Evaluation of Foot-and-Mouth Disease (FMD) Virus Asia1 Genotype-V as an FMD Vaccine Candidate: Study on Vaccine Antigen Production Yield and Inactivation Kinetics

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.


Introduction
Foot-and-mouth disease (FMD) is a highly contagious disease affecting cloven-hoofed animals.Its substantial economic impact has increased the concerns of governments worldwide [1].FMD virus (FMDV), an Aphthovirus genus of the Picornaviridae family, is the causative agent of FMD [2].The nucleic acid of FMDV is a single-stranded, positive-sense RNA genome.The genes corresponding to the capsid are translated into a polyprotein, which is cleaved into structural and nonstructural proteins by proteases [1,3,4].Of the seven FMD serotypes (O, A, Asia1, C, and SAT 1-3), types O, A, and Asia1 have been most commonly identified in Asian countries.Type Asia1 is grouped into nine genetic groups (G-I to G-IX) based on nucleotide variations in the VP1 sequence [5].
In South Korea, FMD broke out in February 2000, after the last FMD outbreak in 1934.Due to the overseas antigen bank established in advance, FMD was eliminated early through an emergency vaccination policy.However, two years later, FMD occurred again, but this time, it was successfully controlled through a culling policy.There was no FMD outbreak for a while, but in January 2010, FMD type A occurred, followed by type O in April, and then a massive scale outbreak of FMD type O occurred from November to April of the following year, causing about 3 trillion won in economic damage.Since then, FMD vaccines have been administered nationwide to cattle, pigs, goats etc. FMD occurred every year from 2014 to 2019 and then again in 2023.As FMD outbreaks have been occurring intermittently, the vaccination policy will need to be maintained to protect the domestic livestock industry.
The serotypes of FMD that have occurred in South Korea so far are serotypes O and A, so a vaccine containing a mixture of the two serotypes is being used.Vaccines for the remaining five serotypes (Asia1, C, and SAT 1, SAT 2, SAT 3), which have never occurred in South Korea, are stockpiled in the form of an antigen bank under a contract with a foreign FMD vaccine manufacturing facility, and in the event of a domestic outbreak, the finished product will be delivered to South Korea within six working days according to the terms of the contract.
Meanwhile, the current FMD vaccines are entirely imported from abroad.However, if an FMD vaccine manufacturing facility is built in South Korea in the next 2-3 years, localization of the FMD vaccine will be achieved, so for this purpose, our institute has been developing various types of FMD vaccine seed viruses.However, type Asia1 occurred in North Korea in 2007 and intermittently in neighboring countries [6].Therefore, the risk of type Asia1 introduction into Korea cannot be excluded.Regarding the type Asia1 outbreak, the government of South Korea has stockpiled the type Asia1 vaccine in the form of an overseas antigen bank.Asia1 Shamir, the most representative Asia1 vaccine, is not highly protective against the Asia1/MOG/05 lineage found in North Korea [7].Therefore, a novel candidate vaccine, Asia1/MOG/05-R, was previously constructed [8].
The capsid of FMDV is composed of four proteins called VP1, VP2, VP3, and VP4.Initially, VP0, a combined form of VP43 and VP2, forms protomers with VP3 and VP1; five protomers come together to form the pentamer of 12S, and 12 pentamers come together to form the viral precursor of 75S.Upon entry of the RNA into the capsid, VP0 is cleaved into VP2 and VP4, creating an intact FMDV (146S) [9].When subjected to sucrose density gradient ultracentrifugation, 146S is present in the densest position, 75S in a slightly less dense position, and 12S in an even less dense position.FMDV is degraded to 12S by heat or pH, which may not seem like a big difference since the components are the same, but there is a huge difference in immunogenicity between the 146S and 12S forms.According to a previous report [10], the intact FMDV (146S) is more than 100 times more protective against FMDV than the pentameric form (12S). Therefore, in the process of FMD vaccine manufacturing, the yield of FMDV particle production is very important.The antigen productivity of the FMD vaccine plant is directly related to the economics of the plant, leading to the competitiveness of the FMD vaccine.
In this regard, this study aimed to investigate the antigen productivity and virus inactivation kinetics of Asia1/MOG/05-R to validate whether the novel Asia1 vaccine can be economically employed as a candidate vaccine for antigen banks in South Korea.

