9 research outputs found
Development of a neutralization monoclonal antibody with a broad neutralizing effect against SARS-CoV-2 variants
No full textBackground
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has challenged the effectiveness of current therapeutic regimens. Here, we aimed to develop a potent SARS-CoV-2 antibody with broad neutralizing effect by screening a scFv library with the spike protein receptor-binding domain (RBD) via phage display.
Methods
SKAI-DS84 was identified through phage display, and we performed pseudovirus neutralization assays, authentic virus neutralization assays, and in vivo neutralization efficacy evaluations. Furthermore, surface plasmon resonance (SPR) analysis was conducted to assess the physical characteristics of the antibody, including binding kinetics and measure its affinity for variant RBDs.
Results
The selected clones were converted to human IgG, and among them, SKAI-DS84 was selected for further analyses based on its binding affinity with the variant RBDs. Using pseudoviruses, we confirmed that SKAI-DS84 was strongly neutralizing against wild-type, B.1.617.2, B.1.1.529, and subvariants of SARS-CoV-2. We also tested the neutralizing effect of SKAI-DS84 on authentic viruses, in vivo and observed a reduction in viral replication and improved lung pathology. We performed binding and epitope mapping experiments to understand the mechanisms underlying neutralization and identified quaternary epitopes formed by the interaction between RBDs as the target of SKAI-DS84.
Conclusions
We identified, produced, and tested the neutralizing effect of SKAI-DS84 antibody. Our results highlight that SKAI-DS84 could be a potential neutralizing antibody against SARS-CoV-2 and its variants.This work was supported by the National Research Foundation of Korea (NRF-2021R1F1A1055906, NRF-2014M3A9D5A01075128, 2021M3H9A1030260) grant funded by the Korea government (MSIT). This research was supported by the R&D Project for the Improvement of Science and Industrial Technology in Gwangwon-do (2022-DD-UP-0287
Implant-associated biofilm infection established in an experimental Galleria mellonella model
No full text
The study reports in vivo biofilm infection implemented in an insect model. We mimicked implant-associated biofilm infections in Galleria mellonella larvae using toothbrush bristles and methicillin-resistant Staphylococcus aureus (MRSA). In vivo biofilm formation on bristle was achieved by sequentially injecting a bristle and MRSA into the larval hemocoel. Then, we evaluated the degree of in vivo biofilm infection by monitoring staphylococcal selective mannitol agar media cultured with bacteria harvested from bristles. As a result, it was found that biofilm formation was in progress without any external sign of infection in most of the bristle-bearing larvae for 12 h after MRSA inoculation. In addition, we investigated the antimicrobial activity of the prophenoloxidase (PPO) system in hemolymph and an antimicrobial peptide (AMP) against MRSA biofilm. The activation of the PPO system did not affect the preformed in vitro MRSA biofilms. In contrast, an AMP exerted its activity against in vitro biofilms and interfered with in vivo biofilm formation when injected into bristle-bearing larvae infected with MRSA. Finally, our confocal laser scanning microscopic analysis revealed that the biomass of the in vivo biofilm is greater compared to that of the in vitro biofilm and harbors a distribution of dead cells, which might be bacteria and/or host cells. </p
Laboratory information management system for COVID-19 non-clinical efficacy trial data
No full textBACKGROUND: As the number of large-scale studies involving multiple organizations producing data has steadily increased, an integrated system for a common interoperable format is needed. In response to the coronavirus disease 2019 (COVID-19) pandemic, a number of global efforts are underway to develop vaccines and therapeutics. We are therefore observing an explosion in the proliferation of COVID-19 data, and interoperability is highly requested in multiple institutions participating simultaneously in COVID-19 pandemic research. RESULTS: In this study, a laboratory information management system (LIMS) approach has been adopted to systemically manage various COVID-19 non-clinical trial data, including mortality, clinical signs, body weight, body temperature, organ weights, viral titer (viral replication and viral RNA), and multiorgan histopathology, from multiple institutions based on a web interface. The main aim of the implemented system is to integrate, standardize, and organize data collected from laboratories in multiple institutes for COVID-19 non-clinical efficacy testings. Six animal biosafety level 3 institutions proved the feasibility of our system. Substantial benefits were shown by maximizing collaborative high-quality non-clinical research. CONCLUSIONS: This LIMS platform can be used for future outbreaks, leading to accelerated medical product development through the systematic management of extensive data from non-clinical animal studies
