Enhancing polyethylene degradation: a novel bioprocess approach using Acinetobacter nosocomialis pseudo-resting cells.

Despite the discovery of several bacteria capable of interacting with polymers, the activity of the natural bacterial isolates is limited. Furthermore, there is a lack of knowledge regarding the development of bioprocesses for polyethylene (PE) degradation. Here, we report a bioprocess using pseudo-resting cells for efficient degradation of PE. The bacterial strain Acinetobacter nosocomialis was isolated from PE-containing landfills and characterized using low-density PE (LDPE) surface oxidation when incubated with LDPE. We optimized culture conditions to generate catalytic pseudo-resting cells of A. nosocomialis that are capable of degrading LDPE films in a bioreactor. After 28 days of bioreactor operation using pseudo-resting cells of A. nosocomialis, we observed the formation of holes on the PE film (39 holes per 217 cm2, a maximum diameter of 1440 µm). This study highlights the potential of bacteria as biocatalysts for the development of PE degradation processes. KEY POINTS: • New bioprocess has been proposed to degrade polyethylene (PE). • Process with pseudo-resting cells results in the formation of holes in PE film. • We demonstrated PE degradation using A. nosocomialis as a biocatalyst.

Polarization- and Electrode-Optimized Polyvinylidene Fluoride Films for Harsh Environmental Piezoelectric Nanogenerator Applications.

While piezoelectric nanogenerators have demonstrated the effective conversion of tiny mechanical vibrations to electricity, their performances are rarely examined under harsh environmental conditions. Here, a multilayered polyvinylidene fluoride (PVDF) film-based piezoelectric nanogenerator (ML-PENG) is demonstrated to generate considerable and stable power outputs even at extremely low temperatures and pressures, and under strong UV. Up-/down-polarized PVDF films are alternately stacked, and Ag electrodes are intercalated between the two adjacent films. At -266 °C and 10-5  Torr, the ML-PENG generates an open-circuit voltage of 1.1 V, a short-circuit current density of 8 nA cm-2 , and a power density of 4.4 nW cm-2 . The piezoelectric outputs are quite stable against prolonged illumination of UV, large temperature- and pressure-variations, and excessive mechanical vibrations. The piezoelectric power density is greatly enhanced above the freezing and glass transition temperatures of PVDF and recorded to be 10, 105, and 282 nW cm-2 at -73, 0, and 77 °C, respectively. The ML-PENG generates sufficient power to operate five light-emitting diodes by harvesting biomechanical energy under simulated Martian conditions. This work suggests that polarization- and electrode-optimized ML-PENG can serve as a reliable and economic power source in harsh and inaccessible environments like polar areas of Earth and extraterrestrial Mars.

Rapid Engineering of Foot-and-Mouth Disease Vaccine and Challenge Viruses.

There are seven antigenically distinct serotypes of foot-and-mouth disease virus (FMDV), each of which has intratypic variants. In the present study, we have developed methods to efficiently generate promising vaccines against seven serotypes or subtypes. The capsid-encoding gene (P1) of the vaccine strain O1/Manisa/Turkey/69 was replaced with the amplified or synthetic genes from the O, A, Asia1, C, SAT1, SAT2, and SAT3 serotypes. Viruses of the seven serotype were rescued successfully. Each chimeric FMDV with a replacement of P1 showed serotype-specific antigenicity and varied in terms of pathogenesis in pigs and mice. Vaccination of pigs with an experimental trivalent vaccine containing the inactivated recombinants based on the main serotypes O, A, and Asia1 effectively protected them from virus challenge. This technology could be a potential strategy for a customized vaccine with challenge tools to protect against epizootic disease caused by specific serotypes or subtypes of FMDV.IMPORTANCE Foot-and-mouth disease (FMD) virus (FMDV) causes significant economic losses. For vaccine preparation, the selection of vaccine strains was complicated by high antigenic variation. In the present study, we suggested an effective strategy to rapidly prepare and evaluate mass-produced customized vaccines against epidemic strains. The P1 gene encoding the structural proteins of the well-known vaccine virus was replaced by the synthetic or amplified genes of viruses of seven representative serotypes. These chimeric viruses generally replicated readily in cell culture and had a particle size similar to that of the original vaccine strain. Their antigenicity mirrored that of the original serotype from which their P1 gene was derived. Animal infection experiments revealed that the recombinants varied in terms of pathogenicity. This strategy will be a useful tool for rapidly generating customized FMD vaccines or challenge viruses for all serotypes, especially for FMD-free countries, which have prohibited the import of FMDVs.

