sars cov 2  (Sino Biological)


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    Name:
    Human SARS coronavirus Nucleoprotein NP Gene Lentiviral ORF cDNA expression plasmid C GFPSpark tag
    Description:
    Full length Clone DNA of Human SARS coronavirus SARS CoV Nucleoprotein NP DNA
    Catalog Number:
    VG40143-ACGLN
    Price:
    485.0
    Category:
    cDNA Clone
    Size:
    1Unit
    Product Aliases:
    coronavirus NP cDNA ORF Clone SARS, coronavirus Nucleocapsid cDNA ORF Clone SARS, coronavirus Nucleoprotein cDNA ORF Clone SARS, cov np cDNA ORF Clone SARS, ncov NP cDNA ORF Clone SARS, novel coronavirus NP cDNA ORF Clone SARS, novel coronavirus Nucleocapsid cDNA ORF Clone SARS, novel coronavirus Nucleoprotein cDNA ORF Clone SARS, NP cDNA ORF Clone SARS, Nucleocapsid cDNA ORF Clone SARS, Nucleoprotein cDNA ORF Clone SARS
    Molecule Name:
    NP-CoV,NP,CoV Nucleoprotein,
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    Structured Review

    Sino Biological sars cov 2
    Human SARS coronavirus Nucleoprotein NP Gene Lentiviral ORF cDNA expression plasmid C GFPSpark tag
    Full length Clone DNA of Human SARS coronavirus SARS CoV Nucleoprotein NP DNA
    https://www.bioz.com/result/sars cov 2/product/Sino Biological
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 - by Bioz Stars, 2021-04
    95/100 stars

    Images

    1) Product Images from "Cross-reactive antibody response between SARS-CoV-2 and SARS-CoV infections"

    Article Title: Cross-reactive antibody response between SARS-CoV-2 and SARS-CoV infections

    Journal: bioRxiv

    doi: 10.1101/2020.03.15.993097

    Human serological responses to SARS-CoV-2. (A) Schematic diagram of the SARS-CoV-2 spike protein. Locations of secretion signal peptide (SP), N-terminal domain (NTD), receptor-binding domain (RBD), S1/S2 cleavage site, fusion peptide (FP), S2’ cleavage site, internal fusion peptide (IFP), heptad repeat 1 (HR1), heptad repeat 1 (HR2), transmembrane domain (TM), and cytoplasmic domain (CP) are indicated. Regions corresponding to the S1, S2, S2’ subunits, and ectodomain are also indicated. (B) Binding of plasma from healthy donors and SARS-CoV-2 infected patients to SARS-CoV-2 spike protein, SARS-CoV-2 RBD protein, SARS-CoV-2 S2 subunit, SARS-CoV spike protein and SARS-CoV RBD protein were measured by ELISA. The mean OD 450 values calculated after testing each plasma sample in triplicate are shown. (C) Neutralization activities of plasma from SARS-CoV-2 infected patients to SARS-CoV-2 and SARS-CoV viruses were measured. Dashed line represents the lower detection limit. Black lines indicate mean +/- standard deviation. (B-C) Grey: plasma samples from healthy donors. Orange: plasma samples from SARS-CoV-2-infected patients. Blue: plasma samples from SARS-CoV-infected patients.
    Figure Legend Snippet: Human serological responses to SARS-CoV-2. (A) Schematic diagram of the SARS-CoV-2 spike protein. Locations of secretion signal peptide (SP), N-terminal domain (NTD), receptor-binding domain (RBD), S1/S2 cleavage site, fusion peptide (FP), S2’ cleavage site, internal fusion peptide (IFP), heptad repeat 1 (HR1), heptad repeat 1 (HR2), transmembrane domain (TM), and cytoplasmic domain (CP) are indicated. Regions corresponding to the S1, S2, S2’ subunits, and ectodomain are also indicated. (B) Binding of plasma from healthy donors and SARS-CoV-2 infected patients to SARS-CoV-2 spike protein, SARS-CoV-2 RBD protein, SARS-CoV-2 S2 subunit, SARS-CoV spike protein and SARS-CoV RBD protein were measured by ELISA. The mean OD 450 values calculated after testing each plasma sample in triplicate are shown. (C) Neutralization activities of plasma from SARS-CoV-2 infected patients to SARS-CoV-2 and SARS-CoV viruses were measured. Dashed line represents the lower detection limit. Black lines indicate mean +/- standard deviation. (B-C) Grey: plasma samples from healthy donors. Orange: plasma samples from SARS-CoV-2-infected patients. Blue: plasma samples from SARS-CoV-infected patients.

    Techniques Used: Binding Assay, Infection, Enzyme-linked Immunosorbent Assay, Neutralization, Standard Deviation

    Mouse serological response to SARS-CoV-2 and SARS-CoV. (A-D) Binding of plasma from OC43-CoV-immunized mice, SARS-CoV-immunized mice, SARS-CoV-infected mice and mock-immunized mice against (A) SARS-CoV-2 spike protein, (B) SARS-CoV-2 RBD protein, (C) SARS-CoV spike protein and (D) SARS-CoV RBD protein were measured by ELISA. Since both SARS-CoV spike protein and SARS-CoV-2 spike contained a C-terminal foldon domain, binding of plasma from mice immunized with SARS-CoV spike protein plasma was not tested against spike proteins from SARS-CoV and SARS-CoV-2. The mean OD 450 values calculated after testing each plasma sample in triplicate are shown. (E-F) Neutralization activities of plasma from mice infected or immunized by SARS-CoV-2 or SARS-CoV to (E) SARS-CoV-2 virus or (F) SARS-CoV virus were measured. Dashed line represents the lower detection limit. Black lines indicate mean +/- standard deviation.
    Figure Legend Snippet: Mouse serological response to SARS-CoV-2 and SARS-CoV. (A-D) Binding of plasma from OC43-CoV-immunized mice, SARS-CoV-immunized mice, SARS-CoV-infected mice and mock-immunized mice against (A) SARS-CoV-2 spike protein, (B) SARS-CoV-2 RBD protein, (C) SARS-CoV spike protein and (D) SARS-CoV RBD protein were measured by ELISA. Since both SARS-CoV spike protein and SARS-CoV-2 spike contained a C-terminal foldon domain, binding of plasma from mice immunized with SARS-CoV spike protein plasma was not tested against spike proteins from SARS-CoV and SARS-CoV-2. The mean OD 450 values calculated after testing each plasma sample in triplicate are shown. (E-F) Neutralization activities of plasma from mice infected or immunized by SARS-CoV-2 or SARS-CoV to (E) SARS-CoV-2 virus or (F) SARS-CoV virus were measured. Dashed line represents the lower detection limit. Black lines indicate mean +/- standard deviation.

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

    2) Product Images from "A single dose of recombinant VSV-∆G-spike vaccine provides protection against SARS-CoV-2 challenge"

    Article Title: A single dose of recombinant VSV-∆G-spike vaccine provides protection against SARS-CoV-2 challenge

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20228-7

    A single-dose i.m. rVSV-∆G-spike vaccine safety and efficacy in hamsters following SARS-CoV-2 challenge. a Body weight changes of mock-vaccinated hamsters ( n = 4), and hamsters vaccinated with rVSV-∆G-spike ranging from 10 4 to 10 8 pfu/hamster ( n = 10, n = 11, n = 11, n = 11, n = 10, for each vaccinated group, respectively). b NT 50 values of i.m. vaccinated hamsters’ sera (10 4 –10 8 pfu/hamster) against SARS-CoV-2 ( n = 4 for 10 4 , 10 6 , 10 7 , and 10 8 , n = 3 for 10 5 ). Means and SEM are indicated below the graph. c Representative immunofluorescence images of Vero E6 cells infected with SARS-CoV-2 (upper panels) or uninfected (lower panels), labeled with serum from either naive (left panel) or rVSV-∆G-spike (10 6 pfu/hamster) i.m. vaccinated hamsters (right panel). Representative images of three experiments are presented. Scale bars: 50 µm. d Body weight changes of hamsters infected with SARS-CoV-2, and hamsters vaccinated with 10 4 –10 8 pfu/hamster and infected with 5 × 10 6 pfu/hamster 25 days post vaccination. Arrow indicates 5 dpi—hamsters were sacrificed and lungs were removed for viral load. For vaccinated groups 10 4 –10 7 : days 0–5 ( n = 12), days 6–12 ( n = 9), 10 8 group: days 0–5 ( n = 11), day 6–12 (n = 8). For unvaccinated infected: days 0–5 ( n = 14), days 6–12 ( n = 12). Statistical significance was determined using two-tailed one unpaired t -test per row, with correction for multiple comparisons using Holm–Sidak method. p
    Figure Legend Snippet: A single-dose i.m. rVSV-∆G-spike vaccine safety and efficacy in hamsters following SARS-CoV-2 challenge. a Body weight changes of mock-vaccinated hamsters ( n = 4), and hamsters vaccinated with rVSV-∆G-spike ranging from 10 4 to 10 8 pfu/hamster ( n = 10, n = 11, n = 11, n = 11, n = 10, for each vaccinated group, respectively). b NT 50 values of i.m. vaccinated hamsters’ sera (10 4 –10 8 pfu/hamster) against SARS-CoV-2 ( n = 4 for 10 4 , 10 6 , 10 7 , and 10 8 , n = 3 for 10 5 ). Means and SEM are indicated below the graph. c Representative immunofluorescence images of Vero E6 cells infected with SARS-CoV-2 (upper panels) or uninfected (lower panels), labeled with serum from either naive (left panel) or rVSV-∆G-spike (10 6 pfu/hamster) i.m. vaccinated hamsters (right panel). Representative images of three experiments are presented. Scale bars: 50 µm. d Body weight changes of hamsters infected with SARS-CoV-2, and hamsters vaccinated with 10 4 –10 8 pfu/hamster and infected with 5 × 10 6 pfu/hamster 25 days post vaccination. Arrow indicates 5 dpi—hamsters were sacrificed and lungs were removed for viral load. For vaccinated groups 10 4 –10 7 : days 0–5 ( n = 12), days 6–12 ( n = 9), 10 8 group: days 0–5 ( n = 11), day 6–12 (n = 8). For unvaccinated infected: days 0–5 ( n = 14), days 6–12 ( n = 12). Statistical significance was determined using two-tailed one unpaired t -test per row, with correction for multiple comparisons using Holm–Sidak method. p

    Techniques Used: Immunofluorescence, Infection, Labeling, Two Tailed Test

    Th1 and Th2 isotytpe analysis of rVSV-∆G-spike induced antibodies. rVSV-∆G-spike vaccinated (10 7 pfu/mouse, n = 7) C57BL/6J mice sera analysis for a NT 50 values against SARS-CoV-2 as determined by PRNT, and b levels of S2P specific binding antibodies: total IgG, IgG2c, and IgG1 as determined by ELISA. Statistical significance was determined using one-way ANOVA nonparametric test, with Kruskal–Wallis test: **** p
    Figure Legend Snippet: Th1 and Th2 isotytpe analysis of rVSV-∆G-spike induced antibodies. rVSV-∆G-spike vaccinated (10 7 pfu/mouse, n = 7) C57BL/6J mice sera analysis for a NT 50 values against SARS-CoV-2 as determined by PRNT, and b levels of S2P specific binding antibodies: total IgG, IgG2c, and IgG1 as determined by ELISA. Statistical significance was determined using one-way ANOVA nonparametric test, with Kruskal–Wallis test: **** p

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

    Viral load analysis in organs of rVSV-∆G-spike vaccinated hamsters following SARS-CoV-2 challenge. a Body weight changes of SARS-CoV-2 infected hamsters (5 × 10 6 pfu) 25 days post vaccination with 1 × 10 6 pfu, or unvaccinated. Arrows indicate 3 and 7 dpi—hamsters were sacrificed. Number of animals per group: days 0–3 n = 15, days 4–7 n = 7, days 8–12 n = 3. Statistical significance was performed by using two-tailed one unpaired t -test per row, with correction for multiple comparisons using Holm–Sidak method, * p
    Figure Legend Snippet: Viral load analysis in organs of rVSV-∆G-spike vaccinated hamsters following SARS-CoV-2 challenge. a Body weight changes of SARS-CoV-2 infected hamsters (5 × 10 6 pfu) 25 days post vaccination with 1 × 10 6 pfu, or unvaccinated. Arrows indicate 3 and 7 dpi—hamsters were sacrificed. Number of animals per group: days 0–3 n = 15, days 4–7 n = 7, days 8–12 n = 3. Statistical significance was performed by using two-tailed one unpaired t -test per row, with correction for multiple comparisons using Holm–Sidak method, * p

    Techniques Used: Infection, Two Tailed Test

    Histopathological analysis of rVSV-∆G-spike i.m. vaccinated and infected hamsters’ lungs at 3 and 7 dpi. General histology (H E) and SARS-CoV-2 DAB immunolabeling of naive, unvaccinated infected (5 × 10 6 ), and vaccinated (10 6 pfu) hamsters’ lungs, at 3 and 7 dpi. Lungs were isolated and processed for paraffin embedding from naive ( a , f ), infected (5 × 10 6 pfu) ( b , g for 3 dpi and d , i for 7 dpi) and vaccinated and infected ( c , h for 3 dpi and e , j for 7 dpi). Sections (4 µm) were taken for H E staining ( a – e ) and SARS-CoV-2 DAB immunolabeling ( f – j , positive SARS-CoV-2—brown, hematoxylin counterstaining—blue). An asterisk (*) indicates cellular debris in bronchiolar lumen. Black arrow heads indicate congestion of blood in blood vessels. Black arrows indicate positive stained cells. a – e : scale bar = 200 µm; f – j : scale bar = 20 µm. k Histopathological severity analysis of hamsters lungs of naive, vaccinated, and infected lungs, at 3 and 7 dpi. l Digital morphometric analysis of DAB immunohistochemical staining for SARS-CoV-2 in lungs of naive, infected, and vaccinated lungs, at 3 and 7 dpi. m Tissue/air space analysis of naive, infected, and vaccinated lungs, at 3 and 7 dpi. Data for a – j was taken for five groups. Each group includes four animals. For each animal, five fields were imaged and analyzed. Data for k – m are presented as mean values ± SEM. Statistical analyses for k – m were performed by one-way ANOVA with Tukey’s multiple comparisons test, with p
    Figure Legend Snippet: Histopathological analysis of rVSV-∆G-spike i.m. vaccinated and infected hamsters’ lungs at 3 and 7 dpi. General histology (H E) and SARS-CoV-2 DAB immunolabeling of naive, unvaccinated infected (5 × 10 6 ), and vaccinated (10 6 pfu) hamsters’ lungs, at 3 and 7 dpi. Lungs were isolated and processed for paraffin embedding from naive ( a , f ), infected (5 × 10 6 pfu) ( b , g for 3 dpi and d , i for 7 dpi) and vaccinated and infected ( c , h for 3 dpi and e , j for 7 dpi). Sections (4 µm) were taken for H E staining ( a – e ) and SARS-CoV-2 DAB immunolabeling ( f – j , positive SARS-CoV-2—brown, hematoxylin counterstaining—blue). An asterisk (*) indicates cellular debris in bronchiolar lumen. Black arrow heads indicate congestion of blood in blood vessels. Black arrows indicate positive stained cells. a – e : scale bar = 200 µm; f – j : scale bar = 20 µm. k Histopathological severity analysis of hamsters lungs of naive, vaccinated, and infected lungs, at 3 and 7 dpi. l Digital morphometric analysis of DAB immunohistochemical staining for SARS-CoV-2 in lungs of naive, infected, and vaccinated lungs, at 3 and 7 dpi. m Tissue/air space analysis of naive, infected, and vaccinated lungs, at 3 and 7 dpi. Data for a – j was taken for five groups. Each group includes four animals. For each animal, five fields were imaged and analyzed. Data for k – m are presented as mean values ± SEM. Statistical analyses for k – m were performed by one-way ANOVA with Tukey’s multiple comparisons test, with p

