sars cov 2 s protein  (Sino Biological)


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    Name:
    SARS CoV Spike S1 S2 ECD His Recombinant Protein
    Description:
    A DNA sequence encoding the full lenght ECD of SARS CoV isolate Tor2 spike NP 828851 1 Met1 Pro1195 was expressed with a C terminal polyhistidine tag
    Catalog Number:
    40634-V08B
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus s1 Protein SARS, coronavirus s2 Protein SARS, coronavirus spike Protein SARS, cov spike Protein SARS, ncov RBD Protein SARS, ncov s1 Protein SARS, ncov s2 Protein SARS, ncov spike Protein SARS, novel coronavirus RBD Protein SARS, novel coronavirus s1 Protein SARS, novel coronavirus s2 Protein SARS, novel coronavirus spike Protein SARS, RBD Protein SARS, S1 Protein SARS, s2 Protein SARS, Spike RBD Protein SARS
    Host:
    Baculovirus-Insect Cells
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    Structured Review

    Sino Biological sars cov 2 s protein
    Anti-severe acute respiratory syndrome virus 2 <t>(SARS-CoV-2)</t> effects of aprotinin and SERPINA1/alpha-1 antitrypsin. ( A ) Concentration-dependent effects of aprotinin and SERPINA1/alpha-1 antitrypsin on SARS-CoV-2-induced cytopathogenic effect (CPE) formation determined 48 h post-infection in Caco2 cells infected at a multiplicity of infection (MOI) of 0.01 with the three different SARS-CoV-2 isolates. The viability of the Caco2 cells was 84.3 ± 2.7% relative to the untreated control in the presence of 20 µM of aprotinin. ( B ) Immunostaining for the SARS-CoV-2 S protein in aprotinin- and SERPINA1/alpha-1 antitrypsin-treated Caco2 cells infected at an MOI of 0.01 with the three different SARS-CoV-2 isolates as determined 48 h post-infection. The protease inhibitors were tested at four concentrations in 1:4 dilution steps ranging from 20 to 0.3125 µM. A quantification is provided in Figure S1 . ( C ) Copy numbers of genomic RNA in Caco2 cells infected with different SARS-CoV-2 isolates (MOI of 0.01) in response to treatment with aprotinin or SERPINA1/alpha-1 antitrypsin as determined 48 h post-infection. FFM1, 1/Human/2020/Frankfurt; FFM2, 2/Human/2020/Frankfurt; FFM6, 6/Human/2020/Frankfurt.
    A DNA sequence encoding the full lenght ECD of SARS CoV isolate Tor2 spike NP 828851 1 Met1 Pro1195 was expressed with a C terminal polyhistidine tag
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    sars cov 2 s protein - by Bioz Stars, 2021-04
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    Images

    1) Product Images from "Aprotinin Inhibits SARS-CoV-2 Replication"

    Article Title: Aprotinin Inhibits SARS-CoV-2 Replication

    Journal: Cells

    doi: 10.3390/cells9112377

    Anti-severe acute respiratory syndrome virus 2 (SARS-CoV-2) effects of aprotinin and SERPINA1/alpha-1 antitrypsin. ( A ) Concentration-dependent effects of aprotinin and SERPINA1/alpha-1 antitrypsin on SARS-CoV-2-induced cytopathogenic effect (CPE) formation determined 48 h post-infection in Caco2 cells infected at a multiplicity of infection (MOI) of 0.01 with the three different SARS-CoV-2 isolates. The viability of the Caco2 cells was 84.3 ± 2.7% relative to the untreated control in the presence of 20 µM of aprotinin. ( B ) Immunostaining for the SARS-CoV-2 S protein in aprotinin- and SERPINA1/alpha-1 antitrypsin-treated Caco2 cells infected at an MOI of 0.01 with the three different SARS-CoV-2 isolates as determined 48 h post-infection. The protease inhibitors were tested at four concentrations in 1:4 dilution steps ranging from 20 to 0.3125 µM. A quantification is provided in Figure S1 . ( C ) Copy numbers of genomic RNA in Caco2 cells infected with different SARS-CoV-2 isolates (MOI of 0.01) in response to treatment with aprotinin or SERPINA1/alpha-1 antitrypsin as determined 48 h post-infection. FFM1, 1/Human/2020/Frankfurt; FFM2, 2/Human/2020/Frankfurt; FFM6, 6/Human/2020/Frankfurt.
    Figure Legend Snippet: Anti-severe acute respiratory syndrome virus 2 (SARS-CoV-2) effects of aprotinin and SERPINA1/alpha-1 antitrypsin. ( A ) Concentration-dependent effects of aprotinin and SERPINA1/alpha-1 antitrypsin on SARS-CoV-2-induced cytopathogenic effect (CPE) formation determined 48 h post-infection in Caco2 cells infected at a multiplicity of infection (MOI) of 0.01 with the three different SARS-CoV-2 isolates. The viability of the Caco2 cells was 84.3 ± 2.7% relative to the untreated control in the presence of 20 µM of aprotinin. ( B ) Immunostaining for the SARS-CoV-2 S protein in aprotinin- and SERPINA1/alpha-1 antitrypsin-treated Caco2 cells infected at an MOI of 0.01 with the three different SARS-CoV-2 isolates as determined 48 h post-infection. The protease inhibitors were tested at four concentrations in 1:4 dilution steps ranging from 20 to 0.3125 µM. A quantification is provided in Figure S1 . ( C ) Copy numbers of genomic RNA in Caco2 cells infected with different SARS-CoV-2 isolates (MOI of 0.01) in response to treatment with aprotinin or SERPINA1/alpha-1 antitrypsin as determined 48 h post-infection. FFM1, 1/Human/2020/Frankfurt; FFM2, 2/Human/2020/Frankfurt; FFM6, 6/Human/2020/Frankfurt.

    Techniques Used: Concentration Assay, Infection, Immunostaining

    2) Product Images from "Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development"

    Article Title: Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-021-00558-8

    CTSL cleaves the SARS-CoV-2 spike (S) protein, and this cleavage promotes cell–cell fusion. a Overview of the SARS-CoV-1 and SARS-CoV-2 S1/S2 cleavage sites. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are units of the S2 subunit that function in membrane fusion. b Analysis of CTSL-mediated S-protein cleavage. Purified SARS-CoV-1 or SARS-CoV-2 S protein was incubated in the presence or absence (assay buffer, pH = 5.5) of CTSL (2 or 10 μg/ml in assay buffer, pH = 5 .5) at 37 °C for 1 h. The reaction system of 2 μg/ml CTSL was further supplemented with CTSL inhibitors (20 μM E64d or 20 μM SID 26681509), as indicated. Proteins were subjected to SDS-PAGE and detected by silver staining. Representative data from three independent experiments are shown. c Syncytium-formation assay: Huh7 cells were untransfected (Null) or transfected with plasmid to express the SARS-CoV-2 S protein. Cells were incubated in the presence or absence (PBS, pH = 7.4) of trypsin (2 μg/ml in PBS, pH = 7.4) or in the presence or absence (PBS, pH = 5.8) of CTSL (2 or 4 μg/ml in PBS, pH = 5.8) for 20 min. Images were acquired after an additional 16 h incubation in the medium. (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from seven independent experiments are shown. d Quantitative analysis of syncytia in panel c . n = 7. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± s.e.m. values. * P
    Figure Legend Snippet: CTSL cleaves the SARS-CoV-2 spike (S) protein, and this cleavage promotes cell–cell fusion. a Overview of the SARS-CoV-1 and SARS-CoV-2 S1/S2 cleavage sites. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are units of the S2 subunit that function in membrane fusion. b Analysis of CTSL-mediated S-protein cleavage. Purified SARS-CoV-1 or SARS-CoV-2 S protein was incubated in the presence or absence (assay buffer, pH = 5.5) of CTSL (2 or 10 μg/ml in assay buffer, pH = 5 .5) at 37 °C for 1 h. The reaction system of 2 μg/ml CTSL was further supplemented with CTSL inhibitors (20 μM E64d or 20 μM SID 26681509), as indicated. Proteins were subjected to SDS-PAGE and detected by silver staining. Representative data from three independent experiments are shown. c Syncytium-formation assay: Huh7 cells were untransfected (Null) or transfected with plasmid to express the SARS-CoV-2 S protein. Cells were incubated in the presence or absence (PBS, pH = 7.4) of trypsin (2 μg/ml in PBS, pH = 7.4) or in the presence or absence (PBS, pH = 5.8) of CTSL (2 or 4 μg/ml in PBS, pH = 5.8) for 20 min. Images were acquired after an additional 16 h incubation in the medium. (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from seven independent experiments are shown. d Quantitative analysis of syncytia in panel c . n = 7. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± s.e.m. values. * P

    Techniques Used: Purification, Incubation, SDS Page, Silver Staining, Tube Formation Assay, Transfection, Plasmid Preparation

    3) Product Images from "Generation of Chicken IgY against SARS-COV-2 Spike Protein and Epitope Mapping"

    Article Title: Generation of Chicken IgY against SARS-COV-2 Spike Protein and Epitope Mapping

    Journal: Journal of Immunology Research

    doi: 10.1155/2020/9465398

    Signal analysis of IgY-C against the SARS-CoV-2 Proteome Microarray. A peptide microarray was performed with IgY-C (10 μ g/ml), and the resulting fluorescence intensities were plotted against sequential peptides covering SARS-CoV-2 S protein from N- to C-terminus. The fluorescence intensity plot of IgY-S (10 μ g/ml) was incorporated for comparison. Weak signal peaks from peptide spots not of “epitope signal pattern” can be seen, and some of these peptides contain an N-terminal TD motif. Signal peaks corresponding to motif EIL are present for both IgY-S and IgY-C.
    Figure Legend Snippet: Signal analysis of IgY-C against the SARS-CoV-2 Proteome Microarray. A peptide microarray was performed with IgY-C (10 μ g/ml), and the resulting fluorescence intensities were plotted against sequential peptides covering SARS-CoV-2 S protein from N- to C-terminus. The fluorescence intensity plot of IgY-S (10 μ g/ml) was incorporated for comparison. Weak signal peaks from peptide spots not of “epitope signal pattern” can be seen, and some of these peptides contain an N-terminal TD motif. Signal peaks corresponding to motif EIL are present for both IgY-S and IgY-C.

    Techniques Used: Microarray, Peptide Microarray, Fluorescence

    IgY-S epitope mapping against the SARS-CoV Antigen Microarray. Peptide microarrays were performed with IgY-S at 1 μ g/ml and 10 μ g/ml, and the resulting fluorescence intensities were plotted in relation to sequential peptides covering SARS-CoV-2 S protein from N- to C-terminus. Fluorescence intensity peaks corresponding to the consensus motifs IVAYTMSLG and VDLGDISGI as well as an EIL-like motif are indicated. Weak signal peaks from peptide spots not of “epitope signal pattern” are also shown.
    Figure Legend Snippet: IgY-S epitope mapping against the SARS-CoV Antigen Microarray. Peptide microarrays were performed with IgY-S at 1 μ g/ml and 10 μ g/ml, and the resulting fluorescence intensities were plotted in relation to sequential peptides covering SARS-CoV-2 S protein from N- to C-terminus. Fluorescence intensity peaks corresponding to the consensus motifs IVAYTMSLG and VDLGDISGI as well as an EIL-like motif are indicated. Weak signal peaks from peptide spots not of “epitope signal pattern” are also shown.

    Techniques Used: Microarray, Fluorescence

    Schematic illustrations of SARS-Cov-2 S protein. (a) Locations of IgY-S epitopes in SARS-Cov-2 S. Locations of the five identified IgY-S epitopes are indicated by arrows. SS: signal sequence; NTD: N-terminal domain; RBD: receptor-binding domain; SD1: subdomain 1; SD2: subdomain 2; S1/S2: S1/S2 cleavage region; S2′: S2′ cleavage region; FP: fusion peptide; HR1: heptad repeat 1; CH: central helix; CD: connector domain; HR2: heptad repeat 2. (b) 3D view of SARS-CoV-2 S trimer based on PDB 6VXX [ 37 ], constructed by UCSF Chimera. The S1/S2 cleavage site is indicated by an arrowhead. The yellow strand represents a peptide containing epitope SIIAYTMSL.
    Figure Legend Snippet: Schematic illustrations of SARS-Cov-2 S protein. (a) Locations of IgY-S epitopes in SARS-Cov-2 S. Locations of the five identified IgY-S epitopes are indicated by arrows. SS: signal sequence; NTD: N-terminal domain; RBD: receptor-binding domain; SD1: subdomain 1; SD2: subdomain 2; S1/S2: S1/S2 cleavage region; S2′: S2′ cleavage region; FP: fusion peptide; HR1: heptad repeat 1; CH: central helix; CD: connector domain; HR2: heptad repeat 2. (b) 3D view of SARS-CoV-2 S trimer based on PDB 6VXX [ 37 ], constructed by UCSF Chimera. The S1/S2 cleavage site is indicated by an arrowhead. The yellow strand represents a peptide containing epitope SIIAYTMSL.