Determination of Optimal Conditions for Asia1/MOG/2005-R Proliferation
BHK-21 suspension cells were adapted in Cellvento BHK-200 medium (Merck, Darmstadt, Germany) until they reached a density of around 3 × 10 6 cells/mL for 3.5 days.Then, the Asia1/MOG/05-R was inoculated into the cells at a multiplicity of infection (MOI) of 0.001, 0.005, 0.01, and 0.05 at the time of adding 30% (v/v) of fresh medium.The cells were incubated in a shaking incubator at 37 • C with 5% CO 2 .Viral infection supernatant was obtained by harvesting at the time of 12, 16, 20, and 24 h post-infection (hpi) and then clarification by centrifugation at 3000× g for 20 min at 4 • C to remove cell debris.

Virus Titration
Adherent BHK-21 cells were used to determine FMDV titers via endpoint titration using the Spearman-Kärber calculation.The results of virus titrations are represented as the tissue culture infectious dose affecting 50% of the cultures (TCID 50 ) per mL [12].

Quantification of FMDV Particles
The amount of the FMD vaccine antigen (146S) was measured as previously described [13].Briefly, the viral infection supernatant was treated with chloroform (Merck) at a ratio of 1:1 (v/v) and mixed vigorously for 5 min.The mixture was then centrifuged at 4000× g for 15 min at 4 • C, and then the aqueous phase on top of the organic solvent was collected.Next, the samples were treated with benzonase (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 0.025 units/µL, and they were incubated at 37 • C for 1 h with shaking.Then, the samples were clarified by centrifugation at 16,000× g for 10 min at 4 • C. The FMDV intact particles in the samples were measured by loading the samples onto a high-performance liquid chromatography (Agilent Technologies, Santa Clara, CA, USA) fitted with a TSKgel G4000PWXL column (TOSOH Bioscience, Tokyo, Japan).Finally, the peak area was integrated for the quantification of FMDV particles.

Preparation of Antigen Using A Shaking Flask and 2 L Bioreactor
For flask culture, when the BHK-21 suspension cells with concentrations of 3 × 10 5 cells/mL at volumes of 28 mL, 140 mL, and 700 mL were grown for 3.5 days up to approximately 3 × 10 6 cells/mL in a Cellvento BHK-200 medium, the Asia1/MOG/05-R was inoculated onto the cells at 0.05 MOI with 12 mL, 60 mL, and 300 mL of fresh Cellvento BHK-200 medium in a shaking incubator at 37 • C with 5% CO 2 for 24 h.For the 2 L bioreactor (Sartorius, Göttingen, Germany), 700 mL of the Cellvento BHK-200 cell culture media was initially cultured with a cell density of 3 × 10 5 cells/mL in a 2 L bioreactor.The bioreactor was fitted with probes to monitor temperature and dissolved oxygen at 37 • C and 45% air saturation.The pH was controlled in the range of 7.2-7.4by adding CO 2 and a 0.5 M NaOH solution.The speed of agitation in a bioreactor was maintained at 150 rpm.When the cell density reached around 3 × 10 6 cells/mL, 300 mL of the cell culture medium was added to the bioreactor, and the pH was adjusted to 7.5.Subsequently, Asia/MOG/05-R was added to the bioreactor containing fresh medium at 0.05 MOI.Viral infection supernatant was harvested after 24 h.