    Techniques Used: Infection, Immunolabeling, Isolation, Staining, Immunohistochemistry

    rVSV-∆G-spike design and generation strategy. a A schematic diagram of the genomic organization of WT-VSV (top diagram), and rVSV-∆G-spike (bottom diagram). N nucleoprotein, P phosphoprotein, M matrix, L large polymerase, G glycoprotein, SPIKE SARS-CoV-2 spike. b pVSV-∆G-spike map. c Schematic representation of the generation process of rVSV-∆G-spike vaccine. Infection of BHK-21 cells with MVA-T7, followed by cotransfection with pVSV-∆G-spike, and VSV-system accessory plasmids; transfection of BHK-21 cells with pCAGGS-VSV-G, followed by infection with the supernatant of the primary transfection, to create P1; sequential passaging in Vero E6 cells were performed creating rVSV-∆G-spike.
    Figure Legend Snippet: rVSV-∆G-spike design and generation strategy. a A schematic diagram of the genomic organization of WT-VSV (top diagram), and rVSV-∆G-spike (bottom diagram). N nucleoprotein, P phosphoprotein, M matrix, L large polymerase, G glycoprotein, SPIKE SARS-CoV-2 spike. b pVSV-∆G-spike map. c Schematic representation of the generation process of rVSV-∆G-spike vaccine. Infection of BHK-21 cells with MVA-T7, followed by cotransfection with pVSV-∆G-spike, and VSV-system accessory plasmids; transfection of BHK-21 cells with pCAGGS-VSV-G, followed by infection with the supernatant of the primary transfection, to create P1; sequential passaging in Vero E6 cells were performed creating rVSV-∆G-spike.

    Techniques Used: Infection, Cotransfection, Transfection, Passaging

    Characterization of rVSV-∆G-spike. a A table summarizing the genome analysis of several passages of rVSV-∆G-spike, showing elimination of VSV-G over time, together with increased titer, and formation of plaques. NA not applicable. “-” not evaluated. b Representative immunofluorescence images of Vero E6 cells infected with early passage (P5)-rVSV-∆G-spike, or late passage (P13)-rVSV-∆G-spike, stained with a SARS-CoV-2 antibody (green), and counterstained with DAPI for nuclei staining (blue). Bottom panels show insets at large magnification. Scale bars: 50 µm. rVSV-∆G-spike at P5 formed syncitia, whereas P13 showed individual infected cells, with no evidence of syncitia. c Transmission electron micrographs of P14 rVSV-∆G-spike (top panels) compared to WT-VSV (bottom panels). Right panels show immunogold labeling using gold nanoparticles conjugated antibodies directed to the spikes’ RBD. Data for b and c are representative of four and five experiments, respectively.
    Figure Legend Snippet: Characterization of rVSV-∆G-spike. a A table summarizing the genome analysis of several passages of rVSV-∆G-spike, showing elimination of VSV-G over time, together with increased titer, and formation of plaques. NA not applicable. “-” not evaluated. b Representative immunofluorescence images of Vero E6 cells infected with early passage (P5)-rVSV-∆G-spike, or late passage (P13)-rVSV-∆G-spike, stained with a SARS-CoV-2 antibody (green), and counterstained with DAPI for nuclei staining (blue). Bottom panels show insets at large magnification. Scale bars: 50 µm. rVSV-∆G-spike at P5 formed syncitia, whereas P13 showed individual infected cells, with no evidence of syncitia. c Transmission electron micrographs of P14 rVSV-∆G-spike (top panels) compared to WT-VSV (bottom panels). Right panels show immunogold labeling using gold nanoparticles conjugated antibodies directed to the spikes’ RBD. Data for b and c are representative of four and five experiments, respectively.

    Techniques Used: Immunofluorescence, Infection, Staining, Transmission Assay, Labeling

    Establishment of a golden Syrian hamster SARS-CoV-2 model. a Body weight changes of hamsters infected with 5 × 10 4 ( n = 8), 5 × 10 5 ( n = 8), or 5 × 10 6 ( n = 8) pfu/hamster of SARS-CoV-2, compared to mock-infected hamsters ( n = 4). Table shows days of significant differences, relative to mock infection. Statistical analysis was performed using one unpaired t -test per row, with correction for multiple comparisons using the Holm–Sidak method, p
    Figure Legend Snippet: Establishment of a golden Syrian hamster SARS-CoV-2 model. a Body weight changes of hamsters infected with 5 × 10 4 ( n = 8), 5 × 10 5 ( n = 8), or 5 × 10 6 ( n = 8) pfu/hamster of SARS-CoV-2, compared to mock-infected hamsters ( n = 4). Table shows days of significant differences, relative to mock infection. Statistical analysis was performed using one unpaired t -test per row, with correction for multiple comparisons using the Holm–Sidak method, p

    Techniques Used: Infection

    Antigenic similarity of rVSV-ΔG-spike and SARS-CoV-2. a Immunofluorescent images of Vero E6 cells infected with either WT-VSV (left panel), rVSV-ΔG-spike (middle panel), or SARS-CoV-2, stained with COVID-19 human convalescent serum. Representative images of five experiments are presented. Scale bars: 50 µm. b Correlation analysis of neutralization of rVSV-∆G-spike and SARS-CoV-2 by a panel of sera from COVID-19 convalescent patients. For each serum sample ( n = 12), NT 50 values were determined for neutralization of rVSV-∆G-spike or SARS-CoV-2. The NT 50 values were plotted to determine the correlation between the neutralization assays. Spearman’s correlation r and p values are indicated. Source data are provided as a Source Data file.
    Figure Legend Snippet: Antigenic similarity of rVSV-ΔG-spike and SARS-CoV-2. a Immunofluorescent images of Vero E6 cells infected with either WT-VSV (left panel), rVSV-ΔG-spike (middle panel), or SARS-CoV-2, stained with COVID-19 human convalescent serum. Representative images of five experiments are presented. Scale bars: 50 µm. b Correlation analysis of neutralization of rVSV-∆G-spike and SARS-CoV-2 by a panel of sera from COVID-19 convalescent patients. For each serum sample ( n = 12), NT 50 values were determined for neutralization of rVSV-∆G-spike or SARS-CoV-2. The NT 50 values were plotted to determine the correlation between the neutralization assays. Spearman’s correlation r and p values are indicated. Source data are provided as a Source Data file.

    Techniques Used: Infection, Staining, Neutralization

    3) Product Images from "Human organs-on-chips as tools for repurposing approved drugs as potential influenza and COVID19 therapeutics in viral pandemics"

    Article Title: Human organs-on-chips as tools for repurposing approved drugs as potential influenza and COVID19 therapeutics in viral pandemics

    Journal: bioRxiv

    doi: 10.1101/2020.04.13.039917

    Effects of FDA-approved drugs on pseudotyped SARS-CoV-2 viral entry in Huh-7 cells versus human Airway Chips. ( A ) Graphs showing the inhibitory effects of chloroquine, arbidol, toremifene, clomiphene, amodiaquine, verapamil, and amiodarone when added at 0, 1, or 5 uM to Huh-7 cells infected with SARS-CoV-2pp for 72 h (black bars). The number of pseudoparticles in the infected cells was quantified by measuring luciferase activity; viral entry in untreated cells was set as 100%. VSVpp were tested in parallel to exclude toxic and nonspecific effects of the drugs tested (grey bars). ( B ) The efficacy of the same drugs in human Airway Chips infected with CoV-2pp. Chloroquine, Arbidol, toremifene, clomiphene, amodiaquine, Verapamil, and Amiodarone were delivered into apical and basal channels of the chip at their respective C max in human blood, and ne day later chips were infected with CoV-2pp while in the continued presence of the drugs for 2 more days. The epithelium from the chips were collected for detection of viral pol gene by qRT-PCR; viral entry in untreated chips was set as 100%. *, P
    Figure Legend Snippet: Effects of FDA-approved drugs on pseudotyped SARS-CoV-2 viral entry in Huh-7 cells versus human Airway Chips. ( A ) Graphs showing the inhibitory effects of chloroquine, arbidol, toremifene, clomiphene, amodiaquine, verapamil, and amiodarone when added at 0, 1, or 5 uM to Huh-7 cells infected with SARS-CoV-2pp for 72 h (black bars). The number of pseudoparticles in the infected cells was quantified by measuring luciferase activity; viral entry in untreated cells was set as 100%. VSVpp were tested in parallel to exclude toxic and nonspecific effects of the drugs tested (grey bars). ( B ) The efficacy of the same drugs in human Airway Chips infected with CoV-2pp. Chloroquine, Arbidol, toremifene, clomiphene, amodiaquine, Verapamil, and Amiodarone were delivered into apical and basal channels of the chip at their respective C max in human blood, and ne day later chips were infected with CoV-2pp while in the continued presence of the drugs for 2 more days. The epithelium from the chips were collected for detection of viral pol gene by qRT-PCR; viral entry in untreated chips was set as 100%. *, P

    Techniques Used: Infection, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR

    Characterization of the SARS-CoV-2pp and their entry into Huh-7 cells. ( A ) Western blot analysis of SARS-CoV-2 S protein in the lysate of the HEK293T packaging cell line and in pseudotyped virions in the supernatant showing that both uncleaved full-length (S1+S2; ∼180 kDa) and cleaved forms (∼90 kDa) of the spike protein are present in the virions. A recombinant protein containing the receptor binding region domain from S1 (RBD fragment) was used as a positive control, and results were compared to cellular GAPDH. ( B ) Huh-7 cells were infected with SARS-CoV-2pp for 72 h. Luciferase activity was measured to estimate the number of pseudoparticles in the host cells; pseudoparticles without SARS-CoV-2 spike protein were used as control.
    Figure Legend Snippet: Characterization of the SARS-CoV-2pp and their entry into Huh-7 cells. ( A ) Western blot analysis of SARS-CoV-2 S protein in the lysate of the HEK293T packaging cell line and in pseudotyped virions in the supernatant showing that both uncleaved full-length (S1+S2; ∼180 kDa) and cleaved forms (∼90 kDa) of the spike protein are present in the virions. A recombinant protein containing the receptor binding region domain from S1 (RBD fragment) was used as a positive control, and results were compared to cellular GAPDH. ( B ) Huh-7 cells were infected with SARS-CoV-2pp for 72 h. Luciferase activity was measured to estimate the number of pseudoparticles in the host cells; pseudoparticles without SARS-CoV-2 spike protein were used as control.

    Techniques Used: Western Blot, Recombinant, Binding Assay, Positive Control, Infection, Luciferase, Activity Assay

    Graphs showing the Ct values of viral pol gene detected by qPCR in the human Airway Chips infected with CoV-2pp. Pseudoparticles without the spike protein of SARS-CoV-2 were used as control (Ctrl).
    Figure Legend Snippet: Graphs showing the Ct values of viral pol gene detected by qPCR in the human Airway Chips infected with CoV-2pp. Pseudoparticles without the spike protein of SARS-CoV-2 were used as control (Ctrl).

    Techniques Used: Real-time Polymerase Chain Reaction, Infection

    4) Product Images from "A key linear epitope for a potent neutralizing antibody to SARS-CoV-2 S-RBD"

    Article Title: A key linear epitope for a potent neutralizing antibody to SARS-CoV-2 S-RBD

    Journal: bioRxiv

    doi: 10.1101/2020.09.11.292631

    Assessment of the neutralizing capabilities of the NAbs against SARS-CoV-2 and its mutated S D614G . Neutralizing potency measured by the neutralization assay against authentic SARS-CoV-2 (a), SARS-CoV-2 pseudovirus (b) and its mutated type S D614G (c). Data for each NAb were obtained from a representative neutralization experiment, with three replicates. Data are presented as mean ± SEM.
    Figure Legend Snippet: Assessment of the neutralizing capabilities of the NAbs against SARS-CoV-2 and its mutated S D614G . Neutralizing potency measured by the neutralization assay against authentic SARS-CoV-2 (a), SARS-CoV-2 pseudovirus (b) and its mutated type S D614G (c). Data for each NAb were obtained from a representative neutralization experiment, with three replicates. Data are presented as mean ± SEM.

    Techniques Used: Neutralization

    Epitope mapping of mAbs and the analysis of NAbs from different clusters. (a) Epitope mapping of purified mAbs targeting three independent epitopes (13G9e, 81A11e and CR3022e). The all NAbs detected by authentic SARS-CoV-2 CPE assay or SARS-CoV-2 pseudovirus neutralizing assay were labelled in red and the top 20 NAbs were indicated by orange stars. The combination effects of 13G9 (b) or 58G6 (c) with 07C1, 57F7 and 510H2, and 510A5 (d) with 51A1 and 51D3 against authentic SARS-CoV-2 were quantified by RT-qPCR, analyzing the efficiency of NAbs neutralization.
    Figure Legend Snippet: Epitope mapping of mAbs and the analysis of NAbs from different clusters. (a) Epitope mapping of purified mAbs targeting three independent epitopes (13G9e, 81A11e and CR3022e). The all NAbs detected by authentic SARS-CoV-2 CPE assay or SARS-CoV-2 pseudovirus neutralizing assay were labelled in red and the top 20 NAbs were indicated by orange stars. The combination effects of 13G9 (b) or 58G6 (c) with 07C1, 57F7 and 510H2, and 510A5 (d) with 51A1 and 51D3 against authentic SARS-CoV-2 were quantified by RT-qPCR, analyzing the efficiency of NAbs neutralization.

