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Covid-19 virus variants identified so far are due to viral genetic diversity, genetic evolution, and variable infectivity, suggesting that high infection rates and high mortality rates may be contributed by these mutations. And it has been reported that the targeting strategies for innate immunity should be less vulnerable to viral evolution, variant emergence and resistance. Therefore, the most effective solution to Covid-19 infection has been proposed to prevent and treat severe exacerbation of patients with moderate disease by enhancing human immune responses such as NK cell and T cell. In previous studies, we demonstrated for the first time that gamma-PGA induced significant antitumor activity and antiviral activity by modulating NK cell-mediated cytotoxicity. Especially intranasal administration of gamma-PGA was found to effectively induce protective innate and CTL immune responses against viruses and we found out that gamma-PGA can be an effective treatment for cervical intraepithelial neoplasia 1 through phase 2b clinical trial. In this study, the possibility of gamma-PGA as a Covid-19 immune modulating agent was confirmed by animal experiments infected with Covid-19 viruses. After oral administration of gamma-PGA 300mug/mouse once a day for 5 days in a K18-hACE2 TG mouse model infected with SARS-CoV-2 (NCCP 43326;original strain) and SARS-CoV-2 (NCCP 43390;Delta variant), virus titer and clinical symptom improvement were confirmed. In the RjHan:AURA Syrian hamster model infected with SARS-CoV-2 (NCCP 49930;Delta variant), 350 or 550 mug/head of gamma-PGA was administered orally for 10 days once a day. The virus for infection was administered at 5 x 104 TCID50, and the titer of virus and the improvement of pneumonia lesions were measured to confirm the effectiveness in terms of prevention or treatment. In the mouse model infected with original Covid-19 virus stain, the weight loss was significantly reduced and the survival rate was also improved by the administration of gamma-PGA. And gamma-PGA alleviated the pneumonic lesions and reduced the virus titer of lung tissue in mice infected with delta variant. In the deltavariant virus infected hamster model, gamma-PGA showed statistically significant improvement of weight loss and lung inflammation during administration after infection. This is a promising result for possibility of Covid-19 therapeutics along with the efficacy results of mouse model, suggesting gammaPGA can be therapeutic candidate to modulate an innate immune response for Covid-19.
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Background & Aim: Gene therapies has become recognized for its remarkable clinical benefits in a variety of medical applications, in particular recent approval of an Ad vector-based COVID-19 vaccines have attracted recent global attention. Here, we present key considerations for GMP compliant process development for Coxsackie virus type B3 (CVB3), an oncolytic virus designed for clinical trial in triple-negative breast cancer. Methods, Results & Conclusion(s): CVB3 is a non-enveloped, linear single-strand RNA virus with a size of approximately 27-33 um in diameter. From the initial type using the zonal rotor centrifuge to the advanced type using the tangential flow filtration system and ion chromatograph, we considered the points of the design concept in constructing the manufacturing process. The final design system is constructed as a closed and single-use manufacturing system in which all processes from upstream large-scale cell culture to downstream target purification and concentration steps. In brief, HEK293 cell suspension extended in 3L serum-free medium infected with CVB3, up to 3.6 times 10 to 7 of TCID50 /mL before going to downstream steps, made total 150 mL of final products as 8.43 times 10 to 7 of TCID50/mL concentration. Although further quality control challenges remain that is removal of product-related impurities such as human cellular proteins and residual DNA/RNA to increase virus purity, this concept is effectively applicable even for other types of viruses as GMP manufacturing processes, and would be also important for technology transfer to future commercial production.Copyright © 2023 International Society for Cell & Gene Therapy
