sars cov 2 2019 ncov spike rbd his recombinant protein covid 19 spike rbd research  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike RBD His Recombinant Protein COVID 19 Spike RBD Research
    Description:
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed with a polyhistidine tag at the C terminus
    Catalog Number:
    40592-V08B
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus spike Protein 2019-nCoV, cov spike Protein 2019-nCoV, ncov RBD Protein 2019-nCoV, ncov s1 Protein 2019-nCoV, ncov s2 Protein 2019-nCoV, ncov spike Protein 2019-nCoV, NCP-CoV RBD Protein 2019-nCoV, NCP-CoV s1 Protein 2019-nCoV, NCP-CoV s2 Protein 2019-nCoV, NCP-CoV Spike Protein 2019-nCoV, novel coronavirus RBD Protein 2019-nCoV, novel coronavirus s1 Protein 2019-nCoV, novel coronavirus s2 Protein 2019-nCoV, novel coronavirus spike Protein 2019-nCoV, RBD Protein 2019-nCoV, S1 Protein 2019-nCoV, S2 Protein 2019-nCoV, Spike RBD Protein 2019-nCoV
    Host:
    Baculovirus-Insect Cells
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    Structured Review

    Sino Biological sars cov 2 2019 ncov spike rbd his recombinant protein covid 19 spike rbd research
    Differential DNAm analyses of PBMCs stimulated in vitro with <t>SARS-CoV-2.</t> A. Venn diagrams depicting the overlap of DMCs from the SARS-CoV-2 in vitro stimulated PBMCs. Intraindividual comparisons of differential DNAm were performed in treated vs . untreated PBMCs from four different blood donors (D1-D4) collected before the start of the COVID-19 pandemic (2014-2019). DMCs were defined as a fold change in M-value > |2|. These DMCs were further mapped to their corresponding annotated genes (DMGs, n=542). B shows results from pathway over-representation analyses in PANTHER based on the 542 DMGs originating from the SARS-CoV-2 in vitro stimulated PBMCs compared to non-stimulated PBMCs. Pathways with a nominal p-value
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed with a polyhistidine tag at the C terminus
    https://www.bioz.com/result/sars cov 2 2019 ncov spike rbd his recombinant protein covid 19 spike rbd research/product/Sino Biological
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    Images

    1) Product Images from "Mild SARS-CoV-2 infection modifies DNA methylation of peripheral blood mononuclear cells from COVID-19 convalescents"

    Article Title: Mild SARS-CoV-2 infection modifies DNA methylation of peripheral blood mononuclear cells from COVID-19 convalescents

    Journal: medRxiv

    doi: 10.1101/2021.07.05.21260014

    Differential DNAm analyses of PBMCs stimulated in vitro with SARS-CoV-2. A. Venn diagrams depicting the overlap of DMCs from the SARS-CoV-2 in vitro stimulated PBMCs. Intraindividual comparisons of differential DNAm were performed in treated vs . untreated PBMCs from four different blood donors (D1-D4) collected before the start of the COVID-19 pandemic (2014-2019). DMCs were defined as a fold change in M-value > |2|. These DMCs were further mapped to their corresponding annotated genes (DMGs, n=542). B shows results from pathway over-representation analyses in PANTHER based on the 542 DMGs originating from the SARS-CoV-2 in vitro stimulated PBMCs compared to non-stimulated PBMCs. Pathways with a nominal p-value
    Figure Legend Snippet: Differential DNAm analyses of PBMCs stimulated in vitro with SARS-CoV-2. A. Venn diagrams depicting the overlap of DMCs from the SARS-CoV-2 in vitro stimulated PBMCs. Intraindividual comparisons of differential DNAm were performed in treated vs . untreated PBMCs from four different blood donors (D1-D4) collected before the start of the COVID-19 pandemic (2014-2019). DMCs were defined as a fold change in M-value > |2|. These DMCs were further mapped to their corresponding annotated genes (DMGs, n=542). B shows results from pathway over-representation analyses in PANTHER based on the 542 DMGs originating from the SARS-CoV-2 in vitro stimulated PBMCs compared to non-stimulated PBMCs. Pathways with a nominal p-value

    Techniques Used: In Vitro

    Outline of included participants, experimental procedures as well as statistical and bioinformatic approaches utilised in the present study. CC19 – convalescent COVID-19, Con – non-infected control, DMG – differentially methylated gene, Pre20 – Pre-2020 non-infected control, SFT – symptom-free individuals with SARS-CoV-2-specific T cell response, SMIA – suspension multiplex immunoassay.
    Figure Legend Snippet: Outline of included participants, experimental procedures as well as statistical and bioinformatic approaches utilised in the present study. CC19 – convalescent COVID-19, Con – non-infected control, DMG – differentially methylated gene, Pre20 – Pre-2020 non-infected control, SFT – symptom-free individuals with SARS-CoV-2-specific T cell response, SMIA – suspension multiplex immunoassay.

    Techniques Used: Infection, Methylation, Multiplex Assay

    2) Product Images from "Pomegranate Peel Extract as an Inhibitor of SARS-CoV-2 Spike Binding to Human ACE2 Receptor (in vitro): A Promising Source of Novel Antiviral Drugs"

    Article Title: Pomegranate Peel Extract as an Inhibitor of SARS-CoV-2 Spike Binding to Human ACE2 Receptor (in vitro): A Promising Source of Novel Antiviral Drugs

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2021.638187

    Infection rate of Spike SARS-CoV-2 pseudo-typed lentivirus in HK-2, determined by GFP fluorescence measure. The results are the averages of six independent experiments, expressed as percentages respect to control arbitrarily set as 100%. The error bars represent SDs and the asterisks indicate statistically significant values ( *** p -value is between 0.0001 and 0.001) according to the T -test. GFP, green fluorescent protein; HK-2, human kidney-2 cells (HK-2); SARS-CoV-2, severe acute respiratory syndrome coronavirus-2.
    Figure Legend Snippet: Infection rate of Spike SARS-CoV-2 pseudo-typed lentivirus in HK-2, determined by GFP fluorescence measure. The results are the averages of six independent experiments, expressed as percentages respect to control arbitrarily set as 100%. The error bars represent SDs and the asterisks indicate statistically significant values ( *** p -value is between 0.0001 and 0.001) according to the T -test. GFP, green fluorescent protein; HK-2, human kidney-2 cells (HK-2); SARS-CoV-2, severe acute respiratory syndrome coronavirus-2.

    Techniques Used: Infection, Fluorescence

    3) Product Images from "A novel rapid detection for SARS-CoV-2 spike 1 antigens using human angiotensin converting enzyme 2 (ACE2)"

    Article Title: A novel rapid detection for SARS-CoV-2 spike 1 antigens using human angiotensin converting enzyme 2 (ACE2)

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112715

    Laboratory confirmation of ACE2-based LFIA using clinical samples a) Schematic diagram of COVID-19 test using ACE2-based LFIA. A nasopharyngeal swab from the COVID-19 patient is placed into the UTM. 50 μL of UTM containing the SARS-CoV-2 is mixed with running buffer in a 1:1 (v/v) ratio, and 100 μL of mixed solution is loaded into the LFIA device. After 20 min, the line intensity of the LFIA strip is semi-quantified by the portable analyzer. b) Results of ACE2-based LFA for the detection sensitivity of cultured SARS-CoV-2. Serially diluted virus concentrates (concentration range: 1.07 × 10 8 copies/mL to 5.35 × 10 6 copies/mL) were tested. After 20 min, the LFIA strip was taken with a smartphone and scanned with an image analyzer. The line intensities of the test and control lines were converted to peak histograms. Also, the intensity of the test lines was measured by a portable line analyzer (I L : line intensity of test line). Furthermore, human coronavirus (OC43) was tested as a negative control. c) Bar graph of intensities for test lines measured by the portable analyzer. The limit of detection (LOD) was determined by the mean value of negative controls (0 copies/mL of SARS-CoV-2) plus three times the standard deviation. d) Laboratory confirmation of ACE2-based LFIA compared to the RT-qPCR using clinical samples. i) Nasopharyngeal swab samples of COVID-19 patients (n = 4) and healthy subjects (n = 4) were tested both ACE2-based LFIA and RT-qPCR. Sensitivity was determined by the number of true positive samples divided by the number of positive samples tested. Moreover, specificity was determined by the number of true negative samples divide by the number of negative samples tested. ii) RT-qPCR results for the detection of the SARS-CoV-2 specific gene (Env gene). C t value and their correspondent viral load in the clinical samples were evaluated. e) Results of ACE2-based LFIA on laboratory confirmation using clinical COVID-19 patient samples. Twenty minutes after sample loading, the test line intensities of the LFIA strips were measured with a portable line analyzer. The limit of detection (LOD) was determined by the mean value of negative controls (healthy control) plus three times the standard deviation.
    Figure Legend Snippet: Laboratory confirmation of ACE2-based LFIA using clinical samples a) Schematic diagram of COVID-19 test using ACE2-based LFIA. A nasopharyngeal swab from the COVID-19 patient is placed into the UTM. 50 μL of UTM containing the SARS-CoV-2 is mixed with running buffer in a 1:1 (v/v) ratio, and 100 μL of mixed solution is loaded into the LFIA device. After 20 min, the line intensity of the LFIA strip is semi-quantified by the portable analyzer. b) Results of ACE2-based LFA for the detection sensitivity of cultured SARS-CoV-2. Serially diluted virus concentrates (concentration range: 1.07 × 10 8 copies/mL to 5.35 × 10 6 copies/mL) were tested. After 20 min, the LFIA strip was taken with a smartphone and scanned with an image analyzer. The line intensities of the test and control lines were converted to peak histograms. Also, the intensity of the test lines was measured by a portable line analyzer (I L : line intensity of test line). Furthermore, human coronavirus (OC43) was tested as a negative control. c) Bar graph of intensities for test lines measured by the portable analyzer. The limit of detection (LOD) was determined by the mean value of negative controls (0 copies/mL of SARS-CoV-2) plus three times the standard deviation. d) Laboratory confirmation of ACE2-based LFIA compared to the RT-qPCR using clinical samples. i) Nasopharyngeal swab samples of COVID-19 patients (n = 4) and healthy subjects (n = 4) were tested both ACE2-based LFIA and RT-qPCR. Sensitivity was determined by the number of true positive samples divided by the number of positive samples tested. Moreover, specificity was determined by the number of true negative samples divide by the number of negative samples tested. ii) RT-qPCR results for the detection of the SARS-CoV-2 specific gene (Env gene). C t value and their correspondent viral load in the clinical samples were evaluated. e) Results of ACE2-based LFIA on laboratory confirmation using clinical COVID-19 patient samples. Twenty minutes after sample loading, the test line intensities of the LFIA strips were measured with a portable line analyzer. The limit of detection (LOD) was determined by the mean value of negative controls (healthy control) plus three times the standard deviation.

    Techniques Used: Stripping Membranes, Cell Culture, Concentration Assay, Negative Control, Standard Deviation, Quantitative RT-PCR

    4) Product Images from "Quantum Dot-Conjugated SARS-CoV-2 Spike Pseudo-Virions Enable Tracking of Angiotensin Converting Enzyme 2 Binding and Endocytosis"

    Article Title: Quantum Dot-Conjugated SARS-CoV-2 Spike Pseudo-Virions Enable Tracking of Angiotensin Converting Enzyme 2 Binding and Endocytosis

    Journal: ACS Nano

    doi: 10.1021/acsnano.0c05975

    NP-based inhibition assay. (a) Left: The structure of neutralizing antibody (top) bound to SARS-CoV-2 Spike RBD (bottom, green). Right: Schematic diagram of the inhibition assay depicting blocking of the interaction between RBD and ACE2 and the resulting inhibition of energy transfer from QD to AuNP. (b) PL recovery of QD 514 -RBD in the presence of neutralizing antibody Ab1. (c) Inhibition test using anti-Spike antibody without neutralizing ability, showing almost no PL recovery of QD 514 -RBD. (d) Calculated EC 50 s for neutralizing antibodies Ab1 and Ab2 were 60 nM and 125 nM with R 2 > 99%, respectively.
    Figure Legend Snippet: NP-based inhibition assay. (a) Left: The structure of neutralizing antibody (top) bound to SARS-CoV-2 Spike RBD (bottom, green). Right: Schematic diagram of the inhibition assay depicting blocking of the interaction between RBD and ACE2 and the resulting inhibition of energy transfer from QD to AuNP. (b) PL recovery of QD 514 -RBD in the presence of neutralizing antibody Ab1. (c) Inhibition test using anti-Spike antibody without neutralizing ability, showing almost no PL recovery of QD 514 -RBD. (d) Calculated EC 50 s for neutralizing antibodies Ab1 and Ab2 were 60 nM and 125 nM with R 2 > 99%, respectively.

    Techniques Used: Inhibition, Blocking Assay

    5) Product Images from "Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19"

    Article Title: Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-020-00301-9

    Neutralizing antibody responses to SARS-CoV-2 in COVID-19 recovered patients. a Scores showing the COVID-19 patient serum-mediated inhibition of the SARS-CoV-2 RBD protein binding to ACE2 protein by ELISA. b Pie charts showing the proportions of patients with high ( > 50, green) or low (
    Figure Legend Snippet: Neutralizing antibody responses to SARS-CoV-2 in COVID-19 recovered patients. a Scores showing the COVID-19 patient serum-mediated inhibition of the SARS-CoV-2 RBD protein binding to ACE2 protein by ELISA. b Pie charts showing the proportions of patients with high ( > 50, green) or low (

    Techniques Used: Inhibition, Protein Binding, Enzyme-linked Immunosorbent Assay

    Subtypes of neutralizing antibodies to SARS-CoV-2 S proteins in COVID-19 recovered patients. a Blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus into ACE2/293T cells by patient sera (no depletion) or S1 antibody-depleted sera (S1-Abs depletion) or S2 antibody-depleted sera (S2-Abs depletion). The dashed line indicates the cutoff value (6.749) determined by the ROC curve analysis. HC healthy control, NC negative control. b , c Pie charts showing the proportions of patients with different neutralizing antibody (NAb) subtype responses in the total 25 patients ( b ), 8 severe patients ( c , left panel), and 17 moderate and mild patients ( c , right panel) of pseudovirus neutralization positive. d Blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus into ACE2/293T cells by “S1-NAbs only” patient sera with RBD antibody depletion (RBD-Abs depletion) or without RBD antibody depletion (no depletion). The dashed line indicates the cutoff value (6.034) determined by the ROC curve analysis. HC healthy control, NC negative control. e Pie chart showing the proportions of “S1-NAbs only” patients with RBD-Nab-dependent or -independent antibody response. Error bars in a , d indicate SEM
    Figure Legend Snippet: Subtypes of neutralizing antibodies to SARS-CoV-2 S proteins in COVID-19 recovered patients. a Blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus into ACE2/293T cells by patient sera (no depletion) or S1 antibody-depleted sera (S1-Abs depletion) or S2 antibody-depleted sera (S2-Abs depletion). The dashed line indicates the cutoff value (6.749) determined by the ROC curve analysis. HC healthy control, NC negative control. b , c Pie charts showing the proportions of patients with different neutralizing antibody (NAb) subtype responses in the total 25 patients ( b ), 8 severe patients ( c , left panel), and 17 moderate and mild patients ( c , right panel) of pseudovirus neutralization positive. d Blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus into ACE2/293T cells by “S1-NAbs only” patient sera with RBD antibody depletion (RBD-Abs depletion) or without RBD antibody depletion (no depletion). The dashed line indicates the cutoff value (6.034) determined by the ROC curve analysis. HC healthy control, NC negative control. e Pie chart showing the proportions of “S1-NAbs only” patients with RBD-Nab-dependent or -independent antibody response. Error bars in a , d indicate SEM

    Techniques Used: Blocking Assay, Luciferase, Negative Control, Neutralization

    Antibody responses to SARS-CoV-2 in COVID-19 recovered patients with different symptom severity. a – c ELISA binding assays of 100-fold diluted COVID-19 patient sera to ELISA plates after coating with SARS-CoV-2 S1 ( a ), RBD ( b ), and S2 ( c ) proteins. The dashed lines in a – c represent the average values of the healthy control groups. * P
    Figure Legend Snippet: Antibody responses to SARS-CoV-2 in COVID-19 recovered patients with different symptom severity. a – c ELISA binding assays of 100-fold diluted COVID-19 patient sera to ELISA plates after coating with SARS-CoV-2 S1 ( a ), RBD ( b ), and S2 ( c ) proteins. The dashed lines in a – c represent the average values of the healthy control groups. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay

    6) Product Images from "High throughput surface plasmon resonance imaging method for clinical detection of presence and strength of binding of IgM, IgG and IgA antibodies against SARS-CoV-2 during CoViD-19 infection"

    Article Title: High throughput surface plasmon resonance imaging method for clinical detection of presence and strength of binding of IgM, IgG and IgA antibodies against SARS-CoV-2 during CoViD-19 infection

    Journal: MethodsX

    doi: 10.1016/j.mex.2021.101432

    Six typical SPRi sensorgrams of anti-SARS-CoV-2 IgM, IgG and IgA in serum of CoViD-19 patients measured using the IBIS MX96. Panel A : patient without detectable immunoglobulins. B : patient with mild symptoms. C : patient with only an IgG response. D : patient with undetectable IgM a moderate IgG and a high IgA. Panel E and F show various levels of IgM, IgG and IgA.
    Figure Legend Snippet: Six typical SPRi sensorgrams of anti-SARS-CoV-2 IgM, IgG and IgA in serum of CoViD-19 patients measured using the IBIS MX96. Panel A : patient without detectable immunoglobulins. B : patient with mild symptoms. C : patient with only an IgG response. D : patient with undetectable IgM a moderate IgG and a high IgA. Panel E and F show various levels of IgM, IgG and IgA.