    Techniques Used: Purification, Neutralizing Assay, Quantitative RT-PCR, Neutralization

    Characteristics of potential NAbs to SARS-CoV-2 S-RBD. (a) The heatmap of the selected NAbs binding to either SARS-CoV-2 S1, SARS-CoV-2 S-RBD or SARS-CoV-2 SD614G (left) and the affinity to SARS-CoV-2 S-RBD (right). The NAbs are ranked by their neutralizing potency to authentic SARS-CoV-2. The EC50 values of NAbs are visualized for binding. Data are representative of at least 2 independent experiments performed in technical duplicate. (b) The CDR3H (left) and CDR3L (right) sequences of the top 20 NAbs were aligned and the same amino acid residues were highlighted in yellow. The lengths of CDR3H and CDR3L sequence were labelled beside.
    Figure Legend Snippet: Characteristics of potential NAbs to SARS-CoV-2 S-RBD. (a) The heatmap of the selected NAbs binding to either SARS-CoV-2 S1, SARS-CoV-2 S-RBD or SARS-CoV-2 SD614G (left) and the affinity to SARS-CoV-2 S-RBD (right). The NAbs are ranked by their neutralizing potency to authentic SARS-CoV-2. The EC50 values of NAbs are visualized for binding. Data are representative of at least 2 independent experiments performed in technical duplicate. (b) The CDR3H (left) and CDR3L (right) sequences of the top 20 NAbs were aligned and the same amino acid residues were highlighted in yellow. The lengths of CDR3H and CDR3L sequence were labelled beside.

    Techniques Used: Binding Assay, Sequencing

    5) Product Images from "COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2"

    Article Title: COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008762

    Classification and structure of coronavirus. (A) Classification of coronaviruses: the 7 known HCoVs are shown in green and red. HCoVs in red bind the host receptor ACE2. (B) Schematic of the SARS-CoV-2 structure; the illustration of the virus is adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . (C) Cartoon depicts key features and the trimeric structure of the SARS-CoV-2 S protein. (D) Schematic of SARS-CoV-2 genome (top) and S protein (bottom); annotations are adapted from NCBI (NC_045512.2) and Expasy ( https://covid-19.uniprot.org/uniprotkb/P0DTC2 ), respectively. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; E, envelope; HCoV, Human Coronavirus; HR1/2, heptad repeat 1/2; M, membrane; N, nucleocapsid; Nsp, nonstructural protein; NTD, N-terminal domain; orf, open reading frame; RBD, receptor-binding domain; RBM, receptor-binding motif; RdRp, RNA-dependent RNA polymerase; S protein, spike protein; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2; UTR, untranslated region.
    Figure Legend Snippet: Classification and structure of coronavirus. (A) Classification of coronaviruses: the 7 known HCoVs are shown in green and red. HCoVs in red bind the host receptor ACE2. (B) Schematic of the SARS-CoV-2 structure; the illustration of the virus is adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . (C) Cartoon depicts key features and the trimeric structure of the SARS-CoV-2 S protein. (D) Schematic of SARS-CoV-2 genome (top) and S protein (bottom); annotations are adapted from NCBI (NC_045512.2) and Expasy ( https://covid-19.uniprot.org/uniprotkb/P0DTC2 ), respectively. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; E, envelope; HCoV, Human Coronavirus; HR1/2, heptad repeat 1/2; M, membrane; N, nucleocapsid; Nsp, nonstructural protein; NTD, N-terminal domain; orf, open reading frame; RBD, receptor-binding domain; RBM, receptor-binding motif; RdRp, RNA-dependent RNA polymerase; S protein, spike protein; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2; UTR, untranslated region.

    Techniques Used: Binding Assay

    Structure of the SARS-CoV-2 S protein alone and in complex with ACE2 receptor. (A) Side view of the trimeric SARS-CoV-2 S ectodomain in the prefusion state (PDB: 6VSB). The protomer in green is in the “up” conformation, and the other 2 protomers in red and cyan are in “down” conformation. (B) Top view of the trimeric S protein showing RBDs in red, blue, and green on each protomer. (C) Structure of a single protomer showing the receptor-binding subunit S1 (blue) and the membrane-fusion subunit S2 (green). The furin-like protease site at the boundary of S1/S2 subunits is depicted. (D) The S1 subunit showing the RBM in the CTD region (blue) and the NTD region (brown). The S2 subunit showing the fusion peptide (red), second cleavage site S2′ (black), and HR1 (pink). (E) Structure of the RBD, core subdomain (green), and RBM (blue) (PDB: 6LZG). (F) SARS-CoV-2-RBD:ACE2 receptor polar interface shown by specific residues. (G) Structure of the SARS-CoV-2-RBD in complex with ACE2 receptor (PDB: 6LZG). (H) Structural similarity between the SARS-CoV-RBD:hACE2 (green) and SARS-CoV-2-S-CTD:hACE2 (yellow) complexes. (I) Crystal structure of the SARS-CoV-2-RBD (green) in complex with a monoclonal antibody CR3022 (orange). The RBM and CR3022 binding sites do not overlap and are distantly located on the RBD (PDB: 6W41). The figures were prepared using Pymol. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; hACE2, human ACE2; HR1, heptad repeat 1; NTD, N-terminal domain; PDB, Protein Data Bank; RBD, receptor-binding domain; RBM, receptor-binding motif; S protein, spike protein; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Structure of the SARS-CoV-2 S protein alone and in complex with ACE2 receptor. (A) Side view of the trimeric SARS-CoV-2 S ectodomain in the prefusion state (PDB: 6VSB). The protomer in green is in the “up” conformation, and the other 2 protomers in red and cyan are in “down” conformation. (B) Top view of the trimeric S protein showing RBDs in red, blue, and green on each protomer. (C) Structure of a single protomer showing the receptor-binding subunit S1 (blue) and the membrane-fusion subunit S2 (green). The furin-like protease site at the boundary of S1/S2 subunits is depicted. (D) The S1 subunit showing the RBM in the CTD region (blue) and the NTD region (brown). The S2 subunit showing the fusion peptide (red), second cleavage site S2′ (black), and HR1 (pink). (E) Structure of the RBD, core subdomain (green), and RBM (blue) (PDB: 6LZG). (F) SARS-CoV-2-RBD:ACE2 receptor polar interface shown by specific residues. (G) Structure of the SARS-CoV-2-RBD in complex with ACE2 receptor (PDB: 6LZG). (H) Structural similarity between the SARS-CoV-RBD:hACE2 (green) and SARS-CoV-2-S-CTD:hACE2 (yellow) complexes. (I) Crystal structure of the SARS-CoV-2-RBD (green) in complex with a monoclonal antibody CR3022 (orange). The RBM and CR3022 binding sites do not overlap and are distantly located on the RBD (PDB: 6W41). The figures were prepared using Pymol. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; hACE2, human ACE2; HR1, heptad repeat 1; NTD, N-terminal domain; PDB, Protein Data Bank; RBD, receptor-binding domain; RBM, receptor-binding motif; S protein, spike protein; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Binding Assay

    Cryo-EM structure of RdRp of SARS-CoV-2. (A) The domain architecture of RdRp or nsp12 of SARS-CoV-2 is subdivided into NiRAN, interface, fingers, palm, and thumb subdomains; A–G indicate conserved motifs. (B) The cryo-EM structure of apo-RdRp complex (shown as front view, PDB: 7BV1) consists of nsp12, nsp7 (brown), and 2 chains of nsp8 (nsp8.1 and nsp8.2, both in gray). The nsp8.1 interacts directly with nsp12, whereas the nsp8.2 binds to nsp7, which in turn interacts with nsp12. The RNA template is expected to enter the active site, which is formed by motifs A and C through a groove clamped by motifs F and G. Motif E and the thumb subdomain support the primer strand. The RdRp subdomain color scheme is according to Fig 4A. (C) The cryo-EM structure (in top view) of the RdRp complex bound to RNA (PDB: 6YYT) shows 2 chains of nsp8 stabilizing the extending RNA with their alpha helices. The apo-RdRp complex structure (PDB: 7BV1) is shown for comparison. The active site is expanded to show the RNA molecules coming out of the groove formed by the finger and the thumb subdomains. The figures were prepared using Pymol. cryo-EM, cryo-electron microscopy; NiRAN, nidovirus RdRp-associated nucleotidyltransferase; PDB, Protein Data Bank; RdRp, RNA-dependent RNA polymerase; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Cryo-EM structure of RdRp of SARS-CoV-2. (A) The domain architecture of RdRp or nsp12 of SARS-CoV-2 is subdivided into NiRAN, interface, fingers, palm, and thumb subdomains; A–G indicate conserved motifs. (B) The cryo-EM structure of apo-RdRp complex (shown as front view, PDB: 7BV1) consists of nsp12, nsp7 (brown), and 2 chains of nsp8 (nsp8.1 and nsp8.2, both in gray). The nsp8.1 interacts directly with nsp12, whereas the nsp8.2 binds to nsp7, which in turn interacts with nsp12. The RNA template is expected to enter the active site, which is formed by motifs A and C through a groove clamped by motifs F and G. Motif E and the thumb subdomain support the primer strand. The RdRp subdomain color scheme is according to Fig 4A. (C) The cryo-EM structure (in top view) of the RdRp complex bound to RNA (PDB: 6YYT) shows 2 chains of nsp8 stabilizing the extending RNA with their alpha helices. The apo-RdRp complex structure (PDB: 7BV1) is shown for comparison. The active site is expanded to show the RNA molecules coming out of the groove formed by the finger and the thumb subdomains. The figures were prepared using Pymol. cryo-EM, cryo-electron microscopy; NiRAN, nidovirus RdRp-associated nucleotidyltransferase; PDB, Protein Data Bank; RdRp, RNA-dependent RNA polymerase; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Electron Microscopy

    Phylogenetic relationships in the Coronavirinae subfamily. The subfamily is formed by 4 genera: Alphacoronavirus , Betacoronavirus (lineages A, B, C, and D), Gammacoronavirus , and Deltacoronavirus . We randomly picked 62 SARS-CoV-2 genome sequences, representing 15 different countries, together with other Coronavirinae subfamily members. The phylogenetic tree was created using NgPhylogeny.fr tool. The analysis indicates that SARS-CoV-2 has a close relationship with bat coronavirus RaTG13 and SARS-CoV; therefore, it is classified as a new member of the lineage B Betacoronavirus . SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Phylogenetic relationships in the Coronavirinae subfamily. The subfamily is formed by 4 genera: Alphacoronavirus , Betacoronavirus (lineages A, B, C, and D), Gammacoronavirus , and Deltacoronavirus . We randomly picked 62 SARS-CoV-2 genome sequences, representing 15 different countries, together with other Coronavirinae subfamily members. The phylogenetic tree was created using NgPhylogeny.fr tool. The analysis indicates that SARS-CoV-2 has a close relationship with bat coronavirus RaTG13 and SARS-CoV; therefore, it is classified as a new member of the lineage B Betacoronavirus . SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used:

    Origin and transmission of pathogenic HCoVs. Yellow and red arrows indicate mild and severe infections in humans, respectively. The figure is inspired from Jie Cui and colleagues [ 46 ], and the illustrations of coronaviruses (left) are adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . HCoV, Human Coronavirus; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Origin and transmission of pathogenic HCoVs. Yellow and red arrows indicate mild and severe infections in humans, respectively. The figure is inspired from Jie Cui and colleagues [ 46 ], and the illustrations of coronaviruses (left) are adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . HCoV, Human Coronavirus; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Transmission Assay

    6) Product Images from "Virus-free and live-cell visualizing SARS-CoV-2 cell entry for studies of neutralizing antibodies and compound inhibitors"

    Article Title: Virus-free and live-cell visualizing SARS-CoV-2 cell entry for studies of neutralizing antibodies and compound inhibitors

    Journal: bioRxiv

    doi: 10.1101/2020.07.22.215236

    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 and TMPRSS2 in 293T and H1299 cells stably transfected with different constructs. NT cell, non-transfected 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 pseudo-colored 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 nM, respectively. (F) Comparisons of the fluorescence response (cMFI) of various SARS-CoV-2 probes on 293T-ACE2iRb3 cells. For panel 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.
    Figure Legend 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 and TMPRSS2 in 293T and H1299 cells stably transfected with different constructs. NT cell, non-transfected 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 pseudo-colored 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 nM, respectively. (F) Comparisons of the fluorescence response (cMFI) of various SARS-CoV-2 probes on 293T-ACE2iRb3 cells. For panel 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.