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Intro: Performance of SARS-CoV-2 antigen tests is described relative to RT-PCR cycle threshold (Ct) values. However, Ct values may not be consistent between tests from different manufacturers, or even between runs. Here we correlate the Roche Elecsys SARS-CoV-2 Antigen assay results to a quantitative RT-PCR readout as a more reproducible measure of viral load. Additionally, we look at the relationship between the antigen test results, viral load and infectious titer. Method(s): Longitudinal nasopharyngeal swab samples from patients (N=452) with severe Covid-19 pneumonia collected between 03 April and 28 May 2020 in a randomized, double-blind, placebo controlled, multicenter study to evaluate the safety and efficacy of Tocilizumab (COVACTA), were assessed for SARS-CoV-2 viral load (RNA copies/ mL) and a qualitative and semi-quantitative readout of Elecsys SARS-CoV-2 Antigen assay. Viral culture experiments were performed to determine the infectious titer (TCID50/ mL) in a subset of samples. Agreement analysis was performed to compare the results of the assays. Please note that the current intended use of Elecsys SARS-CoV-2 Antigen assay is the qualitative detection of SARS-CoV-2 antigen. Finding(s): We observed high negative percent agreement between the Elecsys SARS-CoV-2 Antigen assay results and the RT-PCR results, while the positive percent agreement was only high in samples exceeding a certain viral load and at earlier time points from symptom onset. Infectious titer values and both the antigen assay semi-quantitative readout and the quantitative RT-PCR results correlated well. Positive percent agreement of RT-PCR and antigen results in relation to infectious titer was very high in both cases, while negative percent agreement was moderate to low. Conclusion(s): These data show that in patients with high viral load the Elecsys SARS-CoV-2 Antigen assay correlates well qualitatively and quantitatively with the presence of SARS-CoV-2 RNA and infectious virus as determined by RT-PCR and viral culture, respectively.Copyright © 2023
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The re-emergence of SARS-CoV, known as SARS-CoV-2, has proven extremely infectious that has infected a huge population worldwide. SARS-CoV-2 genome is translated into polyproteins that is processed by virus-specific protease enzymes. 3CLprotease is named as the main protease (Mpro) enzyme that cleaves nsp4 to nsp16. This crucial role of Mpro makes this enzyme a prime and promising antiviral target. Till date, there is no effective commercially available drug against COVID-19 and launching a new drug into the market is a complicated and time-consuming process. Therefore, drug repurposing is a new but familiar approach to reduce the time and cost of drug discovery. We have used a high-throughput virtual screening approach to examine FDA approved library, natural compound library, and LOPAC 1280 (Library of Pharmacologically Active Compounds, Sigma-Aldrich, St. Louis, MO) library against Mpro. Primary screening identified potential drug molecules for the target, among which ten molecules were studied further using biophysical and biochemical techniques. SPR was used to validate the binding of inhibitors to purified Mpro and using FRET-based biochemical protease assay these inhibitors were confirmed to have Mpro inhibitory activity. Based on the kinetic studies, the antiviral efficacy of these compounds was further analysed by cell-culture based antiviral assays. Four out of ten molecules inhibited SARS-CoV-2 replication in Vero cells at a concentration range of 12.5 to 50 muM. The antiviral activity was evaluated by RT-PCR assay and TCID50 experiments. The co-crystallization of Mpro in complex with inhibitor for determining their structures is being carried out. Collectively, this study will provide valuable mechanistic and structural insights for development of effective antiviral therapeutics against SARS-CoV-2.