    Techniques Used:

    7) Product Images from "Small-Molecule Inhibitors of the Coronavirus Spike: ACE2 Protein–Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2"

    Article Title: Small-Molecule Inhibitors of the Coronavirus Spike: ACE2 Protein–Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2

    Journal: ACS Infectious Diseases

    doi: 10.1021/acsinfecdis.1c00070

    Concentration-dependent inhibition of SARS-CoV-S1S2 binding to ACE2 by representative compounds of the present study. Concentration–response curves obtained for the inhibition of the PPI between SARS-CoV-S1S2 (His-tagged, 1 μg/mL) and hACE2 (Fc-conjugated, 1 μg/mL) in cell-free ELISA-type assay by selected representative dye and nondye compounds. Data and fit as before ( Figure 3 ). Most compounds including several DRI-C compounds show similar activity against SARS-CoV (i.e., SARS-CoV-1) as against SARS-CoV-2 raising the possibility of broad-spectrum activity.
    Figure Legend Snippet: Concentration-dependent inhibition of SARS-CoV-S1S2 binding to ACE2 by representative compounds of the present study. Concentration–response curves obtained for the inhibition of the PPI between SARS-CoV-S1S2 (His-tagged, 1 μg/mL) and hACE2 (Fc-conjugated, 1 μg/mL) in cell-free ELISA-type assay by selected representative dye and nondye compounds. Data and fit as before ( Figure 3 ). Most compounds including several DRI-C compounds show similar activity against SARS-CoV (i.e., SARS-CoV-1) as against SARS-CoV-2 raising the possibility of broad-spectrum activity.

    Techniques Used: Concentration Assay, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

    Concentration–response curves for binding of CoV spike protein domains to human ACE2 in cell-free ELISA-type assays. Binding curves and corresponding EC 50 ’s are shown for SARS-CoV-2 (RBD and S1), SARS-CoV (S1 S2), and HCoV-NL63 (S1). They were obtained using Fc-conjugated hACE2 coated on the plate and His-tagged S1, S1S2, or RBD added in increasing amounts as shown with the amount bound detected using an anti-His–HRP conjugate (mean ± SD for two experiments in duplicates).
    Figure Legend Snippet: Concentration–response curves for binding of CoV spike protein domains to human ACE2 in cell-free ELISA-type assays. Binding curves and corresponding EC 50 ’s are shown for SARS-CoV-2 (RBD and S1), SARS-CoV (S1 S2), and HCoV-NL63 (S1). They were obtained using Fc-conjugated hACE2 coated on the plate and His-tagged S1, S1S2, or RBD added in increasing amounts as shown with the amount bound detected using an anti-His–HRP conjugate (mean ± SD for two experiments in duplicates).

    Techniques Used: Concentration Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

    Concentration-dependent inhibition of SARS-CoV-2 pseudovirus entry (BacMam) into hACE2 expressing host cells by selected compounds. Quantification of entry of pseudoviruses bearing the SARS-CoV-2 S protein (plus green fluorescent protein reporters; BacMam-based) in ACE2 (plus red fluorescence)-expressing host cells (HEK293T). Representative images (bottom row) and their quantification for pseudovirus (green) and ACE2 expression (red) using ImageJ (top row) are shown from one experiment for CgRd and DRI-C23041 in (A) and (B), respectively; average data from three experiments fitted with typical concentration–response curves are shown in (C). The amount of green present is proportional with the number of infected cells as green fluorescence is expressed only in pseudovirus infected cells, while amount of red is proportional with the number of ACE2-expressing cells. The organic dye CgRd (A), but especially DRI-C23041 (B) showed concentration-dependent inhibition with activities corresponding to low micromolar IC 50 values, whereas hydroxychloroquine (HCQ) showed no effect (C).
    Figure Legend Snippet: Concentration-dependent inhibition of SARS-CoV-2 pseudovirus entry (BacMam) into hACE2 expressing host cells by selected compounds. Quantification of entry of pseudoviruses bearing the SARS-CoV-2 S protein (plus green fluorescent protein reporters; BacMam-based) in ACE2 (plus red fluorescence)-expressing host cells (HEK293T). Representative images (bottom row) and their quantification for pseudovirus (green) and ACE2 expression (red) using ImageJ (top row) are shown from one experiment for CgRd and DRI-C23041 in (A) and (B), respectively; average data from three experiments fitted with typical concentration–response curves are shown in (C). The amount of green present is proportional with the number of infected cells as green fluorescence is expressed only in pseudovirus infected cells, while amount of red is proportional with the number of ACE2-expressing cells. The organic dye CgRd (A), but especially DRI-C23041 (B) showed concentration-dependent inhibition with activities corresponding to low micromolar IC 50 values, whereas hydroxychloroquine (HCQ) showed no effect (C).

    Techniques Used: Concentration Assay, Inhibition, Expressing, Fluorescence, Infection

    Concentration-dependent inhibition of SARS-CoV-2 pseudovirus (VSV-Δ G ) entry into hACE2/Furin expressing host cells by selected compounds. Entry of VSV-ΔG pseudoviruses bearing the SARS-CoV-2 S protein (plus GFP reporters) in ACE2/Furin overexpressing host cells (Vero-E6) was quantified via GFP fluorescence in a live imaging system (Incucyte). CgRd and DRI-C23041 showed concentration-dependent inhibition with IC 50 values consistent with the previous assay ( Figure 7 ), whereas the negative control sunset yellow (SY FD C #6) showed no significant effect.
    Figure Legend Snippet: Concentration-dependent inhibition of SARS-CoV-2 pseudovirus (VSV-Δ G ) entry into hACE2/Furin expressing host cells by selected compounds. Entry of VSV-ΔG pseudoviruses bearing the SARS-CoV-2 S protein (plus GFP reporters) in ACE2/Furin overexpressing host cells (Vero-E6) was quantified via GFP fluorescence in a live imaging system (Incucyte). CgRd and DRI-C23041 showed concentration-dependent inhibition with IC 50 values consistent with the previous assay ( Figure 7 ), whereas the negative control sunset yellow (SY FD C #6) showed no significant effect.

    Techniques Used: Concentration Assay, Inhibition, Expressing, Fluorescence, Imaging, Negative Control

    Concentration-dependent inhibition of SARS-CoV-2-S-RBD binding to ACE2 by compounds of the present study. Concentration–response curves obtained for the inhibition of the PPI between SARS-CoV-2-RBD (His-tagged, 0.5 μg/mL) and hACE2 (Fc-conjugated, 1 μg/mL) in cell-free ELISA-type assay with dye (A) and nondye (B) compounds tested. The promiscuous PPI inhibitor erythrosine B (ErB) and the food colorant FD C yellow no. 6 (sunset yellow, SY) were included as a positive and negative controls, respectively. Data are mean ± SD from two experiments in duplicates and were fitted with standard sigmoid curves for IC 50 determination. Estimated IC 50 ’s are shown in the legend indicating that while suramin and chloroquine were completely inactive (IC 50 > 500 μM), several of our in-house compounds including organic dyes (CgRd, DV1, and others) as well as proprietary DRI-C compounds (e.g., DRI-C23041, DRI-C91005) showed promising activity, some even at submicromolar levels (IC 50
    Figure Legend Snippet: Concentration-dependent inhibition of SARS-CoV-2-S-RBD binding to ACE2 by compounds of the present study. Concentration–response curves obtained for the inhibition of the PPI between SARS-CoV-2-RBD (His-tagged, 0.5 μg/mL) and hACE2 (Fc-conjugated, 1 μg/mL) in cell-free ELISA-type assay with dye (A) and nondye (B) compounds tested. The promiscuous PPI inhibitor erythrosine B (ErB) and the food colorant FD C yellow no. 6 (sunset yellow, SY) were included as a positive and negative controls, respectively. Data are mean ± SD from two experiments in duplicates and were fitted with standard sigmoid curves for IC 50 determination. Estimated IC 50 ’s are shown in the legend indicating that while suramin and chloroquine were completely inactive (IC 50 > 500 μM), several of our in-house compounds including organic dyes (CgRd, DV1, and others) as well as proprietary DRI-C compounds (e.g., DRI-C23041, DRI-C91005) showed promising activity, some even at submicromolar levels (IC 50

    Techniques Used: Concentration Assay, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

    Identification of the binding partner by protein thermal shift. Differential scanning fluorimetry assay indicating SARS-CoV-2 RBD and not ACE2 as the binding partner of the present SMI compounds. The presence of Congo red (top) or DRI-C23041 (bottom) at 10 μM caused clear shifts in the melting temperature of the protein for RBD as indicated by the derivatives d F /d T (left; purple vs blue line), but not for hACE2 (right) (smaller insets are normalized fluorescence F data).
    Figure Legend Snippet: Identification of the binding partner by protein thermal shift. Differential scanning fluorimetry assay indicating SARS-CoV-2 RBD and not ACE2 as the binding partner of the present SMI compounds. The presence of Congo red (top) or DRI-C23041 (bottom) at 10 μM caused clear shifts in the melting temperature of the protein for RBD as indicated by the derivatives d F /d T (left; purple vs blue line), but not for hACE2 (right) (smaller insets are normalized fluorescence F data).

    Techniques Used: Binding Assay, Fluorimetry Assay, Fluorescence

    8) Product Images from "SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques"

    Article Title: SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques

    Journal: bioRxiv

    doi: 10.1101/2021.04.05.438479

    SARS-CoV-2 vaccine-elicited functional antibody responses in infant rhesus macaques. (A): The ACE2 blocking assay was performed at 1:10 plasma dilution and data are reported as %ACE2 blocking. (B-C) : Neutralization capacity was measured using a pseudovirus assay (B) and whole virus assay (C) ; results are expressed as reciprocal 80% inhibitory dilution (ID 80 ). Different symbols represent individual animals ( Table S1 ).
    Figure Legend Snippet: SARS-CoV-2 vaccine-elicited functional antibody responses in infant rhesus macaques. (A): The ACE2 blocking assay was performed at 1:10 plasma dilution and data are reported as %ACE2 blocking. (B-C) : Neutralization capacity was measured using a pseudovirus assay (B) and whole virus assay (C) ; results are expressed as reciprocal 80% inhibitory dilution (ID 80 ). Different symbols represent individual animals ( Table S1 ).

    Techniques Used: Functional Assay, Blocking Assay, Neutralization

    Spike-specific T cell responses in LN following SARS-CoV-2 vaccination. Intracellular cytokine staining as in Figure 5 in lymph node biopsy samples at week 6 to measure Spike-specific T cell responses. Panels A and B show mRNA-LNP and Protein+3M-052+SE CD4 + T-cell responses, respectively. Panels C and D represent CD8 + T cell cytokine responses from mRNA-LNP and Protein+3M-052+SE animals, respectively. Symbols and the legend mirror those from Figure 6 (see also Table S1 ). Dotted lines represent 2 standard deviations from SARS-CoV-2 naïve LN samples.
    Figure Legend Snippet: Spike-specific T cell responses in LN following SARS-CoV-2 vaccination. Intracellular cytokine staining as in Figure 5 in lymph node biopsy samples at week 6 to measure Spike-specific T cell responses. Panels A and B show mRNA-LNP and Protein+3M-052+SE CD4 + T-cell responses, respectively. Panels C and D represent CD8 + T cell cytokine responses from mRNA-LNP and Protein+3M-052+SE animals, respectively. Symbols and the legend mirror those from Figure 6 (see also Table S1 ). Dotted lines represent 2 standard deviations from SARS-CoV-2 naïve LN samples.

    Techniques Used: Staining

    Spike-specific CD8 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining was performed as described in Figure 5 at weeks 0, 6, 4, 8, and 14 to assess CD8 + T-cell responses. Panels A, B, C and D show responses detected in PBMC from the mRNA-LNP group at weeks 4, 6, 8, and 14, respectively. Panels E, F, G and H show responses in Protein+3M-052+SE vaccinees at weeks 4, 6, 8 and 14, respectively. The legend and symbols used mirror those from Figure 6 (see also Table S1 ). Dotted lines represent the cut-off for cytokine-positive responses, determined as 2 standard deviations above median values of SARS-CoV-2 naïve animals.
    Figure Legend Snippet: Spike-specific CD8 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining was performed as described in Figure 5 at weeks 0, 6, 4, 8, and 14 to assess CD8 + T-cell responses. Panels A, B, C and D show responses detected in PBMC from the mRNA-LNP group at weeks 4, 6, 8, and 14, respectively. Panels E, F, G and H show responses in Protein+3M-052+SE vaccinees at weeks 4, 6, 8 and 14, respectively. The legend and symbols used mirror those from Figure 6 (see also Table S1 ). Dotted lines represent the cut-off for cytokine-positive responses, determined as 2 standard deviations above median values of SARS-CoV-2 naïve animals.

    Techniques Used: Staining

    Weight gain of SARS-CoV-2 immunized infant rhesus macaques. Longitudinal weight data for all female (top) and male (bottom) animals from this study with their respective symbol shapes and colors ( Table S1 ) overlaid with historical weight data from age and sex-matched infant rhesus monkeys housed outdoors at the CNPRC, Davis California (open black circles-female; filled gray circles-male).
    Figure Legend Snippet: Weight gain of SARS-CoV-2 immunized infant rhesus macaques. Longitudinal weight data for all female (top) and male (bottom) animals from this study with their respective symbol shapes and colors ( Table S1 ) overlaid with historical weight data from age and sex-matched infant rhesus monkeys housed outdoors at the CNPRC, Davis California (open black circles-female; filled gray circles-male).

    Techniques Used:

    Polyfunctional CD4 + T cells in SARS-CoV-2 immunized rhesus macaques at week 14. Panels A and B show the frequencies of CD4 + T cells that co-produced IL-17 and IFN-γ in response to stimulation with spike protein overlapping peptides in animals of the mRNA-LNP or Protein-3M-052-SE vaccine group, respectively. Individual symbols represent individual animals in each group ( Table S1 ).
    Figure Legend Snippet: Polyfunctional CD4 + T cells in SARS-CoV-2 immunized rhesus macaques at week 14. Panels A and B show the frequencies of CD4 + T cells that co-produced IL-17 and IFN-γ in response to stimulation with spike protein overlapping peptides in animals of the mRNA-LNP or Protein-3M-052-SE vaccine group, respectively. Individual symbols represent individual animals in each group ( Table S1 ).

    Techniques Used: Produced

    Characterization of Spike-specific B cell responses two weeks post boost. CD20 + CD27 + memory B cells that co-stained with fluorochrome-conjugated SARS-CoV-2 spike protein in mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccinees in blood ( A - B ) or LN ( C - D ). Frequencies are expressed as percent of total memory B cells. The gating strategy is provided in Supplementary Figure S8. ( E–F) portray antibody secreting cell (ASC) as measured by B cell ELISpot in PBMC from mRNA-LNP or Protein+3M-052-SE vaccinees, while (G–H) contain mRNA-LNP and Protein+3M-052-SE ASC responses, respectively, in LN at W6. Different symbols represent individual animals ( Table S1 ). Solid lines represent median values.
    Figure Legend Snippet: Characterization of Spike-specific B cell responses two weeks post boost. CD20 + CD27 + memory B cells that co-stained with fluorochrome-conjugated SARS-CoV-2 spike protein in mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccinees in blood ( A - B ) or LN ( C - D ). Frequencies are expressed as percent of total memory B cells. The gating strategy is provided in Supplementary Figure S8. ( E–F) portray antibody secreting cell (ASC) as measured by B cell ELISpot in PBMC from mRNA-LNP or Protein+3M-052-SE vaccinees, while (G–H) contain mRNA-LNP and Protein+3M-052-SE ASC responses, respectively, in LN at W6. Different symbols represent individual animals ( Table S1 ). Solid lines represent median values.

    Techniques Used: Staining, Enzyme-linked Immunospot

    Study Design: evaluation of immunogenicity of two SARS-CoV-2 vaccines in infant rhesus macaques. Infant rhesus macaques (median age of 2.2 months at study initiation) were immunized at 0 and 4 weeks with either 30 µg mRNA encoding S-2P (Vaccine Research Center, NIH) in lipid nanoparticles (mRNA-LNP) or 15 µg S-2P protein formulated with 3M-052 adjuvant, a TLR7/8 agonist, as a stable emulsion (3M-052-SE). Each group consisted of 8 animals. Blood and saliva samples were collected at weeks 0, 4, 6, 8, 14, 18 and 22, and lymph node biopsies were obtained at week 6.
    Figure Legend Snippet: Study Design: evaluation of immunogenicity of two SARS-CoV-2 vaccines in infant rhesus macaques. Infant rhesus macaques (median age of 2.2 months at study initiation) were immunized at 0 and 4 weeks with either 30 µg mRNA encoding S-2P (Vaccine Research Center, NIH) in lipid nanoparticles (mRNA-LNP) or 15 µg S-2P protein formulated with 3M-052 adjuvant, a TLR7/8 agonist, as a stable emulsion (3M-052-SE). Each group consisted of 8 animals. Blood and saliva samples were collected at weeks 0, 4, 6, 8, 14, 18 and 22, and lymph node biopsies were obtained at week 6.

