Journal: medRxiv

Article Title: Development of a rapid SARS-CoV-2 neutralisation test by detecting antibodies that block interaction of spike protein receptor-binding domain (RBD) with angiotensin-converting enzyme 2 (ACE2)

doi: 10.1101/2021.06.03.21255116

Figure Lengend Snippet: 1a: ACE2 immobilised on Biochip. Anti-SARS-CoV-2 neutralising antibodies block SARS-CoV-2 Spike RBD HRP from binding to ACE2 on Biochip. 1b: SARS-CoV-2 Spike RBD immobilised on Biochip. Anti-SARS-CoV-2 neutralising antibodies block ACE2 HRP from binding to SARS-CoV-2 RBD on Biochip.

Article Snippet: Virus neutralisation antibodies were detected in serum and plasma samples using a blocking chemiluminescent Biochip-based immunoassay on the Evidence Investigator Biochip analyser (SARS-CoV-2 Surrogate Virus Neutralising Assay EV4454, Evidence Investigator analyser EV3602, Randox Laboratories Ltd).

Techniques: Blocking Assay, Binding Assay

Journal: medRxiv

Article Title: Development of a rapid SARS-CoV-2 neutralisation test by detecting antibodies that block interaction of spike protein receptor-binding domain (RBD) with angiotensin-converting enzyme 2 (ACE2)

doi: 10.1101/2021.06.03.21255116

Figure Lengend Snippet: Validation of SARS-CoV-2 Biochip pVNT in a dose response manner. Inhibition of SARS-CoV-2 RBD and ACE2 interaction using specified concentrations of SARS-CoV-2 recombinant neutralising antibodies

Article Snippet: Virus neutralisation antibodies were detected in serum and plasma samples using a blocking chemiluminescent Biochip-based immunoassay on the Evidence Investigator Biochip analyser (SARS-CoV-2 Surrogate Virus Neutralising Assay EV4454, Evidence Investigator analyser EV3602, Randox Laboratories Ltd).

Techniques: Inhibition, Recombinant

Journal: medRxiv

Article Title: Development of a rapid SARS-CoV-2 neutralisation test by detecting antibodies that block interaction of spike protein receptor-binding domain (RBD) with angiotensin-converting enzyme 2 (ACE2)

doi: 10.1101/2021.06.03.21255116

Figure Lengend Snippet: Inhibition of SARS-CoV-2 RBD ACE2 interaction by samples (A-D) collected from patients confirmed neutralising antibody seropositive using the SARS-CoV-2 microneuralisation test. A reduction in % inhibition is observed for all patients using the pVNT as sample dilution increases (starting dilution 1:25). Dotted line indicates SARS-CoV-2 Biochip pVNT cut-off at 50% inhibition.

Article Snippet: Virus neutralisation antibodies were detected in serum and plasma samples using a blocking chemiluminescent Biochip-based immunoassay on the Evidence Investigator Biochip analyser (SARS-CoV-2 Surrogate Virus Neutralising Assay EV4454, Evidence Investigator analyser EV3602, Randox Laboratories Ltd).

Techniques: Inhibition

Journal: medRxiv

Article Title: Development of a rapid SARS-CoV-2 neutralisation test by detecting antibodies that block interaction of spike protein receptor-binding domain (RBD) with angiotensin-converting enzyme 2 (ACE2)

doi: 10.1101/2021.06.03.21255116

Figure Lengend Snippet: Testing of 110 SARS-CoV-2 microneutralisation assay (mVNT) confirmed seropositive or seronegative samples on the SARS-CoV-2 Biochip pVNT. Dotted line indicates SARS-CoV-2 Biochip pVNT cut-off at 50% inhibition.

Article Snippet: Virus neutralisation antibodies were detected in serum and plasma samples using a blocking chemiluminescent Biochip-based immunoassay on the Evidence Investigator Biochip analyser (SARS-CoV-2 Surrogate Virus Neutralising Assay EV4454, Evidence Investigator analyser EV3602, Randox Laboratories Ltd).

Techniques: Inhibition

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Schematic overview of the anti-SARS-CoV-2 drug screening procedure. ( a ) ACE2 targeted inhibitors and compounds from the natural product library were applied prior to infection with SARS-CoV-2 (pre-infection treatment) while compounds from the FDA-approved library and the flavonoid library were applied post infection (post- infection treatment). Out of 2191 compounds tested, 121 displayed viral CPE reductions and 7 were selected for further validations using Vero E6 and HuH7 cell lines and hNEC. ( b ) Uninfected cells, infected cells treated with 0.1% DMSO, and infected cells treated with remdesivir were included as controls in the screen. Following treatment with citicoline, pravastatin sodium, tenofovir alafenamide, imatinib mesylate, calcitriol, dexlansoprazole, and prochlorperazine dimaleate, reduced CPE was observed when compared to the DMSO control.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Drug discovery, Infection, Control