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

    The 83H7 mAb inhibits SARS-CoV-2 via the intracellular neutralization pathway. The 293T-ACE2iRb3 cells were incubated with 20 nM of dylight633-labeled mAbs (Ab633) of 36H6, 53G2, 83H7, and 8H6 and an irrelevant control antibody (ctrAb), in the presence or absence of STG (2.5 nM). Live-cell fluorescence image dynamically tracked using a 63x water immersion objective. Five replicate wells were measured for each group, and 16 fields of each well were imaged. Time-series (at 10-min, 1-hour, 2-hour, 3-hour, 5-hour, 7-hour, 9-hour, 11-hour, and 13-hour) analyses of the STG-IVNs (A), STG-IVpMFI (B), Ab633-IVNs (C), Ab633-IVpMFI (D) and the percentage of STG/Ab633 colocalized vesicles to total internalized STG vesicles (E). IVNs, average internalized vesicle numbers; IVpMFI, the average peak MFI of internalized vesicles. (F) Comparisons of the STG-IVA of the internalized STG vesicles among groups co-incubated with various mAbs at 5-hour post-incubation. ** indicates p
    Figure Legend Snippet: The 83H7 mAb inhibits SARS-CoV-2 via the intracellular neutralization pathway. The 293T-ACE2iRb3 cells were incubated with 20 nM of dylight633-labeled mAbs (Ab633) of 36H6, 53G2, 83H7, and 8H6 and an irrelevant control antibody (ctrAb), in the presence or absence of STG (2.5 nM). Live-cell fluorescence image dynamically tracked using a 63x water immersion objective. Five replicate wells were measured for each group, and 16 fields of each well were imaged. Time-series (at 10-min, 1-hour, 2-hour, 3-hour, 5-hour, 7-hour, 9-hour, 11-hour, and 13-hour) analyses of the STG-IVNs (A), STG-IVpMFI (B), Ab633-IVNs (C), Ab633-IVpMFI (D) and the percentage of STG/Ab633 colocalized vesicles to total internalized STG vesicles (E). IVNs, average internalized vesicle numbers; IVpMFI, the average peak MFI of internalized vesicles. (F) Comparisons of the STG-IVA of the internalized STG vesicles among groups co-incubated with various mAbs at 5-hour post-incubation. ** indicates p

    Techniques Used: Neutralization, Incubation, Labeling, Fluorescence

    Evaluation of neutralization potential of human plasmas from convalescent COVID-19 patients by CSBT and CRBT assays. (A) Comparisons of cMFI inhibitions on CSBT and CRBT assays between plasma samples from convalescent COVID-19 patients and healthy control (HC) subjects. The cMFI inhibition (%) at 1:20 dilution was plotted at the left Y-axis. The cutoff values for CSBT and CRBT were inhibition of 25% (median HC value +3.3×SD) on cMFI at 1:20 dilution. (B) Heatmaps showing CSBT and CRBT effects of two-fold serial dilutions of 32 plasmas from convalescent COVID-19 patients. (C) Distributions of the levels of TAb, IgM, IgG, CSBT, CRBT and LVppNAT of convalescent plasma samples. The numbers indicated the average titers at log10. The titers of Ab, IgM, and IgG were expressed as relative S/CO values determined by serial dilution measurements of each sample (maximum reactive dilution fold multiplied by S/CO). The CRBT and CSBT titers were expresses at ID25, whereas the LVppNAT was expressed as ID50. (D) Correlation analyses between the CSBT titer and the CRBT efficiency (at 1:20 dilution), the TAb titer, the IgM titer, the IgG titer, the LVppNAT and the NAT against authentic SARS-CoV-2 virus among convalescent plasmas. The correlation of CSBT titer and neutralization activity against authentic SARS-CoV-2 virus in 12 representative samples (included 11 convalescent COVID-19 plasmas and 1 control sample).
    Figure Legend Snippet: Evaluation of neutralization potential of human plasmas from convalescent COVID-19 patients by CSBT and CRBT assays. (A) Comparisons of cMFI inhibitions on CSBT and CRBT assays between plasma samples from convalescent COVID-19 patients and healthy control (HC) subjects. The cMFI inhibition (%) at 1:20 dilution was plotted at the left Y-axis. The cutoff values for CSBT and CRBT were inhibition of 25% (median HC value +3.3×SD) on cMFI at 1:20 dilution. (B) Heatmaps showing CSBT and CRBT effects of two-fold serial dilutions of 32 plasmas from convalescent COVID-19 patients. (C) Distributions of the levels of TAb, IgM, IgG, CSBT, CRBT and LVppNAT of convalescent plasma samples. The numbers indicated the average titers at log10. The titers of Ab, IgM, and IgG were expressed as relative S/CO values determined by serial dilution measurements of each sample (maximum reactive dilution fold multiplied by S/CO). The CRBT and CSBT titers were expresses at ID25, whereas the LVppNAT was expressed as ID50. (D) Correlation analyses between the CSBT titer and the CRBT efficiency (at 1:20 dilution), the TAb titer, the IgM titer, the IgG titer, the LVppNAT and the NAT against authentic SARS-CoV-2 virus among convalescent plasmas. The correlation of CSBT titer and neutralization activity against authentic SARS-CoV-2 virus in 12 representative samples (included 11 convalescent COVID-19 plasmas and 1 control sample).

    Techniques Used: Neutralization, Inhibition, Serial Dilution, Activity Assay

    Generation and characterization of FP-fused SARS-CoV-2 S proteins. (A) Schematics of STG and RBG constructs. Functional domains are colored. NTD, N-terminal domain; RBD, receptor binding domain; FP, fusion peptide; HR1/2, heptad repeat 1/2; CH, central helix; TM, transmembrane domain; cyt, cytoplasmic tail; TFd, T4 fibritin trimerization motif; mGam, monomeric Gamillus; mNG, mNeonGreen. (B) SDS-PAGE and fluorescence analyses for purified ST-based and RBD-based SARS-CoV-2 S proteins. (C) Size-exclusion chromatogram (SEC) of the purified SARS-CoV2-ST, SARS-CoV2-STG and SARS-CoV2-STN. Data from UV280 detector (upper panel) and fluorescence detector (lower panel) from a G3000 HPLC Column were showed. The molecular weight of SARS-CoV2-STG (or SARS-CoV2-STN) was about 808 kd, which was calculated according to its elution time in referring to the standard curve of determining the molecular weight as shown in Figure S2A and S2B. (D) SPR sensorgrams showing the binding kinetics for SARS-CoV2-STG (upper panel) or SARS-CoV2-RBG (lower panel) with immobilized rACE2 (human). Colored lines represented a global fit of the data using a 1:1 binding model.
    Figure Legend Snippet: Generation and characterization of FP-fused SARS-CoV-2 S proteins. (A) Schematics of STG and RBG constructs. Functional domains are colored. NTD, N-terminal domain; RBD, receptor binding domain; FP, fusion peptide; HR1/2, heptad repeat 1/2; CH, central helix; TM, transmembrane domain; cyt, cytoplasmic tail; TFd, T4 fibritin trimerization motif; mGam, monomeric Gamillus; mNG, mNeonGreen. (B) SDS-PAGE and fluorescence analyses for purified ST-based and RBD-based SARS-CoV-2 S proteins. (C) Size-exclusion chromatogram (SEC) of the purified SARS-CoV2-ST, SARS-CoV2-STG and SARS-CoV2-STN. Data from UV280 detector (upper panel) and fluorescence detector (lower panel) from a G3000 HPLC Column were showed. The molecular weight of SARS-CoV2-STG (or SARS-CoV2-STN) was about 808 kd, which was calculated according to its elution time in referring to the standard curve of determining the molecular weight as shown in Figure S2A and S2B. (D) SPR sensorgrams showing the binding kinetics for SARS-CoV2-STG (upper panel) or SARS-CoV2-RBG (lower panel) with immobilized rACE2 (human). Colored lines represented a global fit of the data using a 1:1 binding model.

    Techniques Used: Construct, Functional Assay, Binding Assay, SDS Page, Fluorescence, Purification, High Performance Liquid Chromatography, Molecular Weight, SPR Assay

    Detection of compound-induced influence on SARS-CoV-2 S-mediated cellular entry. (A) Schematic summary of the possible mechanisms of 11 compound inhibitors involved in the study. CytD, cytochalasin D; MDC, dansylcadaverine; Baf.A1, bafilomycin A1; vRNA, viral RNA. (B) Dose-dependent inhibitions of 11 compounds against SARS-CoV-2 LVpp infection on H1299-ACE2hR cells. All compounds were tested in a 2-fold dilution series, and the initial drug concentrations were begun at their maximal non-cytotoxic concentrations. The initial concentrations were 200 μM for amiloride, MDC and DMSO (as a solvent control); 100 μM for dynasore; 10 μM for filipin, APY0201, YM201636 and tetrandrine; 4 μM for nystatin; 100 nM for Baf.A1 and apilimod. ND, not detected. (C) Confocal images of STG (green channel), ACE2-mRuby3 (red channel), and nucleus (blue channel) in 293T-ACE2iRb3 cells at 5-hour post STG incubation. The cells were pretreated with compounds for 1-hour before STG loading. These pictures were obtained by using Leica gSTED confocal microscopy on cells treated with compounds at their respective initial concentrations as above-mentioned. Scale bar, 10 μm. (D) Quantitative analysis of the influence of entry inhibitors on STG internalization. Dose-dependent influence of various compounds on STG internalization characteristics on 293T-ACE2iRb3 cells at 1-hour (left panels) and 5-hour (right panels) after incubation. All compounds were tested in a 4-fold dilution series (4 gradients for DMSO control, and 5 gradients for others), and the initial drug concentrations were identical with as (B). Three replicate wells were measured for each group, and 16 fields of each well were imaged. For each compound, 5 colored bars from left-to-right orderly displayed the values measured from cells treated with 4-fold serial high-to-low concentrations of compounds. STG-IFR, internalized STG fluorescence intensity ratio; STG-IVA, average area (μm 2 ) of internalized STG vesicles; STG-IVNs, average numbers of internalized STG vesicles per cell; *, p
    Figure Legend Snippet: Detection of compound-induced influence on SARS-CoV-2 S-mediated cellular entry. (A) Schematic summary of the possible mechanisms of 11 compound inhibitors involved in the study. CytD, cytochalasin D; MDC, dansylcadaverine; Baf.A1, bafilomycin A1; vRNA, viral RNA. (B) Dose-dependent inhibitions of 11 compounds against SARS-CoV-2 LVpp infection on H1299-ACE2hR cells. All compounds were tested in a 2-fold dilution series, and the initial drug concentrations were begun at their maximal non-cytotoxic concentrations. The initial concentrations were 200 μM for amiloride, MDC and DMSO (as a solvent control); 100 μM for dynasore; 10 μM for filipin, APY0201, YM201636 and tetrandrine; 4 μM for nystatin; 100 nM for Baf.A1 and apilimod. ND, not detected. (C) Confocal images of STG (green channel), ACE2-mRuby3 (red channel), and nucleus (blue channel) in 293T-ACE2iRb3 cells at 5-hour post STG incubation. The cells were pretreated with compounds for 1-hour before STG loading. These pictures were obtained by using Leica gSTED confocal microscopy on cells treated with compounds at their respective initial concentrations as above-mentioned. Scale bar, 10 μm. (D) Quantitative analysis of the influence of entry inhibitors on STG internalization. Dose-dependent influence of various compounds on STG internalization characteristics on 293T-ACE2iRb3 cells at 1-hour (left panels) and 5-hour (right panels) after incubation. All compounds were tested in a 4-fold dilution series (4 gradients for DMSO control, and 5 gradients for others), and the initial drug concentrations were identical with as (B). Three replicate wells were measured for each group, and 16 fields of each well were imaged. For each compound, 5 colored bars from left-to-right orderly displayed the values measured from cells treated with 4-fold serial high-to-low concentrations of compounds. STG-IFR, internalized STG fluorescence intensity ratio; STG-IVA, average area (μm 2 ) of internalized STG vesicles; STG-IVNs, average numbers of internalized STG vesicles per cell; *, p

    Techniques Used: Infection, Incubation, Confocal Microscopy, Fluorescence

    7) Product Images from "A single dose of recombinant VSV-ΔG-spike vaccine provides protection against SARS-CoV-2 challenge"

    Article Title: A single dose of recombinant VSV-ΔG-spike vaccine provides protection against SARS-CoV-2 challenge

    Journal: bioRxiv

    doi: 10.1101/2020.06.18.160655

    Histopathology and viral load of rVSV-ΔG-spike vaccinated and infected lungs: General histology (H E) and SARS-CoV-2 Immunolabeling of hamster lungs with and without pre vaccination. Lungs were isolated and processed for paraffin embedding from Naïve (A, E, I), vaccinated + infected 10 6 5dpi (B, F, J; C, G, K), and SARS-CoV-2 5×10 6 5dpi (D, H, L) and groups. Sections (5μm) were taken for H E staining (A-H) and SARS-CoV-2 immunolabeling (I-L, DAPI-Blue, SARS-CoV-2-Green). Pictures A-D: magnification-x1, bar= 100μm; Pictures E-H: magnification-x10, bar= 100μm; Pictures I-L: magnification-x60, bar= 10μm. Black arrows indicate patches of focal inflammation, pleural invaginatio and alveolar collapse. “*”-indicates hemorrhagic areas. “#”-indicates edema and protein rich exudates. Black arrow heads indicate pulmonary mononuclear cells. White arrows indicate CoV-2 positiv immunolabeling. Naïve group: n=4, SARS-CoV-2 5×10 6 pfu/animal 5dpi group: n=1, vaccinated+infecte 10 6 pfu/animal 5dpi group: n=2. (M) Tissue/Air space ratio.
    Figure Legend Snippet: Histopathology and viral load of rVSV-ΔG-spike vaccinated and infected lungs: General histology (H E) and SARS-CoV-2 Immunolabeling of hamster lungs with and without pre vaccination. Lungs were isolated and processed for paraffin embedding from Naïve (A, E, I), vaccinated + infected 10 6 5dpi (B, F, J; C, G, K), and SARS-CoV-2 5×10 6 5dpi (D, H, L) and groups. Sections (5μm) were taken for H E staining (A-H) and SARS-CoV-2 immunolabeling (I-L, DAPI-Blue, SARS-CoV-2-Green). Pictures A-D: magnification-x1, bar= 100μm; Pictures E-H: magnification-x10, bar= 100μm; Pictures I-L: magnification-x60, bar= 10μm. Black arrows indicate patches of focal inflammation, pleural invaginatio and alveolar collapse. “*”-indicates hemorrhagic areas. “#”-indicates edema and protein rich exudates. Black arrow heads indicate pulmonary mononuclear cells. White arrows indicate CoV-2 positiv immunolabeling. Naïve group: n=4, SARS-CoV-2 5×10 6 pfu/animal 5dpi group: n=1, vaccinated+infecte 10 6 pfu/animal 5dpi group: n=2. (M) Tissue/Air space ratio.

    Techniques Used: Histopathology, Infection, Immunolabeling, Isolation, Staining

    Surface antigenic similarity of rVSV-ΔG-spike and SARS-CoV-2: (A) Immunofluorescent images of Vero E6 cells infected with either WT-VSV (left panel), rVSV-ΔG-spike (middle panel), or SARS-CoV-2, stained with serum from COVID-19 human convalescent serum (right panel). (B) Correlation analysis of neutralization of rVSV-ΔG-spike and SARS-CoV-2 by a panel of sera from COVID-19 convalescent patients. For each sera (n=12), NT 50 values were determined for neutralization of rVSV-ΔG-spike, or SARS-CoV-2. The NT 50 values were plotted to determine the correlation between th neutralization assays. R 2 =0.911.
    Figure Legend Snippet: Surface antigenic similarity of rVSV-ΔG-spike and SARS-CoV-2: (A) Immunofluorescent images of Vero E6 cells infected with either WT-VSV (left panel), rVSV-ΔG-spike (middle panel), or SARS-CoV-2, stained with serum from COVID-19 human convalescent serum (right panel). (B) Correlation analysis of neutralization of rVSV-ΔG-spike and SARS-CoV-2 by a panel of sera from COVID-19 convalescent patients. For each sera (n=12), NT 50 values were determined for neutralization of rVSV-ΔG-spike, or SARS-CoV-2. The NT 50 values were plotted to determine the correlation between th neutralization assays. R 2 =0.911.