    Techniques Used:

    Spike-specific CD4 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining for IL-2, IL-17, IFN-γ, and TNF-α was performed on PBMC at weeks 0, 6, 4, 8, and 14 to assess T-cell responses to a peptide pool encompassing the entire SARS-CoV-2 spike protein. (A–D) display responses detected in Protein+3M-052+SE vaccinees (blue). (E-H) portray cytokine responses from mRNA-LNP recipients (red). The dashed lines represent week 0 values plus 2 standard deviations and define the cutoff for positive cytokine responses. Different symbols represent individual animals ( Table S1 ).
    Figure Legend Snippet: Spike-specific CD4 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining for IL-2, IL-17, IFN-γ, and TNF-α was performed on PBMC at weeks 0, 6, 4, 8, and 14 to assess T-cell responses to a peptide pool encompassing the entire SARS-CoV-2 spike protein. (A–D) display responses detected in Protein+3M-052+SE vaccinees (blue). (E-H) portray cytokine responses from mRNA-LNP recipients (red). The dashed lines represent week 0 values plus 2 standard deviations and define the cutoff for positive cytokine responses. Different symbols represent individual animals ( Table S1 ).

    Techniques Used: Staining

    S-specific B cell gating strategy. Panel A shows a representative plot from RM7; Panel B shows a naive, age-matched control macaque without SARS-CoV-2 S protein conjugates; Panel C shows the same donor macaque with the SARS-CoV-2 S protein conjugates included.
    Figure Legend Snippet: S-specific B cell gating strategy. Panel A shows a representative plot from RM7; Panel B shows a naive, age-matched control macaque without SARS-CoV-2 S protein conjugates; Panel C shows the same donor macaque with the SARS-CoV-2 S protein conjugates included.

    Techniques Used:

    Th1 and Th2 cytokines in plasma of infant rhesus macaques prior to and following SARS-CoV-2 vaccination. Plasma levels of IFN-γ ( panel A ) IL-2 ( panel B ), IL-4 ( panel C ) and IL-13 ( panel D ) were measured by multiplex Luminex assay. Different symbols represent individual animals in the mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccine groups, respectively ( Table S1 ). Horizontal lines represent median values.
    Figure Legend Snippet: Th1 and Th2 cytokines in plasma of infant rhesus macaques prior to and following SARS-CoV-2 vaccination. Plasma levels of IFN-γ ( panel A ) IL-2 ( panel B ), IL-4 ( panel C ) and IL-13 ( panel D ) were measured by multiplex Luminex assay. Different symbols represent individual animals in the mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccine groups, respectively ( Table S1 ). Horizontal lines represent median values.

    Techniques Used: Multiplex Assay, Luminex

    SARS-CoV-2 vaccine-elicited binding antibody responses in infant rhesus macaques. Plasma and saliva were collected before vaccination (W0), at W4 -just prior to the boost-, two weeks post boost (W6), at W8, W14, W18 and W22 from infant RM vaccinated with 30 µg mRNA encoding S-2P spike protein in lipid nanoparticles (n=8; red) or with 15 µg prefusion SARS-CoV-2 S-2P spike protein formulated with 3M-052 adjuvant (n=8; blue). (A): S-2P protein-specific antibody responses were measured by enzyme-linked immunosorbent assay (ELISA). Serial dilutions of plasma starting at 1:40 were assayed for IgG binding to SARS-CoV-2 spike. Data are reported as log 10 area under the curve (AUC) values. (B): Salivary RBD-specific IgG was measured by binding antigen multiplex assay (BAMA) using serial dilutions of saliva. (C): Antibody epitope specificity measured by BAMA. Plasma was diluted 1:10,000 to measure binding to different domains of the spike protein, including the full-length S protein, S1, RBD, NTD, and S2. Binding antibody responses are reported as log 10 transformed mean fluorescence intensity (MFI) after subtraction of background values. Red or blue lines and symbols represent the mRNA or protein vaccine groups, respectively, with different symbols representing individual animals ( Table S1 ).
    Figure Legend Snippet: SARS-CoV-2 vaccine-elicited binding antibody responses in infant rhesus macaques. Plasma and saliva were collected before vaccination (W0), at W4 -just prior to the boost-, two weeks post boost (W6), at W8, W14, W18 and W22 from infant RM vaccinated with 30 µg mRNA encoding S-2P spike protein in lipid nanoparticles (n=8; red) or with 15 µg prefusion SARS-CoV-2 S-2P spike protein formulated with 3M-052 adjuvant (n=8; blue). (A): S-2P protein-specific antibody responses were measured by enzyme-linked immunosorbent assay (ELISA). Serial dilutions of plasma starting at 1:40 were assayed for IgG binding to SARS-CoV-2 spike. Data are reported as log 10 area under the curve (AUC) values. (B): Salivary RBD-specific IgG was measured by binding antigen multiplex assay (BAMA) using serial dilutions of saliva. (C): Antibody epitope specificity measured by BAMA. Plasma was diluted 1:10,000 to measure binding to different domains of the spike protein, including the full-length S protein, S1, RBD, NTD, and S2. Binding antibody responses are reported as log 10 transformed mean fluorescence intensity (MFI) after subtraction of background values. Red or blue lines and symbols represent the mRNA or protein vaccine groups, respectively, with different symbols representing individual animals ( Table S1 ).

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Transformation Assay, Fluorescence

    Vaccine-elicited SARS-CoV-2 neutralization responses. Panel A shows the ID 50 neutralization titers obtained using the pseudovirus neutralization assay. Panel B depicts the ID 50 neutralization titers in infant and adult rhesus macaques vaccinated with the mRNA-LNP vaccine ( 16 ). Infant RM were vaccinated with 30 µg of mRNA-LNP at week 0 and at week 4. Adult RM followed the same vaccine schedule but received 10 µg or 100 µg of the mRNA-LNP vaccine. Neutralization was assessed with a pseudovirus assay at week 8 (4 weeks after the second vaccination). Panel C shows the ID 50 neutralization titers obtained using the whole virus neutralization assay. Panel D illustrates the correlation of ID 50 (left graph) and ID 80 (right graph) neutralizing titers from both vaccine groups obtained in the pseudovirus or whole virus neutralization assay at week 6. Correlation was assessed using Spearman rank test.
    Figure Legend Snippet: Vaccine-elicited SARS-CoV-2 neutralization responses. Panel A shows the ID 50 neutralization titers obtained using the pseudovirus neutralization assay. Panel B depicts the ID 50 neutralization titers in infant and adult rhesus macaques vaccinated with the mRNA-LNP vaccine ( 16 ). Infant RM were vaccinated with 30 µg of mRNA-LNP at week 0 and at week 4. Adult RM followed the same vaccine schedule but received 10 µg or 100 µg of the mRNA-LNP vaccine. Neutralization was assessed with a pseudovirus assay at week 8 (4 weeks after the second vaccination). Panel C shows the ID 50 neutralization titers obtained using the whole virus neutralization assay. Panel D illustrates the correlation of ID 50 (left graph) and ID 80 (right graph) neutralizing titers from both vaccine groups obtained in the pseudovirus or whole virus neutralization assay at week 6. Correlation was assessed using Spearman rank test.

    Techniques Used: Neutralization

    9) Product Images from "The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection"

    Article Title: The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection

    Journal: bioRxiv

    doi: 10.1101/2021.03.22.436522

    Halofuginone Inhibits infection and replication of authentic SARS-CoV-2. a-b, Authentic SARS-CoV-2 virus infection of Huh7.5 cells treated with Halofuginone. Huh7.5 cells treated with halofuginone pre- or post-infection, or both pre- and post-infection with authentic SARS-CoV-2 virus. Viral titers ( a ) and quantification of viral RNA ( b ) in the infected cells is shown. c, Immunofluorescent quantification of viral nucleocapsid (red) protein in Vero E6 cells treated with Halofuginone and Remdesivir and infected with authentic SARS-CoV-2 virus (nuclei = green). d, Authentic SARS-CoV-2 virus infection of Vero E6 cells treated with Halofuginone and Remdesivir as measured in flow cytometry. e, Quantification of plaque formation and viral RNA in Vero E6 cells treated with Halofuginone and Remdesivir and infected with authentic SARS-CoV-2 virus. f, Rescue experiment of the effect of halofuginone treatment on SARS-CoV-2 infection using excess proline. g , Treatment of Vero E6 cells with halofuginone and enantiomers and their effect on SARS-CoV-2 infection. h, Treatment of Vero E6 cells with modulators of the PRS pathway and their effect on SARS-CoV-2 infection. i, Treatment of Vero E6 cells with modulators of the ISR pathway and their effect on SARS-CoV-2 infection. j, The distribution of proline distribution and density depicted as a proline distribution score for collagens, cholesterol biosynthetic proteins, lysosomal proteins and viral SARS-CoV-2 (SARS2), SARS-CoV-1 (SARS1), MERS-CoV (MERS), HCoV-229E and HCoV-LN63 proteins( ## p ≤ 0.01 collagen s vs. all other protein classes). k, Treatment of Vero E6 cells with AARS inhibitors and their effect on SARS-CoV-2 infection. Data shown as mean ± S.D. Statistics performed by 1-way ANOVA and uncorrected Fisher’s LSD test (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001, ## : p ≤ 0.01.
    Figure Legend Snippet: Halofuginone Inhibits infection and replication of authentic SARS-CoV-2. a-b, Authentic SARS-CoV-2 virus infection of Huh7.5 cells treated with Halofuginone. Huh7.5 cells treated with halofuginone pre- or post-infection, or both pre- and post-infection with authentic SARS-CoV-2 virus. Viral titers ( a ) and quantification of viral RNA ( b ) in the infected cells is shown. c, Immunofluorescent quantification of viral nucleocapsid (red) protein in Vero E6 cells treated with Halofuginone and Remdesivir and infected with authentic SARS-CoV-2 virus (nuclei = green). d, Authentic SARS-CoV-2 virus infection of Vero E6 cells treated with Halofuginone and Remdesivir as measured in flow cytometry. e, Quantification of plaque formation and viral RNA in Vero E6 cells treated with Halofuginone and Remdesivir and infected with authentic SARS-CoV-2 virus. f, Rescue experiment of the effect of halofuginone treatment on SARS-CoV-2 infection using excess proline. g , Treatment of Vero E6 cells with halofuginone and enantiomers and their effect on SARS-CoV-2 infection. h, Treatment of Vero E6 cells with modulators of the PRS pathway and their effect on SARS-CoV-2 infection. i, Treatment of Vero E6 cells with modulators of the ISR pathway and their effect on SARS-CoV-2 infection. j, The distribution of proline distribution and density depicted as a proline distribution score for collagens, cholesterol biosynthetic proteins, lysosomal proteins and viral SARS-CoV-2 (SARS2), SARS-CoV-1 (SARS1), MERS-CoV (MERS), HCoV-229E and HCoV-LN63 proteins( ## p ≤ 0.01 collagen s vs. all other protein classes). k, Treatment of Vero E6 cells with AARS inhibitors and their effect on SARS-CoV-2 infection. Data shown as mean ± S.D. Statistics performed by 1-way ANOVA and uncorrected Fisher’s LSD test (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001, ## : p ≤ 0.01.

    Techniques Used: Infection, Flow Cytometry

    Halofuginone Inhibition of heparan sulfate presentation and Spike protein binding is not dependent of the integrated stress response. a, Chemical structure of halofuginone and negative control compound MAZ1310. Figure shows the interaction of halofuginone with the human prolyl-tRNA synthetase (PRS) active site, as resolved by X-ray crystallography (PDB: 4K88) 48 . b, Chemical structure of ProSA. Graphic shows the interaction of ProSA with human PRS (PDB: 5V58) 49 . c, Schematic representation of the mediators of the integrated stress response (ISR). c-d, Treatment of Hep3B cells with modulators of the PRS pathway at 0.5 µM (ProSA at 5 µM) and its effect on ( c ) HS presentation as measured by anti-HS mAb 10E4 binding and ( d ) spike RBD binding ( e ) by flow cytometry. Binding is represented as relative to non-treated control. f, Treatment of Hep3B cells with modulators of the halofuginone with or without 4mM proline and its effect on spike RBD binding by flow cytometry. Binding is represented as relative to non-treated control. g, Treatment of Hep3B cells with modulators of the ISR and its effect on HS presentation as measured by anti-HS mAb 10E4 binding in flow cytometry. h, Treatment of Hep3B cells with modulators of the ISR and its effect on SARS-CoV-2 recombinant RBD protein binding in flow cytometry. Binding is represented as relative to non-treated control. i, The distribution of proline distribution and density depicted as a proline distribution score for collagens, cholesterol biosynthetic proteins, lysosomal proteins, heparan sulfate (HS) biosynthetic proteins, heparan sulfate proteoglycans (HSPG) and viral host factor proteins ( ## p ≤ 0.01 collagens vs. all other protein classes). Data shown as mean ± S.D. Statistics performed by 1-way ANOVA and uncorrected Fisher’s LSD test (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001; # : p ≤ 0.05, ## : p ≤ 0.01).
    Figure Legend Snippet: Halofuginone Inhibition of heparan sulfate presentation and Spike protein binding is not dependent of the integrated stress response. a, Chemical structure of halofuginone and negative control compound MAZ1310. Figure shows the interaction of halofuginone with the human prolyl-tRNA synthetase (PRS) active site, as resolved by X-ray crystallography (PDB: 4K88) 48 . b, Chemical structure of ProSA. Graphic shows the interaction of ProSA with human PRS (PDB: 5V58) 49 . c, Schematic representation of the mediators of the integrated stress response (ISR). c-d, Treatment of Hep3B cells with modulators of the PRS pathway at 0.5 µM (ProSA at 5 µM) and its effect on ( c ) HS presentation as measured by anti-HS mAb 10E4 binding and ( d ) spike RBD binding ( e ) by flow cytometry. Binding is represented as relative to non-treated control. f, Treatment of Hep3B cells with modulators of the halofuginone with or without 4mM proline and its effect on spike RBD binding by flow cytometry. Binding is represented as relative to non-treated control. g, Treatment of Hep3B cells with modulators of the ISR and its effect on HS presentation as measured by anti-HS mAb 10E4 binding in flow cytometry. h, Treatment of Hep3B cells with modulators of the ISR and its effect on SARS-CoV-2 recombinant RBD protein binding in flow cytometry. Binding is represented as relative to non-treated control. i, The distribution of proline distribution and density depicted as a proline distribution score for collagens, cholesterol biosynthetic proteins, lysosomal proteins, heparan sulfate (HS) biosynthetic proteins, heparan sulfate proteoglycans (HSPG) and viral host factor proteins ( ## p ≤ 0.01 collagens vs. all other protein classes). Data shown as mean ± S.D. Statistics performed by 1-way ANOVA and uncorrected Fisher’s LSD test (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001; # : p ≤ 0.05, ## : p ≤ 0.01).

    Techniques Used: Inhibition, Protein Binding, Negative Control, Binding Assay, Flow Cytometry, Recombinant

    Screen of epigenetic and translational regulatory compounds identify Halofuginone as a potent inhibitor of SARS-CoV-2 Spike HS dependent cellular adhesion. a, Hep3B cells were treated with a library of epigenetic and translational regulatory compounds or heparin lyase (HSase) as a positive control and tested for their interaction with recombinant SARS-CoV-2 RBD protein. b-e, Titration of halofuginone on Hep3B cells ( b ), Vero E6 ( c ), Calu-3 ( d ) and Cacu-2 ( e ) cells (n= 2-4 replicates per condition), and its effect on binding of recombinant SARS-CoV-2 RBD protein Heparin Lyase treatment is included as control for HS dependent adhesion. f-g, effect of halofuginone treatment on the infection of SARS-CoV-2 spike protein pseudotyped virus in Hep3B ( f ) and Vero E6 ( g ) cells (n= 3-4 replicates per condition). h , Authentic SARS-CoV-2 virus infection of Hep3B cells treated with halofuginone. i, Infection of primary human bronchial epithelial cells, grown at an air-liquid interface, treated with halofuginone (Red and black colors represent the two different primary human bronchial epithelial cells isolates used). j , Relative cell viability of primary human bronchial epithelial cells. Data shown as mean ± S.D. Statistics performed by 1-way ANOVA and uncorrected Fisher’s LSD test (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001).
    Figure Legend Snippet: Screen of epigenetic and translational regulatory compounds identify Halofuginone as a potent inhibitor of SARS-CoV-2 Spike HS dependent cellular adhesion. a, Hep3B cells were treated with a library of epigenetic and translational regulatory compounds or heparin lyase (HSase) as a positive control and tested for their interaction with recombinant SARS-CoV-2 RBD protein. b-e, Titration of halofuginone on Hep3B cells ( b ), Vero E6 ( c ), Calu-3 ( d ) and Cacu-2 ( e ) cells (n= 2-4 replicates per condition), and its effect on binding of recombinant SARS-CoV-2 RBD protein Heparin Lyase treatment is included as control for HS dependent adhesion. f-g, effect of halofuginone treatment on the infection of SARS-CoV-2 spike protein pseudotyped virus in Hep3B ( f ) and Vero E6 ( g ) cells (n= 3-4 replicates per condition). h , Authentic SARS-CoV-2 virus infection of Hep3B cells treated with halofuginone. i, Infection of primary human bronchial epithelial cells, grown at an air-liquid interface, treated with halofuginone (Red and black colors represent the two different primary human bronchial epithelial cells isolates used). j , Relative cell viability of primary human bronchial epithelial cells. Data shown as mean ± S.D. Statistics performed by 1-way ANOVA and uncorrected Fisher’s LSD test (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001).