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Background: BST2/Tetherin is an interferon-stimulated gene with antiviral activity against enveloped viruses. Particularly, BST2 tethers virions at their site of assembly, preventing their release and spread. In addition to this primary role, BST2 is as an important bridge between the innate and adaptive immune system, since (i) BST2 routes tethered particles to lysosomes, which generates viral breakdown products that engage pattern recognition receptors;and (ii) trapped virions facilitate antibody-dependent cell-mediated cytotoxicity (ADCC). In turn, viruses have evolved mechanisms to bypass BST2, either by targeting BST2 for proteasomal/lysosomal degradation or by removing BST2 from sites of virion assembly. However, the role of BST2 in SARS-CoV-2 replication, spread, evolution, and pathogenesis remains largely unknown. Method(s): The antiviral potential of BST2 against SARS-CoV-2 was investigated by infecting different SARS-CoV-2 isolates (Hong Kong, Alpha, Beta, Delta, and Omicron) in BST2+ and BST2- cells. Culture supernatants were collected to assess virion production by ELISA and infectivity by TCID50. Infected cells were analyzed by western blot and flow cytometry to examine viral and cellular protein levels, including BST2. Transfection of individual SARS-CoV-2 ORFs and mutagenesis studies allowed us to identify the genes that the virus uses to downregulate BST2. Immunoprecipitation assays revealed protein-protein interactions and changes in ubiquitination patterns. Experiments with proteasomal and lysosomal inhibitors furthered our mechanistic understanding of how SARS-CoV-2 counteracts BST2. Finally, fluorescence microscopy studies uncovered changes in the subcellular distribution of BST2 in SARS-CoV-2 infected cells. Result(s): While BST2 reduces virion release, SARS-CoV-2 has evolved to counteract this effect. Specifically, SARS-CoV-2 uses the Spike to interact with BST2, sequester the protein at perinuclear locations, and ultimately route it for lysosomal degradation. By surveying different SARS-CoV-2 variants of concern (Alpha-Omicron), we found that each variant is more efficient than the previously circulating strain at downregulating BST2 and facilitating virion production, and that mutations in the Spike account for their enhanced BST2 antagonism. Conclusion(s): As part of its adaptation to humans, SARS-CoV-2 is improving its capacity to counteract BST2, highlighting that BST2 antagonism is important for SARS-CoV-2 infectivity and transmission.
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Background: Ensitrelvir is a SARS-CoV-2 3CL protease inhibitor approved in Japan under emergency regulatory approval system as an oral treatment for COVID-19. Here we report the key analysis results of 125 mg group of phase3 part (SCORPIO-SR). Method(s): This study was a multicenter, randomized, double-blind, placebocontrolled study. Regardless of SARS-CoV-2 vaccination status and presence of risk factors for severe disease, patients with mild-to-moderate COVID-19 within 120 hours from onset were randomized to oral administration of ensitrelvir 125 mg (375 mg loading dose on Day1), ensitrelvir 250 mg (750 mg loading dose on Day1), and placebo once daily, for 5 days. The primary endpoint was time to resolution of 5 symptoms of COVID-19 (stuffy or runny nose, sore throat, cough, feeling hot or feverish, and low energy or tiredness), and the key secondary endpoints include change from baseline on Day4 in the amount of SARS-CoV-2 viral RNA and time to first negative of viral titer. The primary population for the primary and key secondary endpoints was patients with <72 hours from onset to randomization. Result(s): Median time to resolution of 5 symptoms was significantly shorter in 125 mg group (n=336, 167.9 hours) than placebo group (n=321, 192.2 hours) (p=0.0407). Mean change of viral RNA levels from baseline (log10 copies/mL) on Day4 was significantly greater in 125 mg group (-2.48) than in placebo group (-1.01) (p< 0.0001). The time to first negative of viral titer was significantly shorter in 125 mg group (n=199, 36.2 hours) compared to placebo group (n=211, 65.3 hours) (p< 0.0001). Mean changes from baseline in viral titers [log10(TCID50)/mL] were significantly greater in 125mg group on Day2 (-0.807, n=196) and Day4 (-1.108, n=197) than in the placebo group (-0.395, n=208 and -0.850, n=207, respectively) (p< 0.0001). (Table Presented) In the patients randomized within 120 hours of onset, median time to resolution of 5 symptoms was 189.7 hours in 125 mg group (n=582) and 200.3 hours in placebo group (n=572) (p=0.4352). No deaths or serious adverse drug reactions were reported in either group, and the incidence of serious adverse events between the two groups was comparable. Conclusion(s): Ensitrelvir demonstrated a significant reduction in the time to resolution of 5 typical symptoms of COVID-19, robust antiviral effects and good tolerability.