    Techniques Used: Sequencing, Binding Assay, Construct

    ELISA assay of IgY-S immunoreactivity. ELISA graph showing IgY-S immunoreactivity against SARS-CoV-2 S. Plates coated with recombinant extracellular S protein of SARS-COV-2 were incubated with IgY-S or IgY-C (control) at a series of dilutions (1 mg/ml of stock concentration for both antibodies), and optical density was read at 450 nm (OD 450 nm). The results were plotted as OD 450 nm readings versus IgY dilutions. Mean ± SD ( n = 3) are presented.
    Figure Legend Snippet: ELISA assay of IgY-S immunoreactivity. ELISA graph showing IgY-S immunoreactivity against SARS-CoV-2 S. Plates coated with recombinant extracellular S protein of SARS-COV-2 were incubated with IgY-S or IgY-C (control) at a series of dilutions (1 mg/ml of stock concentration for both antibodies), and optical density was read at 450 nm (OD 450 nm). The results were plotted as OD 450 nm readings versus IgY dilutions. Mean ± SD ( n = 3) are presented.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Concentration Assay

    IgY-S epitope mapping against the SARS-CoV-2 Proteome Microarray. Peptide microarrays were performed with IgY-S at 1 μ g/ml and 10 μ g/ml, and stained microarrays were scanned, and signals were collected. Fluorescence intensities of peptide spots were generated with the PepSlide Analyzer software and plotted against sequential peptides covering SARS-CoV-2 S protein from N- to C-terminus. Fluorescence intensity peaks of peptides containing the consensus motifs LDPLSET, SIIAYTMSL, QIYKTPP, AIHADQL, DLGDISGIN, and EIL are indicated. Weak signal peaks from peptide spots not of “epitope signal pattern” are also shown.
    Figure Legend Snippet: IgY-S epitope mapping against the SARS-CoV-2 Proteome Microarray. Peptide microarrays were performed with IgY-S at 1 μ g/ml and 10 μ g/ml, and stained microarrays were scanned, and signals were collected. Fluorescence intensities of peptide spots were generated with the PepSlide Analyzer software and plotted against sequential peptides covering SARS-CoV-2 S protein from N- to C-terminus. Fluorescence intensity peaks of peptides containing the consensus motifs LDPLSET, SIIAYTMSL, QIYKTPP, AIHADQL, DLGDISGIN, and EIL are indicated. Weak signal peaks from peptide spots not of “epitope signal pattern” are also shown.

    Techniques Used: Microarray, Staining, Fluorescence, Generated, Software

    4) Product Images from "An Engineered Antibody with Broad Protective Efficacy in Murine Models of SARS and COVID-19"

    Article Title: An Engineered Antibody with Broad Protective Efficacy in Murine Models of SARS and COVID-19

    Journal: bioRxiv

    doi: 10.1101/2020.11.17.385500

    Competitive binding assays. (A) Structure of the SARS-CoV-2 RBD (white) bound to hACE2 (red) (PBD ID: 6M0J) docked with SARS-CoV-2 mAbs S309 (orange, PDB ID: 6WPS) and CR3022 (cyan, PBD ID: 6W41). (B) S309 and CR3022 sandwich binning sensorgram. The traces depict the association (0-180 seconds) of SARS-CoV-2 S to S309 captured on the probe followed by exposure (180-360 seconds) to CR3022. (C) ADG-2 and hACE2-Fc (left), S309 (middle), and CR3022 (right) sandwich binning sensorgrams. The traces depict the association (0-180 seconds) of SARS-CoV-2 S to ADG-2 captured on the probe followed by exposure (180-360 seconds) to hACE2-Fc, S309 IgG, or CR3022 IgG. Additional binding of the competitor protein indicates an unoccupied epitope (non-competitor), while no binding by the competitor protein indicates blocking (competitor) of the epitope by the IgG.
    Figure Legend Snippet: Competitive binding assays. (A) Structure of the SARS-CoV-2 RBD (white) bound to hACE2 (red) (PBD ID: 6M0J) docked with SARS-CoV-2 mAbs S309 (orange, PDB ID: 6WPS) and CR3022 (cyan, PBD ID: 6W41). (B) S309 and CR3022 sandwich binning sensorgram. The traces depict the association (0-180 seconds) of SARS-CoV-2 S to S309 captured on the probe followed by exposure (180-360 seconds) to CR3022. (C) ADG-2 and hACE2-Fc (left), S309 (middle), and CR3022 (right) sandwich binning sensorgrams. The traces depict the association (0-180 seconds) of SARS-CoV-2 S to ADG-2 captured on the probe followed by exposure (180-360 seconds) to hACE2-Fc, S309 IgG, or CR3022 IgG. Additional binding of the competitor protein indicates an unoccupied epitope (non-competitor), while no binding by the competitor protein indicates blocking (competitor) of the epitope by the IgG.

    Techniques Used: Binding Assay, Blocking Assay

    5) Product Images from "Development of humanized tri-specific nanobodies with potent neutralization for SARS-CoV-2"

    Article Title: Development of humanized tri-specific nanobodies with potent neutralization for SARS-CoV-2

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-74761-y

    Tri-specific VHH-Fcs show potent S1 RBD binding and S/ACE2 blocking activity, and favorable developability features. ( a ) Binding of multi-specific and monoclonal VHH-Fcs to SARS-CoV-2 S1 protein at different concentrations was assessed in duplicates using an ELISA method. The binding signal is based on fluorescence, indicated as Relative Fluorescence Units (RFU). Error bars represent standard deviation. The graph was generated by the Prism (GraphPad) software (Prism version 8.4.3. https://www.graphpad.com/scientific-software/prism/ . Requires permission to be used). ( b , c ) Binding kinetic graphs for tri-specific VHH-Fcs were obtained by biolayer interferometry (Gator). B and C represent the graphs for 1B-3F-2A-Fc and 3F-1B-2A-Fc, respectively. ( d ) Blocking of SARS-CoV-2 S/ACE2 interaction by multi-specific and monoclonal VHH-Fcs at different concentrations was assessed in duplicates using an ELISA method. Percent inhibition was calculated based on the blocking signal in RFU for each VHH-Fc treatment. Error bars represent standard deviation. The graph was generated by the Prism (GraphPad) software (Prism version 8.4.3. https://www.graphpad.com/scientific-software/prism/ . Requires permission to be used). ( e ) Developability features examining the biophysical and chemical characteristics of VHH-Fcs using DLS (Dynamic light scattering), DSF (Differential scanning fluorimetry), SLS (Static light scattering). The kinetic values were obtained by biolayer interferometry (Gator).
    Figure Legend Snippet: Tri-specific VHH-Fcs show potent S1 RBD binding and S/ACE2 blocking activity, and favorable developability features. ( a ) Binding of multi-specific and monoclonal VHH-Fcs to SARS-CoV-2 S1 protein at different concentrations was assessed in duplicates using an ELISA method. The binding signal is based on fluorescence, indicated as Relative Fluorescence Units (RFU). Error bars represent standard deviation. The graph was generated by the Prism (GraphPad) software (Prism version 8.4.3. https://www.graphpad.com/scientific-software/prism/ . Requires permission to be used). ( b , c ) Binding kinetic graphs for tri-specific VHH-Fcs were obtained by biolayer interferometry (Gator). B and C represent the graphs for 1B-3F-2A-Fc and 3F-1B-2A-Fc, respectively. ( d ) Blocking of SARS-CoV-2 S/ACE2 interaction by multi-specific and monoclonal VHH-Fcs at different concentrations was assessed in duplicates using an ELISA method. Percent inhibition was calculated based on the blocking signal in RFU for each VHH-Fc treatment. Error bars represent standard deviation. The graph was generated by the Prism (GraphPad) software (Prism version 8.4.3. https://www.graphpad.com/scientific-software/prism/ . Requires permission to be used). ( e ) Developability features examining the biophysical and chemical characteristics of VHH-Fcs using DLS (Dynamic light scattering), DSF (Differential scanning fluorimetry), SLS (Static light scattering). The kinetic values were obtained by biolayer interferometry (Gator).

    Techniques Used: Binding Assay, Blocking Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Standard Deviation, Generated, Software, Inhibition

    6) Product Images from "Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses"

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

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20201181

    VSV-based SARS-CoV-2 pseudotyped viruses . (A) Schematic representation of the rVSVΔG/NG-NanoLuc genome in which G-coding sequences were replaced by an mNeonGreen-2A-NanoLuc luciferase reporter cassette. Infectious virus particles were generated by passaging G complemented rVSVΔG/NG-NanoLuc virus stocks through 293T cells transfected with a plasmid encoding SARS-CoV-2 SΔ19. (B) Infectivity of pseudotyped rVSVΔG/NG-NanoLuc particles on Huh7.5 cells was quantified by measuring luciferase activity (RLU) or the percentage of GFP-positive cells. Mean and range from two technical replicates are plotted. Virus particles generated by passage through cells that were not transfected with SARS-CoV-2 S were used as a control. (C) NanoLuc luciferase activity (RLU) in Huh7.5 cells measured at various times after infection with pseudotyped rVSVΔG/NG-NanoLuc particles. Mean and range from two technical replicates are plotted. (D) Infectivity of pseudotyped rVSVΔG/NG-NanoLuc particles on the indicated cell lines. Infectivity was quantified by measuring NanoLuc luciferase activity (RLU) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. Mean and range deviation from two technical replicates are shown.
    Figure Legend Snippet: VSV-based SARS-CoV-2 pseudotyped viruses . (A) Schematic representation of the rVSVΔG/NG-NanoLuc genome in which G-coding sequences were replaced by an mNeonGreen-2A-NanoLuc luciferase reporter cassette. Infectious virus particles were generated by passaging G complemented rVSVΔG/NG-NanoLuc virus stocks through 293T cells transfected with a plasmid encoding SARS-CoV-2 SΔ19. (B) Infectivity of pseudotyped rVSVΔG/NG-NanoLuc particles on Huh7.5 cells was quantified by measuring luciferase activity (RLU) or the percentage of GFP-positive cells. Mean and range from two technical replicates are plotted. Virus particles generated by passage through cells that were not transfected with SARS-CoV-2 S were used as a control. (C) NanoLuc luciferase activity (RLU) in Huh7.5 cells measured at various times after infection with pseudotyped rVSVΔG/NG-NanoLuc particles. Mean and range from two technical replicates are plotted. (D) Infectivity of pseudotyped rVSVΔG/NG-NanoLuc particles on the indicated cell lines. Infectivity was quantified by measuring NanoLuc luciferase activity (RLU) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. Mean and range deviation from two technical replicates are shown.

    Techniques Used: Luciferase, Generated, Passaging, Transfection, Plasmid Preparation, Infection, Activity Assay

    Generation 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 inactive 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 (gray). (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). Mean and range 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). Mean and range from two technical replicates are 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.
    Figure Legend Snippet: Generation 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 inactive 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 (gray). (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). Mean and range 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). Mean and range from two technical replicates are 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.

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

    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. RRE, HIV-1 Rev response element; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. (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 (RLUs) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. The mean and range of two technical replicates are shown. Target cells 293T/ACE2cl.22 and HT1080/ACE2cl.14 are single-cell clones engineered to express human ACE2 (see Fig. S1 A ). 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 three plasmids depicted in B. (E) Infectivity measurements 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 (percentage of GFP-positive cells). Mean and range from two technical replicates are shown.
    Figure Legend 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. RRE, HIV-1 Rev response element; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. (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 (RLUs) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. The mean and range of two technical replicates are shown. Target cells 293T/ACE2cl.22 and HT1080/ACE2cl.14 are single-cell clones engineered to express human ACE2 (see Fig. S1 A ). 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 three plasmids depicted in B. (E) Infectivity measurements 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 (percentage of GFP-positive cells). Mean and range from two technical replicates are shown.