    Techniques Used: Positive Control, Recombinant, Titration, Binding Assay, Infection

    10) Product Images from "Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2"

    Article Title: Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2

    Journal: Communications Biology

    doi: 10.1038/s42003-021-02029-w

    Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.
    Figure Legend Snippet: Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.

    Techniques Used: Concentration Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

    CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.
    Figure Legend Snippet: CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.

    Techniques Used: Infection, In Vitro, In Vivo, Expressing, Activity Assay, Neutralization, Mouse Assay, Real-time Polymerase Chain Reaction

    CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.
    Figure Legend Snippet: CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.

    Techniques Used: Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Staining, Concentration Assay, Flow Cytometry

    11) Product Images from "Prime-boost vaccination of mice and rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates"

    Article Title: Prime-boost vaccination of mice and rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates

    Journal: Emerging Microbes & Infections

    doi: 10.1080/22221751.2021.1931466

    Characterization of Sad23L-nCoV-S and Ad49L-nCoV-S vaccines. (A) Recombinant adenovirus constructs Sad23L-nCoV-S and Ad49L-nCoV-S carrying the full-length S gene of SARS-CoV-2 under CMV promotor regulation within the deleted E1 region of Sad23L or Ad49L vector. (B) Western blot analysis for the expression of S protein from Sad23L-nCoV-S or Ad49L-nCoV-S infected HEK-293A cell lysates by rabbit polyclonal antibody to RBD. Sad23L-GFP or Ad49L-GFP virus infected cells were used as mock controls. (C) Expression of S protein in HEK-293A cells detected by immunofluorescence staining. (D) Seroprevalence of neutralizing antibody (AdNAb) to Ad5, Ad49L or Sad23L vector in 600 healthy blood donors.
    Figure Legend Snippet: Characterization of Sad23L-nCoV-S and Ad49L-nCoV-S vaccines. (A) Recombinant adenovirus constructs Sad23L-nCoV-S and Ad49L-nCoV-S carrying the full-length S gene of SARS-CoV-2 under CMV promotor regulation within the deleted E1 region of Sad23L or Ad49L vector. (B) Western blot analysis for the expression of S protein from Sad23L-nCoV-S or Ad49L-nCoV-S infected HEK-293A cell lysates by rabbit polyclonal antibody to RBD. Sad23L-GFP or Ad49L-GFP virus infected cells were used as mock controls. (C) Expression of S protein in HEK-293A cells detected by immunofluorescence staining. (D) Seroprevalence of neutralizing antibody (AdNAb) to Ad5, Ad49L or Sad23L vector in 600 healthy blood donors.

    Techniques Used: Recombinant, Construct, Plasmid Preparation, Western Blot, Expressing, Infection, Immunofluorescence, Staining

    12) Product Images from "Structure and computation-guided design of a mutation-integrated trimeric RBD candidate vaccine with broad neutralization against SARS-CoV-2"

    Article Title: Structure and computation-guided design of a mutation-integrated trimeric RBD candidate vaccine with broad neutralization against SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2021.06.18.448958

    Protection of homo-tri-RBD vaccine candidate against live SARS-CoV-2 prototype virus challenge in transgenic mice. The mouse challenge experiments were performed in the ABSL3 facility. All the animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the National Vaccine and Serum Institute of China, and Institutional Animal Care and Use Committee of National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China. A total of 16 female ACE2-TG/IOZ transgenic mice aged 6-10 weeks (purchased from Beijing Huafukang Bioscience Co., Ltd, China) were used for the live SARS-CoV-2 challenge experiment, which were randomly divided into 3 groups: a vaccine group (5 mice), a saline group (5 mice) and a control group (6 mice). The mice in vaccine group were immunized with two intramuscular injections of 2 μg/dose candidate vaccine on Day 0 and Day 21. In saline group and control group, the mice were intramuscularly injected with physiological saline on Day 0 and Day 21. On day 7 after the second vaccination, the blood from the caudal vein of the immunized mice was collected, and serum was separated to measure the titers of specific IgG and neutralizing antibodies using ELISA and pseudo-virus neutralization assay, respectively. a. The titers of specific IgG and pseudo-virus neutralizing antibodies in the sera of the immunized mice compared with those of the mice in the saline and control groups. Data are presented as mean ± SEM. P values were determined by Student’s t-test. On day 14 after the second immunization, the mice in vaccine group (5 mice) and saline group (5 mice) were challenged with 1.5 × 10 5 TCID 50 of live SARS-CoV-2 prototype virus via the intranasal route. The mice in the control group were not subjected to live virus challenge, serving as the blank control. During the challenge experiment, the body weight of the mice was measured every day. b. Comparison of the body weight changes of the mice in the three groups during the challenge experiment. Five days after virus challenge, all mice were euthanized and the lung tissues were collected. The viral RNA was extracted from lung tissues by using a nucleic acid extraction kit (QIAGEN, No. 52906), and the viral load was evaluated by using a 2019-nCoV (N, ORF1ab and S genes) nucleic acid detection kit (PCR-fluorescence probing) purchased from Mabsky (Shenzhen) with product No. 349. c. Comparison of the viral loads in the lung tissues of the mice in the three groups. N, ORF1ab and S viral genes were detected. In addition, the histopathological sections of the lung tissues of the mice were stained with hematoxylin and eosin (HE), and the pathological examination was performed. d. Histopathological examinations of the lung tissues of the mice in the three groups. Left subfigure in each group displays the lung tissue amplified four times, and right subfigure enlarged ten times. Live virus challenge experiment showed that on day 7 after the whole vaccination, high titers of specific IgG and neutralizing antibodies were detected in all mice immunized with homo-tri-RBD vaccine candidate, whereas in the saline and control groups, no antibody response was detected (see panel a ). During the live virus challenge experiment, the body weight of the mice in the saline group also obviously reduced, suggesting possible infections of SARS-CoV-2. While for the mice in vaccine group, the body weights fluctuated within the normal range, which is similar to the mice in control group (see panel b ). This result indicated that homo-tri-RBD candidate vaccine might provide protections against the infection of live SARS-CoV-2. The viral load detections in the lung tissues showed that all the mice in saline group exhibited very high viral loads after live virus challenge, and the average viral load of the five mice reached 2535268 copies/ml, 2563725 copies/ml and 1652809 copies/ml for N, ORF1ab, and S genes, respectively (see panel c ). In contrast, the viral loads in lung tissues for all the mice in the vaccine group were under the detectable limit (
    Figure Legend Snippet: Protection of homo-tri-RBD vaccine candidate against live SARS-CoV-2 prototype virus challenge in transgenic mice. The mouse challenge experiments were performed in the ABSL3 facility. All the animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the National Vaccine and Serum Institute of China, and Institutional Animal Care and Use Committee of National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China. A total of 16 female ACE2-TG/IOZ transgenic mice aged 6-10 weeks (purchased from Beijing Huafukang Bioscience Co., Ltd, China) were used for the live SARS-CoV-2 challenge experiment, which were randomly divided into 3 groups: a vaccine group (5 mice), a saline group (5 mice) and a control group (6 mice). The mice in vaccine group were immunized with two intramuscular injections of 2 μg/dose candidate vaccine on Day 0 and Day 21. In saline group and control group, the mice were intramuscularly injected with physiological saline on Day 0 and Day 21. On day 7 after the second vaccination, the blood from the caudal vein of the immunized mice was collected, and serum was separated to measure the titers of specific IgG and neutralizing antibodies using ELISA and pseudo-virus neutralization assay, respectively. a. The titers of specific IgG and pseudo-virus neutralizing antibodies in the sera of the immunized mice compared with those of the mice in the saline and control groups. Data are presented as mean ± SEM. P values were determined by Student’s t-test. On day 14 after the second immunization, the mice in vaccine group (5 mice) and saline group (5 mice) were challenged with 1.5 × 10 5 TCID 50 of live SARS-CoV-2 prototype virus via the intranasal route. The mice in the control group were not subjected to live virus challenge, serving as the blank control. During the challenge experiment, the body weight of the mice was measured every day. b. Comparison of the body weight changes of the mice in the three groups during the challenge experiment. Five days after virus challenge, all mice were euthanized and the lung tissues were collected. The viral RNA was extracted from lung tissues by using a nucleic acid extraction kit (QIAGEN, No. 52906), and the viral load was evaluated by using a 2019-nCoV (N, ORF1ab and S genes) nucleic acid detection kit (PCR-fluorescence probing) purchased from Mabsky (Shenzhen) with product No. 349. c. Comparison of the viral loads in the lung tissues of the mice in the three groups. N, ORF1ab and S viral genes were detected. In addition, the histopathological sections of the lung tissues of the mice were stained with hematoxylin and eosin (HE), and the pathological examination was performed. d. Histopathological examinations of the lung tissues of the mice in the three groups. Left subfigure in each group displays the lung tissue amplified four times, and right subfigure enlarged ten times. Live virus challenge experiment showed that on day 7 after the whole vaccination, high titers of specific IgG and neutralizing antibodies were detected in all mice immunized with homo-tri-RBD vaccine candidate, whereas in the saline and control groups, no antibody response was detected (see panel a ). During the live virus challenge experiment, the body weight of the mice in the saline group also obviously reduced, suggesting possible infections of SARS-CoV-2. While for the mice in vaccine group, the body weights fluctuated within the normal range, which is similar to the mice in control group (see panel b ). This result indicated that homo-tri-RBD candidate vaccine might provide protections against the infection of live SARS-CoV-2. The viral load detections in the lung tissues showed that all the mice in saline group exhibited very high viral loads after live virus challenge, and the average viral load of the five mice reached 2535268 copies/ml, 2563725 copies/ml and 1652809 copies/ml for N, ORF1ab, and S genes, respectively (see panel c ). In contrast, the viral loads in lung tissues for all the mice in the vaccine group were under the detectable limit (

    Techniques Used: Transgenic Assay, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Neutralization, Polymerase Chain Reaction, Fluorescence, Staining, Amplification, Infection

    Structure-guided design, production, and characterization of the mutI tri-RBD. a. A schematic illustration of the mutI tri-RBD and homo-tri-RBD design schemes. The RBD region comprising the residues 319-537 was truncated from the S protein, and three truncated RBDs were connected end-by-end to construct the trimeric forms of RBD. In mutI tri-RBD, three RBDs were individually derived from three different circulating SARS-CoV-2 strains, i.e., the prototype, Beta (B.1.351) and Kappa (B.1.617.1). In homo-tri-RBD, the three RBD units were all truncated from the prototype strain. In the upper subfigure, the S1 and S2 subunits of the S protein, as well as NTD, RBD, SD1 and SD2 in S1 subunit, are marked. The lower subfigure displays the natural trimeric arrangement of RBDs in the native structure of S trimer. The arrows indicate the direct connections of the N- and C- terminals between different RBDs. b. Structural modeling and MD simulation of the designed homo-tri-RBD (upper subfigure) and mutI tri-RBD (lower subfigure). Time-evolution of the C α root-mean square deviation (RMSD) of the modelled structure during MD simulation, as well as several snapshot conformations in the simulation, is displayed. c. SDS-PAGE profiles of increasing amounts of the recombinant mutI tri-RBD and homo-tri-RBD proteins expressed by HEK293T cells. d. Molecular weight of mutI tri-RBD determined by MALDI-TOF MS. e. Secondary structure contents of mutI tri-RBD protein analyzed by circular dichroism spectrometry. f. Left: The proportions of free sulfhydryl for all the cysteine residues in mutI tri-RBD. Right: The disulfide-linkages in the recombinant mutI tri-RBD protein detected by liquid chromatography-mass spectrometry. Only the disulfide bonds in one RBD unit are listed. g. Differential scanning calorimetry thermograms of the recombinant mutI tri-RBD protein. h. The binding capability of the designed mutI tri-RBD and homo-tri-RBD proteins with two anti-RBD monoclonal nAbs, i.e., MM43 and R117, evaluated by ELISA. As controls, the binding activities with the monoclonal nAbs for the monomeric his-tagged RBDs from the prototype, Beta (B.1.351) and Kappa (B.1.617.1) SARS-CoV-2 strains were also measured. i. The binding profiles of the recombinant mutI tri-RBD with hACE2 detected by surface plasmon resonance assay.
    Figure Legend Snippet: Structure-guided design, production, and characterization of the mutI tri-RBD. a. A schematic illustration of the mutI tri-RBD and homo-tri-RBD design schemes. The RBD region comprising the residues 319-537 was truncated from the S protein, and three truncated RBDs were connected end-by-end to construct the trimeric forms of RBD. In mutI tri-RBD, three RBDs were individually derived from three different circulating SARS-CoV-2 strains, i.e., the prototype, Beta (B.1.351) and Kappa (B.1.617.1). In homo-tri-RBD, the three RBD units were all truncated from the prototype strain. In the upper subfigure, the S1 and S2 subunits of the S protein, as well as NTD, RBD, SD1 and SD2 in S1 subunit, are marked. The lower subfigure displays the natural trimeric arrangement of RBDs in the native structure of S trimer. The arrows indicate the direct connections of the N- and C- terminals between different RBDs. b. Structural modeling and MD simulation of the designed homo-tri-RBD (upper subfigure) and mutI tri-RBD (lower subfigure). Time-evolution of the C α root-mean square deviation (RMSD) of the modelled structure during MD simulation, as well as several snapshot conformations in the simulation, is displayed. c. SDS-PAGE profiles of increasing amounts of the recombinant mutI tri-RBD and homo-tri-RBD proteins expressed by HEK293T cells. d. Molecular weight of mutI tri-RBD determined by MALDI-TOF MS. e. Secondary structure contents of mutI tri-RBD protein analyzed by circular dichroism spectrometry. f. Left: The proportions of free sulfhydryl for all the cysteine residues in mutI tri-RBD. Right: The disulfide-linkages in the recombinant mutI tri-RBD protein detected by liquid chromatography-mass spectrometry. Only the disulfide bonds in one RBD unit are listed. g. Differential scanning calorimetry thermograms of the recombinant mutI tri-RBD protein. h. The binding capability of the designed mutI tri-RBD and homo-tri-RBD proteins with two anti-RBD monoclonal nAbs, i.e., MM43 and R117, evaluated by ELISA. As controls, the binding activities with the monoclonal nAbs for the monomeric his-tagged RBDs from the prototype, Beta (B.1.351) and Kappa (B.1.617.1) SARS-CoV-2 strains were also measured. i. The binding profiles of the recombinant mutI tri-RBD with hACE2 detected by surface plasmon resonance assay.