Inactivation of foot-and-mouth disease virus by citric acid and sodium carbonate with deicers.

Three out of five outbreaks of foot-and-mouth disease (FMD) since 2010 in the Republic of Korea have occurred in the winter. At the freezing temperatures, it was impossible to spray disinfectant on the surfaces of vehicles, roads, and farm premises because the disinfectant would be frozen shortly after discharge and the surfaces of the roads or machines would become slippery in cold weather. In this study, we added chemical deicers (ethylene glycol, propylene glycol, sodium chloride, calcium chloride, ethyl alcohol, and commercial windshield washer fluid) to keep disinfectants (0.2% citric acid and 4% sodium carbonate) from freezing, and we tested their virucidal efficacies under simulated cold temperatures in a tube. The 0.2% citric acid could reduce the virus titer 4 logs at -20°C with all the deicers. On the other hand, 4% sodium carbonate showed little virucidal activity at -20°C within 30 min, although it resisted being frozen with the function of the deicers. In conclusion, for the winter season, we may recommend the use of citric acid (>0.2%) diluted in 30% ethyl alcohol or 25% sodium chloride solvent, depending on its purpose.

Reemergence of foot-and-mouth disease, South Korea, 2000-2011.

Five outbreaks of foot-and-mouth disease have occurred in South Korea during 2000-2011. Macro-analysis of these outbreaks showed a correlation with outbreaks in countries in eastern Asia. Genetic analyses of food-and-mouth disease viruses in South Korea showed a correlation with viruses that are prevalent in neighboring countries.

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

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.

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.

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

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.

Determination of Optimal Antigen Yield and Virus Inactivation Conditions for the Production of the Candidate Foot-and-Mouth Disease Recombinant Vaccine Strain Asia1 Shamir-R in a Bioreactor.

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.

Control of foot-and-mouth disease during 2010-2011 epidemic, South Korea.

An outbreak of foot-and-mouth disease caused by serotype O virus occurred in cattle and pigs in South Korea during November 2010-April 2011. The highest rates of case and virus detection were observed 44 days after the first case was detected. Detection rates declined rapidly after culling and completion of a national vaccination program.

High-yield production of the VP1 structural protein epitope from serotype O foot-and-mouth disease virus in Escherichia coli.

For effective control of foot-and-mouth disease (FMD), the development of rapid diagnostic systems and vaccines are required against its etiological agent, FMD virus (FMDV). To accomplish this, efficient large-scale expression of the FMDV VP1 protein, with high solubility, needs to be optimized. We attempted to produce high levels of a serotype O FMDV VP1 epitope in Escherichia coli. We identified the subtype-independent serotype O FMDV VP1 epitope sequence and used it to construct a glutathione S-transferase (GST) fusion protein. For efficient production of the FMDV VP1 epitope fused to GST (VP1e-GST), four E. coli strains and three temperatures were examined. The conditions yielding the greatest level of VP1e-GST with highest solubility were achieved with E. coli BL21(DE3) at 25 °C. For high-level production, fed-batch cultures were conducted in 5-l bioreactors. When cells were induced at a high density and complex feeding solutions were supplied, approximately 11 g of VP1e-GST was obtained from a 2.9-l culture. Following purification, the VP1 epitope was used to immunize rabbits, and we confirmed that it induced an immune response.

Efficacy of Binary Ethylenimine in the Inactivation of Foot-and-Mouth Disease Virus for 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.

Production of Foot-and-Mouth Disease Type O and A Vaccine Antigens on a Pilot Scale and Determination of Optimal Amount of Antigen for Monovalent 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.

Dectin-1 signaling coordinates innate and adaptive immunity for potent host defense against viral infection.

Background: Most commercial foot-and-mouth disease (FMD) vaccines have various disadvantages, such as low antibody titers, short-lived effects, compromised host defense, and questionable safety. Objectives: To address these shortcomings, we present a novel FMD vaccine containing Dectin-1 agonist, ß-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. Methods: We demonstrated ß-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 ß-D-glucan. Results: ß-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. Conclusion: Our 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.