    Techniques Used: Infection, Staining, Neutralization

    Characterization of rVSV-ΔG-spike: (A) A summary of the genome analysis of several passage of rVSV-ΔG-spike, showing Ct values of VSV-G, VSV-N, and SARS-CoV-2-S indicating the elimination of VSV-G over time, together with increased titer. Also, plaques are formed at late passages. (B) Immunofluorescence images of Vero E6 cells infected with early passage (P5)-rVSV-ΔG-spike, or lat passage (P13)-rVSV-ΔG-spike, stained with a SARS-CoV-2 antibody (green) and DAPI for nuclei stainin (blue). Top and bottom panels show insets at low (10X) and high magnification (25x), respectively. rVSV-ΔG-spike at P5 forms syncytia, whereas P13 show individual infected cells, with no evidence of syncytia. (C) Transmission electron microscopy of rVSV-ΔG-spike (top panel) compared to WT-VSV. Right panel shows immunogold labeling against RBD.
    Figure Legend Snippet: Characterization of rVSV-ΔG-spike: (A) A summary of the genome analysis of several passage of rVSV-ΔG-spike, showing Ct values of VSV-G, VSV-N, and SARS-CoV-2-S indicating the elimination of VSV-G over time, together with increased titer. Also, plaques are formed at late passages. (B) Immunofluorescence images of Vero E6 cells infected with early passage (P5)-rVSV-ΔG-spike, or lat passage (P13)-rVSV-ΔG-spike, stained with a SARS-CoV-2 antibody (green) and DAPI for nuclei stainin (blue). Top and bottom panels show insets at low (10X) and high magnification (25x), respectively. rVSV-ΔG-spike at P5 forms syncytia, whereas P13 show individual infected cells, with no evidence of syncytia. (C) Transmission electron microscopy of rVSV-ΔG-spike (top panel) compared to WT-VSV. Right panel shows immunogold labeling against RBD.

    Techniques Used: Immunofluorescence, Infection, Staining, Transmission Assay, Electron Microscopy, Labeling

    Single-dose rVSV-ΔG-spike vaccine efficacy in hamsters following SARS-CoV-2 challenge. (A) Body weight changes of hamsters infected with SARS-CoV-2 (n=12), and hamsters vaccinated wit rVSV-ΔG-spike and infected with 5×10 6 pfu/hamster (n=10) 25 days post-vaccination, compared to moc hamsters (n=8). p
    Figure Legend Snippet: Single-dose rVSV-ΔG-spike vaccine efficacy in hamsters following SARS-CoV-2 challenge. (A) Body weight changes of hamsters infected with SARS-CoV-2 (n=12), and hamsters vaccinated wit rVSV-ΔG-spike and infected with 5×10 6 pfu/hamster (n=10) 25 days post-vaccination, compared to moc hamsters (n=8). p

    Techniques Used: Infection

    Dose-dependent vaccination of hamsters with rVSV-ΔG-spike. (A) Body weight changes of mock-vaccinated hamsters (n=4), and hamsters vaccinated with rVSV-ΔG-spike ranging from 10 4 to 10 8 pfu/hamster (n=8, n=10, n=10, n=10, n=8, for each vaccinated group, respectively). (B) NT 50 values of neutralization of SARS-CoV-2 by sera from hamsters following i.m. vaccination with rVSV-ΔG-spik ranging from 10 4 to 10 8 pfu/hamster. n=4 for each group. Means and SEM are indicated below the graph.
    Figure Legend Snippet: Dose-dependent vaccination of hamsters with rVSV-ΔG-spike. (A) Body weight changes of mock-vaccinated hamsters (n=4), and hamsters vaccinated with rVSV-ΔG-spike ranging from 10 4 to 10 8 pfu/hamster (n=8, n=10, n=10, n=10, n=8, for each vaccinated group, respectively). (B) NT 50 values of neutralization of SARS-CoV-2 by sera from hamsters following i.m. vaccination with rVSV-ΔG-spik ranging from 10 4 to 10 8 pfu/hamster. n=4 for each group. Means and SEM are indicated below the graph.

    Techniques Used: Neutralization

    Establishment of golden Syrian hamster SARS-CoV-2 model: (A) Body weight changes of hamsters infected with SARS-CoV-2 at either 5×10 4 (n=7), 5×10 5 (n=7), or 5×10 6 (n=10) pfu/hamster, compared to mock-infected hamsters (n=6). p
    Figure Legend Snippet: Establishment of golden Syrian hamster SARS-CoV-2 model: (A) Body weight changes of hamsters infected with SARS-CoV-2 at either 5×10 4 (n=7), 5×10 5 (n=7), or 5×10 6 (n=10) pfu/hamster, compared to mock-infected hamsters (n=6). p

    Techniques Used: Infection

    rVSV-ΔG-spike design and generation strategy: (A) A schematic diagram of the genom organization of WT-VSV genome (top diagram) and rVSV-ΔG-spike (bottom diagram). N: Nucleoprotein, P: Phosphoprotein, M: Matrix, L: Large polymerase, G: Glycoprotein, SPIKE: SARS-CoV-2 spike. (B) pVSV-ΔG-spike map; in red, the inserted S gene. (C) Schematic representation of the generation proces of creating rVSV-ΔG-spike vaccine. Infection of BHK-21 cells with MVA-T7, followed by co-transfecti with pVSV-ΔG-spike, and VSV-system accessory plasmids; Transfection of BHK-21 cells with pCAGGS-VSV-G, followed by infection with the supernatant of the primary transfection to create P1; serial passaging to create rVSV-ΔG-spike.
    Figure Legend Snippet: rVSV-ΔG-spike design and generation strategy: (A) A schematic diagram of the genom organization of WT-VSV genome (top diagram) and rVSV-ΔG-spike (bottom diagram). N: Nucleoprotein, P: Phosphoprotein, M: Matrix, L: Large polymerase, G: Glycoprotein, SPIKE: SARS-CoV-2 spike. (B) pVSV-ΔG-spike map; in red, the inserted S gene. (C) Schematic representation of the generation proces of creating rVSV-ΔG-spike vaccine. Infection of BHK-21 cells with MVA-T7, followed by co-transfecti with pVSV-ΔG-spike, and VSV-system accessory plasmids; Transfection of BHK-21 cells with pCAGGS-VSV-G, followed by infection with the supernatant of the primary transfection to create P1; serial passaging to create rVSV-ΔG-spike.

    Techniques Used: Infection, Transfection, Passaging

    Detection and neutralization of SARS-CoV-2 by sera from hamsters following s.c. vaccinatio with rVSV-ΔG-spike: (A) Immunofluorescence images of Vero E6 cells infected with SARS-CoV-2, stai with sera from either mock-vaccinated hamsters (left panel) or rVSV-ΔG-spike vaccinated-hamsters (right panel). (B) Plaque reduction neutralization test (PRNT) of hamster sera collected from naïve hamster (PBF, n=5) or hamsters vaccinated with rVSV-ΔG-spike 25 days following vaccination (n=5). (C) Bod weight changes of mock-vaccinated hamsters (Mock, n=16), and hamsters vaccinated with rVSV-ΔG-spike (n=8).
    Figure Legend Snippet: Detection and neutralization of SARS-CoV-2 by sera from hamsters following s.c. vaccinatio with rVSV-ΔG-spike: (A) Immunofluorescence images of Vero E6 cells infected with SARS-CoV-2, stai with sera from either mock-vaccinated hamsters (left panel) or rVSV-ΔG-spike vaccinated-hamsters (right panel). (B) Plaque reduction neutralization test (PRNT) of hamster sera collected from naïve hamster (PBF, n=5) or hamsters vaccinated with rVSV-ΔG-spike 25 days following vaccination (n=5). (C) Bod weight changes of mock-vaccinated hamsters (Mock, n=16), and hamsters vaccinated with rVSV-ΔG-spike (n=8).

    Techniques Used: Neutralization, Immunofluorescence, Infection, Plaque Reduction Neutralization Test

    8) Product Images from "Human Mesenchymal Stromal Cells Are Resistant to SARS-CoV-2 Infection under Steady-State, Inflammatory Conditions and in the Presence of SARS-CoV-2-Infected Cells"

    Article Title: Human Mesenchymal Stromal Cells Are Resistant to SARS-CoV-2 Infection under Steady-State, Inflammatory Conditions and in the Presence of SARS-CoV-2-Infected Cells

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2020.09.003

    Evaluation of SARS-CoV-2 Infection of MSCs Evaluation of SARS-CoV-2 infection of MSCs under steady-state and inflammatory conditions and in the presence of SARS-CoV-2-infected Caco-2 cells. SARS-CoV-2 infection is identified by SARS-CoV-2 S protein staining (red). All MSCs and Caco-2 cells experiments were repeated in three independent settings from three BM-MSC donors and three ASC donors, and were performed in three biological replicates each. One representative picture is shown for each condition. (A) Caco-2 cells without SARS-CoV-2; (B) Caco-2 cells with SARS-CoV-2 MOI1; (C) SARS-CoV-2 replication quantified by qPCR detecting high copy numbers in Caco-2 cells infected by SARS-CoV-2; error bars: SD; (D) BM-MSC steady state with SARS-CoV-2 MOI1; (E) ASC steady state with SARS-CoV-2 MOI1; (F) BM-MSC inflammatory conditions with SARS-CoV-2 MOI1; (G) Co-culture BM-MSC:Caco-2 cells (10:1) with SARS-CoV-2 MOI1; BM-MSCs (black star) + Caco-2 cells (white star). Scale bars, 100 μm and 20 μm (inset in G).
    Figure Legend Snippet: Evaluation of SARS-CoV-2 Infection of MSCs Evaluation of SARS-CoV-2 infection of MSCs under steady-state and inflammatory conditions and in the presence of SARS-CoV-2-infected Caco-2 cells. SARS-CoV-2 infection is identified by SARS-CoV-2 S protein staining (red). All MSCs and Caco-2 cells experiments were repeated in three independent settings from three BM-MSC donors and three ASC donors, and were performed in three biological replicates each. One representative picture is shown for each condition. (A) Caco-2 cells without SARS-CoV-2; (B) Caco-2 cells with SARS-CoV-2 MOI1; (C) SARS-CoV-2 replication quantified by qPCR detecting high copy numbers in Caco-2 cells infected by SARS-CoV-2; error bars: SD; (D) BM-MSC steady state with SARS-CoV-2 MOI1; (E) ASC steady state with SARS-CoV-2 MOI1; (F) BM-MSC inflammatory conditions with SARS-CoV-2 MOI1; (G) Co-culture BM-MSC:Caco-2 cells (10:1) with SARS-CoV-2 MOI1; BM-MSCs (black star) + Caco-2 cells (white star). Scale bars, 100 μm and 20 μm (inset in G).

    Techniques Used: Infection, Staining, Real-time Polymerase Chain Reaction, Co-Culture Assay

    9) Product Images from "A single dose of recombinant VSV-ΔG-spike vaccine provides protection against SARS-CoV-2 challenge"

    Article Title: A single dose of recombinant VSV-ΔG-spike vaccine provides protection against SARS-CoV-2 challenge

    Journal: bioRxiv

    doi: 10.1101/2020.06.18.160655

    Histopathology and viral load of rVSV-ΔG-spike vaccinated and infected lungs: General histology (H E) and SARS-CoV-2 Immunolabeling of hamster lungs with and without pre vaccination. Lungs were isolated and processed for paraffin embedding from Naïve (A, E, I), vaccinated + infected 10 6 5dpi (B, F, J; C, G, K), and SARS-CoV-2 5×10 6 5dpi (D, H, L) and groups. Sections (5μm) were taken for H E staining (A-H) and SARS-CoV-2 immunolabeling (I-L, DAPI-Blue, SARS-CoV-2-Green). Pictures A-D: magnification-x1, bar= 100μm; Pictures E-H: magnification-x10, bar= 100μm; Pictures I-L: magnification-x60, bar= 10μm. Black arrows indicate patches of focal inflammation, pleural invaginatio and alveolar collapse. “*”-indicates hemorrhagic areas. “#”-indicates edema and protein rich exudates. Black arrow heads indicate pulmonary mononuclear cells. White arrows indicate CoV-2 positiv immunolabeling. Naïve group: n=4, SARS-CoV-2 5×10 6 pfu/animal 5dpi group: n=1, vaccinated+infecte 10 6 pfu/animal 5dpi group: n=2. (M) Tissue/Air space ratio.
    Figure Legend Snippet: Histopathology and viral load of rVSV-ΔG-spike vaccinated and infected lungs: General histology (H E) and SARS-CoV-2 Immunolabeling of hamster lungs with and without pre vaccination. Lungs were isolated and processed for paraffin embedding from Naïve (A, E, I), vaccinated + infected 10 6 5dpi (B, F, J; C, G, K), and SARS-CoV-2 5×10 6 5dpi (D, H, L) and groups. Sections (5μm) were taken for H E staining (A-H) and SARS-CoV-2 immunolabeling (I-L, DAPI-Blue, SARS-CoV-2-Green). Pictures A-D: magnification-x1, bar= 100μm; Pictures E-H: magnification-x10, bar= 100μm; Pictures I-L: magnification-x60, bar= 10μm. Black arrows indicate patches of focal inflammation, pleural invaginatio and alveolar collapse. “*”-indicates hemorrhagic areas. “#”-indicates edema and protein rich exudates. Black arrow heads indicate pulmonary mononuclear cells. White arrows indicate CoV-2 positiv immunolabeling. Naïve group: n=4, SARS-CoV-2 5×10 6 pfu/animal 5dpi group: n=1, vaccinated+infecte 10 6 pfu/animal 5dpi group: n=2. (M) Tissue/Air space ratio.