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Background: Individuals living with HIV are at increased risk of morbidity and mortality from COVID-19. Furthermore, SARS-CoV-2 infection in immunocompromised HIV infected individuals poses a risk to prolonged infection and viral shedding and the emergence of new variants of concern (VOCs). Using the SIV macaque model for AIDS, we are investigating the hypothesis that immune dysfunction during HIV infection will prolong SARSCoV- 2 viral infection, promote enhanced COVID-19 disease, and accelerate viral evolution. Here, we report the impact of SIV-CoV-2 co-infection on immune responses and pathogenesis. Method(s): Eight female rhesus macaques (aged 7-15 years, 5.5-9.9kg) were infected with SIVmac251 via low dose intravaginal challenge and then inoculated with 6.5x105 TCID50/mL SARS-CoV-2 (WA-1) at 17-34 weeks post-SIV infection via combined intranasal and intratracheal routes. Blood, bronchoalveolar lavage (BAL), stool, and nasal, oral, and rectal swabs were collected pre-infection through 14 days post-infection (DPI) to measure immune responses and viremia. ELISAs, ELISPOT, qRT-PCR, lung pathology, cytokine multiplex, and virus neutralization assays were performed to measure viral loads, pathogenesis, and immune responses. Result(s): Three days post-SARS-CoV-2 infection, we observed a transient decrease in CD4 counts, but there were no changes in clinical symptoms or plasma SIV viral loads. However, SARS-CoV-2 replication persisted in the upper respiratory tract, but not the lower respiratory tract. In addition, SARS-CoV-2 IgG seroconversion was delayed and antigen-specific T-cell responses were dampened. Notably, viral RNA levels in nasal swabs were significantly higher 7-14 DPI in SIV+ compared to previously published results using the same SARS-CoV-2 challenge virus in SIV- rhesus (PMCID: PMC8462335, PMC8829873). In addition, SIV/CoV-2 co-infected animals exhibited elevated levels of myeloperoxidase (MPO), a marker of neutrophil activation and increased lung inflammation. Conclusion(s): Here we provide evidence for the utility of the rhesus macaque in modeling human HIV-SARS-CoV-2 co-infection. Our results suggest that immunosuppression during SIV infection impairs de novo generation of anti-SARS-CoV-2 immunity, that may contribute to prolonged SARS-CoV-2 viral shedding, increased transmission windows, altered disease pathogenesis, and lower protection against subsequent SARS-CoV-2 exposures. Studies in progress will determine if SARS-CoV-2 viral evolution is accelerated in SIV-infected macaques.
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Background: The rapid emergence of the SARS-CoV-2 Omicron variant that evades many therapies illustrates the need for antiviral treatments with high genetic barriers to resistance. The small molecule PAV-104, identified through a moderate-throughput screen involving cell-free protein synthesis, was recently shown to target a subset of host protein assembly machinery in a manner specific to viral assembly with minimal host toxicity. The chemotype shows broad activity against respiratory viral pathogens, including Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Herpesviridae, and Picornaviridae, with low susceptibility to evolutionary escape. Here, we investigated the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). Method(s): Dose-dependent cytotoxicity of PAV-104 in Calu-3 cells was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 Gamma, Delta, and Omicron variants (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the N gene, immunofluorescence assay (IFA) of nucleocapsid (N) protein, and titration of supernatant (TCID50). Transient co-expression of four SARS-CoV-2 structural proteins (N, M, S, E) to produce virus-like particles (VLPs) was used to study the effect of PAV-104 on viral assembly. Drug resin affinity chromatography was performed to study the interaction between PAV-104 and N. Glycerol gradient sedimentation was used to assess N oligomerization. Total RNA-seq and the REACTOME database were used to evaluate PAV-104 effects on the host transcriptome. Result(s): PAV-104 reached 50% cytotoxicity in Calu-3 cells at 3732 nM (Fig.1A). 50 nM PAV-104 inhibited >99% of SARS-CoV-2 infection in Calu-3 cells (p< 0.01) and in primary AECs (p< 0.01) (Fig.1B-E). PAV-104 specifically inhibited SARS-CoV-2 post entry, and suppressed production of SARS-CoV-2 VLPs without affecting viral protein synthesis. PAV-104 interacted with SARS-CoV-2 N and interfered with N oligomerization. Transcriptome analysis revealed that PAV-104 treatment reversed SARS-CoV-2 induction of the interferon and maturation of nucleoprotein signaling pathways. Conclusion(s): PAV-104 is a pan-respiratory virus small molecule inhibitor with promising activity against SARS-CoV-2 in human airway epithelial cells that should be explored in animal models and clinical studies.