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

    Examples of neutralization of HIV-1 and VSV pseudotyped virus particles by mAbs 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 mAb (C144) targeting SARS-CoV-2 S RBD. Images of the entire well of a 96-well plate are shown. (B) Quantification of rVSVΔG/NG-NanoLuc pseudotyped virus infection (measured by flow cytometry (percentage of mNeonGreen-positive cells, green) or by NanoLuc luciferase activity (RLU, blue) in the presence of the indicated concentrations of a human mAb (C102) targeting SARS-CoV-2 S RBD or a control mAb against the Zika virus envelope glycoprotein. Mean and range of two technical replicates are plotted. (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 mAb (C121) targeting SARS-CoV-2 S RBD infectivity was quantified by measuring NanoLuc luciferase levels (RLU). Mean and range of two technical replicates are plotted.
    Figure Legend Snippet: Examples of neutralization of HIV-1 and VSV pseudotyped virus particles by mAbs 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 mAb (C144) targeting SARS-CoV-2 S RBD. Images of the entire well of a 96-well plate are shown. (B) Quantification of rVSVΔG/NG-NanoLuc pseudotyped virus infection (measured by flow cytometry (percentage of mNeonGreen-positive cells, green) or by NanoLuc luciferase activity (RLU, blue) in the presence of the indicated concentrations of a human mAb (C102) targeting SARS-CoV-2 S RBD or a control mAb against the Zika virus envelope glycoprotein. Mean and range of two technical replicates are plotted. (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 mAb (C121) targeting SARS-CoV-2 S RBD infectivity was quantified by measuring NanoLuc luciferase levels (RLU). Mean and range of two technical replicates are plotted.

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

    7) Product Images from "Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development"

    Article Title: Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-021-00558-8

    CTSL cleaves the SARS-CoV-2 spike (S) protein, and this cleavage promotes cell–cell fusion. a Overview of the SARS-CoV-1 and SARS-CoV-2 S1/S2 cleavage sites. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are units of the S2 subunit that function in membrane fusion. b Analysis of CTSL-mediated S-protein cleavage. Purified SARS-CoV-1 or SARS-CoV-2 S protein was incubated in the presence or absence (assay buffer, pH = 5.5) of CTSL (2 or 10 μg/ml in assay buffer, pH = 5 .5) at 37 °C for 1 h. The reaction system of 2 μg/ml CTSL was further supplemented with CTSL inhibitors (20 μM E64d or 20 μM SID 26681509), as indicated. Proteins were subjected to SDS-PAGE and detected by silver staining. Representative data from three independent experiments are shown. c Syncytium-formation assay: Huh7 cells were untransfected (Null) or transfected with plasmid to express the SARS-CoV-2 S protein. Cells were incubated in the presence or absence (PBS, pH = 7.4) of trypsin (2 μg/ml in PBS, pH = 7.4) or in the presence or absence (PBS, pH = 5.8) of CTSL (2 or 4 μg/ml in PBS, pH = 5.8) for 20 min. Images were acquired after an additional 16 h incubation in the medium. (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from seven independent experiments are shown. d Quantitative analysis of syncytia in panel c . n = 7. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± s.e.m. values. * P
    Figure Legend Snippet: CTSL cleaves the SARS-CoV-2 spike (S) protein, and this cleavage promotes cell–cell fusion. a Overview of the SARS-CoV-1 and SARS-CoV-2 S1/S2 cleavage sites. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are units of the S2 subunit that function in membrane fusion. b Analysis of CTSL-mediated S-protein cleavage. Purified SARS-CoV-1 or SARS-CoV-2 S protein was incubated in the presence or absence (assay buffer, pH = 5.5) of CTSL (2 or 10 μg/ml in assay buffer, pH = 5 .5) at 37 °C for 1 h. The reaction system of 2 μg/ml CTSL was further supplemented with CTSL inhibitors (20 μM E64d or 20 μM SID 26681509), as indicated. Proteins were subjected to SDS-PAGE and detected by silver staining. Representative data from three independent experiments are shown. c Syncytium-formation assay: Huh7 cells were untransfected (Null) or transfected with plasmid to express the SARS-CoV-2 S protein. Cells were incubated in the presence or absence (PBS, pH = 7.4) of trypsin (2 μg/ml in PBS, pH = 7.4) or in the presence or absence (PBS, pH = 5.8) of CTSL (2 or 4 μg/ml in PBS, pH = 5.8) for 20 min. Images were acquired after an additional 16 h incubation in the medium. (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from seven independent experiments are shown. d Quantitative analysis of syncytia in panel c . n = 7. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± s.e.m. values. * P

    Techniques Used: Purification, Incubation, SDS Page, Silver Staining, Tube Formation Assay, Transfection, Plasmid Preparation

    8) Product Images from "SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness"

    Article Title: SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness

    Journal: bioRxiv

    doi: 10.1101/2020.06.11.145920

    A single dose of mRNA-1273 elicits robust antibody responses. BALB/cJ mice were immunized with 0.1 (blue), 1 μg (red), or 10 μg (purple) of mRNA-1273. Sera were collected 2 (open circles) and 4 (closed circles) weeks post-immunization and assessed for SARS-CoV-2 S-specific total IgG by ELISA (a) and neutralizing antibodies against homotypic SARS-CoV-2 pseudovirus (b). (c) S-specific IgG2a and IgG1 were also measured by ELISA, and IgG2a to IgG1 subclass ratios were calculated. Dotted line = assay limit of detection. (a-b) Doses were compared 4 weeks post-boost, and timepoints were compared within each dose level.
    Figure Legend Snippet: A single dose of mRNA-1273 elicits robust antibody responses. BALB/cJ mice were immunized with 0.1 (blue), 1 μg (red), or 10 μg (purple) of mRNA-1273. Sera were collected 2 (open circles) and 4 (closed circles) weeks post-immunization and assessed for SARS-CoV-2 S-specific total IgG by ELISA (a) and neutralizing antibodies against homotypic SARS-CoV-2 pseudovirus (b). (c) S-specific IgG2a and IgG1 were also measured by ELISA, and IgG2a to IgG1 subclass ratios were calculated. Dotted line = assay limit of detection. (a-b) Doses were compared 4 weeks post-boost, and timepoints were compared within each dose level.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    mRNA-1273 elicits robust binding and neutralizing antibody responses in multiple mouse strains. BALB/cJ (a, d), C57BL/6J (b, e), or B6C3F1/J (c, f) mice were immunized at weeks 0 and 3 weeks with 0.01 (green), 0.1 (blue), or 1 μg (red) of mRNA-1273. Sera were collected 2 weeks post-prime (open circles) and 2 weeks post-boost (closed circles) and assessed for SARS-CoV-2 S-specific IgG by ELISA (a-c), and, for post-boost sera, neutralizing antibodies against homotypic SARS-CoV-2 pseudovirus (d-f). Dotted line = assay limit of detection. (a-c) Timepoints were compared within each dose level, and doses were compared post-boost.
    Figure Legend Snippet: mRNA-1273 elicits robust binding and neutralizing antibody responses in multiple mouse strains. BALB/cJ (a, d), C57BL/6J (b, e), or B6C3F1/J (c, f) mice were immunized at weeks 0 and 3 weeks with 0.01 (green), 0.1 (blue), or 1 μg (red) of mRNA-1273. Sera were collected 2 weeks post-prime (open circles) and 2 weeks post-boost (closed circles) and assessed for SARS-CoV-2 S-specific IgG by ELISA (a-c), and, for post-boost sera, neutralizing antibodies against homotypic SARS-CoV-2 pseudovirus (d-f). Dotted line = assay limit of detection. (a-c) Timepoints were compared within each dose level, and doses were compared post-boost.

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

    mRNA-1273 and SAS-adjuvanted S-2P protein elicit both IgG2a and IgG1 subclass S-binding antibodies. BALB/cJ (a-c) or C57BL/6J (d-f) mice were immunized at weeks 0 and 3 with 0.01 (green), 0.1 (blue), or 1 μg (red) of mRNA-1273 SARS-CoV-2 S-2P protein adjuvanted with SAS. Sera were collected 2 weeks post-boost and assessed by ELISA for SARS-CoV-2 S-specific IgG1 and IgG2a or IgG2c for BALB/cJ and C57BL/6J mice, respectively. Endpoint titers (a-b, d-e) and endpoint titer ratios of IgG2a to IgG1 (c) and IgG2c to IgG1 (f) were calculated. For mice for which endpoint titers did not reach the lower limit of detection (dotted line), ratios were not calculated (N/A). IgG1 and IgG2a/c (a-b, d-e) and immunogens (c, f) were compared at each dose level.
    Figure Legend Snippet: mRNA-1273 and SAS-adjuvanted S-2P protein elicit both IgG2a and IgG1 subclass S-binding antibodies. BALB/cJ (a-c) or C57BL/6J (d-f) mice were immunized at weeks 0 and 3 with 0.01 (green), 0.1 (blue), or 1 μg (red) of mRNA-1273 SARS-CoV-2 S-2P protein adjuvanted with SAS. Sera were collected 2 weeks post-boost and assessed by ELISA for SARS-CoV-2 S-specific IgG1 and IgG2a or IgG2c for BALB/cJ and C57BL/6J mice, respectively. Endpoint titers (a-b, d-e) and endpoint titer ratios of IgG2a to IgG1 (c) and IgG2c to IgG1 (f) were calculated. For mice for which endpoint titers did not reach the lower limit of detection (dotted line), ratios were not calculated (N/A). IgG1 and IgG2a/c (a-b, d-e) and immunogens (c, f) were compared at each dose level.

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

    Immunizations with mRNA-1273 and S-2P protein, delivered with TLR4 agonist, elicit S-specific Th1-biased T cell responses. B6C3F1/J mice were immunized at weeks 0 and 3 with 0.01, 0.1, or 1 μg of mRNA-1273 or SAS-adjuvanted SARS-CoV-2 S-2P protein. Sera were collected 2 weeks post-boost and assessed by ELISA for SARS-CoV-2 S-specific IgG1 and IgG2a/c. Endpoint titers (a-b) and endpoint titer ratios of IgG2a/c to IgG1 (c) were calculated. For mice for which endpoint titers did not reach the lower limit of detection (dotted line), ratios were not calculated (N/A). (d-g) Seven weeks post-boost, splenocytes were isolated from 5 mice per group and re-stimulated with no peptides or pools of overlapping peptides from SARS-CoV-2 S protein in the presence of a protein transport inhibitor cocktail. After 6 hours, intracellular cytokine staining (ICS) was performed to quantify CD4+ and CD8+ T cell responses. Cytokine expression in the presence of no peptides was considered background and subtracted from the responses measured from the S1 and S2 peptide pools for each individual mouse. (d-e) CD4+ T cells expressing IFN-γ, TNFα, IL-2, IL-4 and IL-5 in response to the S1 (d) and S2 (e) peptide pools. (f-g) CD8+ T cells expressing IFN-γ, TNF-α, and IL-2 in response to the S1 (f) and S2 (g) peptide pools. IgG1 and IgG2a/c (a-b) and immunogens (c) were compared at each dose level. (d-g) For each cytokine, all comparisons were compared to naïve mice.
    Figure Legend Snippet: Immunizations with mRNA-1273 and S-2P protein, delivered with TLR4 agonist, elicit S-specific Th1-biased T cell responses. B6C3F1/J mice were immunized at weeks 0 and 3 with 0.01, 0.1, or 1 μg of mRNA-1273 or SAS-adjuvanted SARS-CoV-2 S-2P protein. Sera were collected 2 weeks post-boost and assessed by ELISA for SARS-CoV-2 S-specific IgG1 and IgG2a/c. Endpoint titers (a-b) and endpoint titer ratios of IgG2a/c to IgG1 (c) were calculated. For mice for which endpoint titers did not reach the lower limit of detection (dotted line), ratios were not calculated (N/A). (d-g) Seven weeks post-boost, splenocytes were isolated from 5 mice per group and re-stimulated with no peptides or pools of overlapping peptides from SARS-CoV-2 S protein in the presence of a protein transport inhibitor cocktail. After 6 hours, intracellular cytokine staining (ICS) was performed to quantify CD4+ and CD8+ T cell responses. Cytokine expression in the presence of no peptides was considered background and subtracted from the responses measured from the S1 and S2 peptide pools for each individual mouse. (d-e) CD4+ T cells expressing IFN-γ, TNFα, IL-2, IL-4 and IL-5 in response to the S1 (d) and S2 (e) peptide pools. (f-g) CD8+ T cells expressing IFN-γ, TNF-α, and IL-2 in response to the S1 (f) and S2 (g) peptide pools. IgG1 and IgG2a/c (a-b) and immunogens (c) were compared at each dose level. (d-g) For each cytokine, all comparisons were compared to naïve mice.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Isolation, Staining, Expressing

    Dose-dependent mRNA-1273-elicited antibody responses reveal strong positive correlation between binding and neutralization titers. BALB/cJ mice were immunized at weeks 0 and 3 weeks with various doses (0.0025 − 20 μg) of mRNA-1273. (a-b) Sera were collected 2 weeks post-boost and assessed for SARS-CoV-2 S-specific IgG by ELISA (a) and neutralizing antibodies against homotypic SARS-CoV-2 pseudovirus (b). (a-b) All doses were compared to 20 μg dose.
    Figure Legend Snippet: Dose-dependent mRNA-1273-elicited antibody responses reveal strong positive correlation between binding and neutralization titers. BALB/cJ mice were immunized at weeks 0 and 3 weeks with various doses (0.0025 − 20 μg) of mRNA-1273. (a-b) Sera were collected 2 weeks post-boost and assessed for SARS-CoV-2 S-specific IgG by ELISA (a) and neutralizing antibodies against homotypic SARS-CoV-2 pseudovirus (b). (a-b) All doses were compared to 20 μg dose.

