The in vitro and in vivo efficacy of CT-P59 against Gamma, Delta and its associated variants of SARS-CoV-2.

The SARS-CoV-2 variant is rapidly spreading across the world and causes to resurge infections. We previously reported that CT-P59 presented its in vivo potency against Beta variants, despite its reduced activity in cell experiments. Yet, it remains uncertain to exert the antiviral effect of CT-P59 on Gamma, Delta and its associated variants (L452R). To tackle this question, we carried out cell tests and animal studies. CT-P59 showed neutralization against Gamma, Delta, Epsilon, and Kappa variants in cells, with reduced susceptibility. The mouse challenge experiments with Gamma and Delta variants substantiated in vivo potency of CT-P59 showing symptom remission and virus abrogation in the respiratory tract. Collectively, cell and animal studies showed that CT-P59 is effective against Gamma and Delta variants infection, hinting that CT-P59 has therapeutic potential for patients infected with Gamma, Delta and its associated variants.

Therapeutic effect of CT-P59 against SARS-CoV-2 South African variant.

The global circulation of newly emerging variants of SARS-CoV-2 is a new threat to public health due to their increased transmissibility and immune evasion. Moreover, currently available vaccines and therapeutic antibodies were shown to be less effective against new variants, in particular, the South African (SA) variant, termed 501Y.V2 or B.1.351. To assess the efficacy of the CT-P59 monoclonal antibody against the SA variant, we sought to perform as in vitro binding and neutralization assays, and in vivo animal studies. CT-P59 neutralized B.1.1.7 variant to a similar extent as to wild type virus. CT-P59 showed reduced binding affinity against a RBD (receptor binding domain) triple mutant containing mutations defining B.1.351 (K417N/E484K/N501Y) also showed reduced potency against the SA variant in live virus and pseudovirus neutralization assay systems. However, in vivo ferret challenge studies demonstrated that a therapeutic dosage of CT-P59 was able to decrease B.1.351 viral load in the upper and lower respiratory tracts, comparable to that observed for the wild type virus. Overall, although CT-P59 showed reduced in vitro neutralizing activity against the SA variant, sufficient antiviral effect in B.1.351-infected animals was confirmed with a clinical dosage of CT-P59, suggesting that CT-P59 has therapeutic potential for COVID-19 patients infected with SA variant.

A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein.

Vaccines and therapeutics are urgently needed for the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we screen human monoclonal antibodies (mAb) targeting the receptor binding domain (RBD) of the viral spike protein via antibody library constructed from peripheral blood mononuclear cells of a convalescent patient. The CT-P59 mAb potently neutralizes SARS-CoV-2 isolates including the D614G variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/RBD shows that CT-P59 blocks interaction regions of RBD for angiotensin converting enzyme 2 (ACE2) receptor with an orientation that is notably different from previously reported RBD-targeting mAbs. Furthermore, therapeutic effects of CT-P59 are evaluated in three animal models (ferret, hamster, and rhesus monkey), demonstrating a substantial reduction in viral titer along with alleviation of clinical symptoms. Therefore, CT-P59 may be a promising therapeutic candidate for COVID-19.

Comparable Immune Function Inhibition by the Infliximab Biosimilar CT-P13: Implications for Treatment of Inflammatory Bowel Disease.

BACKGROUND AND AIMS: CT-P13 is the first biosimilar monoclonal antibody to infliximab, and was recently approved in the European Union, Japan, Korea, and USA for all six indications of infliximab. However, studies directly assessing the biologic activity of CT-P13 versus inflximab in the context of inflammatory bowel disease [IBD] are still scanty. In the present study, we aimed to compare the biological activities of CT-P13 and infliximab with specific focus on intestinal cells so as to gain insight into the potential biosimilarity of these two agents for treatment of IBD. METHODS: CT-P13 and infliximab were investigated and compared by in vitro experiments for their neutralisation ability of soluble tumour necrosis factor alpha [sTNFα] and membrane-bound tumour necrosis factor alpha [mTNFα], suppression of cytokine release by reverse signalling, induction of regulatory macrophages and wound healing, and antibody-dependent cell cytotoxicity [ADCC]. RESULTS: CT-P13 showed similar biological activities to infliximab as gauged by neutralisation of soluble TNFα, as well as blockade of apoptosis and suppression of pro-inflammatory cytokines in intestinal Caco-2 cells. Infliximab and CT-P13 equally induced apoptosis and outside-to-inside signals through transmembrane TNFα [tmTNFα]. Moreover, regulatory macrophage induction and ensuing wound healing were similarly exerted by CT-P13 and infliximab. However, neither CT-P13 nor infliximab exerted any significant ADCC of ex vivo-stimulated peripheral blood monocytes or lamina propria mononuclear cells from IBD patients. CONCLUSIONS: These findings indicate that CT-P13 and infliximab exert highly similar biological activities in intestinal cells, and further support a mechanistic comparability of these two drugs in the treatment of IBD.