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Background: Natural killer (NK) cells play a critical role in control of viral infections. However, empirical evidence thus far has been unclear on the role of NK cells in pathogenesis and control of SARS-CoV-2 infection with some research suggesting NK cell accumulation as beneficial while others indicate it as deleterious. To address this crucial deficit in understanding, we employed a non-human primate infection model with a validated experimental NK cell depletion technique. Method(s): A total of 12 experimentally naive (75% female) cynomolgus macaques (CM) of Cambodian origin were used in this study. Six CM were NK cell-depleted using an anti-IL-15 neutralizing antibody, while six controls received placebo, prior to intranasal and intratracheal challenge with the SARS-CoV-2 Delta variant at a TCID50 of 1X105. The cohort was monitored for five weeks with scheduled blood, colorectal (CR) biopsies, and lymph node (LN) collections. Total envelope and sub-genomic viral loads (VL) were measured in the nasal cavity, throat, and bronchoalveolar lavage (BAL). 23-color flow cytometry, pathology, and 27-plex inflammatory analyte Luminex analyses were conducted. Statistical tests used were Mann-Whitney U and Spearman's Correlation. Result(s): Control CM exhibited an increase in the frequency of circulating NK cells, reaching a peak at 10 days post-infection (DPI) and returning to baseline by 22DPI. Simultaneously, NK cells expressing activation and tissue retention marker, CD69, also significantly increased. Cytotoxic NK cells were positively associated with VL (r=0.66;p=0.02), suggestive of a virus-induced mobilization. Total experimental NK cell ablation was verified in blood, CR, and LN of NK celldepleted CM, which had higher VL compared to controls in all tissues evaluated, reaching significance at 10DPI (p=0.01) and demonstrated a longer duration of viremia. Although Luminex measures were similar in plasma, BAL samples from NK cell-depleted CM had universally higher concentrations of inflammatory mediators, most notably a 25-fold higher concentration of IFN-alpha compared to controls. Lung pathology scores were also higher in NK cell-depleted CM with increased evidence of fibrosis, syncytia, pneumocyte hyperplasia, and endothelialitis. Conclusion(s): Overall, we find significant and conclusive evidence for NK cell-mediated control of SARS-CoV-2 virus replication and disease pathology. These data suggest adjunct therapies for infection could largely benefit from NK cell-targeted approaches.
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Background: Serum virus-neutralization (VNT)capacity is an important parameter of immunological response in COVID-19 recovered individuals and it can also be used to predict the effectiveness of therapy with COVID-19 convalescent plasma. The most commonly used tests to assess virus-neutralization are those that use native SARS-CoV- 2 (cVNT), SARS-CoV- 2 spike pseudotyped lentivirus-like particles (pVNT), and also inhibition of recombinant RBD binding to ACE2 receptor in vitro (surrogate VNT, sVNT). The aim of this study was to determine the interchangeability of different approaches in the evaluation of the virus-neutralization activity of convalescent serum samples. Method(s): Serum samples (n = 111) were collected 10-36 days after recovery between May and September 2020. The SARS-CoV- 2 strain PMVL-12/ 2020 was used in cVNT at 100 doses of TCID50. In pVNT SARS-CoV- 2 pseudotyped HIV-1 based virus-like particles with GFP reporter gene were used. sVNT was performed with a kit from Xema Co., Russia. Result(s): A very strong correlation was observed between cVNT and pVNT results (Spearman's r = 0.841). The correlation of cVNT and pVNT with the sVNT was only moderate (r = 0.674 and 0.696, respectively). This is consistent with the fact that sVNT detects only RBD blocking antibodies, which are the main but not the only inhibitors of viral infection. Serum samples were also tested for RBD-specific IgG, IgM, IgA antibodies by ELISA. A good correlation was found between the cVNT, pVNT, sVNT results and the RBD-specific IgG (r = 0.669, 0.620, and 0.643, respectively), that confirms a crucial role of specific IgG antibodies in virus-neutralization. RBD-specific IgA showed a moderate correlation with the neutralization capacity of sera from recovered individuals (r = 0.563, 0.583, 0.544). Correlation of approximately the same level was observed between cVNT, pVNT, and RBD-specific IgM (r = 0.663, 0.615), but not sVNT and RBD-specific IgM (r = 0.395). Conclusion(s): This study demonstrated the possibility of using the safe and relatively simple pseudotyped virus-neutralization test instead of cVNT to assess the sera virus-neutralizing capacity. sVNT may be efficiently used in screening studies.