    Techniques Used: Construct, Derivative Assay, SDS Page, Recombinant, Molecular Weight, Liquid Chromatography, Mass Spectrometry, Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay

    Immune responses against SARS-CoV-2 prototype strain elicited by mutI tri-RBD in mice compared with those induced by homo- tri-RBD. a. Dose-dependent responses of RBD-specific IgG against SARS-CoV-2 prototype strain elicited by mutI tri-RBD. The mice were immunized with two-shot or three-shot injections, and for each shot, three different doses were used including low (0.125 μg/dose), middle (0.5 μg/dose) and high (2.0 μg/dose) doses, respectively. The levels of IgG were detected with ELISA by using monomeric RBD of prototype strain. b. The levels of RBD-specific IgG against the prototype strain induced by mutI tri-RBD compared with those induced by homo-tir-RBD. c. Dose-dependent responses of neutralizing antibodies against SARS-CoV-2 pseudo-virus of the prototype strain elicited by mutI tri-RBD. The titers of neutralizing antibodies were assessed by using the pseudo-virus neutralization assays. d. The titers of neutralizing antibodies against the pseudo-virus of prototype strain induced by mutI tri-RBD in contrast with those induced by homo-tri-RBD. e. Dose-dependent responses of neutralizing antibodies against the live prototype SARS-CoV-2 virus elicited by mutI tri-RBD. The titers of neutralizing antibodies were assessed by using the live virus neutralization assays. f. The titers of neutralizing antibodies against the live virus of prototype strain induced by mutI tri-RBD in contrast with those induced by homo-tri-RBD. Data are presented as means±SEMs. P values were calculated with Student’s t-test. **P
    Figure Legend Snippet: Immune responses against SARS-CoV-2 prototype strain elicited by mutI tri-RBD in mice compared with those induced by homo- tri-RBD. a. Dose-dependent responses of RBD-specific IgG against SARS-CoV-2 prototype strain elicited by mutI tri-RBD. The mice were immunized with two-shot or three-shot injections, and for each shot, three different doses were used including low (0.125 μg/dose), middle (0.5 μg/dose) and high (2.0 μg/dose) doses, respectively. The levels of IgG were detected with ELISA by using monomeric RBD of prototype strain. b. The levels of RBD-specific IgG against the prototype strain induced by mutI tri-RBD compared with those induced by homo-tir-RBD. c. Dose-dependent responses of neutralizing antibodies against SARS-CoV-2 pseudo-virus of the prototype strain elicited by mutI tri-RBD. The titers of neutralizing antibodies were assessed by using the pseudo-virus neutralization assays. d. The titers of neutralizing antibodies against the pseudo-virus of prototype strain induced by mutI tri-RBD in contrast with those induced by homo-tri-RBD. e. Dose-dependent responses of neutralizing antibodies against the live prototype SARS-CoV-2 virus elicited by mutI tri-RBD. The titers of neutralizing antibodies were assessed by using the live virus neutralization assays. f. The titers of neutralizing antibodies against the live virus of prototype strain induced by mutI tri-RBD in contrast with those induced by homo-tri-RBD. Data are presented as means±SEMs. P values were calculated with Student’s t-test. **P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Neutralization

    The neutralizing antibody responses against 22 various SARS-CoV-2 pseudo-virus strains elicited by mutI tri-RBD compared with those induced by homo-tri-RBD. a. Sensitivities to the neutralization of homo-tri-RBD immunized sera for the various pseudo-typed variants compared with that of the prototype strain. In this figure, GMT of the prototype strain is taken as reference, and GMT ratios between the variants and the prototype strain are displayed. Each serum sample is presented as a dot in the plot, and each serum sample was tested against all these variants. Data are presented as means±SEMs. b. Neutralizing antibody GMTs against the various pseudo-typed strains induced by mutI tri-RBD (red color) compared with those elicited by homo-tri-RBD (blue color). Numbers in the red box indicate the GMT ratios of mutI tri-RBD to homo-tri-RBD. P values were calculated with Student’s t-test. *P
    Figure Legend Snippet: The neutralizing antibody responses against 22 various SARS-CoV-2 pseudo-virus strains elicited by mutI tri-RBD compared with those induced by homo-tri-RBD. a. Sensitivities to the neutralization of homo-tri-RBD immunized sera for the various pseudo-typed variants compared with that of the prototype strain. In this figure, GMT of the prototype strain is taken as reference, and GMT ratios between the variants and the prototype strain are displayed. Each serum sample is presented as a dot in the plot, and each serum sample was tested against all these variants. Data are presented as means±SEMs. b. Neutralizing antibody GMTs against the various pseudo-typed strains induced by mutI tri-RBD (red color) compared with those elicited by homo-tri-RBD (blue color). Numbers in the red box indicate the GMT ratios of mutI tri-RBD to homo-tri-RBD. P values were calculated with Student’s t-test. *P

    Techniques Used: Neutralization

    Immune responses against the Beta (B.1.351) strain of SARS-CoV-2 elicited by mutI tri-RBD in mice compared with those induced by homo-tri-RBD. a. Dose-dependent responses of RBD-specific IgG against the Beta (B.1.351) SARS-CoV-2 strain elicited by mutI tri-RBD. The levels of IgG were detected with ELISA by using monomeric RBD of Beta (B.1.351) strain. b. The levels of RBD-specific IgG against the Beta (B.1.351) strain induced by mutI tri-RBD compared with those induced by homo-tir-RBD. c. Dose-dependent responses of neutralizing antibodies against SARS-CoV-2 pseudo-virus of the Beta (B.1.351) strain elicited by mutI tri-RBD. The titers of neutralizing antibodies were assessed by using the pseudo-virus neutralization assays. d. The titers of neutralizing antibodies against the pseudo-virus of Beta (B.1.351) strain induced by mutI tri-RBD in contrast with those induced by homo-tri-RBD. e. Dose-dependent responses of neutralizing antibodies against the live Beta (B.1.351) SARS-CoV-2 virus strain elicited by mutI tri-RBD. The titers of neutralizing antibodies were assessed by using the live virus neutralization assays. f. The titers of neutralizing antibodies against the live virus of Beta (B.1.351) strain induced by mutI tri-RBD in contrast with those induced by homo-tri-RBD. Data are presented as means±SEMs. P values were calculated with Student’s t-test. *P
    Figure Legend Snippet: Immune responses against the Beta (B.1.351) strain of SARS-CoV-2 elicited by mutI tri-RBD in mice compared with those induced by homo-tri-RBD. a. Dose-dependent responses of RBD-specific IgG against the Beta (B.1.351) SARS-CoV-2 strain elicited by mutI tri-RBD. The levels of IgG were detected with ELISA by using monomeric RBD of Beta (B.1.351) strain. b. The levels of RBD-specific IgG against the Beta (B.1.351) strain induced by mutI tri-RBD compared with those induced by homo-tir-RBD. c. Dose-dependent responses of neutralizing antibodies against SARS-CoV-2 pseudo-virus of the Beta (B.1.351) strain elicited by mutI tri-RBD. The titers of neutralizing antibodies were assessed by using the pseudo-virus neutralization assays. d. The titers of neutralizing antibodies against the pseudo-virus of Beta (B.1.351) strain induced by mutI tri-RBD in contrast with those induced by homo-tri-RBD. e. Dose-dependent responses of neutralizing antibodies against the live Beta (B.1.351) SARS-CoV-2 virus strain elicited by mutI tri-RBD. The titers of neutralizing antibodies were assessed by using the live virus neutralization assays. f. The titers of neutralizing antibodies against the live virus of Beta (B.1.351) strain induced by mutI tri-RBD in contrast with those induced by homo-tri-RBD. Data are presented as means±SEMs. P values were calculated with Student’s t-test. *P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Neutralization

    13) Product Images from "Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2"

    Article Title: Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2

    Journal: Communications Biology

    doi: 10.1038/s42003-021-02029-w

    Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.
    Figure Legend Snippet: Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.

    Techniques Used: Concentration Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

    CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.
    Figure Legend Snippet: CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.

    Techniques Used: Infection, In Vitro, In Vivo, Expressing, Activity Assay, Neutralization, Mouse Assay, Real-time Polymerase Chain Reaction

    CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.
    Figure Legend Snippet: CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.

    Techniques Used: Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Staining, Concentration Assay, Flow Cytometry

    14) Product Images from "Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity"

    Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity

    Journal: JCI Insight

    doi: 10.1172/jci.insight.142386

    Comparison of seroconversion in patients with COVID-19 and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS 2017–2019 (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.
    Figure Legend Snippet: Comparison of seroconversion in patients with COVID-19 and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS 2017–2019 (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Detection of serum binding antibodies against SARS-CoV-2 proteins in patients with PCR-confirmed COVID-19 and healthy samples. ( A ) Timeline of COVID-19 diagnosis/ICU admittance, serum sample collection, and convalescent plasma (CP) administration. Time 0 is defined as day of COVID-19 diagnosis (PCR positive for SARS-CoV-2) and ICU admittance. Blood collections are denoted in gray and CP administration is denoted in pink. Patients were stratified based on current status (recovered, hospitalized, or deceased). Patient 29 from our cohort had symptoms but was PCR negative for SARS-CoV-2; this sample was not included in figures since there was no proof of disease. ( B ) Schematic of SARS-CoV-2 viral structure (top panel) and antigens assayed (bottom panel). S-protein, light orange; envelope protein, yellow; membrane glycoprotein, dark orange; RNA, blue; N-protein, green. Absorbance normalized to the respective no antigen control for each sample at 450 nm plotted for S-RBD (left panel), and N-protein (right panel), antigen coating with the most recent (or only) SARS-CoV-2 samples not treated with CP ( n = 21) and healthy samples collected in 2017–2019 (HS 2017–2019, n = 104 for S-RBD, n = 103 for N-protein) and 2020 (HS 2020, n = 308). Data are presented with each dot representing the mean normalized absorbance for a given serum sample; the red bar depicts the median ± interquartile range of all samples. HS, healthy sample; NC (line), negative control cutoff (see Methods). Kruskal-Wallis with Dunn’s multiple-comparisons test performed. **** P
    Figure Legend Snippet: Detection of serum binding antibodies against SARS-CoV-2 proteins in patients with PCR-confirmed COVID-19 and healthy samples. ( A ) Timeline of COVID-19 diagnosis/ICU admittance, serum sample collection, and convalescent plasma (CP) administration. Time 0 is defined as day of COVID-19 diagnosis (PCR positive for SARS-CoV-2) and ICU admittance. Blood collections are denoted in gray and CP administration is denoted in pink. Patients were stratified based on current status (recovered, hospitalized, or deceased). Patient 29 from our cohort had symptoms but was PCR negative for SARS-CoV-2; this sample was not included in figures since there was no proof of disease. ( B ) Schematic of SARS-CoV-2 viral structure (top panel) and antigens assayed (bottom panel). S-protein, light orange; envelope protein, yellow; membrane glycoprotein, dark orange; RNA, blue; N-protein, green. Absorbance normalized to the respective no antigen control for each sample at 450 nm plotted for S-RBD (left panel), and N-protein (right panel), antigen coating with the most recent (or only) SARS-CoV-2 samples not treated with CP ( n = 21) and healthy samples collected in 2017–2019 (HS 2017–2019, n = 104 for S-RBD, n = 103 for N-protein) and 2020 (HS 2020, n = 308). Data are presented with each dot representing the mean normalized absorbance for a given serum sample; the red bar depicts the median ± interquartile range of all samples. HS, healthy sample; NC (line), negative control cutoff (see Methods). Kruskal-Wallis with Dunn’s multiple-comparisons test performed. **** P

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Negative Control

    15) Product Images from "Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation"

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

    Graphical illustrations of the COVID-19 related immunoassays that were performed with our microfluidic chemiluminescent ELISA platform, including (A) affinity evaluation of calibration antibodies, (B) detection of circulating anti-SARS-CoV-2 S1 IgG in serum samples, and (C) detection of SARS-CoV-2 antigens such as S1 and N protein.
    Figure Legend Snippet: Graphical illustrations of the COVID-19 related immunoassays that were performed with our microfluidic chemiluminescent ELISA platform, including (A) affinity evaluation of calibration antibodies, (B) detection of circulating anti-SARS-CoV-2 S1 IgG in serum samples, and (C) detection of SARS-CoV-2 antigens such as S1 and N protein.

    Techniques Used: Chemiluminescent ELISA

    16) Product Images from "Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies"

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    Journal: bioRxiv

    doi: 10.1101/2021.04.03.438258

    ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of SARS-CoV-2 S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.
    Figure Legend Snippet: ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of SARS-CoV-2 S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.

    Techniques Used: Over Expression, Activity Assay, Neutralization, Infection, Purification, Labeling, Protein Binding, Flow Cytometry, Quantitative RT-PCR, Luciferase

    SARS-CoV-2 live virus neutralization. HEK293T cells stably expressing ACE2, SIGLEC1, DC-SIGN or L-SIGN were infected with SARS-CoV-2 at MOI 0.02 in the presence of the indicated mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and positive cells were quantified.
    Figure Legend Snippet: SARS-CoV-2 live virus neutralization. HEK293T cells stably expressing ACE2, SIGLEC1, DC-SIGN or L-SIGN were infected with SARS-CoV-2 at MOI 0.02 in the presence of the indicated mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and positive cells were quantified.

    Techniques Used: Neutralization, Stable Transfection, Expressing, Infection

    RBM mAbs trigger the fusogenic rearrangmement of the S protein and promote membrane fusion. a, MAbs or soluble ACE2 were incubated for 1 hour with native-like soluble prefusion S trimer of SARS-CoV-2 to track by negative stain EM imaging the fusogenic rearrangement of soluble S trimers visible as rosettes. b , Cell-cell fusion of CHO cells expressing SARS-CoV-2 S (CHO-S) on the plasma membrane in the absence (upper panel) or presence of 5 μg/ml of S2E12 mAb (lower panel) as detected by immuno-fluorescence. Nuclei stained with Hoechst dye; cytoplasm stained with CellTracker Green. ( c ), CHO-S cell-cell fusion mediated by different spike-specific mAbs quantified as described in Methods. d , Structures of 11 Fab-RBD complexes related to mAbs used in (c) (RBD orientation is fixed) and of ACE2-RBD as determined by a combination of X-ray crystallography and cryo-EM analysis (PDBs, Extended Data Table 1 ). Shown in parentheses the RBD antigenic site as defined according to Piccoli et al. 3 e , Inhibition of S2E12-induced cell-cell fusion performed as in (c) by a fixed amount (15 μg/ml) of indicated mAbs. f , Trans-fusion of S-positive CHO cells with S-negative fluorescently-labelled CHO cells. Staining as in (b).
    Figure Legend Snippet: RBM mAbs trigger the fusogenic rearrangmement of the S protein and promote membrane fusion. a, MAbs or soluble ACE2 were incubated for 1 hour with native-like soluble prefusion S trimer of SARS-CoV-2 to track by negative stain EM imaging the fusogenic rearrangement of soluble S trimers visible as rosettes. b , Cell-cell fusion of CHO cells expressing SARS-CoV-2 S (CHO-S) on the plasma membrane in the absence (upper panel) or presence of 5 μg/ml of S2E12 mAb (lower panel) as detected by immuno-fluorescence. Nuclei stained with Hoechst dye; cytoplasm stained with CellTracker Green. ( c ), CHO-S cell-cell fusion mediated by different spike-specific mAbs quantified as described in Methods. d , Structures of 11 Fab-RBD complexes related to mAbs used in (c) (RBD orientation is fixed) and of ACE2-RBD as determined by a combination of X-ray crystallography and cryo-EM analysis (PDBs, Extended Data Table 1 ). Shown in parentheses the RBD antigenic site as defined according to Piccoli et al. 3 e , Inhibition of S2E12-induced cell-cell fusion performed as in (c) by a fixed amount (15 μg/ml) of indicated mAbs. f , Trans-fusion of S-positive CHO cells with S-negative fluorescently-labelled CHO cells. Staining as in (b).

    Techniques Used: Incubation, Staining, Imaging, Expressing, Fluorescence, Cryo-EM Sample Prep, Inhibition

    S309 or a cocktail of S309 and S2E12 provide robust in vivo protection against SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount of mAb(s) 48 hours before intra-nasal challenge with SARS-CoV-2. ( a-b ) Quantification of viral RNA in the lungs 4 days post-infection. ( c-d ) Quantification of replicating virus in lung homogenates harvested 4 days post infection using a TCID50 assay. ( e-f ) Histopathological score of the lung tissue was assessed 4 days post infection. ( g-h ) Efficacy plots based on the correlation between the level of serum antibody measured at the time of infection and the level of SARS-CoV2 (viral RNA) measured in lungs on day 4 after infection. The dotted lines represents EC50 and EC90 for viral reduction (EC90 of S309 alone vs S309+S2E12: 9 vs 11 μg/ml, respectively).
    Figure Legend Snippet: S309 or a cocktail of S309 and S2E12 provide robust in vivo protection against SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount of mAb(s) 48 hours before intra-nasal challenge with SARS-CoV-2. ( a-b ) Quantification of viral RNA in the lungs 4 days post-infection. ( c-d ) Quantification of replicating virus in lung homogenates harvested 4 days post infection using a TCID50 assay. ( e-f ) Histopathological score of the lung tissue was assessed 4 days post infection. ( g-h ) Efficacy plots based on the correlation between the level of serum antibody measured at the time of infection and the level of SARS-CoV2 (viral RNA) measured in lungs on day 4 after infection. The dotted lines represents EC50 and EC90 for viral reduction (EC90 of S309 alone vs S309+S2E12: 9 vs 11 μg/ml, respectively).