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Summary of compounds identified from the primary screen that exhibit potential antiviral effects against SARS-CoV-2 infection.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Infection, Reverse Transcription, Reflux

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Validation of primary hits. For validating compounds with activity pre-infection, Vero E6 cells were first pre-treated with increasing concentrations of ( a ) citicoline, ( b ) pravastatin sodium and ( c ) tenofovir alafenamide prior to infection with SARS-CoV-2. Similarly, Huh7 cells were pre-treated with ( d ) citicoline, ( e ) pravastatin sodium and ( f ) tenofovir alafenamide and subsequently infected with SARS-CoV-2. For post-infection treatment validation, Vero E6 cells were infected with SARS-CoV-2 and treated with increasing concentrations of ( g ) imatinib mesylate, ( h ) calcitriol, ( i ) dexlansoprazole and ( j ) prochlorperazine dimaleate. Similarly, HuH7 cells were also infected then treated with a range of concentrations of ( k ) imatinib mesylate, ( l ) calcitriol, ( m ) dexlansoprazole and ( n ) prochlorperazine dimaleate. The primary and secondary exes correspond to the viral titre and relative cell viability, and the dashed line represents the CC 50 cut-off for cell viability. One-way ANOVA and Dunnett’s post-test were used to determine statistical differences, with * denoting p < 0.05, ** denoting p < 0.01 and *** denoting p < 0.001. Error bars represent the standard deviation observed from the means of triplicates performed for both cell viability and dose-dependent inhibition studies.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Biomarker Discovery, Activity Assay, Infection, Standard Deviation, Inhibition

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Validation of primary hits in hNECs. Selected primary hits were further validated in hNECs. Cells were either pre-treated with 10 μM citicoline prior to SARS-CoV-2 infection or treated following SARS-CoV-2 infection with 10 μM imatinib mesylate and 10 μM calcitriol. One-way ANOVA and Dunnett’s post-test were used to determine statistical differences, with ** denoting that p < 0.01. Error bars represent the standard deviation observed from the means of triplicates performed for dose-dependent inhibition studies.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Biomarker Discovery, Infection, Standard Deviation, Inhibition

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: SARS-CoV-2 and calcitriol modulate vitamin D Receptor (VDR), 24-hydroxylase (24(OH)ase), and cathelicidin (LL-37) in Vero E6 and Huh7 cells. In the first part of the experiment, Vero E6 and Huh7 cells were either mock-infected with media, or SARS-CoV-2 infected and harvested at indicated timepoints in the absence of calcitriol for baseline VDR, 24(OH)ase, and LL-37 mRNA expression. Vero E6 expression of ( a ) VDR, ( b ) 24(OH)ase and ( c ) LL-37 and Huh7 expression of ( d ) VDR, ( e ) 24(OH)ase and ( f ) LL-37 were as shown. In the second part of the experiment, Vero E6 and Huh7 cells were either mock-infected with media or SARS-CoV-2 for 1 h as above, then treated with 0.01, 0.1, 1, 10 or 20 µM calcitriol following infection. At 48 h post infection, the cells were harvested to check for VDR, 24(OH)ase, and LL-37 mRNA expression. Vero E6 mRNA expression of ( g ) VDR, ( i ) 24(OH)ase and ( k ) LL-37 and Huh7 expression of ( h ) VDR, ( j ) 24(OH)ase and ( l ) LL-37 were as shown. Data are represented as relative quantity to 0.1% DMSO control, or as relative quantity to mock-infected control. One-way ANOVA and Dunnett’s post-test and paired t -test were used to determine statistical differences, with * denoting p < 0.05, ** denoting p < 0.01 and *** denoting p < 0.001. Error bars represent the standard deviation observed from the means of triplicates performed for mRNA expression studies.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Infection, Expressing, Control, Standard Deviation

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Western blot analysis of SARS-CoV-2 spike and VDR proteins in Vero E6 cells. ( a ) Vero E6 cells are either mock infected with media (−) or with SARS-CoV-2 (+) for 1 h. After 1 h post-infection, the cells are then either untreated (−) or treated (+) with 20 µM calcitriol for 6-, 24- and 48- hours before harvesting of total cell lysate. ( b ) Densitometry quantitation of protein expression levels is shown as fold changes to beta-actin. Full-length blots are shown in .