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Rationale: To establish a novel SARS-CoV-2 human challenge model enabling controlled investigation of pathogenesis, correlates of protection and efficacy testing of interventions. Method(s): Thirty-six healthy 18-29-year-old subjects, without evidence of previous infection or vaccination, received 10 TCID50 of a wild-type virus (SARS-CoV-2/human/GBR/484861/2020) intranasally. Following inoculation, subjects resided in a high-containment quarantine, with 24-hour medical monitoring. The study's main objectives were to identify a virus dose that induced well-tolerated infection in >50% of subjects and assess virus and symptoms over time. AEs and longitudinal disease profiles are presented. Result(s): Eighteen of thirty-four evaluable (~53%) subjects became infected and developed serum antibodies. Viral load rose steeply and peaked ~5 days post-inoculation (PI). Virus was first detected in the throat but rose to significantly higher levels in the nose, peaking at ~8.87 log10 copies/ml (median, 95% CI [8.41,9.53]). Viable virus was recoverable from the nose up to ~10 days PI, on average. Mild-to-moderate symptoms were reported by 16 (89%) infected subjects, beginning 2-4 days PI. Anosmia or dysosmia developed in 15 (83%) subjects. Results from lateral flow tests were associated with viable virus and modelling showed that twice-weekly rapid antigen tests could diagnose infection before 70-80% of viable virus had been generated. There were no overt lung function changes, CT abnormalities, or SAEs. Conclusion(s): This novel SARS-CoV-2 challenge of 18-29-year-olds was considered safe. Viral kinetics over the course of primary infection was established, with implications for public health recommendations and strategies to impact transmission.
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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.
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The C-terminal domain (CTD) of porcine deltacoronavirus S1 subunit is the main region which induces the neutralizing antibody. S1-CTD was expressed by HEK-293T eukaryotic expression system and purified, and porcine ileal epithelium cells membrane proteins were extracted to investigate porcine host proteins that interact with it. Thirty-two suspected interacting host proteins were obtained by co-inmunprecipitation (Co-IP) and mass spectrometry. Eukaryotic expression plasmid of KIF1 binding protein (KIFBP) was constructed, and the interaction between KIFBP and S1-CTD was identified by Co-IP and laser confocal microscopy. All results proved that KIFBP interacted with S1-CTD and co-located in cytoplasm. Further research indicated that overexpression of KIFBP could effectively reduce the viral mRNA level and the viral titer in which the mRNA level decreased by about 70%, and the viral titer decreased by 101.6TCID50. In conclusion, a host protein KIFBP interacting with PDCoV S1-CTD was screened and identified in this study which provides a theoretical basis for understanding the pathogenesis of PDCoV.
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.