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

    Flow cytometry panel to quantify SARS-CoV-2 S-specific T cells in mice. (a) A hierarchical gating strategy was used to unambiguously identify single, viable CD4+ and CD8+ T cells. Gating summary of SARS-CoV-2 S-specific (b-c) CD4 (b-c) and (d-e) CD8 (d-e) T cells elicited by 1.0 and 0.01 μg mRNA-1273 immunization. Antigen-specific T cell responses following peptide pool re-stimulation were defined as CD44 hi /cytokine + . Concatenated files shown were generated using the same number of randomly selected events from each animal across the different stimulation conditions using FlowJo software, v1
    Figure Legend Snippet: Flow cytometry panel to quantify SARS-CoV-2 S-specific T cells in mice. (a) A hierarchical gating strategy was used to unambiguously identify single, viable CD4+ and CD8+ T cells. Gating summary of SARS-CoV-2 S-specific (b-c) CD4 (b-c) and (d-e) CD8 (d-e) T cells elicited by 1.0 and 0.01 μg mRNA-1273 immunization. Antigen-specific T cell responses following peptide pool re-stimulation were defined as CD44 hi /cytokine + . Concatenated files shown were generated using the same number of randomly selected events from each animal across the different stimulation conditions using FlowJo software, v1

    Techniques Used: Flow Cytometry, Mouse Assay, Generated, Software

    mRNA-1273 elicits Th1-skewed responses compared to S protein adjuvanted with alum. BALB/c mice were immunized at weeks 0 and 2 weeks with 1 (red) or 10 μg (purple) of mRNA-1273 or 10 μg of SARS-CoV-2 S protein adjuvanted with alum hydrogel (orange). (a-b) Sera were collected 2 weeks post-boost and assessed by ELISA for SARS-CoV-2 S-specific IgG1 and IgG2a. Endpoint titers (a) and endpoint titer ratios of IgG2a to IgG1 (b) were calculated. (c-d) Splenocytes were also collected 4 weeks post-boost to evaluate IFN-γ IL-4, IL-5, and IL-13 cytokine levels secreted by T cells re-stimulated with S1 (c) and S2 (d) peptide pools, measured by Luminex. Dotted line = assay limit of detection. IgG1 and IgG2a/c (a) were compared at each dose level. (c-d) For cytokines, all comparisons were compared to PBS-immunized mice.
    Figure Legend Snippet: mRNA-1273 elicits Th1-skewed responses compared to S protein adjuvanted with alum. BALB/c mice were immunized at weeks 0 and 2 weeks with 1 (red) or 10 μg (purple) of mRNA-1273 or 10 μg of SARS-CoV-2 S protein adjuvanted with alum hydrogel (orange). (a-b) Sera were collected 2 weeks post-boost and assessed by ELISA for SARS-CoV-2 S-specific IgG1 and IgG2a. Endpoint titers (a) and endpoint titer ratios of IgG2a to IgG1 (b) were calculated. (c-d) Splenocytes were also collected 4 weeks post-boost to evaluate IFN-γ IL-4, IL-5, and IL-13 cytokine levels secreted by T cells re-stimulated with S1 (c) and S2 (d) peptide pools, measured by Luminex. Dotted line = assay limit of detection. IgG1 and IgG2a/c (a) were compared at each dose level. (c-d) For cytokines, all comparisons were compared to PBS-immunized mice.

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

    9) Product Images from "Development and effectiveness of Pseudotyped SARS-CoV-2 system as determined by neutralizing efficiency and entry inhibition test in vitro"

    Article Title: Development and effectiveness of Pseudotyped SARS-CoV-2 system as determined by neutralizing efficiency and entry inhibition test in vitro

    Journal: Biosafety and Health

    doi: 10.1016/j.bsheal.2020.08.004

    Identification of SARS-CoV-2 S protein expression and SARS-CoV-2 pseudotyped virus Construction and identification of S expressing plasmid. SARS-CoV-2 S protein gene was inserted in the pCDNA3.1 vector. Immunofluorescence assay for S protein expression in pcDNA3.1-SARS-CoV-2 S plasmid. The expression was determined using mouse pAb against SARS-CoV-2 S protein and convalescent serum samples from COVID-19 patients. Identification of S protein expression in SARS-CoV-2 pseudotyped virus by immunoblot assay. Bands corresponding to SARS-CoV-2 S and HIV-1 p24 proteins were detected at the same sample line in the gel.
    Figure Legend Snippet: Identification of SARS-CoV-2 S protein expression and SARS-CoV-2 pseudotyped virus Construction and identification of S expressing plasmid. SARS-CoV-2 S protein gene was inserted in the pCDNA3.1 vector. Immunofluorescence assay for S protein expression in pcDNA3.1-SARS-CoV-2 S plasmid. The expression was determined using mouse pAb against SARS-CoV-2 S protein and convalescent serum samples from COVID-19 patients. Identification of S protein expression in SARS-CoV-2 pseudotyped virus by immunoblot assay. Bands corresponding to SARS-CoV-2 S and HIV-1 p24 proteins were detected at the same sample line in the gel.

    Techniques Used: Expressing, Plasmid Preparation, Immunofluorescence

    10) Product Images from "Monoclonal antibodies for the S2 subunit of spike of SARS-CoV-1 cross-react with the newly-emerged SARS-CoV-2"

    Article Title: Monoclonal antibodies for the S2 subunit of spike of SARS-CoV-1 cross-react with the newly-emerged SARS-CoV-2

    Journal: Eurosurveillance

    doi: 10.2807/1560-7917.ES.2020.25.28.2000291

    Monoclonal antibodies expected to target a SARS-CoV-2 S protein S2 fragment, (A) hybridise to the peptide fragment in western blot and (B) recognise cells expressing the peptide as shown by immunofluorescence
    Figure Legend Snippet: Monoclonal antibodies expected to target a SARS-CoV-2 S protein S2 fragment, (A) hybridise to the peptide fragment in western blot and (B) recognise cells expressing the peptide as shown by immunofluorescence

    Techniques Used: Western Blot, Expressing, Immunofluorescence

    Antibodies expected to target SARS-CoV-2 S protein, (A) hybridise to the denatured protein in western blot, (B) bind to the protein in ELISA and (C) recognise cells expressing the protein as shown by immunofluorescence
    Figure Legend Snippet: Antibodies expected to target SARS-CoV-2 S protein, (A) hybridise to the denatured protein in western blot, (B) bind to the protein in ELISA and (C) recognise cells expressing the protein as shown by immunofluorescence

    Techniques Used: Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence

    11) Product Images from "Oral delivery of SARS-CoV-2 DNA vaccines using attenuated Salmonella typhimurium as a carrier in rat"

    Article Title: Oral delivery of SARS-CoV-2 DNA vaccines using attenuated Salmonella typhimurium as a carrier in rat

    Journal: bioRxiv

    doi: 10.1101/2020.07.23.217174

    pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP plasmid map.
    Figure Legend Snippet: pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP plasmid map.

    Techniques Used: Plasmid Preparation

    Micrographs of 293T cells transfected with pSARS-CoV-2-S (X 100). (A1, A2) 293T cells transfected with pSARS-CoV-2-S (pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP) at 48 hours after transfection. A1 fluorescence micrograph with GFP expression in cells and light micrograph with the same visual field as A2. (B) 293T cells transfected with pSARS-CoV-2-S at 48 hours after transfection. The SARS-CoV-2-S protein showed about 141 kDa.
    Figure Legend Snippet: Micrographs of 293T cells transfected with pSARS-CoV-2-S (X 100). (A1, A2) 293T cells transfected with pSARS-CoV-2-S (pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP) at 48 hours after transfection. A1 fluorescence micrograph with GFP expression in cells and light micrograph with the same visual field as A2. (B) 293T cells transfected with pSARS-CoV-2-S at 48 hours after transfection. The SARS-CoV-2-S protein showed about 141 kDa.

    Techniques Used: Transfection, Fluorescence, Expressing

    Humoral responses to SARS-CoV-2-S protein antigen in the rat after immunizationon day 0, day 14, and day 28 with Salmonella carrying the control vector or pSARS-CoV-2-S (as described in the methods). (A) After immunization with the control vector, test SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent test mean OD450 nm values (mean±SD) for each of 9 rats, or (B) After immunization with the pSARS-CoV-2-S vector, SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent mean OD450 nm values (3 times measurement, mean±SD) for each of 9 rats.
    Figure Legend Snippet: Humoral responses to SARS-CoV-2-S protein antigen in the rat after immunizationon day 0, day 14, and day 28 with Salmonella carrying the control vector or pSARS-CoV-2-S (as described in the methods). (A) After immunization with the control vector, test SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent test mean OD450 nm values (mean±SD) for each of 9 rats, or (B) After immunization with the pSARS-CoV-2-S vector, SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent mean OD450 nm values (3 times measurement, mean±SD) for each of 9 rats.

    Techniques Used: Plasmid Preparation, Binding Assay

    12) 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: bioRxiv

    doi: 10.1101/2020.09.28.311480

    Biodistribution of Sad23L-nCoV-S and Ad49L-nCoV-S vaccines in inoculated animals. ( A ) Serum NAb titers to Sad23L and Ad49L vectors were measured in macaques immunized by prime-boost inoculation with two vaccines at 4 week interval, or ( B ) in C57BL/6 and BALB/c mice 4 weeks post prime only or prime-boost vaccination with two vaccines or vectorial controls. ( C ) Nested-PCR amplification of Sad23L or Ad49L-hexon gene (500bp) in tissues of C57BL/6 mice 4 weeks after inoculation by prime only or prime-boost immunization with Sad23L-nCoV-S and Ad49L-nCoV-S vaccines. ( D ) Expression of S protein in splenocytes and hepatocytes of tissue frozen sections from vaccine immunized or control C57BL/6 mice by immunofluorescence staining with a human monoclonal antibody to SARS-CoV-2 S and DAPI.
    Figure Legend Snippet: Biodistribution of Sad23L-nCoV-S and Ad49L-nCoV-S vaccines in inoculated animals. ( A ) Serum NAb titers to Sad23L and Ad49L vectors were measured in macaques immunized by prime-boost inoculation with two vaccines at 4 week interval, or ( B ) in C57BL/6 and BALB/c mice 4 weeks post prime only or prime-boost vaccination with two vaccines or vectorial controls. ( C ) Nested-PCR amplification of Sad23L or Ad49L-hexon gene (500bp) in tissues of C57BL/6 mice 4 weeks after inoculation by prime only or prime-boost immunization with Sad23L-nCoV-S and Ad49L-nCoV-S vaccines. ( D ) Expression of S protein in splenocytes and hepatocytes of tissue frozen sections from vaccine immunized or control C57BL/6 mice by immunofluorescence staining with a human monoclonal antibody to SARS-CoV-2 S and DAPI.

    Techniques Used: Mouse Assay, Nested PCR, Amplification, Expressing, Immunofluorescence, Staining

    Examination of SARS-CoV-2 S protein in the tissues of Sad23L-nCoV-S and Ad49L-nCoV-S immunized mice. Spleen, liver, Lung and muscle tissues of immunized C57BL/6 mice were examined by immunofluorescence staining with a human monoclonal antibody to SARS-CoV-2 S and DAPI.
    Figure Legend Snippet: Examination of SARS-CoV-2 S protein in the tissues of Sad23L-nCoV-S and Ad49L-nCoV-S immunized mice. Spleen, liver, Lung and muscle tissues of immunized C57BL/6 mice were examined by immunofluorescence staining with a human monoclonal antibody to SARS-CoV-2 S and DAPI.