    Techniques Used: In Vivo, Injection, Infection, TCID50 Assay

    SIGLEC1, DC-SIGN and L-SIGN modulate neutralizing activity by different classes of antibodies. a-d , Neutralization of infection by authentic SARS-CoV-2 pre-incubated with indicated mAbs of HEK293T cell lines stably overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2. Infection was measured by immunostaining at 24 hours for the SARS-CoV-2 nucleoprotein. e , Summary of the mechanisms of action of different classes of spike-specific mAbs based on this and previous studies. *, mAb-mediated inhibition of fusion between CHO-spike cells and ACE2+ Vero-E6 cells; **, based on mAb-dependent activation of human FcγRs performed with a bioluminescent reporter assay as in 27 . æ , S2X58 binds to open RDB due to a confomational clash with neighboring NTD
    Figure Legend Snippet: SIGLEC1, DC-SIGN and L-SIGN modulate neutralizing activity by different classes of antibodies. a-d , Neutralization of infection by authentic SARS-CoV-2 pre-incubated with indicated mAbs of HEK293T cell lines stably overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2. Infection was measured by immunostaining at 24 hours for the SARS-CoV-2 nucleoprotein. e , Summary of the mechanisms of action of different classes of spike-specific mAbs based on this and previous studies. *, mAb-mediated inhibition of fusion between CHO-spike cells and ACE2+ Vero-E6 cells; **, based on mAb-dependent activation of human FcγRs performed with a bioluminescent reporter assay as in 27 . æ , S2X58 binds to open RDB due to a confomational clash with neighboring NTD

    Techniques Used: Activity Assay, Neutralization, Infection, Incubation, Stable Transfection, Immunostaining, Inhibition, Activation Assay, Reporter Assay

    Expression of auxiliary receptors in infected tissues and their role in mediating trans-infection in vitro a, Distribution and expression of ACE2, DC-SIGN, L-SIGN, and SIGLEC1 in the human lung cell atlas. b, Major cell types with detectable SARS-CoV-2 genome in bronchoalverolar lavage fluid and sputum of severe COVID-19 patients. Left panel shows a t-SNE embedding of single-cell gene expression profiles coloured by cell type and sized by viral load (logCPM); right panel, distribution plots by annotated cell type denote the cumulative fraction of cells (y-axis) with detected viral RNA per cell up to the corresponding logCPM value (x-axis). c, Left panel shows a heatmap matrix of counts for cells with detected transcripts for receptor gene(s) on x-axis by SARS-CoV-2 + cell type on y-axis (total n=3,085 cells from 8 subjects in Ren et al. 20 ); right panel, correlation of receptor transcript counts with SARS-CoV-2 RNA counts in macrophages and in secretory cells. Correlation is based on counts (before log transformation), from Ren et al. 22 . d, Trans-infection: HeLa cells transduced with DC-SIGN, L-SIGN or SIGLEC1 were incubated with VSV-SARS-CoV-2, extensively washed and co-cultured with Vero-E6-TMPRSS2 susceptible target cells. Shown is RLU in the presence or absence of target cells. e, Trans-infection performed as in (d). VSV-SARS-CoV-2 viral adsorption was performed in the presence or absence of an anti-SIGLEC1 blocking antibody.
    Figure Legend Snippet: Expression of auxiliary receptors in infected tissues and their role in mediating trans-infection in vitro a, Distribution and expression of ACE2, DC-SIGN, L-SIGN, and SIGLEC1 in the human lung cell atlas. b, Major cell types with detectable SARS-CoV-2 genome in bronchoalverolar lavage fluid and sputum of severe COVID-19 patients. Left panel shows a t-SNE embedding of single-cell gene expression profiles coloured by cell type and sized by viral load (logCPM); right panel, distribution plots by annotated cell type denote the cumulative fraction of cells (y-axis) with detected viral RNA per cell up to the corresponding logCPM value (x-axis). c, Left panel shows a heatmap matrix of counts for cells with detected transcripts for receptor gene(s) on x-axis by SARS-CoV-2 + cell type on y-axis (total n=3,085 cells from 8 subjects in Ren et al. 20 ); right panel, correlation of receptor transcript counts with SARS-CoV-2 RNA counts in macrophages and in secretory cells. Correlation is based on counts (before log transformation), from Ren et al. 22 . d, Trans-infection: HeLa cells transduced with DC-SIGN, L-SIGN or SIGLEC1 were incubated with VSV-SARS-CoV-2, extensively washed and co-cultured with Vero-E6-TMPRSS2 susceptible target cells. Shown is RLU in the presence or absence of target cells. e, Trans-infection performed as in (d). VSV-SARS-CoV-2 viral adsorption was performed in the presence or absence of an anti-SIGLEC1 blocking antibody.

    Techniques Used: Expressing, Infection, In Vitro, Transformation Assay, Transduction, Incubation, Cell Culture, Adsorption, Blocking Assay

    Characterization of SARS-CoV-2-susceptible cell lines. a , SARS-CoV-2 neutralization with 10 μg/ml of S309, S2E12 and S2X33 on Vero E6 or Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection and viral nucleocapsid protein was immunostained. b , Purified, fluorescently-labelled SARS-CoV-2 spike protein or RBD protein was incubated with the indicated cell lines and protein binding was quantified by flow cytometry. c , Correlation analysis between ACE2 transcript levels and maximum antibody neutralization in all SARS-CoV-2-susceptible cell lines.
    Figure Legend Snippet: Characterization of SARS-CoV-2-susceptible cell lines. a , SARS-CoV-2 neutralization with 10 μg/ml of S309, S2E12 and S2X33 on Vero E6 or Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection and viral nucleocapsid protein was immunostained. b , Purified, fluorescently-labelled SARS-CoV-2 spike protein or RBD protein was incubated with the indicated cell lines and protein binding was quantified by flow cytometry. c , Correlation analysis between ACE2 transcript levels and maximum antibody neutralization in all SARS-CoV-2-susceptible cell lines.

    Techniques Used: Neutralization, Infection, Purification, Incubation, Protein Binding, Flow Cytometry

    Role of host effector function in SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount (mg/kg) of hamster IgG2a S309 either wt or Fc silenced (S309-N297A). a , Quantification of viral RNA in the lung 4 days post infection. b, Quantification of replicating virus in the lung 4 days post infection. c, Histopathological score in the lung 4 days post infection. Control animals (white symbols) were injected with 4 mg/kg unrelated control isotype mAb. *, **, ***, **** p
    Figure Legend Snippet: Role of host effector function in SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount (mg/kg) of hamster IgG2a S309 either wt or Fc silenced (S309-N297A). a , Quantification of viral RNA in the lung 4 days post infection. b, Quantification of replicating virus in the lung 4 days post infection. c, Histopathological score in the lung 4 days post infection. Control animals (white symbols) were injected with 4 mg/kg unrelated control isotype mAb. *, **, ***, **** p

    Techniques Used: Injection, Infection

    HeLa cells expressing DC-SIGN are refractory to SARS-CoV-2 infection. 293T or HeLa cells stably expressing DC-SIGN were infected with SARS-CoV-2-Nluc at MOI0.04 in the presence of the indicated antibodies. Infection was analyzed by quantification of luminescent signal at 24 h post infection.
    Figure Legend Snippet: HeLa cells expressing DC-SIGN are refractory to SARS-CoV-2 infection. 293T or HeLa cells stably expressing DC-SIGN were infected with SARS-CoV-2-Nluc at MOI0.04 in the presence of the indicated antibodies. Infection was analyzed by quantification of luminescent signal at 24 h post infection.

    Techniques Used: Expressing, Infection, Stable Transfection

    Characterization of DC-SIGN, L-SIGN and SIGLEC-1 as SARS-CoV-2 attachment factors. a-b, Binding of antibodies targeting DC/-L-SIGN, DC-SIGN, SIGLEC1 or ACE2 on HEK293T stably over-expressing the respective attachment receptors was analyzed by flow cytometry (a) and immunofluorescence analysis (b). c, HEK293T cells over-expressing the respective attachment receptors were infected with VSV-SARS-COV-2 wildtype spike (grey bars) or spike bearing mutations of the B.1.1.7 variant (red bars). Luminescence was analyzed one day post infection.
    Figure Legend Snippet: Characterization of DC-SIGN, L-SIGN and SIGLEC-1 as SARS-CoV-2 attachment factors. a-b, Binding of antibodies targeting DC/-L-SIGN, DC-SIGN, SIGLEC1 or ACE2 on HEK293T stably over-expressing the respective attachment receptors was analyzed by flow cytometry (a) and immunofluorescence analysis (b). c, HEK293T cells over-expressing the respective attachment receptors were infected with VSV-SARS-COV-2 wildtype spike (grey bars) or spike bearing mutations of the B.1.1.7 variant (red bars). Luminescence was analyzed one day post infection.

    Techniques Used: Binding Assay, Stable Transfection, Expressing, Flow Cytometry, Immunofluorescence, Infection, Variant Assay

    DC-SIGN, L-SIGN and SIGLEC1 function as auxiliary receptors for SARS-CoV-2 infection. a, VSV-SARS-CoV-2 pseudovirus infection of HEK293T cells transfected to over-express ACE2 or a panel of selected lectins and published receptor candidates. b, Stable HEK293T cell lines overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2 were infected with authentic SARS-CoV-2 (MOI 0.1), fixed and immunostained at 24 hours for the SARS-CoV-2 nucleocapsid protein (red). c, HEK293T stable cell lines were infected with SARS-CoV-2-Nluc and luciferase levels were quantified at 24 hours. d, Stable cell lines were incubated with different concentrations of anti-SIGLEC1 mAb (clone 7-239) and infected with SARS-CoV-2-Nluc. e, HEK293T, HeLa and MRC5 cells were transiently transduced to overexpress DC-SIGN, L-SIGN, SIGLEC1 or ACE2 and infected with VSV-SARS-CoV-2 pseudovirus. f, Stable cell lines were treated with ACE2 siRNA followed by infection with VSV-SARS-CoV-2 pseudovirus four days post transfection. g, Stable cell lines were incubated with different concentrations of anti-ACE2 goat polyclonal antibodies and infected with VSV-SARS-CoV-2 pseudovirus.
    Figure Legend Snippet: DC-SIGN, L-SIGN and SIGLEC1 function as auxiliary receptors for SARS-CoV-2 infection. a, VSV-SARS-CoV-2 pseudovirus infection of HEK293T cells transfected to over-express ACE2 or a panel of selected lectins and published receptor candidates. b, Stable HEK293T cell lines overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2 were infected with authentic SARS-CoV-2 (MOI 0.1), fixed and immunostained at 24 hours for the SARS-CoV-2 nucleocapsid protein (red). c, HEK293T stable cell lines were infected with SARS-CoV-2-Nluc and luciferase levels were quantified at 24 hours. d, Stable cell lines were incubated with different concentrations of anti-SIGLEC1 mAb (clone 7-239) and infected with SARS-CoV-2-Nluc. e, HEK293T, HeLa and MRC5 cells were transiently transduced to overexpress DC-SIGN, L-SIGN, SIGLEC1 or ACE2 and infected with VSV-SARS-CoV-2 pseudovirus. f, Stable cell lines were treated with ACE2 siRNA followed by infection with VSV-SARS-CoV-2 pseudovirus four days post transfection. g, Stable cell lines were incubated with different concentrations of anti-ACE2 goat polyclonal antibodies and infected with VSV-SARS-CoV-2 pseudovirus.

    Techniques Used: Infection, Transfection, Stable Transfection, Luciferase, Incubation

    Data collection and processing of the S/S2X58 complex cryoEM datasets. a,b , Representative electron micrograph and 2D class averages of SARS-CoV-2 S in complex with the S2X58 Fab embedded in vitreous ice. Scale bar: 400 Å. c , Gold-standard Fourier shell correlation curves for the S2X58-bound SARS-CoV-2 S trimer in one RBD closed (black line) and three RBDs open conformations (gray line). The 0.143 cutoff is indicated by a horizontal dashed line. d, Local resolution maps calculated using cryoSPARC for the SARS-CoV-2 S/S2X58 Fab complex structure with one RBD closed and three RBDs open shown in two orthogonal orientations.
    Figure Legend Snippet: Data collection and processing of the S/S2X58 complex cryoEM datasets. a,b , Representative electron micrograph and 2D class averages of SARS-CoV-2 S in complex with the S2X58 Fab embedded in vitreous ice. Scale bar: 400 Å. c , Gold-standard Fourier shell correlation curves for the S2X58-bound SARS-CoV-2 S trimer in one RBD closed (black line) and three RBDs open conformations (gray line). The 0.143 cutoff is indicated by a horizontal dashed line. d, Local resolution maps calculated using cryoSPARC for the SARS-CoV-2 S/S2X58 Fab complex structure with one RBD closed and three RBDs open shown in two orthogonal orientations.

    Techniques Used:

    17) Product Images from "Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19"

    Article Title: Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-020-00301-9

    Neutralizing antibody responses to SARS-CoV-2 in COVID-19 recovered patients. a Scores showing the COVID-19 patient serum-mediated inhibition of the SARS-CoV-2 RBD protein binding to ACE2 protein by ELISA. b Pie charts showing the proportions of patients with high ( > 50, green) or low (
    Figure Legend Snippet: Neutralizing antibody responses to SARS-CoV-2 in COVID-19 recovered patients. a Scores showing the COVID-19 patient serum-mediated inhibition of the SARS-CoV-2 RBD protein binding to ACE2 protein by ELISA. b Pie charts showing the proportions of patients with high ( > 50, green) or low (

    Techniques Used: Inhibition, Protein Binding, Enzyme-linked Immunosorbent Assay

    Subtypes of neutralizing antibodies to SARS-CoV-2 S proteins in COVID-19 recovered patients. a Blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus into ACE2/293T cells by patient sera (no depletion) or S1 antibody-depleted sera (S1-Abs depletion) or S2 antibody-depleted sera (S2-Abs depletion). The dashed line indicates the cutoff value (6.749) determined by the ROC curve analysis. HC healthy control, NC negative control. b , c Pie charts showing the proportions of patients with different neutralizing antibody (NAb) subtype responses in the total 25 patients ( b ), 8 severe patients ( c , left panel), and 17 moderate and mild patients ( c , right panel) of pseudovirus neutralization positive. d Blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus into ACE2/293T cells by “S1-NAbs only” patient sera with RBD antibody depletion (RBD-Abs depletion) or without RBD antibody depletion (no depletion). The dashed line indicates the cutoff value (6.034) determined by the ROC curve analysis. HC healthy control, NC negative control. e Pie chart showing the proportions of “S1-NAbs only” patients with RBD-Nab-dependent or -independent antibody response. Error bars in a , d indicate SEM
    Figure Legend Snippet: Subtypes of neutralizing antibodies to SARS-CoV-2 S proteins in COVID-19 recovered patients. a Blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus into ACE2/293T cells by patient sera (no depletion) or S1 antibody-depleted sera (S1-Abs depletion) or S2 antibody-depleted sera (S2-Abs depletion). The dashed line indicates the cutoff value (6.749) determined by the ROC curve analysis. HC healthy control, NC negative control. b , c Pie charts showing the proportions of patients with different neutralizing antibody (NAb) subtype responses in the total 25 patients ( b ), 8 severe patients ( c , left panel), and 17 moderate and mild patients ( c , right panel) of pseudovirus neutralization positive. d Blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus into ACE2/293T cells by “S1-NAbs only” patient sera with RBD antibody depletion (RBD-Abs depletion) or without RBD antibody depletion (no depletion). The dashed line indicates the cutoff value (6.034) determined by the ROC curve analysis. HC healthy control, NC negative control. e Pie chart showing the proportions of “S1-NAbs only” patients with RBD-Nab-dependent or -independent antibody response. Error bars in a , d indicate SEM

    Techniques Used: Blocking Assay, Luciferase, Negative Control, Neutralization

    Antibody responses to SARS-CoV-2 in COVID-19 recovered patients with different symptom severity. a – c ELISA binding assays of 100-fold diluted COVID-19 patient sera to ELISA plates after coating with SARS-CoV-2 S1 ( a ), RBD ( b ), and S2 ( c ) proteins. The dashed lines in a – c represent the average values of the healthy control groups. * P
    Figure Legend Snippet: Antibody responses to SARS-CoV-2 in COVID-19 recovered patients with different symptom severity. a – c ELISA binding assays of 100-fold diluted COVID-19 patient sera to ELISA plates after coating with SARS-CoV-2 S1 ( a ), RBD ( b ), and S2 ( c ) proteins. The dashed lines in a – c represent the average values of the healthy control groups. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay

    18) Product Images from "CV2CoV, an enhanced mRNA-based SARS-CoV-2 vaccine candidate, supports higher protein expression and improved immunogenicity in rats"

    Article Title: CV2CoV, an enhanced mRNA-based SARS-CoV-2 vaccine candidate, supports higher protein expression and improved immunogenicity in rats

    Journal: bioRxiv

    doi: 10.1101/2021.05.13.443734

    CV2CoV induces high titres of virus neutralising antibodies (VNT) against SARS-CoV-2 in rats. Female and male Wistar rats (n=6/group) were vaccinated IM with five different doses ranging from 0.5 μg – 40 μg of CV2CoV on day 0 and day 21. Animals (n=4) vaccinated with NaCl (Buffer) served as negative controls. SARS-CoV-2 100% neutralization titres (VNT100) in serum samples taken on day 14, day 21 and day 42 were analysed. Highest dilution step was 1:5120. Each dot represents an individual animal, bars depict the median.
    Figure Legend Snippet: CV2CoV induces high titres of virus neutralising antibodies (VNT) against SARS-CoV-2 in rats. Female and male Wistar rats (n=6/group) were vaccinated IM with five different doses ranging from 0.5 μg – 40 μg of CV2CoV on day 0 and day 21. Animals (n=4) vaccinated with NaCl (Buffer) served as negative controls. SARS-CoV-2 100% neutralization titres (VNT100) in serum samples taken on day 14, day 21 and day 42 were analysed. Highest dilution step was 1:5120. Each dot represents an individual animal, bars depict the median.

    Techniques Used: Neutralization

    CV2CoV induces high titres of cross neutralising antibodies (VNT) against SARS-CoV-2 variants in rats. Female and male Wistar rats (n=6/group) were vaccinated IM with five different doses ranging from 0.5 μg – 40 μg of CV2CoV on day 0 and day 21. Animals (n=4) vaccinated with NaCl (Buffer) served as negative controls. SARS-CoV-2 100% neutralization titres (VNT100) in serum samples taken on day 42 against distinct SARS-CoV-2 variants were analysed as indicated. Highest dilution step was 1:4096. Each dot represents an individual animal, bars depict the median.
    Figure Legend Snippet: CV2CoV induces high titres of cross neutralising antibodies (VNT) against SARS-CoV-2 variants in rats. Female and male Wistar rats (n=6/group) were vaccinated IM with five different doses ranging from 0.5 μg – 40 μg of CV2CoV on day 0 and day 21. Animals (n=4) vaccinated with NaCl (Buffer) served as negative controls. SARS-CoV-2 100% neutralization titres (VNT100) in serum samples taken on day 42 against distinct SARS-CoV-2 variants were analysed as indicated. Highest dilution step was 1:4096. Each dot represents an individual animal, bars depict the median.

