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Background/Purpose: Although assessing the immunogenicity and protective efficacy of acellular pertussis (aP) vaccines via murine model studies faces limitations, preliminary assessments have been achieved by evaluating respiratory challenge and humoral and cellular immunity. Methods: We performed a long-term intranasal respiratory challenge with reference and clinically isolated strains of Bordetella pertussis. Simultaneously, we assessed humoral and cellular immunity for evaluating the immunogenicity of a newly developed tri-component diphtheria-tetanus-aP (DTaP) vaccine. Moreover, comparative assessment was made by performing the same evaluations with a commercially available tri-component DTaP vaccine as the positive control. Results: Both groups showed significantly increased levels of antibodies against pertussis toxin, filamentous hemagglutinin and pertactin, and the levels of interferon-γ and interleukin-10 were significantly increased after two doses of vaccination. Furthermore, since cross cell-mediated immune reactivity between the two vaccines was detected, the possibility of interchangeability was indirectly suggested. Although the positive control group showed significantly higher titers in antibody responses for filamentous hemagglutinin and pertactin compared to the experimental group, anti-pertussis toxin antibody titers of the two groups were not significantly different and the protective efficacy against the clinical and reference strains was maintained in both groups for 18 weeks. Conclusion: The results showed inferior immunogenicity of the new DTaP vaccine compared to a commercial vaccine despite comparable cellular immunity and protective efficacy. Some efforts are necessary for improving immunogenicity against filamentous hemagglutinin and pertactin before conducting human clinical trials. Keywords: Diphtheria-tetanus-acellular pertussis vaccine, Immunogenicity, Efficacy, Mice

Purpose There is no standard method for confirming the immunogenicity of acellular pertussis vaccines. We tried to develop a local standard method for evaluating the immunogenicity of the three-component of acellular pertussis vaccines which was developed by a Korean local company. Materials and methods The developed pertussis antigens (pertussis toxin, filamentous hemagglutinin, pertactin) were evaluated by in-house enzyme-linked immunosorbent assay (ELISA) using 189 negative sera, 25 positive sera, and 73 paired sera (pre- and post-Tdap [tetanus, diphtheria, and acellular pertussis] vaccinated sera). ELISA units were calculated by the reference line method, compared with World Health Organization reference sera, and the cut-off value was calculated using negative sera. Results When compared to National Institute for Biological Standards and Control control antigen (NIBSC) control antigens, the developed pertussis toxin (PT) and filamentous haemagglutinin (FHA) antigens were 203.48 and 61.60 IU/µg, respectively. Each in-house ELISA was established by validating the coefficients of variation % (PT, 11.53%; FHA, 8.60%; pertactin [PRN], 9.86%) obtained from the results of inter- and intra-assay variation. Also, the cut-off values of PT, FHA, and PRN were 11.65, 38.95, and 5.66 EU/mL, respectively. The distributions of antibody levels in paired showed that 93.15% (68/73) in anti-PT IgG, 97.26% (72/73) in anti-FHA IgG, and 100% in anti-PRN IgG were higher than a 100% increase after vaccination. Additionally, the values of 89.04% (65/73) in anti-PT IgG, 97.26% (72/73) in anti-FHA IgG, and 100% in anti-PRN IgG were below each cut-off point. Conclusion We established an in-house ELISA method using self-developed antigens, and these immunoassays have provided a way to standardize measuring the immunogenicity of newly developed vaccines, through single- and dual-serology.

Purpose Although the DTaP and Tdap vaccines used to prevent pertussis have been used for a long time, there is no standard method for measuring pertussis antigens. Therefore, this preliminary study was conducted to develop an enzyme-linked immunosorbent assay method using an animal model for measuring antibodies against pertussis toxin, the most important pertussis pathogenic antigen, in the sera of vaccinated mice. Materials and Methods Bordetella pertussis Tohama phase I was cultured for 24–30 hours, and then pertussis toxin was purified from the culture medium by chromatography. Purified pertussis toxin was diluted in phosphate-buffered saline-coating buffer, and 100 µL of diluted pertussis toxin was added to each well and reacted at room temperature for 4 hours. Positive serum was diluted to 1/1,250–1/80,000 and negative serum was diluted to 1/50 to determine the coating concentration with the optimal signal/noise ratio. Optimal test conditions were confirmed from the dilution factors of the secondary antibody and streptavidin horseradish peroxidase (SA-HRP). Results Optimal conditions were as follows: 4 µg/mL for coating antigen; 1/40,000 for secondary antibody; and 1/1,000 for the SA-HRP dilution factor. Comparison of the sera obtained from mice treated with a developing vaccine and commercial vaccine with National Institute for Biological Standard and Control standard serum under the established conditions showed the following results: 1,300.62, 534.94, and 34.85, respectively. Conclusion The method developed in this study is suitable for measuring anti-pertussis toxin antibodies and may be applicable for clinical sample analysis or indirect diagnosis of pertussis.