Recapitulation of Candidate Systemic Lupus Erythematosus-Associated Variants in Koreans.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects multiple organ systems. Although the etiology of SLE remains unclear, it is widely accepted that genetic factors could be involved in its pathogenesis. A number of genome-wide association studies (GWASs) have identified novel single-nucleotide polymorphisms (SNPs) associated with the risk of SLE in diverse populations. However, not all the SNP candidates identified from non-Asian populations have been validated in Koreans. In this study, we aimed to replicate the SNPs that were recently discovered in the GWAS; these SNPs have not been validated in Koreans or have only been replicated in Koreans with an insufficient sample size to conclude any association. For this, we selected five SNPs (rs1801274 in FCGR2A and rs2286672 in PLD2, rs887369 in CXorf21, rs9782955 in LYST, and rs3794060 in NADSYN1). Through the replication study with 656 cases and 622 controls, rs1801274 in FCGR2A was found to be significantly associated with SLE in Koreans (odds ratio, 1.26, 95% confidence interval, 1.06 to 1.50; p = 0.01 in allelic model). This association was also significant in two other models (dominant and recessive). The other four SNPs did not show a significant association. Our data support that FCGR polymorphisms play important roles in the susceptibility to SLE in diverse populations, including Koreans.

Draft genome sequence of non-shiga toxin-producing Escherichia coli O157 NCCP15738.

BACKGROUND: The non-shiga toxin-producing Escherichia coli (non-STEC) O157 is a pathogenic strain that cause diarrhea but does not cause hemolytic-uremic syndrome, or hemorrhagic colitis. Here, we present the 5-Mb draft genome sequence of non-STEC O157 NCCP15738, which was isolated from the feces of a Korean patient with diarrhea, and describe its features and the structural basis for its genome evolution. RESULTS: A total of 565-Mbp paired-end reads were generated using the Illumina-HiSeq 2000 platform. The reads were assembled into 135 scaffolds throughout the de novo assembly. The assembled genome size of NCCP15738 was 5,005,278 bp with an N50 value of 142,450 bp and 50.65 % G+C content. Using Rapid Annotation using Subsystem Technology analysis, we predicted 4780 ORFs and 31 RNA genes. The evolutionary tree was inferred from multiple sequence alignment of 45 E. coli species. The most closely related neighbor of NCCP15738 indicated by whole-genome phylogeny was E. coli UMNK88, but that indicated by multilocus sequence analysis was E. coli DH1(ME8569). CONCLUSIONS: A comparison between the NCCP15738 genome and those of reference strains, E. coli K-12 substr. MG1655 and EHEC O157:H7 EDL933 by bioinformatics analyses revealed unique genes in NCCP15738 associated with lysis protein S, two-component signal transduction system, conjugation, the flagellum, nucleotide-binding proteins, and metal-ion binding proteins. Notably, NCCP15738 has a dual flagella system like that in Vibrio parahaemolyticus, Aeromonas spp., and Rhodospirillum centenum. The draft genome sequence and the results of bioinformatics analysis of NCCP15738 provide the basis for understanding the genomic evolution of this strain.

Quantifying fenobucarb residue levels in beef muscles using liquid chromatography-tandem mass spectrometry and QuEChERS sample preparation.

This paper describes a comparison of the properties of the three versions of the QuEChERS method (quick, easy, cheap, effective, rugged and safe) - the original (unbuffered), acetate-buffered, and citrate-buffered methods - for the determination of fenobucarb residues in beef muscles via liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI(+)-MS/MS). The recovery results were good for all the versions; however, the acetate-buffered version gave higher and more consistent recoveries for fenobucarb than the other versions. Performance characteristics, such as linearity, accuracy, and precision were determined. Matrix-matched standard calibration was used for quantification, obtaining recoveries in the range of 83.7-93.4% with relative standard deviations of <5%, at two spiking levels: 10 and 40 µg/kg. The limits of detection (LOD) and quantification (LOQ) were estimated to be 1.5 and 5 µg/kg, respectively. Finally, the method was applied to the analysis of 15 market samples, and no residues were found over the limit of quantification. The method developed was found able to determine the analyte with satisfactory intensity and accuracy.

Development of QuEChERS-based extraction and liquid chromatography-tandem mass spectrometry method for quantifying flumethasone residues in beef muscle.