    Techniques Used: Neutralization

    19) Product Images from "The Characterization of Disease Severity Associated IgG Subclasses Response in COVID-19 Patients"

    Article Title: The Characterization of Disease Severity Associated IgG Subclasses Response in COVID-19 Patients

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.632814

    IgG1 and IgG3 were the main subclasses induced in COVID-19 patients and related with disease severity. IgG1 (A) and IgG3 (B) responses to NP, S, and RBD and neutralizing antibody (C) against SARS-CoV-2 pseudo-virus with luciferase reporter gene in the healthy controls ( n = 11) and the severe ( n = 24), moderate ( n = 54), mild ( n = 35), asymptomatic ( n = 10) patients were detected. Differences of medium values between groups were analyzed by Mann-Whitney U -test. Significant correlations among Severity, NAb and IgG subclasses (D) including anti-NP IgG1, IgG3, anti-S IgG1, IgG3, anti-RBD IgG1, IgG3 were shown. Spearman correlation coefficient was calculated. A two-tailed P value
    Figure Legend Snippet: IgG1 and IgG3 were the main subclasses induced in COVID-19 patients and related with disease severity. IgG1 (A) and IgG3 (B) responses to NP, S, and RBD and neutralizing antibody (C) against SARS-CoV-2 pseudo-virus with luciferase reporter gene in the healthy controls ( n = 11) and the severe ( n = 24), moderate ( n = 54), mild ( n = 35), asymptomatic ( n = 10) patients were detected. Differences of medium values between groups were analyzed by Mann-Whitney U -test. Significant correlations among Severity, NAb and IgG subclasses (D) including anti-NP IgG1, IgG3, anti-S IgG1, IgG3, anti-RBD IgG1, IgG3 were shown. Spearman correlation coefficient was calculated. A two-tailed P value

    Techniques Used: Luciferase, MANN-WHITNEY, Two Tailed Test

    IgA, IgG, and IgM antibodies responses in COVID-19 ranging from asymptomatic to severe patients. Serum samples collected from COVID-19 patients were used for detecting IgA, IgG, and IgM levels to NP (A) , S (B) , and RBD (C) antigens of SARS-CoV-2 via ELISA. Antibody titers of the healthy controls ( n = 11) and the severe ( n = 24), moderate ( n = 54), mild ( n = 35), asymptomatic ( n = 10) patients were shown in (A–C) . Mann-Whitney U -test was used to compare differences of medium values between groups, a two-tailed P value
    Figure Legend Snippet: IgA, IgG, and IgM antibodies responses in COVID-19 ranging from asymptomatic to severe patients. Serum samples collected from COVID-19 patients were used for detecting IgA, IgG, and IgM levels to NP (A) , S (B) , and RBD (C) antigens of SARS-CoV-2 via ELISA. Antibody titers of the healthy controls ( n = 11) and the severe ( n = 24), moderate ( n = 54), mild ( n = 35), asymptomatic ( n = 10) patients were shown in (A–C) . Mann-Whitney U -test was used to compare differences of medium values between groups, a two-tailed P value

    Techniques Used: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Two Tailed Test

    20) Product Images from "SARS-CoV-2–Specific Antibody Detection for Seroepidemiology: A Multiplex Analysis Approach Accounting for Accurate Seroprevalence"

    Article Title: SARS-CoV-2–Specific Antibody Detection for Seroepidemiology: A Multiplex Analysis Approach Accounting for Accurate Seroprevalence

    Journal: The Journal of Infectious Diseases

    doi: 10.1093/infdis/jiaa479

    Discrimination of COVID-19 patients with varying severity from a cross-sectional population panel and ILI patients. A , Individuals from the cross-sectional panel aged 3–90 years (n = 224), ILI patients with noncoronavirus (n = 75), and non-SARS-CoV-2 seasonal coronavirus-infected ILI patients (n = 109) were compared to hospitalized and nonhospitalized COVID-19 patients. Median concentration and 95% confidence intervals and statistical results (adjusted P values of Tukey multiple comparison) between the groups are shown. B , Laboratory-confirmed viral infections (see Supplementary Table 2 ) and concentration data of ILI patients are shown to confirm that the assay discriminates SARS-CoV-2–specific antibodies from antibodies induced by various laboratory-confirmed viral infections. Abbreviations: AU, arbitrary unit; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleoprotein; non-HCoV, noncoronavirus; RBD, receptor binding domain; RSV, respiratory syncytial virus; S1, spike protein subunit 1.
    Figure Legend Snippet: Discrimination of COVID-19 patients with varying severity from a cross-sectional population panel and ILI patients. A , Individuals from the cross-sectional panel aged 3–90 years (n = 224), ILI patients with noncoronavirus (n = 75), and non-SARS-CoV-2 seasonal coronavirus-infected ILI patients (n = 109) were compared to hospitalized and nonhospitalized COVID-19 patients. Median concentration and 95% confidence intervals and statistical results (adjusted P values of Tukey multiple comparison) between the groups are shown. B , Laboratory-confirmed viral infections (see Supplementary Table 2 ) and concentration data of ILI patients are shown to confirm that the assay discriminates SARS-CoV-2–specific antibodies from antibodies induced by various laboratory-confirmed viral infections. Abbreviations: AU, arbitrary unit; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleoprotein; non-HCoV, noncoronavirus; RBD, receptor binding domain; RSV, respiratory syncytial virus; S1, spike protein subunit 1.

    Techniques Used: Infection, Concentration Assay, Binding Assay

    21) Product Images from "Aerosol Exposure of Cynomolgus Macaques to SARS-CoV-2 Results in More Severe Pathology than Existing Models"

    Article Title: Aerosol Exposure of Cynomolgus Macaques to SARS-CoV-2 Results in More Severe Pathology than Existing Models

    Journal: bioRxiv

    doi: 10.1101/2021.04.27.441510

    Comparison of significant fever responses during SARS-CoV-2 infection of NHPs. A) Fever-hr B) Maximum daily temperature elevation C) Daily percent TEsig i.e. percent of the 24-hr daily time period when body temperatures were significantly elevated. All data are shown as the group mean ± SEM. A non-parametric one-way ANOVA (Kruskal-Wallis test) was performed for each day.
    Figure Legend Snippet: Comparison of significant fever responses during SARS-CoV-2 infection of NHPs. A) Fever-hr B) Maximum daily temperature elevation C) Daily percent TEsig i.e. percent of the 24-hr daily time period when body temperatures were significantly elevated. All data are shown as the group mean ± SEM. A non-parametric one-way ANOVA (Kruskal-Wallis test) was performed for each day.

    Techniques Used: Infection

    Comparison of human COVID-19 and SARS-CoV-2 disease in NHPs. The size of the NHPs shows the relative response for that group.
    Figure Legend Snippet: Comparison of human COVID-19 and SARS-CoV-2 disease in NHPs. The size of the NHPs shows the relative response for that group.

    Techniques Used:

    Respiratory pathology in CM exposed to SARS-CoV-2 by the AE route. Images shown from animal CM AE 1. A. Thoracic cavity: Multiple fibrinous adhesions between left cranial and caudal lung lobes and the thoracic wall. B. Lung, left caudal lung lobe, peripheral: Multifocal, moderate, interstitial pneumonia with pleural fibrin, 2x, H E. C. Lung, higher magnification of boxed area in B: Multifocal moderate lymphohistiocytic interstitial pneumonia with type II pneumocyte hyperplasia, intra-alveolar fibrin deposition (black arrow), septal fibrosis, pleuritis and pleural fibrin, 10x, H E. D. Lung: ISH positive in areas of inflammation, 20x, RNA probe for SARS-CoV-2.
    Figure Legend Snippet: Respiratory pathology in CM exposed to SARS-CoV-2 by the AE route. Images shown from animal CM AE 1. A. Thoracic cavity: Multiple fibrinous adhesions between left cranial and caudal lung lobes and the thoracic wall. B. Lung, left caudal lung lobe, peripheral: Multifocal, moderate, interstitial pneumonia with pleural fibrin, 2x, H E. C. Lung, higher magnification of boxed area in B: Multifocal moderate lymphohistiocytic interstitial pneumonia with type II pneumocyte hyperplasia, intra-alveolar fibrin deposition (black arrow), septal fibrosis, pleuritis and pleural fibrin, 10x, H E. D. Lung: ISH positive in areas of inflammation, 20x, RNA probe for SARS-CoV-2.

    Techniques Used: In Situ Hybridization

    Evidence of clinical disease in radiographs from SARS-CoV-2-infected NHPs. A. Representative radiographs are shown from each group. All images are ventrodorsal. The white dashed lines shown in the Day 6 PE images from RM IT/IN, CM AE, and CM IT/IN outline infiltrates and opacity present in those images; no dashed lines are shown in RM AE due to the absence of lesions. Baseline radiographs were obtained prior to challenge (Day -2). B. Average radiographic scores per group over the course of the study. Box-and-whisker plot showing the range of the data. A non-parametric one-way ANOVA (Kruskal-Wallis test) was performed for each day.
    Figure Legend Snippet: Evidence of clinical disease in radiographs from SARS-CoV-2-infected NHPs. A. Representative radiographs are shown from each group. All images are ventrodorsal. The white dashed lines shown in the Day 6 PE images from RM IT/IN, CM AE, and CM IT/IN outline infiltrates and opacity present in those images; no dashed lines are shown in RM AE due to the absence of lesions. Baseline radiographs were obtained prior to challenge (Day -2). B. Average radiographic scores per group over the course of the study. Box-and-whisker plot showing the range of the data. A non-parametric one-way ANOVA (Kruskal-Wallis test) was performed for each day.

    Techniques Used: Infection, Whisker Assay

    Clinical pathology and serological responses to SARS-CoV-2 exposure. A-C. Percent change from baseline for A) platelets, B) CRP, and C) CK over the course of the study. The dashed line at y=0 represents the baseline, which is defined as the average of the results from Days -2 and 0. Values greater than or equal to a 25% change from baseline (dashed lines in A) were considered noteworthy. Data are shown as mean ± SEM. D-E. IgG (D) and IgA (E) results from the Euroimmun SARS-CoV-2 ELISA kits. Results are shown as the signal to noise ratio. The bottom and top dotted lines represent the assay cutoffs for negative and positive results respectively, with the gray shaded region representing indeterminate results. The mean is denoted by a colored dash for each group F) PRNT80 geometric mean titer (GMT) for RM versus CM in serum obtained at the terminal time point (Day 9/10 PE), with each animal represented as a dot and color corresponding to experimental group. The dotted line represents the assay cutoff for positive results (PRNT80 of 20). Error bars represent the geometric SD. Unpaired t test was performed, with * p
    Figure Legend Snippet: Clinical pathology and serological responses to SARS-CoV-2 exposure. A-C. Percent change from baseline for A) platelets, B) CRP, and C) CK over the course of the study. The dashed line at y=0 represents the baseline, which is defined as the average of the results from Days -2 and 0. Values greater than or equal to a 25% change from baseline (dashed lines in A) were considered noteworthy. Data are shown as mean ± SEM. D-E. IgG (D) and IgA (E) results from the Euroimmun SARS-CoV-2 ELISA kits. Results are shown as the signal to noise ratio. The bottom and top dotted lines represent the assay cutoffs for negative and positive results respectively, with the gray shaded region representing indeterminate results. The mean is denoted by a colored dash for each group F) PRNT80 geometric mean titer (GMT) for RM versus CM in serum obtained at the terminal time point (Day 9/10 PE), with each animal represented as a dot and color corresponding to experimental group. The dotted line represents the assay cutoff for positive results (PRNT80 of 20). Error bars represent the geometric SD. Unpaired t test was performed, with * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Infection of RM and CM with SARS-CoV-2 by AE or IT/IN exposure. A. Study design. Red droplets denote phlebotomy days. Baseline sampling points are represented by green diamonds, while dark blue represents post-infection sampling points. The blue gradient provides a visual representation of the peak responses observed from each assay/measurement. B-D. Detection of viral RNA by qRT-PCR (B), infectious virus by plaque assay (C), and subgenomic RNA by real-time RT-PCR in NP swabs. Data is shown as the group mean ± SEM.
    Figure Legend Snippet: Infection of RM and CM with SARS-CoV-2 by AE or IT/IN exposure. A. Study design. Red droplets denote phlebotomy days. Baseline sampling points are represented by green diamonds, while dark blue represents post-infection sampling points. The blue gradient provides a visual representation of the peak responses observed from each assay/measurement. B-D. Detection of viral RNA by qRT-PCR (B), infectious virus by plaque assay (C), and subgenomic RNA by real-time RT-PCR in NP swabs. Data is shown as the group mean ± SEM.

    Techniques Used: Infection, Sampling, Quantitative RT-PCR, Plaque Assay

    The cellular targets of SARS-CoV-2 and inflammatory infiltrates in the lungs of CM AE NHPs. A. The spike (S) protein of SARS-CoV-2 (green) was detected in pneumocytes (red) labelled by anti-Pan-cytokeratin antibody (red). B. The nucleoprotein (NP) of SARS-CoV-2 (green) was detected in CD68+ macrophages (red). C–H. In comparison to uninfected control lung tissue, CD3+ T cells (green in C and D), CD45+ leukocytes (red in C and D), CD68+ macrophages (red in E and F), and Ki67+ proliferating cells (green in E and F), MPO+ polymorphonuclear cells (neutrophils, eosinophils, and basophils, green in G and H), expression of the type 1 interferon-induced GTP-binding protein Mx1 (a readout of type 1 interferon response, red in G and H) in the lungs of CM AE NHPs exposed to SARS-CoV-2. I–J. Alveolar spaces were congested by CD68+ macrophages (green, arrows) in some areas of the lungs of CM AE animals. Lung epithelium is stained outlined by anti-Pan-cytokeratin antibody (red). Nucleus was stained by DAPI (blue).
    Figure Legend Snippet: The cellular targets of SARS-CoV-2 and inflammatory infiltrates in the lungs of CM AE NHPs. A. The spike (S) protein of SARS-CoV-2 (green) was detected in pneumocytes (red) labelled by anti-Pan-cytokeratin antibody (red). B. The nucleoprotein (NP) of SARS-CoV-2 (green) was detected in CD68+ macrophages (red). C–H. In comparison to uninfected control lung tissue, CD3+ T cells (green in C and D), CD45+ leukocytes (red in C and D), CD68+ macrophages (red in E and F), and Ki67+ proliferating cells (green in E and F), MPO+ polymorphonuclear cells (neutrophils, eosinophils, and basophils, green in G and H), expression of the type 1 interferon-induced GTP-binding protein Mx1 (a readout of type 1 interferon response, red in G and H) in the lungs of CM AE NHPs exposed to SARS-CoV-2. I–J. Alveolar spaces were congested by CD68+ macrophages (green, arrows) in some areas of the lungs of CM AE animals. Lung epithelium is stained outlined by anti-Pan-cytokeratin antibody (red). Nucleus was stained by DAPI (blue).

    Techniques Used: Expressing, Binding Assay, Staining

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    Article Snippet: Antibody responses in plasma and saliva using Suspension Multiplex Immunoassay (SMIA) MagPlex-C microspheres (Luminex Corp., Austin, TX, USA) were used for the coupling of antigens according to the manufacturer’s protocol as previously described ( ). .. Briefly, 200 µl of the stock microsphere solution (1.25 × 107 beads/ml) were coupled by adding 10 μg of recombinant SARS-CoV-2 Spike protein RBD His-Tag (#40592-V08B, SinoBiological Inc., USA). .. After the coupling, beads were incubated in phosphate buffered saline (PBS: 0.15 M sodium chloride, 10 mM sodium phosphate, pH 7.4) containing 0.05% (v/v) Tween 20 (PBS-T) for 15 min on a rocking shaker at RT.