Genetic characterization and pathogenicity in a mouse model of newly isolated bat-originated mammalian orthoreovirus in South Korea
No full textMammalian orthoreoviruses (MRVs) infect a wide range of hosts, including humans, livestock, and wildlife. In the present study, we isolated a novel Mammalian orthoreovirus from the intestine of a microbat (Myotis aurascens) and investigated its biological and pathological characteristics. Phylogenetic analysis indicated that the new isolate was serotype 2, sharing the segments with those from different hosts. Our results showed that it can infect a wide range of cell lines from different mammalian species, including human, swine, and non-human primate cell lines. Additionally, media containing trypsin, yeast extract, and tryptose phosphate broth promoted virus propagation in primate cell lines and most human cell lines, but not in A549 and porcine cell lines. Mice infected with this strain via the intranasal route, but not via the oral route, exhibited weight loss and respiratory distress. The virus is distributed in a broad range of organs and causes lung damage. In vitro and in vivo experiments also suggested that the new virus could be a neurotropic infectious strain that can infect a neuroblastoma cell line and replicate in the brains of infected mice. Additionally, it caused a delayed immune response, as indicated by the high expression levels of cytokines and chemokines only at 14 days post-infection (dpi). These data provide an important understanding of the genetics and pathogenicity of mammalian orthoreoviruses in bats at risk of spillover infections.IMPORTANCEMammalian orthoreoviruses (MRVs) have a broad range of hosts and can cause serious respiratory and gastroenteritis diseases in humans and livestock. Some strains infect the central nervous system, causing severe encephalitis. In this study, we identified BatMRV2/SNU1/Korea/2021, a reassortment of MRV serotype 2, isolated from bats with broad tissue tropism, including the neurological system. In addition, it has been shown to cause respiratory syndrome in mouse models. The given data will provide more evidence of the risk of mammalian orthoreovirus transmission from wildlife to various animal species and the sources of spillover infections. Mammalian orthoreoviruses (MRVs) have a broad range of hosts and can cause serious respiratory and gastroenteritis diseases in humans and livestock. Some strains infect the central nervous system, causing severe encephalitis. In this study, we identified BatMRV2/SNU1/Korea/2021, a reassortment of MRV serotype 2, isolated from bats with broad tissue tropism, including the neurological system. In addition, it has been shown to cause respiratory syndrome in mouse models. The given data will provide more evidence of the risk of mammalian orthoreovirus transmission from wildlife to various animal species and the sources of spillover infections.Y
Establishment of multicenter COVID-19 therapeutics preclinical test system in Republic of Korea
No full textThroughout the recent COVID-19 pandemic, South Korea led national efforts to develop vaccines and thera-peutics for SARS-CoV-2. The project proceeded as follows: 1) evaluation system setup (including Animal Biosafety Level 3 (ABSL3) facility alliance, standardized nonclinical evaluation protocol, and laboratory infor-mation management system), 2) application (including committee review and selection), and 3) evaluation (including expert judgment and reporting). After receiving 101 applications, the selection committee reviewed pharmacokinetics, toxicity, and efficacy data and selected 32 final candidates. In the nonclinical efficacy test, we used golden Syrian hamsters and human angiotensin-converting enzyme 2 transgenic mice under a cytokeratin 18 promoter to evaluate mortality, clinical signs, body weight, viral titer, neutralizing antibody presence, and histopathology. These data indicated eight new drugs and one repositioned drug having significant efficacy for COVID-19. Three vaccine and four antiviral drugs exerted significant protective activities against SARS-CoV-2 pathogenesis. Additionally, two anti-inflammatory drugs showed therapeutic effects on lung lesions and weight loss through their mechanism of action but did not affect viral replication. Along with systematic veri-fication of COVID-19 animal models through large-scale studies, our findings suggest that ABSL3 multicenter alliance and nonclinical evaluation protocol standardization can promote reliable efficacy testing against COVID-19, thus expediting medical product development.Y
Additional file 1 of Development of a neutralization monoclonal antibody with a broad neutralizing effect against SARS-CoV-2 variants
No full textAdditional file 1. Additional material and figures
SIRT1 ubiquitination is regulated by opposing activities of APC/C-Cdh1 and AROS during stress-induced premature senescence