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Western Blot, Infection, Quantitation Assay, Expressing

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: In vivo study of calcitriol treatment. 8-week old K18-hACE2 male and female mice were separated into treatment and non-treatment groups. Treatment study group has been given 3 days pre-treatment and 5 days post-treatment of calcitriol (5 μg/kg) via intraperitoneal injection. The non-treatment group has been given a PBS as a treatment control. Next, 10 3 PFU of SARS-CoV-2 (L, Alpha, and Beta variants) were inoculated through intranasal delivery to all groups after pre-treatment. Physiological parameters: body weight ( a – c ), survival rate ( d – f ), and physiological conditions were monitored after infections. For comparison between individual physiological conditions, 5 dpi infected mice were compared between treatment groups of all three variant infected mice ( g – i ). Scoring of physiological conditions was based on five criteria: appearance of the mouse coat, level of consciousness, activity level, eye condition, and respiratory quality. To compare the severity of damage in mouse tissues after infection, left lung lobes from 4 dpi mice were processed for histological analyses and scored based on six criteria to determine severity ( j – l ). The six criteria are inflammatory cell infiltration, haemorrhage, oedema, bronchial epithelial cell damage, degeneration of alveolar epithelial cells, and parenchymal wall expansion. For viral load determination, right lung, brain, liver, and spleen tissues from the same 4 dpi mice were harvested and homogenised for virus titration ( m – o ). Data is not shown for liver and spleen tissues. Statistical significance is determined with a two-tailed unpaired t -test, with * denoting p < 0.05. Survival group: variant L: n = 6 (treatment and no treatment); Alpha: n = 6 (treatment), n = 4 (no treatment); Beta: n = 6 (treatment), n = 5 (no treatment). 4 dpi group: variant L: n = 7 (treatment and no treatment); Alpha and Beta: n = 5 each (treatment and no treatment). Mock infection, n = 3.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: In Vivo, Injection, Control, Comparison, Infection, Variant Assay, Activity Assay, Virus, Titration, Two Tailed Test

Journal: Scientific Reports

Article Title: Immunogenicity and protection efficacy of a COVID-19 DNA vaccine encoding spike protein with D614G mutation and optimization of large-scale DNA vaccine production

doi: 10.1038/s41598-024-64690-5

Figure Lengend Snippet: In silico analyses of vaccine antigen. ( A ) Variations detected in Spike gene of 20 SARS-CoV-2 isolates collected from various provinces of Türkiye. The map in figure was generated by Microsoft 365 Excel. ( B ) A1841G mutation causes D614G amino acid alteration ( C ) 3D image of human ACE2 receptor docked with Wuhan Spike protein and coSpikeD614G vaccine antigen. Purple color: Spike protein; blue color: receptor binding domain (RBD) of Spike protein; green color: human ACE2 receptor; red color: receptor binding motif of human ACE2 protein.

Article Snippet: Neutralizing antibody responses in vaccinated mice sera were analyzed using an ELISA-based SARS-CoV-2 surrogate Virus Neutralization Test (sVNT) which is a S1 protein/ACE2 ligand binding assay) (AffinityImmuno, Canada) according to the manufacturer’s protocol.

Techniques: In Silico, Generated, Mutagenesis, Binding Assay

Journal: Scientific Reports

Article Title: Immunogenicity and protection efficacy of a COVID-19 DNA vaccine encoding spike protein with D614G mutation and optimization of large-scale DNA vaccine production

doi: 10.1038/s41598-024-64690-5

Figure Lengend Snippet: Animal studies and humoral immune response elicited by pcoSpikeD614G DNA vaccine. ( A ) Brief timeline of animal studies. BALB/c mice were immunized to assess the immunogenicity and K18-hACE2 transgenic mice were immunized to determine protective efficacy conferred by pcoSpikeD614G DNA vaccine ( B ) Western blot image shows the presence of anti-S1 and anti S1 + S2 antibody responses in sera of vaccinated BALB/c mice. Lane 1: Protein ladder; Lanes 2 and 3: recombinant S1 protein probed with pooled pcoSpikeD614G vaccinated mice sera immunized thrice through ID + EP and IM routes. Red arrowheads show the recombinant S1 proteins with a size of over \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sim$$\end{document} ∼ 76.41 kDa due to glycosylation; Lanes 4 and 5: recombinant S1 + S2 protein pooled with pcoSpikeD614G vaccinated mice sera immunized thrice through ID + EP and IM routes. Blue arrowheads show the recombinant S1 + S2 proteins with a size of over \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sim$$\end{document} ∼ 138.5 kDa due to glycosylation; Lane 6–7 and 8–9: recombinant S1 and S1 + S2 proteins probed with pooled vaccinated mice sera administered with empty pVAX1. Original Western blot images have been shown in Supplementary file named S5 Figure ( C ) The anti-S1 IgG kinetics derived from mice immunized with pcoSpikeD614G administered through ID + EP and IM routes show significant increase at day 70 compared to controls ( D ) IgG2a/IgG1 polarization was assessed at days 0 and 70 for empty pVAX1 and pcoSpikeD614G and IgG2a responses were significantly higher ( E ) The ratio of IgG2a/IgG1 is slightly higher in pcoSpikeD614G administered through IM route ( F ) SARS-CoV-2 Surrogate Virus Neutralization Test showed that inhibition potential of 1:10 diluted sera obtained from vaccinated mice at day 70 was significantly higher than controls ( G ) SARS-CoV-2 50% neutralization titers (VNT50) in sera of in pcoSpikeD614G immunized mice collated on day 70. Data are presented as GMT ± SD.