FMDV Inactivation Kinetics
Dissolving bromoethylamine hydrobromide (Sigma-Aldrich) in 10 mL of 0.2 N sodium hydroxide solution (Sigma-Aldrich) resulted in binary ethylenimine (BEI) with a concentration of 0.1 M.Then, the solution was incubated in a shaking incubator at 100 rpm at 37 • C for 1 h.The final pH was adjusted to approximately 8.5-9.The solution was prepared just before experiment.For inactivation kinetics of the samples, 100 mL of the viral infection supernatant was inactivated by adding BEI from 0.5 mM to 3 mM and then was incubated in an incubator at 26 • C and 37 • C at the shaking speed of 75 rpm for 24 h.Next, each sample (12 mL) was collected at the interval of 1 h up to 6 h and 24 h post BEI treatment.Finally, 10% volume of 1 M sodium thiosulfate (Daejung Chemicals, Siheung-si, Korea) to a final concentration of 2% (v/v) was added to neutralize the residual BEI after experiment.

Animal Experiment
The purified FMDV Asia1/MOG/05-R antigen (15 µg per dose) derived from the 2 L bioreactor was mixed with 1% saponin (Sigma-Aldrich) and 10% aluminum hydroxide gel (General Chemical, Moorestown, NJ, USA) to prepare a monovalent vaccine.Next, the ISA 206 VG adjuvant (Seppic, Paris, France), pre-warmed at 30 • C, was added at a ratio of 1:1, resulting in a 2 mL/dose of experimental vaccine.The mixtures were incubated at 20 • C for 1 h in a water bath without light exposure and stored at 4 • C until use.Two-month-old pigs (n = 5) that had not been previously vaccinated against FMD were immunized twice at the four-week interval with the monovalent Asia1/MOG/05-R vaccine.The control group comprised three unvaccinated pigs.Blood samples were collected at 0, 14, 21, 28, 35, 42, 49, and 56 days post-vaccination.Animal experiments in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of the Animal and Plant Quarantine Agency (IACUC No. 2023-761).

Virus Neutralization Test
The virus neutralization (VN) test was performed as described in the WOAH terrestrial manual [14].Sera were inactivated at 56 • C for 30 min prior to testing.Starting from 1/4 dilution, 50 µL of two-fold serially diluted sera were mixed with 50 µL of each virus containing 100 TCID 50 .After incubation at 37 • C for 1 h, 50 µL of LFBK cells (0.5 × 10 6 cells/mL) were added to each well.The plates were sealed and incubated at 37 • C with 5% CO 2 for 2-3 days.The VN titer was calculated as the reciprocal of the maximum dilution of serum that neutralized the 100 TCID 50 of the FMDV and expressed as a log 10 value.

Statistical Analysis
Each experiment was conducted in triplicate, and the means and standard deviations of all values are presented.Statistical data were analyzed using GraphPad Prism version 9 (GraphPad Software, La Jolla, CA, USA) for visual representation.Statistical significance was assessed using two-way ANOVA and set at p < 0.05.

Optimization of Conditions for Asia1/MOG/05-R Proliferation
The FMDV titer and antigen yield were measured according to the Asia1/MOG/05-R concentration and virus infection time to determine the optimal conditions for antigen production using Asia1/MOG/05-R.The viral titer remained constant at approximately 10 8 TCID 50 /mL under all conditions (Figure 1).However, the amount of antigen increased with a longer virus infection time, peaking at 24 hpi.In addition, the amount of antigen increased with increasing viral concentration.Finally, the optimal condition for antigen production was achieved with a virus concentration of 0.05 MOI and 24 hpi, resulting in an antigen yield of 3.7 µg/mL.

Antigen Yield According to pH Adjustment of the Asia1/MOG/05-R
The difference in antigen yield was investigated by changing the medium pH from 6.0 to 9.0 during the Asia1/MOG/05-R inoculation phase.The results showed 0 μg/mL, 1.86 μg/mL, 3.55 μg/mL, 3.98 μg/mL, 3.84 μg/mL, 3.50 μg/mL, and 2.95 μg/mL at pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively (Figure 2).The amount of antigen was lowest at pH 6.5 and highest at pH 7.5, followed by a decreasing trend with increasing pH.Finally, the optimal condition for Asia1/MOG/05-R antigen production was a medium pH of 7.5 at the time of virus inoculation.