No full textAbstract SIRT1, a member of the mammalian sirtuin family, is a nicotinamide adenosine dinucleotide (NAD)-dependent deacetylase with key roles in aging-related diseases and cellular senescence. However, the mechanism by which SIRT1 protein homeostasis is controlled under senescent conditions remains elusive. Here, we revealed that SIRT1 protein is significantly downregulated due to ubiquitin-mediated proteasomal degradation during stress-induced premature senescence (SIPS) and that SIRT1 physically associates with anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ubiquitin ligase. Ubiquitin-dependent SIRT1 degradation is stimulated by the APC/C coactivator Cdh1 and not by the coactivator Cdc20. We found that Cdh1 depletion impaired the SIPS-promoted downregulation of SIRT1 expression and reduced cellular senescence, likely through SIRT1-driven p53 inactivation. In contrast, AROS, a SIRT1 activator, reversed the SIRT1 degradation induced by diverse stressors and antagonized Cdh1 function through competitive interactions with SIRT1. Furthermore, our data indicate opposite roles for Cdh1 and AROS in the epigenetic regulation of the senescence-associated secretory phenotype genes IL-6 and IL-8. Finally, we demonstrated that pinosylvin restores downregulated AROS (and SIRT1) expression levels in bleomycin-induced mouse pulmonary senescent tissue while repressing bleomycin-promoted Cdh1 expression. Overall, our study provides the first evidence of the reciprocal regulation of SIRT1 stability by APC/C-Cdh1 and AROS during stress-induced premature senescence, and our findings suggest pinosylvin as a potential senolytic agent for pulmonary fibrosis
Laboratory information management system for COVID-19 non-clinical efficacy trial data
No full textBackground: As the number of large-scale studies involving multiple organizations producing data has steadily increased, an integrated system for a common interoperable format is needed. In response to the coronavirus disease 2019 (COVID-19) pandemic, a number of global efforts are underway to develop vaccines and therapeutics. We are therefore observing an explosion in the proliferation of COVID-19 data, and interoperability is highly requested in multiple institutions participating simultaneously in COVID-19 pandemic research.
Results: In this study, a laboratory information management system (LIMS) approach has been adopted to systemically manage various COVID-19 non-clinical trial data, including mortality, clinical signs, body weight, body temperature, organ weights, viral titer (viral replication and viral RNA), and multiorgan histopathology, from multiple institutions based on a web interface. The main aim of the implemented system is to integrate, standardize, and organize data collected from laboratories in multiple institutes for COVID-19 non-clinical efficacy testings. Six animal biosafety level 3 institutions proved the feasibility of our system. Substantial benefits were shown by maximizing collaborative high-quality non-clinical research.
Conclusions: This LIMS platform can be used for future outbreaks, leading to accelerated medical product development through the systematic management of extensive data from non-clinical animal studies.ope
Rapid discovery and classification of inhibitors of coronavirus infection by pseudovirus screen and amplified luminescence proximity homogeneous assay
No full textTo identify potent antiviral compounds, we introduced a high-throughput screen platform that can rapidly classify hit compounds according to their target. In our platform, we performed a compound screen using a lentivirus-based pseudovirus presenting a spike protein of coronavirus, and we evaluated the hit compounds using an amplified luminescence proximity homogeneous assay (alpha) test with purified host receptor protein and the receptor binding domain of the viral spike. With our screen platform, we were able to identify both spike-specific compounds (class I) and broad-spectrum antiviral compounds (class II). Among the hit compounds, thiosemicarbazide was identified to be selective to the interaction between the viral spike and its host cell receptor, and we further optimized the binding potency of thiosemicarbazide through modification of the pyridine group. Among the class II compounds, we found raloxifene and amiodarone to be highly potent against human coronaviruses including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. In particular, using analogs of the benzothiophene moiety, which is also present in raloxifene, we have identified benzothiophene as a novel structural scaffold for broad-spectrum antivirals. This work highlights the strong utility of our screen platform using a pseudovirus assay and an alpha test for rapid identification of potential antiviral compounds and their mechanism of action, which can lead to the accelerated development of therapeutics against newly emerging viral infections
