ABSTRACT
Background & Aim: The new UCOE models we have recently developed, tested on many cell groups (including mouse ES and human iPS cells) and human mAb recombinant production studies as well, shows a powerful resistance to DNA methylation- mediated silencing and provides a higher and stable transfection profile. By the urgent need of vaccine development for COVID-19 during the pandemic, in this study we aimed to produce a potential recombinant vaccine by using the new generation UCOEs models of our own design. Methods, Results & Conclusion(s): Existing new-generation UCOE models and standard plasmid vectors to be used as control group were provided. Then, the sequences related to the PCR method were amplified for sufficient stock generation and cloning experiments. Verification in the plasmid vector was carried out in gel electrophoresis. Transfection of 293T cells was performed with clone plasmids carrying antigen genes and plasmids carrying genetic information of lentivirus units for the production of lentiviral vectors. Afterwards, 293T cells produced lentiviral vectors carrying antigen genes. Harvesting of these vectors was carried out during 48th and 72nd hours. Afterwards, CHO cells were transduced with appropriate quantity of lentiviral vectors. Isolation and purification of targeted proteins from the relevant medium were performed by HPLC and Q-TOF methods. A part of the spike and nucleocapsid gene sequences of COVID-19 were firstly cloned into our UCOE models. These UCOEs plasmids were then transferred into 293T cells along with plasmids carrying the genes that will form the lentivirus vectors (LVs). After harvesting and calculation of LV vector titers, the cloned vectors were then transfected into the CHO cells which the targeted recombinant production of the antigen proteins will be carried out. Antigenic structures were then isolated from the culture medium of CHO cells in following days for confirmation. Using HPLC and qTOF mass spectrometer methods, these structures in the medium were confirmed to be the units of spike and nucleocapsid proteins of the COVID-19 virus. In order to produce large amount of the recombinant antigens, the culture was then carried out with bioreactors in liters. At the final stage, these recombinantly produced antigen proteins were tested on rats to measure their immunogenic responses, and the study recently been completed successfully as a potential recombinant vaccine against COVID-19.Copyright © 2023 International Society for Cell & Gene Therapy
ABSTRACT
Finding effective and safe medicines to fight SARS-CoV-2 infection is an urgent task. RPH-137 is an original trap fusion protein against SARS-CoV-2 virus. It comprises the angiotensin-converting enzyme type 2 extracellular domain and the human IgG1 Fc fragment. The aim of the study was to carry out a preclinical evaluation of the efficacy of RPH-137 and molnupiravir against SARS-CoV-2 infection. Material(s) and Method(s): the authors analysed RPH-137 expressed in a stable CHO cell line and molnupiravir used as an active pharmaceutical ingredient. Drug-mediated inhibition of virus-induced cytotoxicity was assessed in Vero cell culture. In vivo efficacy assessments were performed in Syrian hamsters. The animals were infected intranasally with SARS-CoV-2 (PIK35 clinical isolate) in the dose of 5 log TCID50. The authors evaluated body weight measurements, lung-body weight ratios, and lung histopathology findings and determined viral RNA levels in oropharyngeal swabs by RT-PCR using the amplification cycle threshold (Ct). The statistical analyses involved one- and two-way ANOVA, Student's t-test, and Mann-Whitney test. Result(s): RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells;the EC50 values of RPH-137 amounted to 4.69 mug/mL (21.3 nM) and 16.24 mug/mL (73.8 nM) for 50 TCID50 and 200 TCID50, respectively, whereas the EC50 values of molnupiravir were 0.63 mug/mL (1900 nM) for both doses. Intramuscular RPH-137 (30 and 80 mg/kg) had no effect on the infection process in Syrian hamsters. The comparison with the challenge control group showed that intraperitoneal RPH-137 (100 mg/kg) had statistically significant effects on a number of parameters, including a 27% reduction in inflammation and a 30% reduction in the total lesion area of the lungs by Day 7. Intragastric molnupiravir (300 mg/kg twice daily) significantly inhibited SARS-CoV-2 infection. Conclusion(s): both RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells. In Syrian hamsters, molnupiravir demonstrated a more pronounced inhibition of SARS-CoV-2 than RPH-137. However, RPH-137 had statistically significant effects on a range of parameters. This offers additional perspectives for further research.Copyright © 2023 Safety and Risk of Pharmacotherapy. All rights reserved.