    Techniques Used: Histopathology, Infection, Immunolabeling, Isolation, Staining

    Surface antigenic similarity of rVSV-ΔG-spike and SARS-CoV-2: (A) Immunofluorescent images of Vero E6 cells infected with either WT-VSV (left panel), rVSV-ΔG-spike (middle panel), or SARS-CoV-2, stained with serum from COVID-19 human convalescent serum (right panel). (B) Correlation analysis of neutralization of rVSV-ΔG-spike and SARS-CoV-2 by a panel of sera from COVID-19 convalescent patients. For each sera (n=12), NT 50 values were determined for neutralization of rVSV-ΔG-spike, or SARS-CoV-2. The NT 50 values were plotted to determine the correlation between th neutralization assays. R 2 =0.911.
    Figure Legend Snippet: Surface antigenic similarity of rVSV-ΔG-spike and SARS-CoV-2: (A) Immunofluorescent images of Vero E6 cells infected with either WT-VSV (left panel), rVSV-ΔG-spike (middle panel), or SARS-CoV-2, stained with serum from COVID-19 human convalescent serum (right panel). (B) Correlation analysis of neutralization of rVSV-ΔG-spike and SARS-CoV-2 by a panel of sera from COVID-19 convalescent patients. For each sera (n=12), NT 50 values were determined for neutralization of rVSV-ΔG-spike, or SARS-CoV-2. The NT 50 values were plotted to determine the correlation between th neutralization assays. R 2 =0.911.

    Techniques Used: Infection, Staining, Neutralization

    Characterization of rVSV-ΔG-spike: (A) A summary of the genome analysis of several passage of rVSV-ΔG-spike, showing Ct values of VSV-G, VSV-N, and SARS-CoV-2-S indicating the elimination of VSV-G over time, together with increased titer. Also, plaques are formed at late passages. (B) Immunofluorescence images of Vero E6 cells infected with early passage (P5)-rVSV-ΔG-spike, or lat passage (P13)-rVSV-ΔG-spike, stained with a SARS-CoV-2 antibody (green) and DAPI for nuclei stainin (blue). Top and bottom panels show insets at low (10X) and high magnification (25x), respectively. rVSV-ΔG-spike at P5 forms syncytia, whereas P13 show individual infected cells, with no evidence of syncytia. (C) Transmission electron microscopy of rVSV-ΔG-spike (top panel) compared to WT-VSV. Right panel shows immunogold labeling against RBD.
    Figure Legend Snippet: Characterization of rVSV-ΔG-spike: (A) A summary of the genome analysis of several passage of rVSV-ΔG-spike, showing Ct values of VSV-G, VSV-N, and SARS-CoV-2-S indicating the elimination of VSV-G over time, together with increased titer. Also, plaques are formed at late passages. (B) Immunofluorescence images of Vero E6 cells infected with early passage (P5)-rVSV-ΔG-spike, or lat passage (P13)-rVSV-ΔG-spike, stained with a SARS-CoV-2 antibody (green) and DAPI for nuclei stainin (blue). Top and bottom panels show insets at low (10X) and high magnification (25x), respectively. rVSV-ΔG-spike at P5 forms syncytia, whereas P13 show individual infected cells, with no evidence of syncytia. (C) Transmission electron microscopy of rVSV-ΔG-spike (top panel) compared to WT-VSV. Right panel shows immunogold labeling against RBD.

    Techniques Used: Immunofluorescence, Infection, Staining, Transmission Assay, Electron Microscopy, Labeling

    Single-dose rVSV-ΔG-spike vaccine efficacy in hamsters following SARS-CoV-2 challenge. (A) Body weight changes of hamsters infected with SARS-CoV-2 (n=12), and hamsters vaccinated wit rVSV-ΔG-spike and infected with 5×10 6 pfu/hamster (n=10) 25 days post-vaccination, compared to moc hamsters (n=8). p
    Figure Legend Snippet: Single-dose rVSV-ΔG-spike vaccine efficacy in hamsters following SARS-CoV-2 challenge. (A) Body weight changes of hamsters infected with SARS-CoV-2 (n=12), and hamsters vaccinated wit rVSV-ΔG-spike and infected with 5×10 6 pfu/hamster (n=10) 25 days post-vaccination, compared to moc hamsters (n=8). p

    Techniques Used: Infection

    Dose-dependent vaccination of hamsters with rVSV-ΔG-spike. (A) Body weight changes of mock-vaccinated hamsters (n=4), and hamsters vaccinated with rVSV-ΔG-spike ranging from 10 4 to 10 8 pfu/hamster (n=8, n=10, n=10, n=10, n=8, for each vaccinated group, respectively). (B) NT 50 values of neutralization of SARS-CoV-2 by sera from hamsters following i.m. vaccination with rVSV-ΔG-spik ranging from 10 4 to 10 8 pfu/hamster. n=4 for each group. Means and SEM are indicated below the graph.
    Figure Legend Snippet: Dose-dependent vaccination of hamsters with rVSV-ΔG-spike. (A) Body weight changes of mock-vaccinated hamsters (n=4), and hamsters vaccinated with rVSV-ΔG-spike ranging from 10 4 to 10 8 pfu/hamster (n=8, n=10, n=10, n=10, n=8, for each vaccinated group, respectively). (B) NT 50 values of neutralization of SARS-CoV-2 by sera from hamsters following i.m. vaccination with rVSV-ΔG-spik ranging from 10 4 to 10 8 pfu/hamster. n=4 for each group. Means and SEM are indicated below the graph.

    Techniques Used: Neutralization

    Establishment of golden Syrian hamster SARS-CoV-2 model: (A) Body weight changes of hamsters infected with SARS-CoV-2 at either 5×10 4 (n=7), 5×10 5 (n=7), or 5×10 6 (n=10) pfu/hamster, compared to mock-infected hamsters (n=6). p
    Figure Legend Snippet: Establishment of golden Syrian hamster SARS-CoV-2 model: (A) Body weight changes of hamsters infected with SARS-CoV-2 at either 5×10 4 (n=7), 5×10 5 (n=7), or 5×10 6 (n=10) pfu/hamster, compared to mock-infected hamsters (n=6). p

    Techniques Used: Infection

    rVSV-ΔG-spike design and generation strategy: (A) A schematic diagram of the genom organization of WT-VSV genome (top diagram) and rVSV-ΔG-spike (bottom diagram). N: Nucleoprotein, P: Phosphoprotein, M: Matrix, L: Large polymerase, G: Glycoprotein, SPIKE: SARS-CoV-2 spike. (B) pVSV-ΔG-spike map; in red, the inserted S gene. (C) Schematic representation of the generation proces of creating rVSV-ΔG-spike vaccine. Infection of BHK-21 cells with MVA-T7, followed by co-transfecti with pVSV-ΔG-spike, and VSV-system accessory plasmids; Transfection of BHK-21 cells with pCAGGS-VSV-G, followed by infection with the supernatant of the primary transfection to create P1; serial passaging to create rVSV-ΔG-spike.
    Figure Legend Snippet: rVSV-ΔG-spike design and generation strategy: (A) A schematic diagram of the genom organization of WT-VSV genome (top diagram) and rVSV-ΔG-spike (bottom diagram). N: Nucleoprotein, P: Phosphoprotein, M: Matrix, L: Large polymerase, G: Glycoprotein, SPIKE: SARS-CoV-2 spike. (B) pVSV-ΔG-spike map; in red, the inserted S gene. (C) Schematic representation of the generation proces of creating rVSV-ΔG-spike vaccine. Infection of BHK-21 cells with MVA-T7, followed by co-transfecti with pVSV-ΔG-spike, and VSV-system accessory plasmids; Transfection of BHK-21 cells with pCAGGS-VSV-G, followed by infection with the supernatant of the primary transfection to create P1; serial passaging to create rVSV-ΔG-spike.

    Techniques Used: Infection, Transfection, Passaging

    Detection and neutralization of SARS-CoV-2 by sera from hamsters following s.c. vaccinatio with rVSV-ΔG-spike: (A) Immunofluorescence images of Vero E6 cells infected with SARS-CoV-2, stai with sera from either mock-vaccinated hamsters (left panel) or rVSV-ΔG-spike vaccinated-hamsters (right panel). (B) Plaque reduction neutralization test (PRNT) of hamster sera collected from naïve hamster (PBF, n=5) or hamsters vaccinated with rVSV-ΔG-spike 25 days following vaccination (n=5). (C) Bod weight changes of mock-vaccinated hamsters (Mock, n=16), and hamsters vaccinated with rVSV-ΔG-spike (n=8).
    Figure Legend Snippet: Detection and neutralization of SARS-CoV-2 by sera from hamsters following s.c. vaccinatio with rVSV-ΔG-spike: (A) Immunofluorescence images of Vero E6 cells infected with SARS-CoV-2, stai with sera from either mock-vaccinated hamsters (left panel) or rVSV-ΔG-spike vaccinated-hamsters (right panel). (B) Plaque reduction neutralization test (PRNT) of hamster sera collected from naïve hamster (PBF, n=5) or hamsters vaccinated with rVSV-ΔG-spike 25 days following vaccination (n=5). (C) Bod weight changes of mock-vaccinated hamsters (Mock, n=16), and hamsters vaccinated with rVSV-ΔG-spike (n=8).

    Techniques Used: Neutralization, Immunofluorescence, Infection, Plaque Reduction Neutralization Test

    10) Product Images from "A Modular Microarray Imaging System for Highly Specific COVID-19 Antibody Testing"

    Article Title: A Modular Microarray Imaging System for Highly Specific COVID-19 Antibody Testing

    Journal: bioRxiv

    doi: 10.1101/2020.05.22.111518

    Fluorescence images and data analysis of CoVAM probed with positive sera. ( a ) Four exemplary fluorescence images acquired with the Array Cam 400-S. ( b ) Corresponding TinyArray images. ( c ) Background-subtracted median fluorescence intensities obtained for each microarray spot with the Array Cam 400-S and the TinyArray imager that were normalized and plotted against each other; linear regressions were performed and R 2 values were calculated. ( d ) Heat maps of 9 SARS-CoV-2-positive and 10 negative control samples generated from the Array Cam 400-S (top row) and TinyArray imager data (bottom row). Gray/black/red colors indicate low/medium/high antibody prevalence. ( e,f ) Statistical analysis of the seven SARS-CoV-2 antigens in the CoVAM for positive sera for the ArrayCam and the TinyArray imager data. Scale bar, 2 mm.
    Figure Legend Snippet: Fluorescence images and data analysis of CoVAM probed with positive sera. ( a ) Four exemplary fluorescence images acquired with the Array Cam 400-S. ( b ) Corresponding TinyArray images. ( c ) Background-subtracted median fluorescence intensities obtained for each microarray spot with the Array Cam 400-S and the TinyArray imager that were normalized and plotted against each other; linear regressions were performed and R 2 values were calculated. ( d ) Heat maps of 9 SARS-CoV-2-positive and 10 negative control samples generated from the Array Cam 400-S (top row) and TinyArray imager data (bottom row). Gray/black/red colors indicate low/medium/high antibody prevalence. ( e,f ) Statistical analysis of the seven SARS-CoV-2 antigens in the CoVAM for positive sera for the ArrayCam and the TinyArray imager data. Scale bar, 2 mm.

    Techniques Used: Fluorescence, Chick Chorioallantoic Membrane Assay, Microarray, Negative Control, Generated

    11) Product Images from "SARS-CoV-2 infection induces germinal center responses with robust stimulation of CD4 T follicular helper cells in rhesus macaques"

    Article Title: SARS-CoV-2 infection induces germinal center responses with robust stimulation of CD4 T follicular helper cells in rhesus macaques

    Journal: bioRxiv

    doi: 10.1101/2020.07.07.191007

    Humoral responses to SARS-CoV-2 are dominated by IgG antibodies Concentrations of (A) IgM, (B) IgG, and (C) IgA antibodies specific for S1, S2, and N proteins were measured by BAMA or ELISA in serum of macaques infused with human COVID-19 convalescent plasma (CP; blue symbols) or naive plasma (NP; red symbols) and control non-infused animals (black symbols). The dashed line represents the median pre-infection (day 0) concentration for all animals. (D) The magnitude of the IgM, IgG and IgA antibody responses in animals that were not given human convalescent plasma was determined by dividing post-infection concentrations by those measured on day 0 in each animal. Geometric mean fold increases with SEM are shown. (E) Correlations between day 10 levels of S1-specific IgG and IgM, N-specific IgA and IgG, and pseudovirus neutralizing antibody titers and anti-RBD IgG antibodies measured by ELISA.
    Figure Legend Snippet: Humoral responses to SARS-CoV-2 are dominated by IgG antibodies Concentrations of (A) IgM, (B) IgG, and (C) IgA antibodies specific for S1, S2, and N proteins were measured by BAMA or ELISA in serum of macaques infused with human COVID-19 convalescent plasma (CP; blue symbols) or naive plasma (NP; red symbols) and control non-infused animals (black symbols). The dashed line represents the median pre-infection (day 0) concentration for all animals. (D) The magnitude of the IgM, IgG and IgA antibody responses in animals that were not given human convalescent plasma was determined by dividing post-infection concentrations by those measured on day 0 in each animal. Geometric mean fold increases with SEM are shown. (E) Correlations between day 10 levels of S1-specific IgG and IgM, N-specific IgA and IgG, and pseudovirus neutralizing antibody titers and anti-RBD IgG antibodies measured by ELISA.