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    Blocking Assay:

    Article Title: A novel pseudovirus‐based mouse model of SARS-CoV-2 infection to test COVID-19 interventions
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    Incubation:

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    Sino Biological sars cov 2 spike protein
    A replication-competent <t>VSV/SARS-CoV-2</t> chimera. A . Schematic representation of the rVSV/SARS-CoV-2/GFP genome in which G-encoding sequences were replaced by SARS-CoV-2 SΔ18 coding sequences. GFP-encoding sequences were introduced between the SARS-CoV-2 SΔ18 and L open reading frames. B . Representative images of 293T/ACE2(B) cells infected with the indicated volumes of plaque purified, adapted derivatives (2E1 and 1D7) of VSV/SARS-CoV-2/GFP following passage in the same cell line. Left and center images show contents of an entire well of a 96-well plate, the right image shows expanded view of the boxed areas containing individual plaques. C . Infectivity measurements of rVSV/SARS-CoV-2/GFP virus stocks on 293T/ACE2(B) or control 293T cells, quantified by measuring % GFP-positive cells at 16h after infection. Average and standard deviation from two technical replicates is shown. D . Schematic representation of the adaptive changes acquired in rVSV/SARS-CoV-2/GFP during passage. Changes in 1D7 and 2E1 are shown in blue and red, respectively.
    Sars Cov 2 Spike Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological sars cov 2 2019 ncov spike rbd his recombinant protein covid 19 spike rbd research
    Differential DNAm analyses of PBMCs stimulated in vitro with <t>SARS-CoV-2.</t> A. Venn diagrams depicting the overlap of DMCs from the SARS-CoV-2 in vitro stimulated PBMCs. Intraindividual comparisons of differential DNAm were performed in treated vs . untreated PBMCs from four different blood donors (D1-D4) collected before the start of the COVID-19 pandemic (2014-2019). DMCs were defined as a fold change in M-value > |2|. These DMCs were further mapped to their corresponding annotated genes (DMGs, n=542). B shows results from pathway over-representation analyses in PANTHER based on the 542 DMGs originating from the SARS-CoV-2 in vitro stimulated PBMCs compared to non-stimulated PBMCs. Pathways with a nominal p-value
    Sars Cov 2 2019 Ncov Spike Rbd His Recombinant Protein Covid 19 Spike Rbd Research, supplied by Sino Biological, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A replication-competent VSV/SARS-CoV-2 chimera. A . Schematic representation of the rVSV/SARS-CoV-2/GFP genome in which G-encoding sequences were replaced by SARS-CoV-2 SΔ18 coding sequences. GFP-encoding sequences were introduced between the SARS-CoV-2 SΔ18 and L open reading frames. B . Representative images of 293T/ACE2(B) cells infected with the indicated volumes of plaque purified, adapted derivatives (2E1 and 1D7) of VSV/SARS-CoV-2/GFP following passage in the same cell line. Left and center images show contents of an entire well of a 96-well plate, the right image shows expanded view of the boxed areas containing individual plaques. C . Infectivity measurements of rVSV/SARS-CoV-2/GFP virus stocks on 293T/ACE2(B) or control 293T cells, quantified by measuring % GFP-positive cells at 16h after infection. Average and standard deviation from two technical replicates is shown. D . Schematic representation of the adaptive changes acquired in rVSV/SARS-CoV-2/GFP during passage. Changes in 1D7 and 2E1 are shown in blue and red, respectively.

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: A replication-competent VSV/SARS-CoV-2 chimera. A . Schematic representation of the rVSV/SARS-CoV-2/GFP genome in which G-encoding sequences were replaced by SARS-CoV-2 SΔ18 coding sequences. GFP-encoding sequences were introduced between the SARS-CoV-2 SΔ18 and L open reading frames. B . Representative images of 293T/ACE2(B) cells infected with the indicated volumes of plaque purified, adapted derivatives (2E1 and 1D7) of VSV/SARS-CoV-2/GFP following passage in the same cell line. Left and center images show contents of an entire well of a 96-well plate, the right image shows expanded view of the boxed areas containing individual plaques. C . Infectivity measurements of rVSV/SARS-CoV-2/GFP virus stocks on 293T/ACE2(B) or control 293T cells, quantified by measuring % GFP-positive cells at 16h after infection. Average and standard deviation from two technical replicates is shown. D . Schematic representation of the adaptive changes acquired in rVSV/SARS-CoV-2/GFP during passage. Changes in 1D7 and 2E1 are shown in blue and red, respectively.

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Infection, Purification, Standard Deviation

    Examples of neutralization of HIV-1 and VSV pseudotyped virus particles by monoclonal antibodies targeting SARS-CoV-2 S. A . Images of Huh7.5 cells following infection with rVSVΔG/NG-NanoLuc pseudotyped virus (∼10 3 IU/well) in the presence of the indicated concentrations of a human monoclonal antibody (C144) targeting SARS-CoV-2 S RBD. B . Quantification of rVSVΔG/NG-NanoLuc pseudotyped virus infection (measured by flow cytometry (% mNeonGreen positive cells, green) or by NanoLuc luciferase activity (RLU, blue) in the presence of the indicated concentrations of a human monoclonal antibody (C102) targeting SARS-CoV-2 S RBD, or a control monoclonal antibody against the Zika virus envelope glycoprotein. C . Quantification of HIV-1 NL ΔEnv-NanoLuc or CCNanoLuc/GFP pseudotyped virus infection on the indicated cell lines in the presence of the indicated concentrations of a human monoclonal antibody (C121) targeting SARS-CoV-2 S RBD Infectivity was quantified by measuring NanoLuc luciferase levels (RLU).

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: Examples of neutralization of HIV-1 and VSV pseudotyped virus particles by monoclonal antibodies targeting SARS-CoV-2 S. A . Images of Huh7.5 cells following infection with rVSVΔG/NG-NanoLuc pseudotyped virus (∼10 3 IU/well) in the presence of the indicated concentrations of a human monoclonal antibody (C144) targeting SARS-CoV-2 S RBD. B . Quantification of rVSVΔG/NG-NanoLuc pseudotyped virus infection (measured by flow cytometry (% mNeonGreen positive cells, green) or by NanoLuc luciferase activity (RLU, blue) in the presence of the indicated concentrations of a human monoclonal antibody (C102) targeting SARS-CoV-2 S RBD, or a control monoclonal antibody against the Zika virus envelope glycoprotein. C . Quantification of HIV-1 NL ΔEnv-NanoLuc or CCNanoLuc/GFP pseudotyped virus infection on the indicated cell lines in the presence of the indicated concentrations of a human monoclonal antibody (C121) targeting SARS-CoV-2 S RBD Infectivity was quantified by measuring NanoLuc luciferase levels (RLU).

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Neutralization, Infection, Flow Cytometry, Luciferase, Activity Assay

    Measurement of neutralization activity in COVID19 convalescent donor plasma. A . Plasma neutralization of SARS-CoV-2: serial 5-fold dilutions of plasma samples from convalescent donors were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using VeroE6 target cells, expressed as % infected cells by immunostaining. B . Plasma neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP-positive cells, as indicated. C . Correlation between NT 50 values for each of the 20 plasmas for each of the surrogate viruses (x-axis) and NT 50 values for the same plasmas for SARS-CoV-2 (y-axis).

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: Measurement of neutralization activity in COVID19 convalescent donor plasma. A . Plasma neutralization of SARS-CoV-2: serial 5-fold dilutions of plasma samples from convalescent donors were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using VeroE6 target cells, expressed as % infected cells by immunostaining. B . Plasma neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP-positive cells, as indicated. C . Correlation between NT 50 values for each of the 20 plasmas for each of the surrogate viruses (x-axis) and NT 50 values for the same plasmas for SARS-CoV-2 (y-axis).

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Neutralization, Activity Assay, Incubation, Infection, Immunostaining, Luciferase

    Generation of and HIV-1 pseudotype infection of ACE2-expressing cell lines. A . 293T cells were stably transduced with a lentivirus vector CSIB, expressing either wild type ACE2 or catalytically active mutant ACE2*. Following selection, cells were used as uncloned bulk populations (B) or single cell clones were isolated. Flow cytometry histograms show staining with an antibody against huACE2 (purple) or an isotype control (grey). B . HT1080 cells were stably transduced as in A and a single cell clone used throughout this study is shown, stained as in A. C . Infectivity of CCNanoLuc/GFP viruses, pseudotyped with either full length or C-terminally truncated SARS-CoV and SARS-CoV-2 S proteins on 293T/ACE2*(B) cells. Virus particles generated in the absence of an S protein (No S) were used as background controls. Infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. D . Infectivity of HIV-1 NL ΔEnv-NanoLuc in the various cell lines. Virus generated in the absence of S is used as a background control and infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. E . Same as D except that CCNanoLuc/GFP virus was used F . Effect of virus ultracentrifugation on the infectivity of HIV-1-based pseudotyped virus particles. 293T/ACE2*(B) cells were infected with equivalent doses of unconcentrated HIV-1 NL ΔEnv-NanoLuc, or the same virus that had be pelleted through 20% sucrose and then diluted to the original volume.

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: Generation of and HIV-1 pseudotype infection of ACE2-expressing cell lines. A . 293T cells were stably transduced with a lentivirus vector CSIB, expressing either wild type ACE2 or catalytically active mutant ACE2*. Following selection, cells were used as uncloned bulk populations (B) or single cell clones were isolated. Flow cytometry histograms show staining with an antibody against huACE2 (purple) or an isotype control (grey). B . HT1080 cells were stably transduced as in A and a single cell clone used throughout this study is shown, stained as in A. C . Infectivity of CCNanoLuc/GFP viruses, pseudotyped with either full length or C-terminally truncated SARS-CoV and SARS-CoV-2 S proteins on 293T/ACE2*(B) cells. Virus particles generated in the absence of an S protein (No S) were used as background controls. Infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. D . Infectivity of HIV-1 NL ΔEnv-NanoLuc in the various cell lines. Virus generated in the absence of S is used as a background control and infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. E . Same as D except that CCNanoLuc/GFP virus was used F . Effect of virus ultracentrifugation on the infectivity of HIV-1-based pseudotyped virus particles. 293T/ACE2*(B) cells were infected with equivalent doses of unconcentrated HIV-1 NL ΔEnv-NanoLuc, or the same virus that had be pelleted through 20% sucrose and then diluted to the original volume.

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Infection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Mutagenesis, Selection, Clone Assay, Isolation, Flow Cytometry, Staining, Generated, Luciferase, Activity Assay, Standard Deviation

    Measurement of neutralization potency of human monoclonal antibodies. A . Neutralization of SARS-CoV-2: the indicated concentrations of monoclonal antibodies were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using Vero E6 target cells, expressed as % infected cells, by immunostaining B . Monoclonal antibody neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP positive cells, as indicated. C . Correlation between IC 50 values for each of the 15 monoclonal antibodies for each of the surrogate viruses (x-axis) and IC 50 values for the same antibodies for SARS-CoV-2 (y-axis).

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: Measurement of neutralization potency of human monoclonal antibodies. A . Neutralization of SARS-CoV-2: the indicated concentrations of monoclonal antibodies were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using Vero E6 target cells, expressed as % infected cells, by immunostaining B . Monoclonal antibody neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP positive cells, as indicated. C . Correlation between IC 50 values for each of the 15 monoclonal antibodies for each of the surrogate viruses (x-axis) and IC 50 values for the same antibodies for SARS-CoV-2 (y-axis).

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Neutralization, Incubation, Infection, Immunostaining, Luciferase

    Two-plasmid and three-plasmid HIV-1-based pseudotyped viruses. A . Schematic representation of the modified HIV-1 NL ΔEnv-NanoLuc genome in which a deletion in env was introduced and Nef-coding sequences were replaced by those encoding a NanoLuc luciferase reporter. Infectious virus particles were generated by cotransfection of pHIV-1 NL4 ΔEnv-NanoLuc and a plasmid encoding the SARS-CoV-2 S lacking the 19 amino acids at the C-terminus of the cytoplasmic tail (SΔ19). B . Schematic representation of constructs used to generate SARS-CoV-2 S pseudotyped HIV-1-based particles in which HIV-1 NL GagPol, an HIV-1 reporter vector (pCCNanoLuc/GFP) encoding both NanoLuc luciferase and EGFP reporter and the SARS-CoV-2 SΔ19 are each expressed on separate plasmids. C . Infectivity measurements of HIV-1 NL ΔEnv-NanoLuc particles (generated using the plasmids depicted in A) on the indicated cell lines. Infectivity was quantified by measuring NanoLuc luciferase activity (Relative Light Units, RLU) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. The mean and standard deviation of two technical replicates is shown. Target cells 293T/ACE2cl.22 and HT1080/ACE2cl.14 are single-cell clones engineered to express human ACE2 (see Fig S1A ). Virus particles generated in the absence of viral envelope glycoproteins were used as background controls. D . Same as, C but viruses were generated using the 3 plasmids depicted in B. E . Infectivity meaurements of CCNanoLuc/GFP containing SARS-CoV-2 pseudotyped particles generated using plasmids depicted in B on 293ACE2*(B) cells, quantified by measuring NanoLuc luciferase activity (RLU) or GFP levels (% of GFP positive cells). Mean and standard deviation from two technical replicates is shown.

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: Two-plasmid and three-plasmid HIV-1-based pseudotyped viruses. A . Schematic representation of the modified HIV-1 NL ΔEnv-NanoLuc genome in which a deletion in env was introduced and Nef-coding sequences were replaced by those encoding a NanoLuc luciferase reporter. Infectious virus particles were generated by cotransfection of pHIV-1 NL4 ΔEnv-NanoLuc and a plasmid encoding the SARS-CoV-2 S lacking the 19 amino acids at the C-terminus of the cytoplasmic tail (SΔ19). B . Schematic representation of constructs used to generate SARS-CoV-2 S pseudotyped HIV-1-based particles in which HIV-1 NL GagPol, an HIV-1 reporter vector (pCCNanoLuc/GFP) encoding both NanoLuc luciferase and EGFP reporter and the SARS-CoV-2 SΔ19 are each expressed on separate plasmids. C . Infectivity measurements of HIV-1 NL ΔEnv-NanoLuc particles (generated using the plasmids depicted in A) on the indicated cell lines. Infectivity was quantified by measuring NanoLuc luciferase activity (Relative Light Units, RLU) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. The mean and standard deviation of two technical replicates is shown. Target cells 293T/ACE2cl.22 and HT1080/ACE2cl.14 are single-cell clones engineered to express human ACE2 (see Fig S1A ). Virus particles generated in the absence of viral envelope glycoproteins were used as background controls. D . Same as, C but viruses were generated using the 3 plasmids depicted in B. E . Infectivity meaurements of CCNanoLuc/GFP containing SARS-CoV-2 pseudotyped particles generated using plasmids depicted in B on 293ACE2*(B) cells, quantified by measuring NanoLuc luciferase activity (RLU) or GFP levels (% of GFP positive cells). Mean and standard deviation from two technical replicates is shown.

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Plasmid Preparation, Modification, Luciferase, Generated, Cotransfection, Construct, Infection, Activity Assay, Standard Deviation, Clone Assay

    Differential DNAm analyses of PBMCs stimulated in vitro with SARS-CoV-2. A. Venn diagrams depicting the overlap of DMCs from the SARS-CoV-2 in vitro stimulated PBMCs. Intraindividual comparisons of differential DNAm were performed in treated vs . untreated PBMCs from four different blood donors (D1-D4) collected before the start of the COVID-19 pandemic (2014-2019). DMCs were defined as a fold change in M-value > |2|. These DMCs were further mapped to their corresponding annotated genes (DMGs, n=542). B shows results from pathway over-representation analyses in PANTHER based on the 542 DMGs originating from the SARS-CoV-2 in vitro stimulated PBMCs compared to non-stimulated PBMCs. Pathways with a nominal p-value

    Journal: medRxiv

    Article Title: Mild SARS-CoV-2 infection modifies DNA methylation of peripheral blood mononuclear cells from COVID-19 convalescents

    doi: 10.1101/2021.07.05.21260014

    Figure Lengend Snippet: Differential DNAm analyses of PBMCs stimulated in vitro with SARS-CoV-2. A. Venn diagrams depicting the overlap of DMCs from the SARS-CoV-2 in vitro stimulated PBMCs. Intraindividual comparisons of differential DNAm were performed in treated vs . untreated PBMCs from four different blood donors (D1-D4) collected before the start of the COVID-19 pandemic (2014-2019). DMCs were defined as a fold change in M-value > |2|. These DMCs were further mapped to their corresponding annotated genes (DMGs, n=542). B shows results from pathway over-representation analyses in PANTHER based on the 542 DMGs originating from the SARS-CoV-2 in vitro stimulated PBMCs compared to non-stimulated PBMCs. Pathways with a nominal p-value

    Article Snippet: Briefly, 200 µl of the stock microsphere solution (1.25 × 107 beads/ml) were coupled by adding 10 μg of recombinant SARS-CoV-2 Spike protein RBD His-Tag (#40592-V08B, SinoBiological Inc., USA).

    Techniques: In Vitro

    Outline of included participants, experimental procedures as well as statistical and bioinformatic approaches utilised in the present study. CC19 – convalescent COVID-19, Con – non-infected control, DMG – differentially methylated gene, Pre20 – Pre-2020 non-infected control, SFT – symptom-free individuals with SARS-CoV-2-specific T cell response, SMIA – suspension multiplex immunoassay.

    Journal: medRxiv

    Article Title: Mild SARS-CoV-2 infection modifies DNA methylation of peripheral blood mononuclear cells from COVID-19 convalescents

    doi: 10.1101/2021.07.05.21260014

    Figure Lengend Snippet: Outline of included participants, experimental procedures as well as statistical and bioinformatic approaches utilised in the present study. CC19 – convalescent COVID-19, Con – non-infected control, DMG – differentially methylated gene, Pre20 – Pre-2020 non-infected control, SFT – symptom-free individuals with SARS-CoV-2-specific T cell response, SMIA – suspension multiplex immunoassay.

    Article Snippet: Briefly, 200 µl of the stock microsphere solution (1.25 × 107 beads/ml) were coupled by adding 10 μg of recombinant SARS-CoV-2 Spike protein RBD His-Tag (#40592-V08B, SinoBiological Inc., USA).

    Techniques: Infection, Methylation, Multiplex Assay