Antigen Yield According to pH Adjustment of the Asia1/MOG/05-R
The difference in antigen yield was investigated by changing the medium pH from 6.0 to 9.0 during the Asia1/MOG/05-R inoculation phase.The results showed 0 µg/mL, 1.86 µg/mL, 3.55 µg/mL, 3.98 µg/mL, 3.84 µg/mL, 3.50 µg/mL, and 2.95 µg/mL at pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively (Figure 2).The amount of antigen was lowest at pH 6.5 and highest at pH 7.5, followed by a decreasing trend with increasing pH.Finally, the optimal condition for Asia1/MOG/05-R antigen production was a medium pH of 7.5 at the time of virus inoculation.

Antigen Yield According to pH Adjustment of the Asia1/MOG/05-R
The difference in antigen yield was investigated by changing the medium pH from 6.0 to 9.0 during the Asia1/MOG/05-R inoculation phase.The results showed 0 μg/mL, 1.86 μg/mL, 3.55 μg/mL, 3.98 μg/mL, 3.84 μg/mL, 3.50 μg/mL, and 2.95 μg/mL at pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively (Figure 2).The amount of antigen was lowest at pH 6.5 and highest at pH 7.5, followed by a decreasing trend with increasing pH.Finally, the optimal condition for Asia1/MOG/05-R antigen production was a medium pH of 7.5 at the time of virus inoculation.

Comparison of Antigen Yield According to Production Scale
The amount of antigen for the Asia1/MOG/05-R was evaluated by culturing in the volumes of 40 mL, 200 mL, and 1000 mL, resulting in 4.1 µg/mL, 3.85 µg/mL, and 3.82 µg/mL, respectively.Viral titers were >10 7 TCID 50 /mL, regardless of the volume of viral proliferation (Figure 3).When the Asia1/MOG/05-R antigen was produced in a bioreactor based on the optimal conditions established at the flask scale, the virus titer exceeded 10 7 TCID 50 /mL, and the antigen yield was 4.1 µg/mL.

Comparison of Antigen Yield According to Production Scale
The amount of antigen for the Asia1/MOG/05-R was evaluated by culturing in the volumes of 40 mL, 200 mL, and 1000 mL, resulting in 4.1 μg/mL, 3.85 μg/ mL, and 3.82 μg/mL, respectively.Viral titers were >10 7 TCID50/mL, regardless of the volume of viral proliferation (Figure 3).When the Asia1/MOG/05-R antigen was produced in a bioreactor based on the optimal conditions established at the flask scale, the virus titer exceeded 10 7 TCID50/mL, and the antigen yield was 4.1 μg/mL.

BEI Inactivation Kinetics of the Asia1/MOG/05-R
The inactivation kinetics of the virus were investigated using the Asia1/MOG/05-R infection supernatant obtained from the flask.The inactivation rate was greater at 37 °C than at 26 °C, and the higher the BEI concentration, the greater the inactivation rate.While the viral titer was estimated to be <10 −7 TCID50/mL with linear regression within 24 h with 2 mM BEI at 26 °C, inactivation was completed by reducing the viral titer to 10 −7 TCID50/mL within 24 h with 0.5 mM BEI at 37 °C (Figure 4).The amount of antigen was measured immediately before and 6 h and 24 h after BEI inactivation.Treatment with 3 mM BEI showed antigen yields of 5.0 and 5.1 μg/mL at 26 °C and 37 °C, respectively, from the initial antigen yield of 5.7 μg/mL.However, this loss was not due to BEI treatment because the samples without BEI treatment also showed equivalent amounts of antigen (5.2 μg/mL) under the same conditions (Table 1).