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

    SARS-CoV-2 infection induces germinal center responses in mediastinal lymph nodes. (A) Gating strategy for identifying CD4 T cells in lung; red overlay represents paired CD4 subset from blood (either CD95- (naive) or CD95+ as indicated). (B) Scatter plot shows expression of Granzyme B, PD-1, CXCR3, CCR6 on CD69- and CD69+ subsets in lung and CD95+ CD4 T cells in blood. (C) Gating strategy for identification of GC T fh cells, GC B cells and FDCs. (D) Relative expression of Bcl-6, CD21, CD140b, and CXCR3 within GC cell subsets. (E) Frequency of GC T fh cells, GC B cells, FDCs significantly higher in mediastinal lymph node (*p
    Figure Legend Snippet: SARS-CoV-2 infection induces germinal center responses in mediastinal lymph nodes. (A) Gating strategy for identifying CD4 T cells in lung; red overlay represents paired CD4 subset from blood (either CD95- (naive) or CD95+ as indicated). (B) Scatter plot shows expression of Granzyme B, PD-1, CXCR3, CCR6 on CD69- and CD69+ subsets in lung and CD95+ CD4 T cells in blood. (C) Gating strategy for identification of GC T fh cells, GC B cells and FDCs. (D) Relative expression of Bcl-6, CD21, CD140b, and CXCR3 within GC cell subsets. (E) Frequency of GC T fh cells, GC B cells, FDCs significantly higher in mediastinal lymph node (*p

    Techniques Used: Infection, Expressing

    SARS-CoV-2 infection leads to a rapid and transient shift in innate immune responses and increases the number CD4 T follicular helper cells in peripheral blood. (A) Experimental design. Indian-origin rhesus macaques were inoculated with SARS-CoV-2 (SARS-CoV-2/human/USA/CA-CZB-59×002/2020) via the intranasal (IN), intratracheal (IT) and ocular route. Twenty-four hours later, animals were infused with either COVID-19 convalescent human plasma (I+CP; blue symbols), or normal plasma (I+NP; red symbols) (both at 4ml/kg), and four animals did not receive any plasma (infected; black symbols). Blood was sampled over the course of infection and tissues were collected at necropsy (11-14 DPI) for immune profiling. (B) Mean viral RNA (+range) in each of the groups within nasal washes (C) Flow plot illustrating gating strategy to identify innate immune subsets in whole blood. (D ) Kinetics of innate immune responses (*p
    Figure Legend Snippet: SARS-CoV-2 infection leads to a rapid and transient shift in innate immune responses and increases the number CD4 T follicular helper cells in peripheral blood. (A) Experimental design. Indian-origin rhesus macaques were inoculated with SARS-CoV-2 (SARS-CoV-2/human/USA/CA-CZB-59×002/2020) via the intranasal (IN), intratracheal (IT) and ocular route. Twenty-four hours later, animals were infused with either COVID-19 convalescent human plasma (I+CP; blue symbols), or normal plasma (I+NP; red symbols) (both at 4ml/kg), and four animals did not receive any plasma (infected; black symbols). Blood was sampled over the course of infection and tissues were collected at necropsy (11-14 DPI) for immune profiling. (B) Mean viral RNA (+range) in each of the groups within nasal washes (C) Flow plot illustrating gating strategy to identify innate immune subsets in whole blood. (D ) Kinetics of innate immune responses (*p

    Techniques Used: Infection

    CD4 T fh cells targeting the spike (S) and nucleocapsid (N) are generated following SARS-CoV-2 infection (A) Gating strategy for identifying SARS-CoV-2 specific CD4 T cells in spleen following stimulation with peptide megapools (B) Scatter plot showing AIM+ CD4 subsets; naive, CXCR5-, CXCR5+, and CXCR5+ PD-1 ++ GC T fh cells. The dashed line represents undetectable responses assigned a value of 0.01% ( C ) Cytokine profiles (IFN-γ, IL-2, TNFα, IL-17, IL-21) of CXCR5+, CXCR5-, and CD8+CD95+ T cells in spleen following PMA/Ionomycin stimulation. ( D ) Pie chart demonstrates polyfunctionality of T cell subsets following SARS-CoV-2 infection. (E ) Gating strategy for identifying SARS-CoV-2 specific CD4 T cells in PBMCs. (F) AIM+ CXCR5- and CXCR5+ CD4 subsets in PBMCs at Day 7. Black squares denote SARS-CoV-2 unexposed animals. Circles denote infected and triangles denote infected+infused animals.
    Figure Legend Snippet: CD4 T fh cells targeting the spike (S) and nucleocapsid (N) are generated following SARS-CoV-2 infection (A) Gating strategy for identifying SARS-CoV-2 specific CD4 T cells in spleen following stimulation with peptide megapools (B) Scatter plot showing AIM+ CD4 subsets; naive, CXCR5-, CXCR5+, and CXCR5+ PD-1 ++ GC T fh cells. The dashed line represents undetectable responses assigned a value of 0.01% ( C ) Cytokine profiles (IFN-γ, IL-2, TNFα, IL-17, IL-21) of CXCR5+, CXCR5-, and CD8+CD95+ T cells in spleen following PMA/Ionomycin stimulation. ( D ) Pie chart demonstrates polyfunctionality of T cell subsets following SARS-CoV-2 infection. (E ) Gating strategy for identifying SARS-CoV-2 specific CD4 T cells in PBMCs. (F) AIM+ CXCR5- and CXCR5+ CD4 subsets in PBMCs at Day 7. Black squares denote SARS-CoV-2 unexposed animals. Circles denote infected and triangles denote infected+infused animals.

    Techniques Used: Generated, Infection

    12) Product Images from "Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 in Vitro"

    Article Title: Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 in Vitro

    Journal: bioRxiv

    doi: 10.1101/2020.12.02.408112

    Potency of selected HCV PI against SARS-CoV-2 was confirmed in Huh7.5 cells. Huh7.5 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir and simeprevir, as described in Materials and Methods. After 70-74 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of 7 replicates ± SEM and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay as described in Materials and Methods. Data points (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance of 12 nontreated controls. Sigmoidal concentration response curves were fitted and CC50 values were determined, as shown in Supplementary Figure 4. The blue stippled line represents the drug concentrations at which DMSO is expected to induce cytotoxicity with reduction of cell viability to
    Figure Legend Snippet: Potency of selected HCV PI against SARS-CoV-2 was confirmed in Huh7.5 cells. Huh7.5 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir and simeprevir, as described in Materials and Methods. After 70-74 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of 7 replicates ± SEM and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay as described in Materials and Methods. Data points (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance of 12 nontreated controls. Sigmoidal concentration response curves were fitted and CC50 values were determined, as shown in Supplementary Figure 4. The blue stippled line represents the drug concentrations at which DMSO is expected to induce cytotoxicity with reduction of cell viability to

    Techniques Used: Infection, Incubation, Immunostaining, Concentration Assay, Colorimetric Assay

    At equipotent concentrations, simeprevir but not boceprevir synergized with remdesivir to completely suppress viral infection. VeroE6 cells seeded the previous day in T25 flasks were infected with SARS-CoV-2 followed by treatment with 0.4-fold EC50 of remdesivir (REM) or 1-fold EC50 of PI boceprevir (BOC) or simeprevir (SIM), or a combination of remdesivir with either PI, including an infected, nontreated culture serving as a positive control for viral spread, as described in Materials and Methods. Treatment was administered immediately after infection and subsequently at the indicated timepoints when cells were split, as described in Materials and Methods. Left panel, the % of SARS-CoV-2 infected cells on the specified days post infection was determined by immunostaining. Right panel, replicate cultures were derived following cell splitting and treatment and immunostained for the SARS-CoV-2 Spike protein and counterstained with Hoechst dye and images were acquired, as described in Materials and Methods. *Culture was terminated, or infection data not recorded, due to virus induced cell death. # Culture was maintained for a total of 15 days without indication of infection (no observation of single SARS-CoV-2 Spike protein positive cells).
    Figure Legend Snippet: At equipotent concentrations, simeprevir but not boceprevir synergized with remdesivir to completely suppress viral infection. VeroE6 cells seeded the previous day in T25 flasks were infected with SARS-CoV-2 followed by treatment with 0.4-fold EC50 of remdesivir (REM) or 1-fold EC50 of PI boceprevir (BOC) or simeprevir (SIM), or a combination of remdesivir with either PI, including an infected, nontreated culture serving as a positive control for viral spread, as described in Materials and Methods. Treatment was administered immediately after infection and subsequently at the indicated timepoints when cells were split, as described in Materials and Methods. Left panel, the % of SARS-CoV-2 infected cells on the specified days post infection was determined by immunostaining. Right panel, replicate cultures were derived following cell splitting and treatment and immunostained for the SARS-CoV-2 Spike protein and counterstained with Hoechst dye and images were acquired, as described in Materials and Methods. *Culture was terminated, or infection data not recorded, due to virus induced cell death. # Culture was maintained for a total of 15 days without indication of infection (no observation of single SARS-CoV-2 Spike protein positive cells).

    Techniques Used: Infection, Positive Control, Immunostaining, Derivative Assay

    Comparison of barrier to escape for HCV PI at equipotent concentrations. VeroE6 cells seeded the previous day in T25 flasks were infected with SARS-CoV-2 followed by treatment with 1-fold EC50 of PI boceprevir, telaprevir, narlaprevir, simeprevir, paritaprevir, grazoprevir, vaniprevir, danoprevir, asunaprevir and faldaprevir, which were administered immediately after infection and on day 1, 3, 5 and 7 post infection when cells were split, as described in Materials and Methods. Left panel, the % of SARS-CoV-2 infected cells on the specified days post infection, was determined by immunostaining. Right panel, replicate cultures were derived following cell splitting and treatment and immunostained for the SARS-CoV-2 Spike protein and counterstained with Hoechst dye and images were acquired, as descried in Materials and Methods. Cultures summarized in this figure are derived from different experimental setups, each including an infected nontreated control culture, which showed viral spread comparable to that in the depicted representative culture. *Culture was terminated, or infection data not recorded, due to virus induced cell death. **Culture was terminated due to drug induced cytotoxicity, possibly enhanced by viral infection.
    Figure Legend Snippet: Comparison of barrier to escape for HCV PI at equipotent concentrations. VeroE6 cells seeded the previous day in T25 flasks were infected with SARS-CoV-2 followed by treatment with 1-fold EC50 of PI boceprevir, telaprevir, narlaprevir, simeprevir, paritaprevir, grazoprevir, vaniprevir, danoprevir, asunaprevir and faldaprevir, which were administered immediately after infection and on day 1, 3, 5 and 7 post infection when cells were split, as described in Materials and Methods. Left panel, the % of SARS-CoV-2 infected cells on the specified days post infection, was determined by immunostaining. Right panel, replicate cultures were derived following cell splitting and treatment and immunostained for the SARS-CoV-2 Spike protein and counterstained with Hoechst dye and images were acquired, as descried in Materials and Methods. Cultures summarized in this figure are derived from different experimental setups, each including an infected nontreated control culture, which showed viral spread comparable to that in the depicted representative culture. *Culture was terminated, or infection data not recorded, due to virus induced cell death. **Culture was terminated due to drug induced cytotoxicity, possibly enhanced by viral infection.

    Techniques Used: Infection, Immunostaining, Derivative Assay

    Analysis of interactions of selected HCV PI with remdesivir. VeroE6 cells seeded in 96-well plates were infected the following day with SARS-CoV-2 followed by treatment with specified concentrations of the linear PI boceprevir (BOC) and narlaprevir (NAR), or the macrocyclic PI simeprevir (SIM), paritaprevir (PAR) and grazoprevir (GRA), or polymerase inhibitor remdesivir (REM), or a combination of these PI and remdesivir, as described in Materials and Methods. After 46-50 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Fractional effect (Fa) values were calculated by relating counts from infected and treated cultures to the mean count from at least 21 infected nontreated cultures and were entered into CompuSyn software. Datapoints are means of 6 to 7 replicates, and for each treatment experiment 6 to 10 datapoints were entered. For each inhibitor combination depicted per row, the following curves were fitted using Compusyn: (A) concentration-Fa curves plotting Fa values ranging from 0.01 to 0.99 against specified inhibitor concentrations. (B) Fa-CI curves plotting CI values ranging from 0 to 2 against Fa values ranging from 0.01 to 0.99. (C) Fa-Log 10 CI curves plotting logarithmic CI values ranging from 0.01 to 100 against Fa values ranging from 0.01 to 0.99. (B and C) Overall, CI values ≥1.1 suggest antagonism “A”, while CI values
    Figure Legend Snippet: Analysis of interactions of selected HCV PI with remdesivir. VeroE6 cells seeded in 96-well plates were infected the following day with SARS-CoV-2 followed by treatment with specified concentrations of the linear PI boceprevir (BOC) and narlaprevir (NAR), or the macrocyclic PI simeprevir (SIM), paritaprevir (PAR) and grazoprevir (GRA), or polymerase inhibitor remdesivir (REM), or a combination of these PI and remdesivir, as described in Materials and Methods. After 46-50 hours incubation SARS-CoV-2 infected cells were visualized by immunostaining for SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Fractional effect (Fa) values were calculated by relating counts from infected and treated cultures to the mean count from at least 21 infected nontreated cultures and were entered into CompuSyn software. Datapoints are means of 6 to 7 replicates, and for each treatment experiment 6 to 10 datapoints were entered. For each inhibitor combination depicted per row, the following curves were fitted using Compusyn: (A) concentration-Fa curves plotting Fa values ranging from 0.01 to 0.99 against specified inhibitor concentrations. (B) Fa-CI curves plotting CI values ranging from 0 to 2 against Fa values ranging from 0.01 to 0.99. (C) Fa-Log 10 CI curves plotting logarithmic CI values ranging from 0.01 to 100 against Fa values ranging from 0.01 to 0.99. (B and C) Overall, CI values ≥1.1 suggest antagonism “A”, while CI values

    Techniques Used: Infection, Incubation, Immunostaining, Software, Concentration Assay

    Potency of a panel of HCV PI and an HCV NS4A inhibitor against SARS-CoV-2 in VeroE6 cells. VeroE6 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir, narlaprevir, simeprevir, paritaprevir, grazoprevir, glecaprevir, voxilaprevir, vaniprevir, danoprevir, deldeprevir, asunaprevir and faldaprevir, as well as HCV NS4A inhibitor ACH-806, as described in Materials and Methods. After 46-50 hours of incubation, SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of counts from 7 replicate cultures ± standard errors of the means (SEM) and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay, as described in Materials and Methods. Datapoints (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance from 12 replicate nontreated control cultures. Sigmoidal concentration response curves were fitted and CC50 values were determined as shown in Supplementary Figure 3. The red / blue stippled line represents the drug concentrations at which DMSO is expected to induce antiviral effects with reduction of residual infectivity to
    Figure Legend Snippet: Potency of a panel of HCV PI and an HCV NS4A inhibitor against SARS-CoV-2 in VeroE6 cells. VeroE6 cells were seeded in 96-well plates and the following day infected with SARS-CoV-2 followed by treatment with specified concentrations of the PI boceprevir, telaprevir, narlaprevir, simeprevir, paritaprevir, grazoprevir, glecaprevir, voxilaprevir, vaniprevir, danoprevir, deldeprevir, asunaprevir and faldaprevir, as well as HCV NS4A inhibitor ACH-806, as described in Materials and Methods. After 46-50 hours of incubation, SARS-CoV-2 infected cells were visualized by immunostaining for the SARS-CoV-2 Spike protein and quantified by automated counting, as described in Materials and Methods. Datapoints (red dots) are means of counts from 7 replicate cultures ± standard errors of the means (SEM) and represent % residual infectivity, determined as % SARS-CoV-2 positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration response curves (red lines) were fitted and EC50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay, as described in Materials and Methods. Datapoints (blue triangles) are means of 3 replicate cultures ± SEM and represent % cell viability relative to mean absorbance from 12 replicate nontreated control cultures. Sigmoidal concentration response curves were fitted and CC50 values were determined as shown in Supplementary Figure 3. The red / blue stippled line represents the drug concentrations at which DMSO is expected to induce antiviral effects with reduction of residual infectivity to