Pertussis toxin (PTx) is an essential component of the acellular pertussis (aP) vaccine. However, because PTx in its native form is considered too toxic for human vaccine use, it must be inactivated into a stable, nontoxic form by treatment with chemical detoxifying agents or by genetic modification. Therefore, testing for the residual PTx in the aP vaccine is a major quality control step for vaccine manufacturers and regulatory authorities. The histamine sensitization test is currently the standard safety test method for all aP vaccines, regardless of the vaccine formula or the detoxification process, except for those with genetically modified PTx. However, test result variability and ethical concerns regarding animal use necessitate an alternative method. In vitro assays based on the biochemical properties of PTx have been considered as potential alternatives to the histamine sensitization test. In this study, the suitability of assays based on the ADP-ribosyltransferase and carbohydrate binding activities of PTx was assessed for PTx after treatment with formaldehyde, glutaraldehyde or both denaturants in sequence. The results indicated a distinctive pattern of the biochemical activities depending on the detoxification methods and storage conditions. These results suggest that although a more careful study is needed, these in vitro biochemical assays can be considered potential alternatives to the histamine sensitization test, as they might provide more specific safety information of aP vaccines.

Coenzyme Q 10 (CoQ 10 ) is an electron carrier in the respiration chain with antioxidant activity. The effects of aeration and agitation rates, dissolved oxygen levels, an electron flux inhibitor (azide), a proton gradient releaser (2,4-dinitrophenol (DNP)), and an oxidative stressor (hydrogen peroxide (H 2 O 2 )) on the intracellular CoQ 10 contents in Agrobacterium tumefaciens ATCC4452 were investigated. With a decrease of dissolved oxygen level from 20 to 5%, the intracellular CoQ 10 content increased about four-fold, yielding 2 mg/g dry cell weight at 5% dissolved oxygen level. Azide significantly increased the intracellular CoQ 10 content, with the highest value of 5.3 mg/g dry cell weight in the presence of 0.45 mM of sodium azide. However, DNP (up to 200 μM) and H 2 O 2 (up to 10 μM) did not affect the intracellular CoQ 10 content, indicating proton gradient release, and oxidative stress do not affect the synthesis of CoQ 10 . These results show that restricted electron flux by limited oxygen supply and the addition of azide increases the intracellular CoQ 10 content, suggesting a feedback regulation of CoQ 10 biosynthesis by its physiological function of electron carrier in the respiration chain.

A newly isolated enantioselective esterase from Pseudomonas fluorescens KCTC 1767, which is currently considered as a biocatalyst for the production of a commercially valuable ( S )-ketoprofen, has revealed a low structural and thermal stability. In order to enhance the stability, directed evolution was attempted on this enantioselective esterase by successive steps of an error prone and staggered extension PCR. After the second round of evolution, the best mutant 6–52 with enhanced thermal stability was selected and analyzed. DNA sequence analyses of 6–52 revealed that the three amino acid residues (L120P, I208V, and T249A) were changed and the mutation L120P was presumed as a structurally important residue due to its presence in all positive variants. The purified mutant 6–52, when incubated at 50 and 55 °C for 2 h, remained its activity over 30 and 10%, respectively, whereas there were no detectable activities in wild-type enzyme. The analysis of 6–52 in the presence of 15% ethanol showed 1.8-fold increase in the activity, compared to that of wild-type enzyme. The K m and V max values of 6–52 were estimated to be slightly increased, leading to 1.2-fold-higher the catalytic efficacy k cat / K m than that of wild-type enzyme. Additionally, the mutant 6–52 was more resistant to high substrate concentrations than that of wild-type enzyme.

A newly isolated gene from Pseudomonas sp. KCTC 10122BP, encoding an esterase with enantioselectivity towards racemic ketoprofen (rac-ketoprofen) ethyl ester, was cloned in Escherichia coli and its nucleotide sequence determined. The deduced amino acid sequence predicted an open reading frame (ORF) encoding a polypeptide of 381 amino acid residues (1143 nucleotides) with a calculated isoelectric point of pH 5.32 and molecular mass of 41,149 Da. The primary structure of the enzyme exhibited a significant level of homology (>31%) with those of related enzymes from various sources and an extreme homology (>81%) with five esterases from the genus Pseudomonas. The enzyme was expressed at a high level in an active form in the soluble fraction and purified to homogeneity by a successive chromatographic procedure. The purified enzyme was determined to be a monomer, plus it exhibited a strict selectivity (>99%) and high activity (2360 units/mg-protein) towards (S)-ketoprofen ethyl ester.

In order to obtain novel strains that hydrolyzed the rac-ketoprofen ethyl ester to an enantiomer of ketoprofen, we enriched strains from broad ecological niches in which the thermostable enzymes were ubiquitously found. The isolated strains were first analyzed to detect the ester-hydrolyzing activity by using a selective plate, and then further screened by activity on the (R,S)-ketoprofen ethyl ester. Twenty-six strains were screened primarily and further compared for the ethyl ester-hydrolyzing activity in terms of conversion yield and enantioselectivity. Consequently, a strain JY144 was isolated as a novel strain that produced a (R)-stereospecific esterase with a high stability and systematically identified as a Bacillus stearothermophilus JY144. The enzyme was indeed stable at a broad range of pH (6.0–9.5) and temperature, up to 65 °C. The optimal temperature and pH for enzymatic conversion were 50 °C and 9.0, respectively. Since the wild-type strain showed a poor cell growth and enzyme activity, we further attempted the cloning of esterase gene into Escherichia coli, with a simple and rapid strategy for selecting the positive clones, and determined its primary structure. The cloned gene had an open reading frame (250 amino acids) with a calculated molecular mass of 27.4 kDa. The primary structure showed a relatively high homology (38–52%) to probable esterases unidentified to date, and thus presumed as a novel family enzyme. In whole cell conversions, the recombinant enzyme had a superior potential than the parent strain, and it could completely convert the rac-ketoprofen ethyl ester to (R)-ketoprofen, with an optical purity of 99%.