    Techniques Used: Mouse Assay, Immunofluorescence, Staining

    13) Product Images from "Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections"

    Article Title: Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20465-w

    Cryo-EM structures of the SARS-CoV-2 S trimer in complex with the 3C1 Fab. a , b S-3C1-F3b cryo-EM map ( a ) and pseudo atomic model ( b ). All the three RBDs are up and each of them binds with a 3C1 Fab. The heavy chain of the 3C1 Fab in medium blue and light chain in violet red. c , d S-3C1-F3a cryo-EM map ( c ) and pseudo atomic model ( d ). There are two up RBDs and one down RBD, with each bound with a 3C1 Fab. e Structural alignment of the three up RBDs of S-3C1-F3b (in color) and the only up RBD from S-open (gray), suggesting 3C1 induced outward tilt of the RBDs within the S trimer. f , g Conformational comparation between S-3C1-F1 and S-open ( f ), as well as between S-3C1-F3a and S-3C1-F2 ( g ). h RBD/3C1 interaction interface (take RBD-3/3C1 of S-3C1-F3b as an example), with major involved structural elements labeled. i ACE2 (coral, PDB: 6M0J) would clash with the heavy chain of 3C1 Fab (blue). They share overlapping epitopes on the RBM (dotted black circle); additionally, the framework of 3C1-VH would clash with ACE2 (dotted black frame), which could be enhanced by the presence of an N-linked glycan at site N322 of ACE2. j 3C1 showed two distinct orientations to bind RBD within S trimer, i.e., adopting orientation 1 to associate with up RBD while orientation 2 with down RBD. k Contact footprint variations of 3C1 on up RBD (left) compared with that on down RBD (right), with unique epitopes indicated by dotted black frame. l – m Potential simultaneous binding of RBD by 2H2 and 3C1 cocktail. In 3C1 orientation 1, 3C1 and 2H2 could have minor clash (indicated by black frame, l ); while in origination 2, there is no clash between 3C1 and 2H2 Fabs ( m ).
    Figure Legend Snippet: Cryo-EM structures of the SARS-CoV-2 S trimer in complex with the 3C1 Fab. a , b S-3C1-F3b cryo-EM map ( a ) and pseudo atomic model ( b ). All the three RBDs are up and each of them binds with a 3C1 Fab. The heavy chain of the 3C1 Fab in medium blue and light chain in violet red. c , d S-3C1-F3a cryo-EM map ( c ) and pseudo atomic model ( d ). There are two up RBDs and one down RBD, with each bound with a 3C1 Fab. e Structural alignment of the three up RBDs of S-3C1-F3b (in color) and the only up RBD from S-open (gray), suggesting 3C1 induced outward tilt of the RBDs within the S trimer. f , g Conformational comparation between S-3C1-F1 and S-open ( f ), as well as between S-3C1-F3a and S-3C1-F2 ( g ). h RBD/3C1 interaction interface (take RBD-3/3C1 of S-3C1-F3b as an example), with major involved structural elements labeled. i ACE2 (coral, PDB: 6M0J) would clash with the heavy chain of 3C1 Fab (blue). They share overlapping epitopes on the RBM (dotted black circle); additionally, the framework of 3C1-VH would clash with ACE2 (dotted black frame), which could be enhanced by the presence of an N-linked glycan at site N322 of ACE2. j 3C1 showed two distinct orientations to bind RBD within S trimer, i.e., adopting orientation 1 to associate with up RBD while orientation 2 with down RBD. k Contact footprint variations of 3C1 on up RBD (left) compared with that on down RBD (right), with unique epitopes indicated by dotted black frame. l – m Potential simultaneous binding of RBD by 2H2 and 3C1 cocktail. In 3C1 orientation 1, 3C1 and 2H2 could have minor clash (indicated by black frame, l ); while in origination 2, there is no clash between 3C1 and 2H2 Fabs ( m ).

    Techniques Used: Labeling, Binding Assay

    A proposed model of stepwise binding of 2H2/3C1 Fabs to the RBD of SARS-CoV-2 S trimer. a 2H2 and 3C1 Fabs appear to follow similar pathway to induce generally comparable conformational transitions of the S trimer to neutralize the virus. RBD-1, RBD-2, and RBD-3 are colored in light green, light blue, and gold, respectively; 2H2 and 3C1 Fab in violent red and medium blue, respectively. Red ellipsoid and black ellipsoid indicate Fab bound to up RBD and down RBD, respectively. The maps of S-2H2 and S-3C1 complexes shown here were generated by lowpass filtering of the corresponding models to 10 Å resolution. b Population distribution for the S-2H2 and S-3C1 dataset.
    Figure Legend Snippet: A proposed model of stepwise binding of 2H2/3C1 Fabs to the RBD of SARS-CoV-2 S trimer. a 2H2 and 3C1 Fabs appear to follow similar pathway to induce generally comparable conformational transitions of the S trimer to neutralize the virus. RBD-1, RBD-2, and RBD-3 are colored in light green, light blue, and gold, respectively; 2H2 and 3C1 Fab in violent red and medium blue, respectively. Red ellipsoid and black ellipsoid indicate Fab bound to up RBD and down RBD, respectively. The maps of S-2H2 and S-3C1 complexes shown here were generated by lowpass filtering of the corresponding models to 10 Å resolution. b Population distribution for the S-2H2 and S-3C1 dataset.

    Techniques Used: Binding Assay, Generated

    Cryo-EM structures of the SARS-CoV-2 S trimer in complex with 2H2 Fab. a , b Side and top views of the S-2H2-F3a cryo-EM map ( a ) and pseudo atomic model ( b ). RBD-1 and RBD-2 are in up configuration, while RBD-3 is down, with each of the RBDs bound with a 2H2 Fab. Protomer 1, 2, and 3 are shown in light green, powder blue, and gold, respectively. This color scheme is followed throughout. Heavy chain and light chain of 2H2 Fab in royal blue and violet red, respectively. c , d Side and top views of the S-2H2-F2 cryo-EM map ( c ) and pseudo atomic model ( d ), with two up RBDs (RBD-1 and RBD-2) each bound with a 2H2 Fab. e , f 2H2 Fab-induced conformational changes of the S trimer. Shown is the structural comparation of RBDs between S-2H2-F1 (in color) and S-open (dim gray) ( e ), and between S-2H2-F3a (in color) and S-2H2-F2 (dim gray) ( f ). g 2H2 Fab mainly binds to the RBM (light sea green surface) of RBD, with major involved structural elements labeled. RBD core is rendered as light green surface. h 2H2 Fab (left) and ACE2 (right, gold, PDB: 6M0J) share overlapping epitopes on RBM (second row) and would clash upon binding to the S trimer. i , j The involved regions/residues forming potential contacts between the light chain (in violent red, i ) or heavy chain (in royal blue, j ) of 2H2 and the RBD-1 of S-2H2-F3a. Asterisks highlight residues also involved in the interactions with ACE2. Note that considering the local resolution limitation in the RBD-2H2 portion of the map due to intrinsic dynamic nature in these regions, we analyzed the potential interactions that fulfill criteria of both
    Figure Legend Snippet: Cryo-EM structures of the SARS-CoV-2 S trimer in complex with 2H2 Fab. a , b Side and top views of the S-2H2-F3a cryo-EM map ( a ) and pseudo atomic model ( b ). RBD-1 and RBD-2 are in up configuration, while RBD-3 is down, with each of the RBDs bound with a 2H2 Fab. Protomer 1, 2, and 3 are shown in light green, powder blue, and gold, respectively. This color scheme is followed throughout. Heavy chain and light chain of 2H2 Fab in royal blue and violet red, respectively. c , d Side and top views of the S-2H2-F2 cryo-EM map ( c ) and pseudo atomic model ( d ), with two up RBDs (RBD-1 and RBD-2) each bound with a 2H2 Fab. e , f 2H2 Fab-induced conformational changes of the S trimer. Shown is the structural comparation of RBDs between S-2H2-F1 (in color) and S-open (dim gray) ( e ), and between S-2H2-F3a (in color) and S-2H2-F2 (dim gray) ( f ). g 2H2 Fab mainly binds to the RBM (light sea green surface) of RBD, with major involved structural elements labeled. RBD core is rendered as light green surface. h 2H2 Fab (left) and ACE2 (right, gold, PDB: 6M0J) share overlapping epitopes on RBM (second row) and would clash upon binding to the S trimer. i , j The involved regions/residues forming potential contacts between the light chain (in violent red, i ) or heavy chain (in royal blue, j ) of 2H2 and the RBD-1 of S-2H2-F3a. Asterisks highlight residues also involved in the interactions with ACE2. Note that considering the local resolution limitation in the RBD-2H2 portion of the map due to intrinsic dynamic nature in these regions, we analyzed the potential interactions that fulfill criteria of both

    Techniques Used: Labeling, Binding Assay

    14) Product Images from "Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development"

    Article Title: Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-021-00558-8

    CTSL cleaves the SARS-CoV-2 spike (S) protein, and this cleavage promotes cell–cell fusion. a Overview of the SARS-CoV-1 and SARS-CoV-2 S1/S2 cleavage sites. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are units of the S2 subunit that function in membrane fusion. b Analysis of CTSL-mediated S-protein cleavage. Purified SARS-CoV-1 or SARS-CoV-2 S protein was incubated in the presence or absence (assay buffer, pH = 5.5) of CTSL (2 or 10 μg/ml in assay buffer, pH = 5 .5) at 37 °C for 1 h. The reaction system of 2 μg/ml CTSL was further supplemented with CTSL inhibitors (20 μM E64d or 20 μM SID 26681509), as indicated. Proteins were subjected to SDS-PAGE and detected by silver staining. Representative data from three independent experiments are shown. c Syncytium-formation assay: Huh7 cells were untransfected (Null) or transfected with plasmid to express the SARS-CoV-2 S protein. Cells were incubated in the presence or absence (PBS, pH = 7.4) of trypsin (2 μg/ml in PBS, pH = 7.4) or in the presence or absence (PBS, pH = 5.8) of CTSL (2 or 4 μg/ml in PBS, pH = 5.8) for 20 min. Images were acquired after an additional 16 h incubation in the medium. (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from seven independent experiments are shown. d Quantitative analysis of syncytia in panel c . n = 7. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± s.e.m. values. * P
    Figure Legend Snippet: CTSL cleaves the SARS-CoV-2 spike (S) protein, and this cleavage promotes cell–cell fusion. a Overview of the SARS-CoV-1 and SARS-CoV-2 S1/S2 cleavage sites. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are units of the S2 subunit that function in membrane fusion. b Analysis of CTSL-mediated S-protein cleavage. Purified SARS-CoV-1 or SARS-CoV-2 S protein was incubated in the presence or absence (assay buffer, pH = 5.5) of CTSL (2 or 10 μg/ml in assay buffer, pH = 5 .5) at 37 °C for 1 h. The reaction system of 2 μg/ml CTSL was further supplemented with CTSL inhibitors (20 μM E64d or 20 μM SID 26681509), as indicated. Proteins were subjected to SDS-PAGE and detected by silver staining. Representative data from three independent experiments are shown. c Syncytium-formation assay: Huh7 cells were untransfected (Null) or transfected with plasmid to express the SARS-CoV-2 S protein. Cells were incubated in the presence or absence (PBS, pH = 7.4) of trypsin (2 μg/ml in PBS, pH = 7.4) or in the presence or absence (PBS, pH = 5.8) of CTSL (2 or 4 μg/ml in PBS, pH = 5.8) for 20 min. Images were acquired after an additional 16 h incubation in the medium. (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from seven independent experiments are shown. d Quantitative analysis of syncytia in panel c . n = 7. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± s.e.m. values. * P

    Techniques Used: Purification, Incubation, SDS Page, Silver Staining, Tube Formation Assay, Transfection, Plasmid Preparation

    15) Product Images from "Aprotinin Inhibits SARS-CoV-2 Replication"

    Article Title: Aprotinin Inhibits SARS-CoV-2 Replication

    Journal: Cells

    doi: 10.3390/cells9112377

    Anti-severe acute respiratory syndrome virus 2 (SARS-CoV-2) effects of aprotinin and SERPINA1/alpha-1 antitrypsin. ( A ) Concentration-dependent effects of aprotinin and SERPINA1/alpha-1 antitrypsin on SARS-CoV-2-induced cytopathogenic effect (CPE) formation determined 48 h post-infection in Caco2 cells infected at a multiplicity of infection (MOI) of 0.01 with the three different SARS-CoV-2 isolates. The viability of the Caco2 cells was 84.3 ± 2.7% relative to the untreated control in the presence of 20 µM of aprotinin. ( B ) Immunostaining for the SARS-CoV-2 S protein in aprotinin- and SERPINA1/alpha-1 antitrypsin-treated Caco2 cells infected at an MOI of 0.01 with the three different SARS-CoV-2 isolates as determined 48 h post-infection. The protease inhibitors were tested at four concentrations in 1:4 dilution steps ranging from 20 to 0.3125 µM. A quantification is provided in Figure S1 . ( C ) Copy numbers of genomic RNA in Caco2 cells infected with different SARS-CoV-2 isolates (MOI of 0.01) in response to treatment with aprotinin or SERPINA1/alpha-1 antitrypsin as determined 48 h post-infection. FFM1, 1/Human/2020/Frankfurt; FFM2, 2/Human/2020/Frankfurt; FFM6, 6/Human/2020/Frankfurt.
    Figure Legend Snippet: Anti-severe acute respiratory syndrome virus 2 (SARS-CoV-2) effects of aprotinin and SERPINA1/alpha-1 antitrypsin. ( A ) Concentration-dependent effects of aprotinin and SERPINA1/alpha-1 antitrypsin on SARS-CoV-2-induced cytopathogenic effect (CPE) formation determined 48 h post-infection in Caco2 cells infected at a multiplicity of infection (MOI) of 0.01 with the three different SARS-CoV-2 isolates. The viability of the Caco2 cells was 84.3 ± 2.7% relative to the untreated control in the presence of 20 µM of aprotinin. ( B ) Immunostaining for the SARS-CoV-2 S protein in aprotinin- and SERPINA1/alpha-1 antitrypsin-treated Caco2 cells infected at an MOI of 0.01 with the three different SARS-CoV-2 isolates as determined 48 h post-infection. The protease inhibitors were tested at four concentrations in 1:4 dilution steps ranging from 20 to 0.3125 µM. A quantification is provided in Figure S1 . ( C ) Copy numbers of genomic RNA in Caco2 cells infected with different SARS-CoV-2 isolates (MOI of 0.01) in response to treatment with aprotinin or SERPINA1/alpha-1 antitrypsin as determined 48 h post-infection. FFM1, 1/Human/2020/Frankfurt; FFM2, 2/Human/2020/Frankfurt; FFM6, 6/Human/2020/Frankfurt.