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Previous data have suggested an antiviral effect of teriflunomide, including against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the agent underlying the ongoing COVID-19 pandemic. We undertook an in vitro investigation to evaluate the inhibitory activity of teriflunomide against SARS-CoV-2 in a cell-based assay. Teriflunomide was added to Vero (kidney epithelial) cells that had been infected with SARS-CoV-2. A nucleocapsid immunofluorescence assay was performed to examine viral inhibition with teriflunomide and any potential cytotoxic effect. The 50% effective concentration (EC(50)) for teriflunomide against SARS-CoV-2 was 15.22 μM. No cytotoxicity was evident for teriflunomide in the Vero cells (i.e., the 50% cytotoxic concentration [CC(50)] was greater than the highest test concentration of 100 μM). The data were supported by additional experiments using other coronaviruses and human cell lines. In the SARS-CoV-2-infected Vero cells, the prodrug leflunomide had an EC(50) of 16.49 μM and a CC(50) of 54.80 μM. Our finding of teriflunomide-mediated inhibition of SARS-CoV-2 infection at double-digit micromolar potency adds to a growing body of evidence for a broad-ranging antiviral effect of teriflunomide.
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The ongoing pandemic caused by the novel coronavirus, SARS-CoV-2, is influencing global health. Moreover, there is a major threat of future coronaviruses affecting the entire world in a similar, or even more dreadful, manner. Therefore, effective and biocompatible therapeutic options against coronaviruses are urgently needed. To address this challenge, medical specialists require a well-informed and safe approach to treating human coronaviruses (HCoVs). Herein, an environmental friendly approach for viral inactivation, based on plasma technology, was considered. A microwave plasma system was employed for the generation of the high amount of gaseous nitric oxide to prepare nitric oxide enriched plasma-activated water (NO-PAW), the effects of which on coronaviruses, have not been reported to date. To determine these effects, alpha-HCoV-229E was used in an experimental model. We found that NO-PAW treatment effectively inhibited coronavirus infection in host lung cells, visualized by evaluating the cytopathic effect and expression level of spike proteins. Interestingly, NO-PAW showed minimal toxicity towards lung host cells, suggesting its potential for therapeutic application. Moreover, this new approach resulted in viral inactivation and greatly improved the gene levels involved in host antiviral responses. Together, our findings provide evidence of an initiation point for further progress toward the clinical development of antiviral treatments, including such coronaviruses. © 2022 The Authors
ABSTRACT
Aggressive diagnostic testing remains an indispensable strategy for health and aged care facilities to prevent the transmission of SARS-CoV-2 in vulnerable populations. The preferred diagnostic platform has shifted towards COVID-19 rapid antigen tests (RATs) to identify the most infectious individuals. As such, RATs are being manufactured faster than at any other time in our history yet lack the relevant quantitative analytics required to inform on absolute analytical sensitivity enabling manufacturers to maintain high batch-to-batch reproducibility, and end-users to accurately compare brands for decision making. Here, we describe a novel reference standard to measure and compare the analytical sensitivity of RATs using a recombinant GFP-tagged nucleocapsid protein (NP-GFP). Importantly, we show that the GFP tag does not interfere with NP detection and provides several advantages affording streamlined protein expression and purification in high yields as well as faster, cheaper and more sensitive quality control measures for post-production assessment of protein solubility and stability. Ten commercial COVID-19 RATs were evaluated and ranked using NP-GFP as a reference standard. Analytical sensitivity data of the selected devices as determined with NP-GFP did not correlate with those reported by the manufacturers using the median tissue culture infectious dose (TCID50) assay. Of note, TCID50 discordance has been previously reported. Taken together, our results highlight an urgent need for a reliable reference standard for evaluation and benchmarking of the analytical sensitivity of RAT devices. NP-GFP is a promising candidate as a reference standard that will ensure that RAT performance is accurately communicated to healthcare providers and the public.
