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.

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.

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.

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.

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.

Genomic and Phenotypic Characterization of Cutibacterium acnes Bacteriophages Isolated from Acne Patients.

Cutibacterium acnes is a pathogen that can cause acne vulgaris, sarcoidosis, endodontic lesions, eye infections, prosthetic joint infections, and prostate cancer. Recently, bacteriophage (phage) therapy has been developed as an alternative to antibiotics. In this study, we attempted to isolate 15 phages specific to C. acnes from 64 clinical samples obtained from patients with acne vulgaris. Furthermore, we sequenced the genomes of these three phages. Bioinformatic analysis revealed that the capsid and tape measure proteins are strongly hydrophobic. To efficiently solubilize the phage particles, we measured the adsorption rate, one-step growth curve, and phage stability using an SMT2 buffer containing Tween 20. Here, we report the genotypic and phenotypic characteristics of the novel C. acnes-specific phages.

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.

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.

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.

LysSAP26, a New Recombinant Phage Endolysin with a Broad Spectrum Antibacterial Activity.

Multidrug-resistant (MDR) bacteria are a major threat to public health. Bacteriophage endolysins (lysins) are a promising alternative treatment to traditional antibiotics. However, the lysins currently under development are still underestimated. Herein, we cloned the lysin from the SAP-26 bacteriophage genome. The recombinant LysSAP26 protein inhibited the growth of carbapenem-resistant Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, oxacillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus faecium with minimum inhibitory concentrations of 5~80 µg/mL. In animal experiments, mice infected with A. baumannii were protected by LysSAP26, with a 40% survival rate. Transmission electron microscopy analysis confirmed that LysSAP26 treatment resulted in the destruction of bacterial cell walls. LysSAP26 is a new endolysin that can be applied to treat MDR A. baumannii, E. faecium, S. aureus, K. pneumoniae, P. aeruginosa, and E. coli infections, targeting both Gram-positive and Gram-negative bacteria.

Antibacterial activities of and biofilm removal by Ablysin, an endogenous lysozyme-like protein originated from Acinetobacter baumannii 1656-2.

OBJECTIVES: Multidrug-resistant (MDR) Acinetobacter baumannii as well as MDR Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and other Enterobacteriaceae ('ESKAPE' pathogens) currently present a major public-health problem. These bacteria are associated with opportunistic infections in intensive care units as well as in immunocompromised patients. There is an urgent need for new alternative antibacterials to control these MDR bacteria. Here we describe the antibacterial action of a novel peptidoglycan hydrolase that targets the bacterial cell wall, identified in the genome of clinical isolate A. baumannii 1656-2. METHODS: We generated a recombinant protein from a sequence encoding a lysozyme-like protein identified in the genome of A. baumannii 1656-2. We named it Ablysin and tested its antibacterial activity and biofilm removal ability targeting ESKAPE pathogens. RESULTS: In vitro application of Ablysin resulted in growth inhibition of the six aforementioned bacterial species, with a highest activity against A. baumannii. Electron microscopy revealed the concentration-dependent (250-2000 µg/mL) rupture of A. baumannii bacterial cells accompanied by elimination of the associated biofilm. CONCLUSIONS: Ablysin represents a potential new class of antibacterial proteins that can be used to target MDR A. baumannii as well as other bacterial species.

Antimicrobial activity of LysSS, a novel phage endolysin, against Acinetobacter baumannii and Pseudomonas aeruginosa.

OBJECTIVES: Multidrug-resistant (MDR) bacteria are a major public-health concern. Bacteriophage endolysins (lysins) can be used as novel antimicrobial agents against bacterial infections. In this study, a novel endolysin (LysSS) containing a lysozyme-like domain was evaluated for its antibacterial activity against various species of bacteria. METHODS: The LysSS-encoding gene was analyzed and cloned and the LysSS recombinant protein was expressed and purified. Purified LysSS was used to determine its antimicrobial activity against various bacterial species in vitro and to measure its protection rate against Acinetobacter baumannii systemic infection in an in vivo murine model. RESULTS: Recombinant LysSS showed activity against MDR A. baumannii, MDR Escherichia coli, MDR Klebsiella pneumoniae, MDR Pseudomonas aeruginosa and Salmonella sp. without pre-treatment with an outer membrane permeabiliser. Moreover, LysSS inhibited the growth of methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentration (MIC) of LysSS against 16 MDR A. baumannii strains ranged from 0.063-0.25mg/mL. LysSS had no cytotoxic effect on A549 human lung cells below 250µg/mL. In an animal model, mice infected with A. baumannii were protected (40% survival rate with 125µg LysSS) by intraperitoneal injection of LysSS. CONCLUSION: The current results demonstrate that LysSS may be a novel and promising antimicrobial agent against MRSA and MDR Gram-negative bacteria, including A. baumannii and P. aeruginosa.

Acinetobacter baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles.