    Techniques Used: Concentration Assay, Infection, Immunostaining

    16) Product Images from "SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2"

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

    Journal: Cell

    doi: 10.1016/j.cell.2020.09.033

    SARS-CoV-2 Spike Ectodomain Protein Binding to Cells Is Differentially Affected by HS from Different Organs and Potently Inhibited by Heparinoids (A) LC-MS/MS disaccharide analysis of HS isolated from human kidney, liver, tonsil, and lung tissue. (B) Inhibition of binding of recombinant SARS-CoV-2 S RBD protein to H1299 cells, using tissue HS. Analysis by flow cytometry. (C) Inhibition of recombinant trimeric SARS-CoV-2 protein (20 μg/mL) binding to H1299 cells, using CHO HS, heparin, MST heparin, and split-glycol heparin. Analysis by flow cytometry. (D) Similar analysis of A549 cells. Curve fitting was performed using non-linear regression and the inhibitor versus response least-squares fit algorithm. IC 50 values are listed in Table 1 . Graphs show representative experiments performed in technical duplicates or triplicates. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).
    Figure Legend Snippet: SARS-CoV-2 Spike Ectodomain Protein Binding to Cells Is Differentially Affected by HS from Different Organs and Potently Inhibited by Heparinoids (A) LC-MS/MS disaccharide analysis of HS isolated from human kidney, liver, tonsil, and lung tissue. (B) Inhibition of binding of recombinant SARS-CoV-2 S RBD protein to H1299 cells, using tissue HS. Analysis by flow cytometry. (C) Inhibition of recombinant trimeric SARS-CoV-2 protein (20 μg/mL) binding to H1299 cells, using CHO HS, heparin, MST heparin, and split-glycol heparin. Analysis by flow cytometry. (D) Similar analysis of A549 cells. Curve fitting was performed using non-linear regression and the inhibitor versus response least-squares fit algorithm. IC 50 values are listed in Table 1 . Graphs show representative experiments performed in technical duplicates or triplicates. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).

    Techniques Used: Protein Binding, Liquid Chromatography with Mass Spectroscopy, Isolation, Inhibition, Binding Assay, Recombinant, Flow Cytometry

    Binding of RBD Protein to Hep3B Mutants, Related to Figure 3 Binding of SARS-CoV-2 S RBD protein (20 μg/mL) to Hep3B mutants. Binding was measured by flow cytometry. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).
    Figure Legend Snippet: Binding of RBD Protein to Hep3B Mutants, Related to Figure 3 Binding of SARS-CoV-2 S RBD protein (20 μg/mL) to Hep3B mutants. Binding was measured by flow cytometry. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).

    Techniques Used: Binding Assay, Flow Cytometry

    SARS-CoV-2 Spike Ectodomain Binding to Cells Is Dependent on Cellular HS (A) Titration of recombinant SARS-CoV-2 spike protein binding to human H1299 cells with and without treatment with a mix of heparin lyases I, II, and III (HSase). (B) Recombinant SARS-CoV-2 spike protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (C) SARS-CoV-2 S RBD protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (D) SARS-CoV-2 spike protein binding (20 μg/mL) to H1299 and A375 cells with and without HSase treatment. (E) Anti-HS (F58-10E4) staining of H1299, A549, Hep3B, and A375 cells with and without HSase treatment. (F) Binding of recombinant SARS-CoV-2 spike protein (20 μg/mL) to Hep3B mutants altered in HS biosynthesis enzymes. Specific enzymes that were lacking in the mutants are listed along the x axis. All values were obtained by flow cytometry. Graphs shows representative experiments performed in technical triplicate. The experiments were repeated at least three times. Statistical analysis by unpaired t test (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S4 .
    Figure Legend Snippet: SARS-CoV-2 Spike Ectodomain Binding to Cells Is Dependent on Cellular HS (A) Titration of recombinant SARS-CoV-2 spike protein binding to human H1299 cells with and without treatment with a mix of heparin lyases I, II, and III (HSase). (B) Recombinant SARS-CoV-2 spike protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (C) SARS-CoV-2 S RBD protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (D) SARS-CoV-2 spike protein binding (20 μg/mL) to H1299 and A375 cells with and without HSase treatment. (E) Anti-HS (F58-10E4) staining of H1299, A549, Hep3B, and A375 cells with and without HSase treatment. (F) Binding of recombinant SARS-CoV-2 spike protein (20 μg/mL) to Hep3B mutants altered in HS biosynthesis enzymes. Specific enzymes that were lacking in the mutants are listed along the x axis. All values were obtained by flow cytometry. Graphs shows representative experiments performed in technical triplicate. The experiments were repeated at least three times. Statistical analysis by unpaired t test (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S4 .

    Techniques Used: Binding Assay, Titration, Recombinant, Protein Binding, Staining, Flow Cytometry

    SARS-CoV-2 Pseudovirus Infection Depends on Heparan Sulfate (A) Left, SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells measured by flow cytometry with and without HSase. Right, heparin and split-glycol heparin inhibit SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells by flow cytometry. Statistical analysis by unpaired t test. (B) Western blot analysis of ACE2 expression in Vero E6 cells compared to A549, H1299, and A375 cells. A representative blot of three extracts is shown for each strain. (C) Infection of Vero E6 cells with SARS-CoV-2 spike protein expressing pseudotyped virus expressing GFP. Infection was done with and without HSase treatment of the cells. Insert shows GFP expression in the infected cells by imaging. Counting was performed by flow cytometry with gating for GFP-positive cells as indicated by “infected.” (D) Quantitative analysis of GFP-positive cells. (E) Infection of Vero E6 cells with SARS-CoV-2 S protein pseudotyped virus expressing luciferase, as measured by the addition of Bright-Glo and detection of luminescence. The figure shows infection experiments done at low and high titer. (F) HSase treatment diminishes infection by SARS-CoV-2 S protein pseudotyped virus (luciferase) at low and high titer. (G) Heparin (0.5 μg/mL) blocks infection with SARS-CoV-2 S protein pseudotyped virus (luciferase). (H) Effect of HSase treatment of Vero E6 cells on the infection of both SARS-CoV-1 S and SARS-CoV-2 S protein pseudotyped virus expressing luciferase. (I) Infection of Hep3B with and without HSase and in Hep3B cells containing mutations in EXT1 , NDST1 , and HS6ST1 / HS6ST2 . Cells were infected with SARS-CoV-2 S protein pseudotyped virus expressing luciferase. All experiments were repeated at least three times. Graphs shows representative experiments performed in technical triplicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001). See also Figure S6 .
    Figure Legend Snippet: SARS-CoV-2 Pseudovirus Infection Depends on Heparan Sulfate (A) Left, SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells measured by flow cytometry with and without HSase. Right, heparin and split-glycol heparin inhibit SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells by flow cytometry. Statistical analysis by unpaired t test. (B) Western blot analysis of ACE2 expression in Vero E6 cells compared to A549, H1299, and A375 cells. A representative blot of three extracts is shown for each strain. (C) Infection of Vero E6 cells with SARS-CoV-2 spike protein expressing pseudotyped virus expressing GFP. Infection was done with and without HSase treatment of the cells. Insert shows GFP expression in the infected cells by imaging. Counting was performed by flow cytometry with gating for GFP-positive cells as indicated by “infected.” (D) Quantitative analysis of GFP-positive cells. (E) Infection of Vero E6 cells with SARS-CoV-2 S protein pseudotyped virus expressing luciferase, as measured by the addition of Bright-Glo and detection of luminescence. The figure shows infection experiments done at low and high titer. (F) HSase treatment diminishes infection by SARS-CoV-2 S protein pseudotyped virus (luciferase) at low and high titer. (G) Heparin (0.5 μg/mL) blocks infection with SARS-CoV-2 S protein pseudotyped virus (luciferase). (H) Effect of HSase treatment of Vero E6 cells on the infection of both SARS-CoV-1 S and SARS-CoV-2 S protein pseudotyped virus expressing luciferase. (I) Infection of Hep3B with and without HSase and in Hep3B cells containing mutations in EXT1 , NDST1 , and HS6ST1 / HS6ST2 . Cells were infected with SARS-CoV-2 S protein pseudotyped virus expressing luciferase. All experiments were repeated at least three times. Graphs shows representative experiments performed in technical triplicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001). See also Figure S6 .

    Techniques Used: Infection, Binding Assay, Flow Cytometry, Western Blot, Expressing, Imaging, Luciferase

    Molecular Modeling of the SARS-Cov-2 Spike RBD Interaction with Heparin (A) A molecular model of SARS CoV-2 S protein trimer (PDB: 6VSB and 6M0J ) rendered with Pymol. ACE2 is shown in blue and the RBD open conformation in green. A set of positively charged residues lies distal to the ACE2 binding site. (B) Electrostatic surface rendering of the SARS-CoV-2 RBD (PDB: 6M17 ) docked with dp4 heparin oligosaccharides. Blue and red surfaces indicate electropositive and electronegative surfaces, respectively. Oligosaccharides are represented using standard CPK format. (C) Mesh surface rendering of the RBD (green) docked with dp4 heparin oligosaccharides (red). (D) Number of contacts between the RBD amino acids and a set of docked heparin dp4 oligosaccharides from (A and B). (E) Calculated energy contributions of each amino acid residue in the RBD that can interact with heparin. (F) Amino acid sequence alignment of the SARS-CoV-1 and SARS-Cov-2 RBD. Red boxes indicate amino acid residues contributing to the electropositive patch in (A and B). Identical residues are shaded dark gray. Conservative substitutions have backgrounds in blue. Non-conserved residues have a white background (G) Structural alignment of SARS-CoV-1 (cyan; PDB: 3BGF ) and SARS-CoV-2 (red; PDB: 6M17 ) RBD. (H) Electrostatic surface rendering of the SARS-CoV-1 and SAR-CoV-2 RBDs. See also Figure S1 .
    Figure Legend Snippet: Molecular Modeling of the SARS-Cov-2 Spike RBD Interaction with Heparin (A) A molecular model of SARS CoV-2 S protein trimer (PDB: 6VSB and 6M0J ) rendered with Pymol. ACE2 is shown in blue and the RBD open conformation in green. A set of positively charged residues lies distal to the ACE2 binding site. (B) Electrostatic surface rendering of the SARS-CoV-2 RBD (PDB: 6M17 ) docked with dp4 heparin oligosaccharides. Blue and red surfaces indicate electropositive and electronegative surfaces, respectively. Oligosaccharides are represented using standard CPK format. (C) Mesh surface rendering of the RBD (green) docked with dp4 heparin oligosaccharides (red). (D) Number of contacts between the RBD amino acids and a set of docked heparin dp4 oligosaccharides from (A and B). (E) Calculated energy contributions of each amino acid residue in the RBD that can interact with heparin. (F) Amino acid sequence alignment of the SARS-CoV-1 and SARS-Cov-2 RBD. Red boxes indicate amino acid residues contributing to the electropositive patch in (A and B). Identical residues are shaded dark gray. Conservative substitutions have backgrounds in blue. Non-conserved residues have a white background (G) Structural alignment of SARS-CoV-1 (cyan; PDB: 3BGF ) and SARS-CoV-2 (red; PDB: 6M17 ) RBD. (H) Electrostatic surface rendering of the SARS-CoV-1 and SAR-CoV-2 RBDs. See also Figure S1 .

