, high-yield vaccine seed strains through targeted epitope exchange.

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.

Since the foot-and-mouth disease (FMD) outbreak in South Korea in 2010–2011, vaccination policies utilizing inactivated FMD vaccines composed of types O and A have been implemented nationwide. However, because type Asia1 occurred in North Korea in 2007 and intermittently in neighboring countries, the risk of type Asia1 introduction cannot be ruled out. This study evaluated the antigen yield and viral inactivation kinetics of the recombinant Asia1 Shamir vaccine strain (Asia1 Shamir-R). When Asia1 Shamir-R was proliferated in shaking flasks (1 L), a 2 L bioreactor (1 L), and a wave bioreactor (25 L), the antigen yields were 7.5 μg/mL, 5.2 μg/mL, and 3.8 μg/mL, respectively. The optimal FMDV inactivation conditions were 2 mM BEI at 26 °C and 1.0 mM BEI at 37 °C. There was no antigen loss due to BEI treatment, and only a decrease in antigen levels was observed during storage. The sera from pigs immunized with antigen derived from a bioreactor exhibited a neutralizing antibody titer of approximately 1/1000 against Asia1 Shamir and Asia1/MOG/05 viruses; therefore, Asia1 Shamir-R is expected to provide sufficient protection against both viruses. If an FMD vaccine production facility is established, this Asia1 Shamir-R can be employed for domestic antigen banks in South Korea.

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.

South Korea has experienced outbreaks of foot-and-mouth disease (FMD) of serotypes O and A, leading to nationwide vaccination with a bivalent vaccine. Since the FMD virus (FMDV) Asia1 group-V genotype occurred in North Korea in 2007, an Asia1/MOG/05 vaccine strain belonging to the Asia1 group-V genotype was developed using a genetic recombination method (Asia1/MOG/05-R). This study aimed to evaluate the antigen productivity and viral inactivation kinetics of Asia1/MOG/05-R to assess its commercial viability. The antigen yield of Asia1/MOG/05-R produced in flasks and bioreactors was approximately 4.0 μg/mL. Binary ethylenimine (BEI) inactivation kinetics of Asia1/MOG/05-R showed that 2 mM and 1.0 mM BEI treatment at 26 °C and 37 °C, respectively, resulted in a virus titer <10−7 TCID50/mL within 24 h, meeting the inactivation kinetics criteria. During incubation at 26 °C and 37 °C, 10% antigen loss occurred, but not due to BEI treatment. When pigs were inoculated twice with the Asia1/MOG/05-R antigen, the virus neutralization titer increased to approximately 1:1000; therefore, it can sufficiently protect against Asia1/MOG/05-R and Asia1 Shamir viruses. The Asia1/MOG/05-R will be useful as a vaccine strain for domestic antigen banks.

BackgroundMost commercial foot-and-mouth disease (FMD) vaccines have various disadvantages, such as low antibody titers, short-lived effects, compromised host defense, and questionable safety.ObjectivesTo address these shortcomings, we present a novel FMD vaccine containing Dectin-1 agonist, β-D-glucan, as an immunomodulatory adjuvant. The proposed vaccine was developed to effectively coordinate innate and adaptive immunity for potent host defense against viral infection.MethodsWe demonstrated β-D-glucan mediated innate and adaptive immune responses in mice and pigs in vitro and in vivo. The expressions of pattern recognition receptors, cytokines, transcription factors, and co-stimulatory molecules were promoted via FMD vaccine containing β-D-glucan.Resultsβ-D-glucan elicited a robust cellular immune response and early, mid-, and long-term immunity. Moreover, it exhibited potent host defense by modulating host’s innate and adaptive immunity.ConclusionOur study provides a promising approach to overcoming the limitations of conventional FMD vaccines. Based on the proposed vaccine’s safety and efficacy, it represents a breakthrough among next-generation FMD vaccines.

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.