Acinetobacter baumannii is an important nosocomial pathogen that causes a high morbidity and mortality rate in infected patients, but pathogenic mechanisms of this microorganism regarding the secretion and delivery of virulence factors to host cells have not been characterized. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs) that play a role in the delivery of virulence factors to host cells. A. baumannii has been shown to secrete OMVs when cultured in vitro, but the role of OMVs in A. baumannii pathogenesis is not well elucidated. In the present study, we evaluated the secretion and delivery of virulence factors of A. baumannii to host cells via the OMVs and assessed the cytotoxic activity of outer membrane protein A (AbOmpA) packaged in the OMVs. A. baumannii ATCC 19606(T) secreted OMVs during in vivo infection as well as in vitro cultures. Potential virulence factors, including AbOmpA and tissue-degrading enzymes, were associated with A. baumannii OMVs. A. baumannii OMVs interacted with lipid rafts in the plasma membranes and then delivered virulence factors to host cells. The OMVs from A. baumannii ATCC 19606(T) induced apoptosis of host cells, whereas this effect was not detected in the OMVs from the ΔompA mutant, thereby reflecting AbOmpA-dependent host cell death. The N-terminal region of AbOmpA(22-170) was responsible for host cell death. In conclusion, the OMV-mediated delivery of virulence factors to host cells may well contribute to pathogenesis during A. baumannii infection.

Emergence of vancomycin-intermediate Staphylococcus aureus from predominant methicillin-resistant S. aureus clones in a Korean hospital.

The genetic and epidemiological features of four vancomycin-intermediate Staphylococcus aureus (VISA) isolates obtained from a Korean hospital were evaluated in this study. The VISA isolates were genotyped as sequence type (ST) 5-staphylococcal cassette chromosome mec (SCCmec) II variant (n=2) and ST239-SCCmec III (n=2), which were derived from the predominant methicillin-resistant S. aureus (MRSA) clones in Korean hospitals. One VISA isolate was acquired during vancomycin treatment, whereas three VISA isolates were obtained from the patients who had not previously been exposed to glycopeptides. As VISA is likely to arise from the predominant MRSA clones and may then possibly spread between patients, the emergence of VISA should be monitored with great care in hospitals.

Emergence of a new mutation and its accumulation in the topoisomerase IV gene confers high levels of resistance to fluoroquinolones in Escherichia coli isolates.

Mutations in DNA gyrase and topoisomerase IV genes are the main mechanisms of resistance to quinolones. In this study, we determined mutations in gyrA, gyrB, parC and parE among 57 ciprofloxacin-resistant Escherichia coli isolates from a South Korean hospital and analysed the relationship between the minimal inhibitory concentrations (MICs) of fluoroquinolones and mutations in the topoisomerase IV gene. All ciprofloxacin-resistant E. coli isolates carried double mutations in gyrA and at least a single mutation in parC; some isolates also carried a single mutation in parE. The most common mutations were S83L and D87N in gyrA, S80I in parC and S458A in parE, which accounted for 25% of isolates. Single mutations in parE at L445I, S458P and S458W were identified for the first time. Double mutations in parC and a combination of single mutations in parC and parE significantly increased the MIC values of fluoroquinolones. In vitro induction of resistance to ciprofloxacin showed that double mutations in gyrA were a prerequisite to conferring a resistant phenotype to fluoroquinolones, and an additional mutation in the topoisomerase IV gene increased the MIC values of ciprofloxacin. In conclusion, emergence of a new mutation in parC and parE and its accumulation induces high levels of resistance to fluoroquinolones in E. coli.

Serum resistance of Acinetobacter baumannii through the binding of factor H to outer membrane proteins.

Bacteremia is a common systemic disease caused by Acinetobacter baumannii, an important hospital-acquired pathogen among critically ill patients. The complement system is central to innate immune defense against invading bacteria in the blood. The present study investigated the susceptibility of clinical A. baumannii isolates to normal human sera (NHS), and determined the resistance mechanism of A. baumannii against complement-mediated lysis. The survival of A. baumannii isolates from bacteremic patients was significantly decreased in undiluted NHS, but they were resistant to 40% NHS. The alternative complement pathway was responsible for the direct killing of bacteria. The main regulator of the alternative complement pathway, factor H, bound to the surface of live A. baumannii treated with NHS. Factor H interacted with the outer membrane proteins with molecular sizes of 38 (AbOmpA), 32, and 24 kDa. The isogenic AbOmpA(-) mutant was highly susceptible to NHS in comparison with the wild-type A. baumannii strain, suggesting that AbOmpA was an important complement regulator-acquiring surface protein. These results indicate that A. baumannii evades complement attack through the acquisition of factor H to their surface.

Genetic basis of resistance to aminoglycosides in Acinetobacter spp. and spread of armA in Acinetobacter baumannii sequence group 1 in Korean hospitals.

A total of 75 Acinetobacter isolates resistant to all available aminoglycosides obtained from 2 Korean hospitals were studied for the genetic basis of resistance to aminoglycosides. The MIC(50) and MIC(90) of Acinetobacter baumannii isolates (n = 61) to amikacin, gentamicin, isepamycin spectinomycin, streptomycin, and tobramycin were higher than those of Acinetobacter genomic species 13TU isolates (n = 14). Genes encoding aminoglycoside-modifying enzymes, ant(3")-Ia, aac(6')-Ib, aph(3')-1a, aac(3)-Ia, and aph(3')-VI, and 16S ribosomal RNA (rRNA) methylase armA were detected. ant(3")-Ia and aac(6')-Ib were commonly detected in both Acinetobacter spp., but armA and aph(3")-Ia were only detected in A. baumannii. armA was located on the plasmids. A. baumannii isolates carrying armA were classified into 7 pulsotypes but belonged to sequence group 1. The combination of aminoglycoside-modifying enzymes is responsible for the moderate-level resistance to aminoglycosides in Acinetobacter genomic species 13TU, whereas armA is responsible for the high-level resistance to aminoglycosides in A. baumannii sequence group 1.