A rapid, specific, and sensitive method based on liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) in the positive ion mode using multiple reaction monitoring (MRM) was developed and validated to quantify flumethasone residues in beef muscle. Methods were compared between the original as well as the EN quick, easy, cheap, effective, rugged, and safe (QuEChERS)-based extraction. Good linearity was achieved at concentration levels of 5-30 µg/kg. Estimated recovery rates at spiking levels of 5 and 10 µg/kg ranged from 72.1 to 84.6%, with relative standard deviations (RSDs)<7%. The results of the inter-day study, which was performed by fortifying beef muscle samples (n=18) on 3 separate days, showed an accuracy of 93.4-94.4%. The precision (expressed as relative standard deviation values) for the inter-day variation at two levels of fortification (10 and 20 µg/kg) was 1.9-5.2%. The limit of detection (LOD) and limit of quantitation (LOQ) were 1.7 and 5 µg/kg, at signal-to-noise ratios (S/Ns) of 3 and 10, respectively. The method was successfully applied to analyze real samples obtained from large markets throughout the Korean Peninsula. The method proved to be sensitive and reliable and, thus, rendered an appropriate means for residue analysis studies.

Determination of PDE-5 inhibitors and appetite suppressants in adulterated dietary supplements using LC/PDA and LC/MS.

There have been a number of reports of dietary supplements contaminated with illegal adulterants that threaten consumers' health because of their adverse pharmacological effects. In the present study, a convenient and economic method was developed to detect illegal pharmaceutics, such as PDE-5 inhibitor and appetite suppressants, using liquid chromatography (LC)/photodiode array (PDA) for screening and LC/mass spectrometry (MS) for successive confirmation. Target peaks were identified by comparison of their chromatographic retention times and PDA spectra with those of synthetic standards and finally confirmed by LC/MS. As a result, tadalafil, a PDE-5 inhibitor, and N-desmethylsibutramine, a derivative of sibutramine, were detected in various dietary supplements at concentrations of 13.5-21.9 mg and 3.0 mg per single dose, respectively. The present study will contribute to the development of an analytical method enabling rapid screening of a variety of health foods, and the result suggests that consumers should be aware of serious health risks related to these illegal compounds.

Determination of Oxyclozanide in Beef and Milk using High-Performance Liquid Chromatography System with UV Detector.

This study was developed and validated for the determination of oxyclozanide residue concentrations in beef and commercial milk, using high-performance liquid chromatography system. Oxyclozanide was successfully separated on a reverse phase column (Xbridge-C(18), 4.6×250 mm, 5 µm) with a mobile phase composed of acetonitrile and 0.1% phosphoric acid (60:40, v/v%). This analytical procedure involved a deproteinization process using acetonitrile for beef and 2% formic acid in acetonitrile for commercial milk, dehydration by adding sodium sulfate to the liquid analytical sample, and a defatting process using n-hexane; after these steps, the extract was exposed to a stream of nitrogen dryness. The final extracted sample was dissolved in the mobile phase and filtered using a 0.45 µm syringe filter. This method had good selectivity and recovery (70.70±7.90-110.79±14.95%) from the matrices. The LOQs ranged from 9.7 to 9.8 µg/kg for beef and commercial milk. The recoveries met the standards set by the CODEX guideline.

Simultaneous multiresidue analysis of 41 pesticide residues in cooked foodstuff using QuEChERS: Comparison with classical method.

The principal objective of this study was to develop a simple multiresidue method involving a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method for the identification and quantification of 41 pesticide residues in cooked foodstuffs including cooked potatoes, radishes, and rice using GC-µECD. The analytes were subsequently confirmed via GC-MS. The results were then compared using the classical method established by the KFDA. The quantitation of individual pesticides was based on matrix-matched calibration curves with a correlation coefficient in excess of 0.993 for the 41 pesticides selected herein. Using QuEChERS, the mean recoveries ranged between 68.6 and 130.0% for the majority of the tested pesticides; however, the classical method exhibited low recoveries for dichlofluanid, tetraconazole, oxadixyl, fenbuconazloe, and paclobutrazol. After QuEChERS, the LODs and LOQs ranged between 0.004 and 0.3µg/kg and 0.0125 and 1.0µg/kg, respectively. The proposed method was applied successfully to determine the residue levels in cooked foodstuffs, and none of the samples contained detectable amounts of pesticide residues.

Detection of hepatitis a virus from oyster by nested PCR using efficient extraction and concentration method.

The molecular methods using polymerase chain reaction have been proposed as useful tools for the identification of viral pathogens in food and water. However, the PCR-based methods are highly dependent on the methods of virus concentration and nucleic acid purification due to the low sensitivity of PCR in the presence of PCR inhibitors. We developed TPTT [tris elution buffer-PEG-TRIzol-poly(dT) magnetic bead] protocol in order to detect hepatitis A virus (HAV) inoculated in oyster digestive glands. The detection limit of HAV precipitated with zirconium hydroxide was 10(5) fold less sensitive in a nested PCR than that precipitated the HAV supernatant twice with PEG/NaCl (16% polyethylene glycol 6,000, 0.525 M NaCl) in a 1:2 (v/v) ratio, which provided an efficient detection of 0.0148 PFU/g from approximately 0.05 g of oyster homogenate. This method is efficient for potential use in the detection of HAV from shellfish and is more sensitive than most currently published tests.