    Techniques Used: Binding Assay, Sequencing

    17) Product Images from "Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2"

    Article Title: Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2

    Journal: Genes & Diseases

    doi: 10.1016/j.gendis.2020.07.006

    Detection of SARS-CoV-2 spike (S) protein expression and localization. (A) Schematic illustration of the SARS-CoV-2 full-length spike (S-FL) and mutant S variants. The RBD (receptor binding domain) is in subunit S1; the FP (fusion peptide), HR1 (heptad repeat 1), HR2 (heptad repeat 2), TM (transmembrane domain), and CT (cytoplasmic tail) are in subunit S2. The endoplasmic reticulum retrieval signals (“KxHxx” motif) in the CT domain of S-FL were destroyed in S-Mut protein. The C-terminal 19 amino acids were lacking in S-C19del. (B) Detection of SARS-CoV-2 S expression in HKE293T cells by Western blot using the anti-RBD monoclonal antibody. Cells were transfected with pS-FL, pS-Mut, and pS-C19del plasmids or with an empty vector. (C) Detection of SARS-CoV-2 S subcellular localization in HKE293T cells by confocal microscopy. Cells were grown on glass coverslips for 24 h preceding transfection of plasmids encoding S protein variants. The cells were harvested and labeled with the corresponding antibodies. Calreticulin, ER marker. Nuclei were counterstained with DAPI. Bar = 20 μm.
    Figure Legend Snippet: Detection of SARS-CoV-2 spike (S) protein expression and localization. (A) Schematic illustration of the SARS-CoV-2 full-length spike (S-FL) and mutant S variants. The RBD (receptor binding domain) is in subunit S1; the FP (fusion peptide), HR1 (heptad repeat 1), HR2 (heptad repeat 2), TM (transmembrane domain), and CT (cytoplasmic tail) are in subunit S2. The endoplasmic reticulum retrieval signals (“KxHxx” motif) in the CT domain of S-FL were destroyed in S-Mut protein. The C-terminal 19 amino acids were lacking in S-C19del. (B) Detection of SARS-CoV-2 S expression in HKE293T cells by Western blot using the anti-RBD monoclonal antibody. Cells were transfected with pS-FL, pS-Mut, and pS-C19del plasmids or with an empty vector. (C) Detection of SARS-CoV-2 S subcellular localization in HKE293T cells by confocal microscopy. Cells were grown on glass coverslips for 24 h preceding transfection of plasmids encoding S protein variants. The cells were harvested and labeled with the corresponding antibodies. Calreticulin, ER marker. Nuclei were counterstained with DAPI. Bar = 20 μm.

    Techniques Used: Expressing, Mutagenesis, Binding Assay, Western Blot, Transfection, Plasmid Preparation, Confocal Microscopy, Labeling, Marker

    Detection of SARS-CoV-2 S pseudotyped virus infectivity. (A) Schematic representation of the pseudovirus production and neutralization assay and the applications of the pseudovirus. (B) HEK293T and 293T-ACE2 cells were infected with lentiviruses pseudotyped with vesicular stomatitis virus G (VSV-G) and SARS-CoV-2 S protein variants. The y -axis shows the relative luminescence units (RLU) detected at 48 h post-pseudovirus inoculation. (C) Optimization of the incubation time for pseudovirus luciferase assay. Luciferase activities were measured 24–72 h post-virus infection. For this purpose, 72 h was chosen as the optimized incubation time. The data are presented as the means ± standard deviations (SDs) of three independent biological replicates.
    Figure Legend Snippet: Detection of SARS-CoV-2 S pseudotyped virus infectivity. (A) Schematic representation of the pseudovirus production and neutralization assay and the applications of the pseudovirus. (B) HEK293T and 293T-ACE2 cells were infected with lentiviruses pseudotyped with vesicular stomatitis virus G (VSV-G) and SARS-CoV-2 S protein variants. The y -axis shows the relative luminescence units (RLU) detected at 48 h post-pseudovirus inoculation. (C) Optimization of the incubation time for pseudovirus luciferase assay. Luciferase activities were measured 24–72 h post-virus infection. For this purpose, 72 h was chosen as the optimized incubation time. The data are presented as the means ± standard deviations (SDs) of three independent biological replicates.

    Techniques Used: Infection, Neutralization, Incubation, Luciferase

    Related Articles

    Expressing:

    Article Title: Prime-boost vaccination of mice and Rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates
    Article Snippet: Western blottingHEK-293A cells were infected with Sad23L-nCoV-S and Ad49L-nCoV-S strains, respectively, and Sad23L-GFP and Ad49L-GFP vectorial viruses were used as mock control. .. The expression of SARS-CoV-2 S protein was analyzed by Western blotting with rabbit polyclonal antibody to SARS-CoV-2 RBD (Sino Biological, China) and heat-inactivated human serum samples from Chinese COVID-19 infected patients. ..

    Article Title: Human organs-on-chips as tools for repurposing approved drugs as potential influenza and COVID19 therapeutics in viral pandemics
    Article Snippet: Chloroquine was dissolved in water to a stock concentration of 10 mM; all other tested drugs were dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 10 mM. .. PlasmidsPlasmid expressing the spike protein of SARS-CoV-2 (pCMV3-SARS-CoV2-Spike) was purchased from Sino Biological Inc. (Beijing, China). pCMV-VSVG, pNL4-3.Luc.R-E-, and pAdvantage were obtained from Addgene, NIH AIDS Reagent Program, and Promega, respectively. .. All plasmids used for transfection were amplified using the Maxiprep Kit (Promega) according to the manufacturer’s instructions.

    Western Blot:

    Article Title: Prime-boost vaccination of mice and Rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates
    Article Snippet: Western blottingHEK-293A cells were infected with Sad23L-nCoV-S and Ad49L-nCoV-S strains, respectively, and Sad23L-GFP and Ad49L-GFP vectorial viruses were used as mock control. .. The expression of SARS-CoV-2 S protein was analyzed by Western blotting with rabbit polyclonal antibody to SARS-CoV-2 RBD (Sino Biological, China) and heat-inactivated human serum samples from Chinese COVID-19 infected patients. ..

    Infection:

    Article Title: Prime-boost vaccination of mice and Rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates
    Article Snippet: Western blottingHEK-293A cells were infected with Sad23L-nCoV-S and Ad49L-nCoV-S strains, respectively, and Sad23L-GFP and Ad49L-GFP vectorial viruses were used as mock control. .. The expression of SARS-CoV-2 S protein was analyzed by Western blotting with rabbit polyclonal antibody to SARS-CoV-2 RBD (Sino Biological, China) and heat-inactivated human serum samples from Chinese COVID-19 infected patients. ..

    Article Title: Targeting pentose phosphate pathway for SARS-CoV-2 therapy
    Article Snippet: Immunohistochemistry stainingViral infection was assessed by staining of SARS-CoV-2 spike protein. .. 24h post infection, cells were fixed with acetone:methanol (40:60) solution followed by incubation with a primary monoclonal antibody directed against the spike protein of SARS-CoV-2 (1:1500, Sinobiological). .. Primary antibody was detected with a peroxidase-conjugated anti-rabbit secondary antibody (1:1000, Dianova), followed by addition of AEC substrate.

    Recombinant:

    Article Title: Mucin-type O-glycosylation Landscapes of SARS-CoV-2 Spike Proteins
    Article Snippet: Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials were purchased from Fresh Bioscience (Shanghai, China). .. Recombinant SARS-CoV-2 S protein (S1+S2 ECD, His tag) expressed by insect cells (High Five) via a baculovirus, and S protein (S1, His tag) expressed by human embryonic kidney (HEK293) cells were purchased from Sino Biological (Beijing, China). .. Sequencing-grade trypsin and Glu-C were obtained from Enzyme & Spectrum (Beijing, China).

    Article Title: SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development
    Article Snippet: Hygromycin (Cat: V900372) were purchased from Sigma-Aldrich. .. SARS-CoV neutralizing antibodies were generated from mice (M103, M127) or rabbits (R314, R301, R325, R302, R258, R348) immunized with recombinant S1 protein of SARS-CoV. .. Luciferase assay system (Cat: E1501) was purchased from Promega.

    Article Title: A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells
    Article Snippet: Substrates were purchased from the following vendors: Z-FR-AMC (EMD Millipore), GF-AMC (MP Biomedicals), and Z-LR-AMC (Enzo Life Sciences, Inc.). .. Recombinant SARS-CoV-1 spike protein was obtained from SinoBiological and SARS-CoV-2 spike was obtained from Genscript and Acro Biosystems. .. SARS-CoV-2 protease assays Recombinant SARS-CoV-2 3CLpro (100 nM) was assayed at 25°C in either (a) in 30 μL reaction volumes containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 5 % glycerol, 0.01% Tween 20 100 μM of Mu-HSSKLQ-AMC (Sigma-Aldrich, SCP0224), in 384-well black plates in triplicate or (b) in 50 μL reaction mixtures containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 10% DMSO, and 25-50 nM 3CLpro in 96-well plates (Greiner, flat-bottom half volume, clear black plates), using the FRET-based substrate Abz-SAVLQSGFRK(DNP)-NH2 wherein peptidolysis was measured at 320/420 nm (ex/em) (Biotek® Synergy M2) in the presence and absence of 0-50 μM K777 in duplicate.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Monoclonal antibodies for the S2 subunit of spike of SARS-CoV-1 cross-react with the newly-emerged SARS-CoV-2
    Article Snippet: Images were acquired with Olympus CKX53 microscope using Olympus (Tokyo, Japan) LCAch N 20×/0.40 iPC objective lens and Olympus DP27 colour camera with Olympus cellSens software. .. ELISA Whole ectodomain of SARS-CoV-2 S protein with His Tag (Sino Biological Inc., Beijing, China; catalogue number: 40589-B08V1) was diluted with coating buffer (0.1 M NaHCO3 , 34 mM Na2 CO3) and a total of 20 ng of protein was loaded into individual wells of a 96 well plate (Nunc, Roskilde, Denmark) and allowed to coat overnight at 4 °C. .. Plates were then washed four times with 0.05% Tween 20 in PBS (PBST) and blocked with 5% bovine serum albumin (BSA)/PBST for 30 min before murine antibodies serially diluted with blocking buffer were added to desired wells for 1 hour.

    Generated:

    Article Title: SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development
    Article Snippet: Hygromycin (Cat: V900372) were purchased from Sigma-Aldrich. .. SARS-CoV neutralizing antibodies were generated from mice (M103, M127) or rabbits (R314, R301, R325, R302, R258, R348) immunized with recombinant S1 protein of SARS-CoV. .. Luciferase assay system (Cat: E1501) was purchased from Promega.

    Mouse Assay:

    Article Title: SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development
    Article Snippet: Hygromycin (Cat: V900372) were purchased from Sigma-Aldrich. .. SARS-CoV neutralizing antibodies were generated from mice (M103, M127) or rabbits (R314, R301, R325, R302, R258, R348) immunized with recombinant S1 protein of SARS-CoV. .. Luciferase assay system (Cat: E1501) was purchased from Promega.

    Incubation:

    Article Title: Targeting pentose phosphate pathway for SARS-CoV-2 therapy
    Article Snippet: Immunohistochemistry stainingViral infection was assessed by staining of SARS-CoV-2 spike protein. .. 24h post infection, cells were fixed with acetone:methanol (40:60) solution followed by incubation with a primary monoclonal antibody directed against the spike protein of SARS-CoV-2 (1:1500, Sinobiological). .. Primary antibody was detected with a peroxidase-conjugated anti-rabbit secondary antibody (1:1000, Dianova), followed by addition of AEC substrate.