BEI Inactivation Kinetics of the Asia1/MOG/05-R
The inactivation kinetics of the virus were investigated using the Asia1/MOG/05-R infection supernatant obtained from the flask.The inactivation rate was greater at 37 • C than at 26 • C, and the higher the BEI concentration, the greater the inactivation rate.While the viral titer was estimated to be <10 −7 TCID 50 /mL with linear regression within 24 h with 2 mM BEI at 26 • C, inactivation was completed by reducing the viral titer to 10 −7 TCID 50 /mL within 24 h with 0.5 mM BEI at 37 • C (Figure 4).The amount of antigen was measured immediately before and 6 h and 24 h after BEI inactivation.Treatment with 3 mM BEI showed antigen yields of 5.0 and 5.1 µg/mL at 26 • C and 37 • C, respectively, from the initial antigen yield of 5.7 µg/mL.However, this loss was not due to BEI treatment because the samples without BEI treatment also showed equivalent amounts of antigen (5.2 µg/mL) under the same conditions (Table 1).Pigs were inoculated with a 15 µg/dose of the Asia1/MOG/05-R antigens produced from the bioreactor.VN titers against Asia1/MOG/05-R and Asia1 Shamir viruses were approximately 1:45 or lower after the first vaccination.However, after the second immunization, the VN titers increased and reached approximately 1:1000 (Figure 5).There was no significant difference in the VN titers between the Asia1/MOG/05-R and Asia1 Shamir viruses.

Immunogenicity of the Asia1/MOG/05-R in Pigs
Pigs were inoculated with a 15 μg/dose of the Asia1/MOG/05-R antigens produced from the bioreactor.VN titers against Asia1/MOG/05-R and Asia1 Shamir viruses were approximately 1:45 or lower after the first vaccination.However, after the second immunization, the VN titers increased and reached approximately 1:1000 (Figure 5).There was no significant difference in the VN titers between the Asia1/MOG/05-R and Asia1 Shamir viruses.

Discussion
A recombinant Asia1/MOG/05 vaccine (Asia1/MOG/05-R) was developed at our institute [8].To evaluate the feasibility of using Asia1/MOG/05-R in antigen banks, its antigen productivity and virus inactivation kinetics were investigated.In addition, the immunogenicity of the antigen produced in the bioreactor was evaluated in pigs.
The optimal conditions for Asia1/MOG/05-R antigen production were determined using the Cellvento BHK-200 medium, which does not require medium exchange during the virus inoculation stage.FMDV titers were equivalent at approximately 10 8 TCID 50 /mL under all conditions.However, the antigen yield was higher at higher virus concentrations and longer virus infection times.There was a difference in antigen yield even at similar titers; however, the reason for this is unclear.Meanwhile, the efficacy of the FMD vaccine depends on the 146S intact virus particle content rather than the virus titer; therefore, antigen content is important.Other researchers have reported that high viral titers but low antigen amounts lead to low immunogenicity in animals [15].

Figure 1 .
Figure 1.Optimization of the conditions for producing the Asia1/MOG/05-R antigen.The BHK-21 cells were inoculated with Aisa1/MOG/05-R at different virus infection doses and cultivation times, and then antigen yield and titers were measured.Data are presented as the mean ± standard deviation from three independent experiments.hpi: hours post-infection, ** p < 0.01, and **** p < 0.001.

Figure 2 .
Figure 2. Determination of optimal pH for producing the Asia1/MOG/05-R antigen.The antigen yield was measured according to the medium pH at the virus inoculation stage.Data are presented as the mean ± standard deviation from three independent experiments.* p < 0.05, ** p < 0.01.

Figure 1 .
Figure 1.Optimization of the conditions for producing the Asia1/MOG/05-R antigen.The BHK-21 cells were inoculated with Aisa1/MOG/05-R at different virus infection doses and cultivation times, and then antigen yield and titers were measured.Data are presented as the mean ± standard deviation from three independent experiments.hpi: hours post-infection, ** p < 0.01, and **** p < 0.001.

Figure 1 .
Figure 1.Optimization of the conditions for producing the Asia1/MOG/05-R antigen.The BHK-21 cells were inoculated with Aisa1/MOG/05-R at different virus infection doses and cultivation times, and then antigen yield and titers were measured.Data are presented as the mean ± standard deviation from three independent experiments.hpi: hours post-infection, ** p < 0.01, and **** p < 0.001.