Foot-and-mouth disease (FMD) vaccines must be produced in a biosafety level 3 facility, so the FMD virus (FMDV) must be completely inactivated after amplification. The inactivation kinetics of FMDV during vaccine antigen production were assessed by evaluating whether the viral titer dropped below 10−7 TCID50/mL within 24 h of binary ethyleneimine (BEI) treatment. This study dealt with four FMD vaccine candidate strains for the efficacy of BEI treatment at different concentrations and temperatures to determine the optimal inactivation condition of each virus. Two domestic isolates, O/SKR/Boeun/2017 (O BE) and A/SKR/Yeoncheon/2017 (A YC), and two recombinant viruses, PAK/44/2008 (O PA-2) and A22/Iraq/24/64 (A22 IRQ), were investigated. The O BE and A22 IRQ required 2 mM BEI at 26 °C and 0.5 mM BEI at 37 °C for complete inactivation. The O PA-2 and A YC required 2 mM BEI at 26 °C and 1 mM BEI at 37 °C. Crucially, the yield of FMD virus particles (146S) in the viral infection supernatant was higher (>4.0 µg/mL) than those previously reported; additionally, there was little antigen loss, even after 24 h of treatment with 3 mM BEI. Overall, it is considered economical to produce FMD vaccines using these four kinds of viruses; therefore, these candidate strains will be prioritized for the manufacture of FMD vaccines in South Korea.

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease that affects cloven-hoofed animals and often causes enormous economic loss in the livestock industry. The capsid of FMD virus (FMDV) consists of four structural proteins. Initially, one copy each of the proteins VP0, VP3, and VP1 are folded together into a protomer, and five copies of the protomer compose a pentamer. Finally, 12 pentamers are assembled into an icosahedral capsid. At the maturation stage during RNA encapsidation, VP0 is cleaved into VP4 and VP2. The mechanism underlying VP0 maturation remains unclear. While monoclonal antibodies (mAbs) against VP2 have been developed in previous studies, a mAb specific to VP0 has not yet been reported. In this study, we generated VP0-specific mAbs by immunizing mice with peptides spanning the C-terminal amino acids of VP4 and N-terminal amino acids of VP2. We verified that these mAbs displayed specificity to VP0 with no reactivity to VP4 or VP2. Therefore, these mAbs could prove useful in identifying the role of VP0 in FMDV replication and elucidating the mechanism underlying VP0 cleavage into VP4 and VP2.

Purpose Patients with self-limited epilepsy with centrotemporal spikes (SLECTS) rarely experience generalized tonic-clonic seizures (GTCS) after remission, and post-remission GTCS has not been thoroughly described in earlier studies. Herein, we describe the clinical and electrographic features of GTCS after a substantial period of seizure freedom in patients with SLECTS. Methods This study included six patients (three boys and three girls) diagnosed with SLECTS who later developed GTCS after or near remission. Medical records, including clinical data and serial electroencephalography (EEG) recordings, were retrospectively reviewed for all patients. Results Patients’ age at SLECTS onset ranged from 5.2 to 10.2 years (mean, 8.4 years), while seizure cessation was achieved between 8 and 12.2 years. During SLECTS, typical centrotemporal spikes were observed in all patients, and generalized spike-and-wave discharges were observed in three patients. The age at the first episode of subsequent GTCS ranged from 14.4 to 17.3 years (mean, 15.8 years), constituting an average interval of 5.6 years after the last episode of seizures (range, 4.1 to 8.1 years). EEG at subsequent episodes of GTCS revealed generalized discharges in two patients, focal discharges in two other patients, and normal discharges in the remaining two patients. Two patients had multiple episodes of GTCS. Conclusion Although rare, GTCS may occur near or after remission in patients with SLECTS, and clinicians should be aware of this. Subsequent GTCS may be a manifestation of idiopathic generalized epilepsy. However, large-scale studies are needed to determine the nature of such episodes of GTCS and their associated risk factors.

Foot-and-mouth disease (FMD) is an economically important and highly infectious viral disease, predominantly controlled by vaccination. The removal of non-structural proteins (NSPs) is very important in the process of FMD vaccine production, because vaccinated and naturally infected animals can be distinguished by the presence of NSP antibodies in the FMD serological surveillance. A previous study reported that 3AB protein, a representative of NSPs, was removed by chloroform treatment. Therefore, in this study, the causes of 3AB removal and factors affecting the effect of chloroform were investigated. As a result, the effectiveness of chloroform differed depending on the virus production medium and was eliminated by detergents. In addition, it was found that 3AB protein removal by chloroform is due to the transmembrane domain of the N-terminal region (59–76 amino acid domain). Further, industrial applicability was verified by applying the chloroform treatment process to scale-up FMD vaccine antigen production. A novel downstream process using ultrafiltration instead of polyethylene glycol precipitation for high-purity FMD vaccine antigen production was established. This result will contribute toward simplifying the conventional process of manufacturing FMD vaccine antigens and ultimately reducing the time and cost of vaccine production.