Article Snippet: Neutralizing antibody responses in vaccinated mice sera were analyzed using an ELISA-based SARS-CoV-2 surrogate Virus Neutralization Test (sVNT) which is a S1 protein/ACE2 ligand binding assay) (AffinityImmuno, Canada) according to the manufacturer’s protocol.

Techniques: Transgenic Assay, Western Blot, Recombinant, Derivative Assay, Virus, Neutralization, Inhibition

Journal: Scientific Reports

Article Title: Immunogenicity and protection efficacy of a COVID-19 DNA vaccine encoding spike protein with D614G mutation and optimization of large-scale DNA vaccine production

doi: 10.1038/s41598-024-64690-5

Figure Lengend Snippet: Protection conferred by pcoSpikeD614G DNA vaccine in K18-hACE2 transgenic mice challenged by SARS-CoV-2. ( A ) Gross pathological scoring of lungs to evaluate the level of pneumonia. Healthy lungs were scored as 0, edema-hyperemia between 0.5 and 1, pneumonia lesions were between 1.5 and 5 and dead mice were scored as 5. ( B ) Mean Ct values of RT-qPCR targeting NC gene of SARS‐CoV‐2 derived from the lungs of mice. The virus load is represented by Crossing point threshold (Ct) values. The Ct value of a negative PCR sample is accepted as 40 ( C ) Histopathology scoring of lungs to evaluate the level of inflammation status. Absence of inflammation is represented by 0 and various level of inflammation were scored between 1 and 3. Three lungs from the control group and one lung from the mice group immunized with pcoSpikeD614G administered through IM route were not scored ( D, E ) In the group of mice immunized with pcoSpikeD614G administered through IM route, images show normal lungs at magnifications of 4 × and 10 × . Arrow shows alveoli with normal morphology ( F, G ) In the group of mice immunized with pcoSpikeD614G administered through ID + EP route, images show normal lungs at magnifications of 4 × and 10 × . Arrow shows alveoli with normal morphology. ( H, I ) In the control group of mice, images show lungs with interstitial inflammatory cell infiltration at magnifications of 4 × and 10 × . Asterisks indicate areas of lymphocyte infiltration in the interalveolar spaces. ( J ) Kaplan–Meier Survival analysis after intranasal instillation of 10 5 TCID 50 virus for 3 consecutive days.

Article Snippet: Neutralizing antibody responses in vaccinated mice sera were analyzed using an ELISA-based SARS-CoV-2 surrogate Virus Neutralization Test (sVNT) which is a S1 protein/ACE2 ligand binding assay) (AffinityImmuno, Canada) according to the manufacturer’s protocol.

Techniques: Transgenic Assay, Quantitative RT-PCR, Derivative Assay, Virus, Histopathology

Journal: Cytokine

Article Title: Is IFN expression by NK cells a hallmark of severe COVID-19?

doi: 10.1016/j.cyto.2022.155971

Figure Lengend Snippet: List of peptides chosen in this study, through prediction studies and Spike peptide pool.

Article Snippet: SARS-CoV-2 virus-specific CD4 and CD8 peptides ( ) ( patent number: BR 10 2022 005518 1 ), resuspended in H 2 O (MiliQ), pooled (Pool CoV-2, which contained peptides from the spike protein and non-spike proteins, and Pool Spike Cov-2, which contained peptides from the spike protein only, ) were synthesized (GenOne Biotechnologies, Rio de Janeiro, Brazil) lyophilized and stored at −20 °C until use.

Techniques:

Journal: Cytokine

Article Title: Is IFN expression by NK cells a hallmark of severe COVID-19?

doi: 10.1016/j.cyto.2022.155971

Figure Lengend Snippet: Ki-67 and TIM-3 expression by CD3 - CD56 + NK cells in healthy individuals (HC), mild (mild CoV), and severe (severe CoV) COVID-19 recovered patients.

Article Snippet: SARS-CoV-2 virus-specific CD4 and CD8 peptides ( ) ( patent number: BR 10 2022 005518 1 ), resuspended in H 2 O (MiliQ), pooled (Pool CoV-2, which contained peptides from the spike protein and non-spike proteins, and Pool Spike Cov-2, which contained peptides from the spike protein only, ) were synthesized (GenOne Biotechnologies, Rio de Janeiro, Brazil) lyophilized and stored at −20 °C until use.

Techniques: Expressing, Control