ABSTRACT
Finding effective and safe medicines to fight SARS-CoV-2 infection is an urgent task. RPH-137 is an original trap fusion protein against SARS-CoV-2 virus. It comprises the angiotensin-converting enzyme type 2 extracellular domain and the human IgG1 Fc fragment. The aim of the study was to carry out a preclinical evaluation of the efficacy of RPH-137 and molnupiravir against SARS-CoV-2 infection. Materials and methods: the authors analysed RPH-137 expressed in a stable CHO cell line and molnupiravir used as an active pharmaceutical ingredient. Drug-mediated inhibition of virus-induced cytotoxicity was assessed in Vero cell culture. In vivo efficacy assessments were performed in Syrian hamsters. The animals were infected intranasally with SARS-CoV-2 (PIK35 clinical isolate) in the dose of 5 log TCID50. The authors evaluated body weight measurements, lung-body weight ratios, and lung histopathology findings and determined viral RNA levels in oropharyngeal swabs by RT-PCR using the amplification cycle threshold (Ct). The statistical analyses involved one- and two-way ANOVA, Student's t-test, and Mann–Whitney test. Results: RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells;the EC50 values of RPH-137 amounted to 4.69 μg/mL (21.3 nM) and 16.24 μg/mL (73.8 nM) for 50 TCID50 and 200 TCID50, respectively, whereas the EC50 values of molnupiravir were 0.63 μg/mL (1900 nM) for both doses. Intramuscular RPH-137 (30 and 80 mg/kg) had no effect on the infection process in Syrian hamsters. The comparison with the challenge control group showed that intraperitoneal RPH-137 (100 mg/kg) had statistically significant effects on a number of parameters, including a 27% reduction in inflammation and a 30% reduction in the total lesion area of the lungs by Day 7. Intragastric molnupiravir (300 mg/kg twice daily) significantly inhibited SARS-CoV-2 infection. Conclusions: both RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells. In Syrian hamsters, molnupiravir demonstrated a more pronounced inhibition of SARS-CoV-2 than RPH-137. However, RPH-137 had statistically significant effects on a range of parameters. This offers additional perspectives for further research.
ABSTRACT
Aim To explore the feasibility of the micro- dynamic chromogenic method for quantitative detection of bacterial endotoxin in recombinant novel coronavirus vaccine ( CHO cell).Methods The micro-dynamic color method of Limulus reagent was used to establish a bacterial endotoxin standard curve.The dilution factor was determined through interference pre -experiment, the recoverv rate of the endotoxin added to the test so- J lution was determined, and the interference test to complete the quantitative detection test of the bacterial endotoxin content in the test product was performed, and the results were compared with those of the gel-clot method.Results Hie linear range of the concentration of the standard curve was 0.02 to 2.0 EU * mL 1 , and the regression equation of the standard curve was lgT =-0.302 7 lgC +2.858 7( r = 0.998 9).When recombinant novel coronavirus vaccine ( CHO cell) was cliluted 40 times or below, the micro -dynamic chromogenic reagent did not interfere with the bacterial endotoxin agglutination reaction, and the recovery rate was 50% to 200%.The test results were consistent with the gel- clot method.Conclusions The micro-dynamic chromogenic method can be used for the quantitative detection of bacterial endotoxins in recombinant novel coronavirus vaccine ( CHO cell) with accurate results, high sensitivity, and process monitoring. Copyright © 2022 Publication Centre of Anhui Medical University. All rights reserved.
ABSTRACT
Since November 2019, the COVID-19 pandemic has been going on around the world, according to the WHO, more 5.5 million people have died. The main strategy for developing therapeutic antibodies is to obtain human viral neutralizing antibodies directed to the receptor-binding domains (RBD) of the SARS-CoV-2 S-protein. However, it is known that the immune response of humans and mice to different antigens is different, therefore, studies of B-cell epitopes of SARS-CoV-2 S-protein with mouse monoclonal antibodies may allow us to find new virus neutralizing epitopes. Eighteen monoclonal antibodies (mAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were obtained using hybridoma technology from mice immunized with inactivated SARS-CoV-2. ELISA demonstrated that selected 16 mAbs bound recombinant spike (S) protein and 2 mAbs bound recombinant nucleocapsid (N) protein. The equilibrium dissociation constants of the obtained mAbs against S protein ranged from 0.08 to 10 nM. Three mAbs bound recombinant RBD of S protein, the equilibrium dissociation constants of the mAbs against RBD ranged from 0.2 to 3 nM. Anti-RBD mAbs did not neutralize SARS-CoV-2 in the plaque reduction neutralization test. mAbs RS2 demonstrated a dose-dependent inhibition of plaque formation after infection with SARS-CoV-2. The kD and IC50 values for this antibody were 0.2 nM and 400 mcg/ml, respectively. To determine the S protein region responsible for binding to mAb RS2 S1, S2 and RBD subunit of S protein SARS-CoV-2 were expressed in CHO cells. Unfortunately, the localization of the epitope recognized by neutralizing mAb RS2 was not identified using ELISA or western blot analysis. Moreover, mAb RS2 do not recognized full sized recombinant S-protein in western blot analysis. The obtained results demonstrated that the epitope recognized by neutralizing mAb RS2 were discontinuous and have quaternary structure.