Development of Monoclonal Antibody to Specifically Recognize VP0 but Not VP4 and VP2 of Foot-and-Mouth Disease Virus.

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.

Comparison of High-Performance Liquid Chromatography with Sucrose Density Gradient Ultracentrifugation for the Quantification of Foot-and-Mouth Disease Vaccine Antigens.

Foot-and-mouth disease (FMD) causes substantial economic losses in the livestock industry. The protective immunizing component of the FMD virus (FMDV) is a ribonucleoprotein particle with a sedimentation coefficient of 146S. Size-exclusion high-performance liquid chromatography (SE-HPLC) was introduced to replace sucrose density gradient ultracentrifugation (SDG), which is the gold standard for the quantification of FMDV 146S particles. SE-HPLC showed a pattern similar to that of SDG; however, the two methods resulted in different quantities for the same amount of 146S particles. This study aimed to identify the reason for this disparity and adjust the difference between the two methods by employing a standard material. While SE-HPLC displayed all the virus particles in the peak fraction by SDS-PAGE and Western blotting, the virus particles were widely dispersed in multiple fractions, including peak fractions in the SDG. To adjust the difference between the two methods, a stable surrogate virus, bovine enterovirus, was devised to draw a standard curve, and the gap was reduced to <10%. To our knowledge, this is the first report to provide experimental evidence on the difference between SDG and SE-HPLC for the quantification of FMDV particles.

Factors Involved in Removing the Non-Structural Protein of Foot-and-Mouth Disease Virus by Chloroform and Scale-Up Production of High-Purity Vaccine Antigens.

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.

Production of a Foot-and-Mouth Disease Vaccine Antigen Using Suspension-Adapted BHK-21 Cells in a Bioreactor.

The baby hamster kidney-21 (BHK-21) cell line is a continuous cell line used to propagate foot-and-mouth disease (FMD) virus for vaccine manufacturing. BHK-21 cells are anchorage-dependent, although suspension cultures would enable rapid growth in bioreactors, large-scale virus propagation, and cost-effective vaccine production with serum-free medium. Here, we report the successful adaptation of adherent BHK-21 cells to growth in suspension to a viable cell density of 7.65 × 106 cells/mL on day 3 in serum-free culture medium. The suspension-adapted BHK-21 cells showed lower adhesion to five types of extracellular matrix proteins than adherent BHK-21 cells, which contributed to the suspension culture. In addition, a chemically defined medium (selected by screening various prototype media) led to increased FMD virus production yields in the batch culture, even at a cell density of only 3.5 × 106 cells/mL. The suspension BHK-21 cell culture could be expanded to a 200 L bioreactor from a 20 mL flask, which resulted in a comparable FMD virus titer. This platform technology improved virus productivity, indicating its potential for enhancing FMD vaccine production.

Development of a Potent Stabilizer for Long-Term Storage of Foot-and-Mouth Disease Vaccine Antigens.

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.

Validation of Pretreatment Methods for the In-Process Quantification of Foot-and-Mouth Disease Vaccine Antigens.

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is controlled by vaccine policy in many countries. For vaccine potency, the content of intact virus particles (146S antigens) is critical, and the sucrose density gradient (SDG) fractionation is the gold standard for the quantification of 146S antigens. However, this method has several drawbacks. Although size-exclusion high-performance liquid chromatography (SE-HPLC) was introduced to replace the classic method, its application is generally confined to purified samples owing to the interfering signals. Therefore, we aimed to develop optimal pretreatment methods for SE-HPLC quantification in less purified samples. Crude virus infection supernatant (CVIS) and semi-purified samples with PEG precipitation (PEG-P) were used. Chloroform pretreatment was essential to remove a high level of non-specific signals in CVIS, whereas it caused loss of 146S antigens without the distinctive removal of non-specific signals in PEG-P. Benzonase pretreatment was required to improve the resolution of the target peak in the chromatogram for both CVIS and PEG-P. Through spiking tests with pure 146S antigens, it was verified that the combined pretreatment with chloroform and benzonase was optimal for the CVIS, while the sole pretreatment of benzonase was beneficial for PEG-P.