To isolate novel strains expressing an esterase that hydrolyzed the rac-ketoprofen ethyl ester to (S)-ketoprofen in the stereospecific manner, we screened broad ecological niches and soil samples in which the activity was expected to be found. Thousands of microbial strains were tested to determine their ester-hydrolyzing activity by using an agar plate containing insoluble tributyrin as an indicative substrate, and then further screened by activity on the (R, S)-ketoprofen ethyl ester. Twenty-eight strains were screened primarily and compared with respect to the potential to ketoprofen ethyl ester-hydrolyzing activity in terms of conversion yield and chiral specificity. Consequently, a strain S34 was isolated as a best producer and finally identified as a Pseudomonas sp. S34. We first formulated the optimal medium for the high level production of the enzyme, and as a preliminary experiment for enzymatic resolution, we characterized the fractionated enzyme. The enzyme with ketoprofen ethyl ester-hydrolyzing activity to (S)-ketoprofen showed a high degree of enantioselectivity (>94%) and was mainly found in cell extracts, whereas no distinct activity was detected in culture broth. The optimum pH and temperature of the enzyme were 9.5 and 35 °C, respectively. The activity of the enzyme was markedly increased (four-fold) by addition of a non-ionic detergent Triton X-100 and, resultantly, a high activity toward ketoprofen ethyl ester (52 U/mg) was found. The small-scale conversion of (R, S)-ketoprofen ethyl ester to (S)-ketoprofen using the partially purified enzymes was completed in 28 h, with optical purity of 99% and yield of 47%.

Metagenomes from various environmental soils were screened using alpha-naphthyl acetate and Fast Blue RR for a novel ester-hydrolyzing enzyme on Escherichia coli. Stepwise fragmentations and subcloning of the initial insert DNA (30-40 kb) using restriction enzymes selected to exclude already known esterases with subsequent screenings resulted in a positive clone with a 2.5-kb DNA fragment. The cloned sequence included an open reading frame consisting of 1089 bp, designated as est25, encoding a protein of 363 amino acids with a molecular mass of about 38.3 kDa. Amino acid sequence analysis revealed only moderate identity (or = 48%) to the known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases, such as HGGG (residues 124-127), GxSxG (residues 199-203), and the putative catalytic triad composed of Ser201, Asp303, and His333. Est25 was functionally overexpressed in a soluble form in E. coli with optimal activity at pH 7.0 and 25 degrees C. The purified Est25 exhibited hydrolyzing activity toward p-nitrophenyl (NP)-fatty acyl esters with short-length acyl chains (or = C6) with the highest activity toward p-NP-acetate (Km=1.0 mM and Vmax = 63.7 U/mg), but not with chain lengthsor = C8, demonstrating that Est25 is an esterase originated most likely from a mesophilic microorganism in soils. Est25 efficiently hydrolyzed (R,S)-ketoprofen ethyl ester with Km of 16.4 mM and Vmax of 59.1 U/mg with slight enantioselectivity toward (R)-ketoprofen ethyl ester. This study demonstrates that functional screening combined with the sequential uses of restriction enzymes to exclude already known enzymes is a useful approach for isolating novel enzymes from a metagenome.

The ester-hydrolyzing enzyme families, including lipase and esterase, mediated a broad range of reactions and, thus, were able to act on a variety of ester compounds that are found naturally or exploited industrially. With the increasing demand for pharmacological use, attempts to produce an enantiomer (S)-ketoprofen from the corresponding ethyl ester have recently been proliferating, but information about the structure and function of related enzymes has not been reported to date in detail. Here, we reported the construction, expression, and one-step purification of a potential esterase in Escherichia coli with a hexahistidine tag at its N-terminus. The expression level of the enzyme was more than 20% of the total protein in E. coli, resulting in approximately 1.2mg of the purified proteins by an affinity resin, Ni-NTA, from a 0.2L of bacterial culture in a single step. As typical properties, its innate traits that revealed favorable reactions at alkaline pH and high activity to the triglycerides composed of short chain fatty acids (C(6)) supported the enzyme to be an esterase. The enzyme was determined to be a monomer with a calculated molecular mass of 42 kDa and showed quite a high activity to rac-ketoprofen ethyl ester (27,000 U), with strict selectivity to (S)-enantiomer (99% ee(p)). The small-scale conversion using the recombinant enzyme strongly suggested the enzyme to be useful for enzyme-mediated chiral resolution of (S)-ketoprofen.