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    Sino Biological sars cov 2 rbd protein
    Immunogenicity evaluation of a single mRNA-RBD vaccination. a – c Groups of BALB/c mice ( n = 6) were immunized with a single injection of mRNA-RBD at different doses or with a placebo via the i.m. route. Sera at 4 weeks post immunization were collected. <t>SARS-CoV-2</t> RBD-specific IgG ( a ) and neutralizing antibody titers in sera against pseudovirus ( b ) and live virus ( c ) infection were determined. d – h C57BL/6 mice ( n = 6) were inoculated with a single mRNA-RBD vaccination or a placebo. Serum samples were collected from mice at 4 weeks following vaccination. RBD-specific IgG titers and pseudovirus-neutralizing antibodies were measured as shown in d and e , respectively. f An ELISPOT assay was performed to evaluate the capacity of splenocytes to secrete IFNγ following re-stimulation with SARS-CoV-2 RBD peptide pools. g , h An ICS assay was conducted to quantify the proportions of IFNγ-secreting CD8 + ( g ) and CD4 + ( h ) T cells. mRNA-RBD-L indicates the low dose (2 μg). mRNA-RBD-H indicates the high dose (15 μg). HCS represents human convalescent sera. Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; mRNA-RBD-L vaccinated animals = blue triangles; mRNA-RBD-H vaccinated animals = red squares; HCS = brown circles; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.
    Sars Cov 2 Rbd Protein, 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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    95
    Sino Biological anti s2
    N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. A . Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] − and [N] − 293T cells. All 9 viruses were applied at equal titer to stable 293T/ACE2. B - C . O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. D . Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. E . Western blot of Spike protein using <t>anti-S2</t> Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] − 293T products due to truncation of glycan biosynthesis. F . Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] − 293Ts is almost fully proteolyzed during viral production (red arrowhead). * P
    Anti S2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Sino Biological sars cov2 s1
    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with <t>SARS‐CoV2‐RBG</t> and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and <t>SARS‐CoV2‐S1</t> proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.
    Sars Cov2 S1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Immunogenicity evaluation of a single mRNA-RBD vaccination. a – c Groups of BALB/c mice ( n = 6) were immunized with a single injection of mRNA-RBD at different doses or with a placebo via the i.m. route. Sera at 4 weeks post immunization were collected. SARS-CoV-2 RBD-specific IgG ( a ) and neutralizing antibody titers in sera against pseudovirus ( b ) and live virus ( c ) infection were determined. d – h C57BL/6 mice ( n = 6) were inoculated with a single mRNA-RBD vaccination or a placebo. Serum samples were collected from mice at 4 weeks following vaccination. RBD-specific IgG titers and pseudovirus-neutralizing antibodies were measured as shown in d and e , respectively. f An ELISPOT assay was performed to evaluate the capacity of splenocytes to secrete IFNγ following re-stimulation with SARS-CoV-2 RBD peptide pools. g , h An ICS assay was conducted to quantify the proportions of IFNγ-secreting CD8 + ( g ) and CD4 + ( h ) T cells. mRNA-RBD-L indicates the low dose (2 μg). mRNA-RBD-H indicates the high dose (15 μg). HCS represents human convalescent sera. Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; mRNA-RBD-L vaccinated animals = blue triangles; mRNA-RBD-H vaccinated animals = red squares; HCS = brown circles; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2

    doi: 10.1038/s41467-021-21037-2

    Figure Lengend Snippet: Immunogenicity evaluation of a single mRNA-RBD vaccination. a – c Groups of BALB/c mice ( n = 6) were immunized with a single injection of mRNA-RBD at different doses or with a placebo via the i.m. route. Sera at 4 weeks post immunization were collected. SARS-CoV-2 RBD-specific IgG ( a ) and neutralizing antibody titers in sera against pseudovirus ( b ) and live virus ( c ) infection were determined. d – h C57BL/6 mice ( n = 6) were inoculated with a single mRNA-RBD vaccination or a placebo. Serum samples were collected from mice at 4 weeks following vaccination. RBD-specific IgG titers and pseudovirus-neutralizing antibodies were measured as shown in d and e , respectively. f An ELISPOT assay was performed to evaluate the capacity of splenocytes to secrete IFNγ following re-stimulation with SARS-CoV-2 RBD peptide pools. g , h An ICS assay was conducted to quantify the proportions of IFNγ-secreting CD8 + ( g ) and CD4 + ( h ) T cells. mRNA-RBD-L indicates the low dose (2 μg). mRNA-RBD-H indicates the high dose (15 μg). HCS represents human convalescent sera. Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; mRNA-RBD-L vaccinated animals = blue triangles; mRNA-RBD-H vaccinated animals = red squares; HCS = brown circles; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Article Snippet: Briefly, a monoclonal antibody specific for SARS-CoV-2 RBD protein was pre-coated onto plate wells.

    Techniques: Mouse Assay, Injection, Infection, Enzyme-linked Immunospot, Two Tailed Test

    Duration and long-term protection of humoral response induced by mRNA-RBD. a Passive immunization and challenge schedule. The blue and red arrow indicates the time of vaccination and sera transfer, respectively. b , c Groups of BALB/c mice ( n = 10) received 15 μg of mRNA-RBD or a placebo. Half of the mice per group were euthanized at 8 weeks (short term) post vaccination, and massive sera were collected for further passive immunization. The other mice of the group were bled as desired and eventually euthanized at 26 weeks (long term) post vaccination to collect massive sera for further passive immunization. All serum samples were detected for IgG ( b ) and neutralizing antibodies ( c ) titers. d–e hACE2 transgenic mice ( n = 5) were administered 350 μl per mouse of pooled short- and long-term immune sera and one day later were challenged with 1 × 10 5 FFU of SARS-CoV-2 via the i.n. route. d The hACE2 mice weight change was recorded after challenge. e Virus titers in lung. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; animals for long-term study = blue triangles; animals for short-term study = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2

    doi: 10.1038/s41467-021-21037-2

    Figure Lengend Snippet: Duration and long-term protection of humoral response induced by mRNA-RBD. a Passive immunization and challenge schedule. The blue and red arrow indicates the time of vaccination and sera transfer, respectively. b , c Groups of BALB/c mice ( n = 10) received 15 μg of mRNA-RBD or a placebo. Half of the mice per group were euthanized at 8 weeks (short term) post vaccination, and massive sera were collected for further passive immunization. The other mice of the group were bled as desired and eventually euthanized at 26 weeks (long term) post vaccination to collect massive sera for further passive immunization. All serum samples were detected for IgG ( b ) and neutralizing antibodies ( c ) titers. d–e hACE2 transgenic mice ( n = 5) were administered 350 μl per mouse of pooled short- and long-term immune sera and one day later were challenged with 1 × 10 5 FFU of SARS-CoV-2 via the i.n. route. d The hACE2 mice weight change was recorded after challenge. e Virus titers in lung. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; animals for long-term study = blue triangles; animals for short-term study = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Article Snippet: Briefly, a monoclonal antibody specific for SARS-CoV-2 RBD protein was pre-coated onto plate wells.

    Techniques: Mouse Assay, Transgenic Assay, Two Tailed Test

    Protection efficacy of mRNA-RBD in hACE2 transgenic mice against SARS-CoV-2. a-d Groups of hACE2 transgenic mice ( n = 6) received one (prime group) or two (boost group) doses of mRNA-RBD-H or placebo via the i.m. route. Four weeks post initial vaccination, mice were challenged with 1 × 10 5 FFU of SARS-CoV-2 virus. a Mice immunization and challenge schedule. The blue arrows indicate the time of vaccination. b , c Sera collected at 4 weeks post initial vaccination were examined for IgG ( b ) and neutralizing antibody ( c ) titers. d Mice weight change after challenge. e Virus titers in lungs of challenged mice ( n = 4). f Representative histopathology (H E) of lungs in SARS-CoV-2-infected hACE2 mice (5 dpi). Infiltration of lymphocytes within alveolar spaces is indicated by yellow arrows. Scale bar, 100 μm. g Representative immunohistochemistry (IHC) of lung tissues with SARS-CoV-2 N-specific monoclonal antibodies. Virus is indicated by yellow arrows. Scale bar, 100 μm. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; one injection-animals = blue triangles; two injections-vaccinated animals = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2

    doi: 10.1038/s41467-021-21037-2

    Figure Lengend Snippet: Protection efficacy of mRNA-RBD in hACE2 transgenic mice against SARS-CoV-2. a-d Groups of hACE2 transgenic mice ( n = 6) received one (prime group) or two (boost group) doses of mRNA-RBD-H or placebo via the i.m. route. Four weeks post initial vaccination, mice were challenged with 1 × 10 5 FFU of SARS-CoV-2 virus. a Mice immunization and challenge schedule. The blue arrows indicate the time of vaccination. b , c Sera collected at 4 weeks post initial vaccination were examined for IgG ( b ) and neutralizing antibody ( c ) titers. d Mice weight change after challenge. e Virus titers in lungs of challenged mice ( n = 4). f Representative histopathology (H E) of lungs in SARS-CoV-2-infected hACE2 mice (5 dpi). Infiltration of lymphocytes within alveolar spaces is indicated by yellow arrows. Scale bar, 100 μm. g Representative immunohistochemistry (IHC) of lung tissues with SARS-CoV-2 N-specific monoclonal antibodies. Virus is indicated by yellow arrows. Scale bar, 100 μm. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; one injection-animals = blue triangles; two injections-vaccinated animals = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Article Snippet: Briefly, a monoclonal antibody specific for SARS-CoV-2 RBD protein was pre-coated onto plate wells.

    Techniques: Transgenic Assay, Mouse Assay, Histopathology, Infection, Immunohistochemistry, Two Tailed Test, Injection

    Construction and characterization of mRNA-RBD vaccine. a Schematic of the mRNA-RBD vaccine design. The SARS-CoV-2 mRNA encodes the signal peptide (SP), receptor-binding domain (RBD) from SARS-CoV-2 strain Wuhan/IVDC-HB-01/2019. b mRNA-RBD was transfected into HEK293T cells. RBD expression in the cell lysate and supernatant was analyzed by western blotting. c Particle size of LNPs by dynamic light scattering. d A representative cryo-electron microscopy image of a LNPs solution following mRNA encapsulation. Scale bar, 100 nm. e Zeta potential for LNPs at pH 4.0 and 7.4. For b and d , two independent experiments were carried out with similar results. For c and e , one representative result from three independent experiments is shown. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2

    doi: 10.1038/s41467-021-21037-2

    Figure Lengend Snippet: Construction and characterization of mRNA-RBD vaccine. a Schematic of the mRNA-RBD vaccine design. The SARS-CoV-2 mRNA encodes the signal peptide (SP), receptor-binding domain (RBD) from SARS-CoV-2 strain Wuhan/IVDC-HB-01/2019. b mRNA-RBD was transfected into HEK293T cells. RBD expression in the cell lysate and supernatant was analyzed by western blotting. c Particle size of LNPs by dynamic light scattering. d A representative cryo-electron microscopy image of a LNPs solution following mRNA encapsulation. Scale bar, 100 nm. e Zeta potential for LNPs at pH 4.0 and 7.4. For b and d , two independent experiments were carried out with similar results. For c and e , one representative result from three independent experiments is shown. Source data are provided as a Source Data file.

    Article Snippet: Briefly, a monoclonal antibody specific for SARS-CoV-2 RBD protein was pre-coated onto plate wells.

    Techniques: Binding Assay, Transfection, Expressing, Western Blot, Electron Microscopy

    N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. A . Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] − and [N] − 293T cells. All 9 viruses were applied at equal titer to stable 293T/ACE2. B - C . O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. D . Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. E . Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] − 293T products due to truncation of glycan biosynthesis. F . Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] − 293Ts is almost fully proteolyzed during viral production (red arrowhead). * P

    Journal: bioRxiv

    Article Title: Inhibition of SARS-CoV-2 viral entry in vitro upon blocking N- and O-glycan elaboration

    doi: 10.1101/2020.10.15.339838

    Figure Lengend Snippet: N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. A . Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] − and [N] − 293T cells. All 9 viruses were applied at equal titer to stable 293T/ACE2. B - C . O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. D . Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. E . Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] − 293T products due to truncation of glycan biosynthesis. F . Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] − 293Ts is almost fully proteolyzed during viral production (red arrowhead). * P

    Article Snippet: Identity of expressed protein and also viral Spike was determined using western blotting with anti-Fc (Jackson), anti-RBD (Sino Biologicals), anti-S2 (Sino Biologicals) and anti-ACE2 (R & D Systems) pAbs.

    Techniques: Modification, Expressing, Mutagenesis, Produced, Generated, Titration, Infection, Western Blot

    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.

    Journal: Small Methods

    Article Title: Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors, Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

    doi: 10.1002/smtd.202001031

    Figure Lengend Snippet: Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.

    Article Snippet: Generation and Production of Antibodies against SARS‐CoV‐2 S Balb/c mice were intraperitoneal immunized with 5 µg of SARS‐CoV2‐RBD (expression in this study, n = 5), SARS‐CoV2‐S1 (Sino Biological, 40591‐V08H, n = 3), and SARS‐CoV2‐S2 (Sino Biological, 40590‐V08B, n = 3), respectively.

    Techniques: Construct, Modification, Derivative Assay, Plasmid Preparation, Western Blot, Stable Transfection, Transfection, Fluorescence, Incubation, Imaging, Inhibition, Recombinant, Binding Assay, Blocking Assay