ABSTRACT
Background At the Urbino hospital, all patients tested with antigen tests for SARS-CoV-2, if positive, are confirmed with a molecular test. Among the molecular tests in use in our laboratory, VitaPCRTM SARS-CoV-2 Assay is the fastest and easiest to use, as it takes about half an hour from preparation to the result and does not require a biosecurity laboratory, because performed from inactivated samples. Aim of the study Given the rapidity of VitaPCRTM SARS-CoV-2 Assay, it was decided to use it for the confirmation of samples found positive to LIAISON SARS-CoV-2 Ag test. This last test is performed by dedicated tubes with inactivating buffer based on detergent, therefore it does not interfere in the processes of extraction and amplification of nucleic acids. Materials and methods For evaluation, 48 samples tested positive with LIAISON SARS-CoV-2 Ag (Diasorin) test were analyzed, coming from various departments of the Urbino hospital, and confirmed by SimplexaTM COVID-19 Direct (Diasorin) molecular test. From the tubes of all the samples, 200 uL were taken and transferred to dedicated tubes for analysis with VitaPCRTM SARS-CoV-2 Assay (Menarini) to be analyzed, after eliminating 2.2 mL of buffer. Results VitaPCRTM SARS-CoV-2 Assay was able to detect SARSCoV- 2 RNA in all 48 tested antigenically positive samples. In particular, even in all positive samples with low values of TCID50/mL (<1000), 11 out of 48, results with Ct<28.8 were found, although not maintaining a direct correspondence between quantity of antigen and quantity of detected RNA. Conclusions The results obtained, despite requiring an increase in the number of cases, demonstrate the potential use of VitaPCRTM SARS-CoV-2 Assay for the confirmation of positive samples directly from the inactivated buffer used for LIAISON SARS-CoV-2 Ag assay. VitaPCRTM instrument, given the speed, simplicity of use and the possibility of use outside biosafety laboratories, it is an excellent diagnostic tool to support the confirmation procedures of samples tested positive for the SARSCoV- 2 N antigen.
ABSTRACT
COVID-19 has resulted in nearly 598 million infections and over 6.46 million deaths since the start of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in 2019. The rapid onset of the pandemic, combined with the emergence of viral variants, crippled many health systems particularly from the perspective of coping with massive diagnostic loads. Shortages of diagnostic kits and capacity forced laboratories to store clinical samples resulting in huge backlogs, the effects of this on diagnostic pickup have not been fully understood. Herein, we investigated the impact of storing SARS-CoV-2 inoculated dry swabs on the detection and viability of four viral strains over a period of 7 days. Viral load, as detected by qRT-PCR, displayed no significant degradation during this time for all viral loads tested. In contrast, there was a ca. 2 log reduction in viral viability as measured by the tissue culture infectious dose (TCID) assay, with 1-3 log viable virus detected on dry swabs after 7 days. When swabs were coated with 102 viral copies of the Omicron variant, no viable virus was detected after 24 hours following storage at 4°C or room temperature. However there was no loss of PCR signal over 7 days. All four strains showed comparable growth kinetics and survival when cultured in Vero E6 cells. Our data provide information on the viability of SARS-CoV-2 on stored swabs in a clinical setting with important implications for diagnostic pickup and laboratory processing protocols. Survival after 7 days of SARS-CoV-2 strains on swabs with high viral loads may impact public health and biosafety practices in diagnostic laboratories.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , Pandemics , Viral LoadABSTRACT
Enrichment of viral infectious titers following its propagation by cell culture is desirable for various experimental studies. The performance of an ultrafiltration (UF) process to concentrate infectious titers of non-enveloped Canine parvovirus 2 (CPV-2) and enveloped Feline coronavirus (FCoV) obtained from cell culture supernatants was evaluated in this study, and compared with ultracentrifugation (UC) process. A mean gain of > 1.0 log10 TCID50/mL was obtained for CPV-2 with UF, which was comparable with the gain obtained by UC. On the other hand, the gain was lower (0.7-1.0 log10 TCID50/mL) for FCoV with UF in contrast to UC (> 2.0 log10 TCID50/mL). However, the lower retentate volume following UC (â¼120 fold) compared to that following UF (â¼10 fold) for either of the viruses suggests a trend of increased infectious titer retention in UF concentrates relative to UC concentrates. The simplistic UF process evaluated here thus has the potential for use in applications requiring increased infectious titers of CPV-2 and FCoV.