Figure 2 .
Figure 2. Determination of optimal pH for producing the Asia1/MOG/05-R antigen.The antigen yield was measured according to the medium pH at the virus inoculation stage.Data are presented as the mean ± standard deviation from three independent experiments.* p < 0.05, ** p < 0.01.

Figure 2 .
Figure 2. Determination of optimal pH for producing the Asia1/MOG/05-R antigen.The antigen yield was measured according to the medium pH at the virus inoculation stage.Data are presented as the mean ± standard deviation from three independent experiments.* p < 0.05, ** p < 0.01.

Figure 3 .
Figure 3.Comparison of antigen yield according to the production scale.Asia1/MOG/05-R was produced by progressively increasing the culture volume 5-fold, and antigen yield and titer were measured at each step.Data are presented as the mean ± standard deviation from three independent experiments.ns: not significant.

Figure 3 .
Figure 3.Comparison of antigen yield according to the production scale.Asia1/MOG/05-R was produced by progressively increasing the culture volume 5-fold, and antigen yield and titer were measured at each step.Data are presented as the mean ± standard deviation from three independent experiments.ns: not significant.

Figure 4 .
Figure 4. Inactivation kinetics of Asia1/MOG/05-R.The supernatant obtained after the Asia1/MOG/05-R was inoculated in the BHK-21 suspension cells was inactivated by each binary ethyleneimine (BEI) concentration, with samples taken at intervals of 1 hour up to 6 h and 24 h.The individual graph was extrapolated with a linear line for the analysis of inactivation kinetics.

Figure 4 .
Figure 4. Inactivation kinetics of Asia1/MOG/05-R.The supernatant obtained after the Asia1/MOG/05-R was inoculated in the BHK-21 suspension cells was inactivated by each binary ethyleneimine (BEI) concentration, with samples taken at intervals of 1 h up to 6 h and 24 h.The individual graph was extrapolated with a linear line for the analysis of inactivation kinetics.

Figure 4 .
Figure 4. Inactivation kinetics of Asia1/MOG/05-R.The supernatant obtained after the Asia1/MOG/05-R was inoculated in the BHK-21 suspension cells was inactivated by each binary ethyleneimine (BEI) concentration, with samples taken at intervals of 1 hour up to 6 h and 24 h.The individual graph was extrapolated with a linear line for the analysis of inactivation kinetics.

Figure 5 .Figure 5 .
Figure 5. Virus neutralization antibody titers post-immunization with the Asia1/MOG/ 05-R vaccine.Virus neutralization tests against the Asia1/MOG/05-R and Asia1 Shamir viruses were Figure 5. Virus neutralization antibody titers post-immunization with the Asia1/MOG/05-R vaccine.Virus neutralization tests against the Asia1/MOG/05-R and Asia1 Shamir viruses were performed using sera collected weekly from pigs immunized twice with the Asia1/MOG/05-R vaccine at the four-week interval.Data are presented as the mean ± standard deviation from three independent experiments.The dotted line indicates 1.65 log10 (1:45) virus neutralization (VN) titer.* p < 0.05, ns: not significant.

Table 1 .
The amount of foot-and-mouth disease vaccine antigen (146S) in the Asia1/MOG/05-R virus supernatants after binary ethylenimine (BEI) treatment at 26 °C and 37 °C for 6 h and 24 h.

Table 1 .
The amount of foot-and-mouth disease vaccine antigen (146S) in the Asia1/MOG/05-R virus supernatants after binary ethylenimine (BEI) treatment at 26 • C and 37 • C for 6 h and 24 h.

Table 1 .
The amount of foot-and-mouth disease vaccine antigen (146S) in the Asia1/MOG/05-R virus supernatants after binary ethylenimine (BEI) treatment at 26 °C and 37 °C for 6 h and 24 h.