    Techniques Used: Infection, Incubation, Immunostaining, Concentration Assay, Colorimetric Assay

    Boceprevir was capable of completely suppressing SARS-CoV-2. VeroE6 cells seeded the previous day in T25 flasks were infected with SARS-CoV-2 followed by treatment with 1-, 1.5-, 2-, 2.5-, 3- and 5-fold EC50 boceprevir, which was administered immediately after infection and subsequently at the indicated timepoints when cells were split, as described in Materials and Methods. Left panel, the % of SARS-CoV-2 infected cells on the specified days post infection was determined by immunostaining. Right panel, replicate cultures were derived following cell splitting and treatment and immunostained for the SARS-CoV-2 Spike protein and counterstained with Hoechst dye and images were acquired, as described in Materials and Methods. Cultures summarized in this figure are derived from different experimental setups, each including an infected nontreated control culture, which showed viral spread comparable to that in the depicted representative culture. *Culture was terminated, or infection data not recorded, due to virus induced cell death. ** Culture was maintained for a total of 17 days without indication of infection (no observation of single SARS-CoV-2 Spike protein positive cells).
    Figure Legend Snippet: Boceprevir was capable of completely suppressing SARS-CoV-2. VeroE6 cells seeded the previous day in T25 flasks were infected with SARS-CoV-2 followed by treatment with 1-, 1.5-, 2-, 2.5-, 3- and 5-fold EC50 boceprevir, which was administered immediately after infection and subsequently at the indicated timepoints when cells were split, as described in Materials and Methods. Left panel, the % of SARS-CoV-2 infected cells on the specified days post infection was determined by immunostaining. Right panel, replicate cultures were derived following cell splitting and treatment and immunostained for the SARS-CoV-2 Spike protein and counterstained with Hoechst dye and images were acquired, as described in Materials and Methods. Cultures summarized in this figure are derived from different experimental setups, each including an infected nontreated control culture, which showed viral spread comparable to that in the depicted representative culture. *Culture was terminated, or infection data not recorded, due to virus induced cell death. ** Culture was maintained for a total of 17 days without indication of infection (no observation of single SARS-CoV-2 Spike protein positive cells).

    Techniques Used: Infection, Immunostaining, Derivative Assay

    13) Product Images from "Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2"

    Article Title: Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2020.12.30.424862

    Screening SARS-CoV-2 antiviral activity using the FDA-approved and bioactive compound libraries. (A) Assay scheme: Cells are treated with DMSO (left panel) or drug (middle and right panels), infected with SARS-CoV-2 or left uninfected (right panel) and incubated for 72-96h to observe cytopathic effect (CPE). CPE is measured by CTG assay, quantifying ATP content in viable cells using luminescence (RLU). The right panel shows the cytotoxicity control, treating cells with drugs but without virus. (B-C) Average luminescence is shown for (B) Vero-E6 at 72h or (C) Calu-3 cells at 96h post-infection. (D) Screen of FDA-approved and bioactive compound libraries on Vero-E6 cells with inhibition of CPE (%) on the y-axis and cell viability (%) on the x-axis normalized to DMSO-treated wells. Red: high priority hits with a cutoff of > 20% inhibition of CPE and > 70% cell viability. (E) As in (D), but on Calu-3 cells, with a cutoff of > 70% inhibition of CPE and > 70% cell viability. (F) Combination of inhibition of CPE (%) on Vero-E6 (y-axis) from (D) and Calu-3 (x-axis) from (E). (G) Gene set enrichment analysis. Distribution of the enrichment score (green line) across compounds annotated to molecular targets (vertical black lines). CDK1, CDK2, GSK-3 p
    Figure Legend Snippet: Screening SARS-CoV-2 antiviral activity using the FDA-approved and bioactive compound libraries. (A) Assay scheme: Cells are treated with DMSO (left panel) or drug (middle and right panels), infected with SARS-CoV-2 or left uninfected (right panel) and incubated for 72-96h to observe cytopathic effect (CPE). CPE is measured by CTG assay, quantifying ATP content in viable cells using luminescence (RLU). The right panel shows the cytotoxicity control, treating cells with drugs but without virus. (B-C) Average luminescence is shown for (B) Vero-E6 at 72h or (C) Calu-3 cells at 96h post-infection. (D) Screen of FDA-approved and bioactive compound libraries on Vero-E6 cells with inhibition of CPE (%) on the y-axis and cell viability (%) on the x-axis normalized to DMSO-treated wells. Red: high priority hits with a cutoff of > 20% inhibition of CPE and > 70% cell viability. (E) As in (D), but on Calu-3 cells, with a cutoff of > 70% inhibition of CPE and > 70% cell viability. (F) Combination of inhibition of CPE (%) on Vero-E6 (y-axis) from (D) and Calu-3 (x-axis) from (E). (G) Gene set enrichment analysis. Distribution of the enrichment score (green line) across compounds annotated to molecular targets (vertical black lines). CDK1, CDK2, GSK-3 p

    Techniques Used: Activity Assay, Infection, Incubation, CTG Assay, Inhibition

    (A) HPMEC, (B) BEAS-2B, (C) HCT-116, (D) LNCaP, (E) HaCaT, (F) RD, (G) NCI-H1437, (H) Huh-7.5.1, (I) Caco-2, (J) A549/hACE2, (K) HBEC-30KT, or (L) A549 cells were infected with SARS-CoV-2 at MOI 0.5 or 0.05 as in figure 1 . Viral titers were analyzed by TCID50 assay at the indicated time points (hours post-infection, hpi). Dashed lines represent limit of detection. Data represent mean ± SEM for n = 2 independent experiments.
    Figure Legend Snippet: (A) HPMEC, (B) BEAS-2B, (C) HCT-116, (D) LNCaP, (E) HaCaT, (F) RD, (G) NCI-H1437, (H) Huh-7.5.1, (I) Caco-2, (J) A549/hACE2, (K) HBEC-30KT, or (L) A549 cells were infected with SARS-CoV-2 at MOI 0.5 or 0.05 as in figure 1 . Viral titers were analyzed by TCID50 assay at the indicated time points (hours post-infection, hpi). Dashed lines represent limit of detection. Data represent mean ± SEM for n = 2 independent experiments.

    Techniques Used: Infection, TCID50 Assay

    Dose response curves of compounds with SARS-CoV-2 antiviral activity. Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with compounds at indicated concentrations. Data show % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.
    Figure Legend Snippet: Dose response curves of compounds with SARS-CoV-2 antiviral activity. Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with compounds at indicated concentrations. Data show % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.

    Techniques Used: Activity Assay, Infection, Inhibition

    (A) Clinical status of compounds tested. (B-C) Remdesivir dose response curves in (B) Vero-E6 and (C) Calu-3 cells showing % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.
    Figure Legend Snippet: (A) Clinical status of compounds tested. (B-C) Remdesivir dose response curves in (B) Vero-E6 and (C) Calu-3 cells showing % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.

    Techniques Used: Inhibition, Infection

    Confirmation and characterization of SARS-CoV-2 antiviral candidate compounds. Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05, treated with the top 12 compounds (shown in Figure 3 ), disulfiram, or apilimod mesylate at indicated concentrations and supernatants were collected at 24 hpi. Viral titers and genome copies were calculated by TCID50 and qRT-PCR, respectively. (A) and (B) protein kinase and protease inhibitors, (C) and (D) anti-inflammatory compounds, (E) and (F) direct-acting antivirals and (G) and (H) other host-targeting compounds. TCID50 data represent mean ± SD for n = 2 independent experiments. Genome copy data represent mean ± SEM for n = 2 technical replicates and are representative of n = 2 independent experiments.
    Figure Legend Snippet: Confirmation and characterization of SARS-CoV-2 antiviral candidate compounds. Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05, treated with the top 12 compounds (shown in Figure 3 ), disulfiram, or apilimod mesylate at indicated concentrations and supernatants were collected at 24 hpi. Viral titers and genome copies were calculated by TCID50 and qRT-PCR, respectively. (A) and (B) protein kinase and protease inhibitors, (C) and (D) anti-inflammatory compounds, (E) and (F) direct-acting antivirals and (G) and (H) other host-targeting compounds. TCID50 data represent mean ± SD for n = 2 independent experiments. Genome copy data represent mean ± SEM for n = 2 technical replicates and are representative of n = 2 independent experiments.

    Techniques Used: Infection, Quantitative RT-PCR

    Permissive cell lines to SARS-CoV2 infection. (A) Vero-E6, (B) Calu-3, (C) Huh-7 and (D) HPMEC/hACE2 cells were seeded in 24-well plates and infected with SARS-CoV-2 at MOI 0.5 or 0.05 at 37°C and 5% C0 2 for 30 minutes. Viral inoculum was then removed, cells were washed once in 1x PBS, and 1 ml of regular media was replaced. At the indicated time points (hours post-infection, hpi), plates were freeze/thawed and viral titers from whole cell lysates were analyzed by TCID50 assay. Dashed line represents limit of detection of the assay. Data represent mean ± SEM for n = 2 independent experiments.
    Figure Legend Snippet: Permissive cell lines to SARS-CoV2 infection. (A) Vero-E6, (B) Calu-3, (C) Huh-7 and (D) HPMEC/hACE2 cells were seeded in 24-well plates and infected with SARS-CoV-2 at MOI 0.5 or 0.05 at 37°C and 5% C0 2 for 30 minutes. Viral inoculum was then removed, cells were washed once in 1x PBS, and 1 ml of regular media was replaced. At the indicated time points (hours post-infection, hpi), plates were freeze/thawed and viral titers from whole cell lysates were analyzed by TCID50 assay. Dashed line represents limit of detection of the assay. Data represent mean ± SEM for n = 2 independent experiments.

    Techniques Used: Infection, TCID50 Assay

    Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with compounds at indicated concentrations. Data show % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.
    Figure Legend Snippet: Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with compounds at indicated concentrations. Data show % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.

    Techniques Used: Infection, Inhibition

    Cell-type specificity of compounds antiviral activity. Huh7, HPMEC/hACE2 and Vero-E6 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with (A) Dinaciclib, (B) BFH772, (C) Budesonide, (D) GC376 sodium, (E) Apilimod mesylate, (F) GKT137831, (G) Cyclosporin A, (H) B02, and (I) Camostat mesylate at indicated concentrations. At 48hpi cells were washed, fixed, and stained with DAPI and for SARS-CoV-2 nucleocapsid protein. Plates were fluorescently imaged and analyzed for nucleocapsid stain per nuclei. Relative infection (full lines) and relative number of cells (dashed lines) are normalized to DMSO-treated wells. Data represent mean ± SEM for n = 4 technical replicates and are representative of n = 3 independent experiments.
    Figure Legend Snippet: Cell-type specificity of compounds antiviral activity. Huh7, HPMEC/hACE2 and Vero-E6 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with (A) Dinaciclib, (B) BFH772, (C) Budesonide, (D) GC376 sodium, (E) Apilimod mesylate, (F) GKT137831, (G) Cyclosporin A, (H) B02, and (I) Camostat mesylate at indicated concentrations. At 48hpi cells were washed, fixed, and stained with DAPI and for SARS-CoV-2 nucleocapsid protein. Plates were fluorescently imaged and analyzed for nucleocapsid stain per nuclei. Relative infection (full lines) and relative number of cells (dashed lines) are normalized to DMSO-treated wells. Data represent mean ± SEM for n = 4 technical replicates and are representative of n = 3 independent experiments.

    Techniques Used: Activity Assay, Infection, Staining

    B02 synergy with remdesivir. (A) Vero-E6 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with 2 μM remdesivir, 10 μM B02, or a combination of 2 μM remdesivir and 10 μM B02 for 72h. CPE inhibition was measured by CTG assay. (B) Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with remdesivir at indicated concentrations in the presence or absence of 10 μM B02 for 96h. CPE inhibition was measured by CTG assay and was normalized to DMSO-treated wells. Data represent mean ± SD for n = 2 technical replicates.
    Figure Legend Snippet: B02 synergy with remdesivir. (A) Vero-E6 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with 2 μM remdesivir, 10 μM B02, or a combination of 2 μM remdesivir and 10 μM B02 for 72h. CPE inhibition was measured by CTG assay. (B) Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with remdesivir at indicated concentrations in the presence or absence of 10 μM B02 for 96h. CPE inhibition was measured by CTG assay and was normalized to DMSO-treated wells. Data represent mean ± SD for n = 2 technical replicates.

    Techniques Used: Infection, Inhibition, CTG Assay

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

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

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

    Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.
    Figure Legend Snippet: Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL
    Figure Legend Snippet: SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL

    Techniques Used: Standard Deviation

    Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Serial Dilution, Standard Deviation, Generated

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

    Techniques Used: Chemiluminescent ELISA

    15) Product Images from "Targeting pentose phosphate pathway for SARS-CoV-2 therapy"

    Article Title: Targeting pentose phosphate pathway for SARS-CoV-2 therapy

    Journal: bioRxiv

    doi: 10.1101/2020.08.19.257022

    Glycolysis and pentose phosphate pathway as host target for antiviral therapy against SARS-CoV-2 (A) Heatmap of changes in protein abundance of components of glycolysis and pentose phosphate pathway in SARS-CoV-2 infected Caco-2 cells at 24h post infection. A Z score transformation was performed such that red and blue represent high and low protein abundance respectively. The plot was performed using the heatmaps2 function of the gplots package of the R suite. (B) Immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM1 and SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT. Caco-2 cells were pre-treated with different concentration of BOT for 24h. The cells were then infected with two different SARS-CoV-2 strains at MOI 0.01. 24h post infection, cells were fixed and stain for spike protein. Representative images of three independent experiments are shown. (C) Dose-response curves of viral inhibition and cell viability in BOT treated cells. Percentage of viral inhibition was evaluated by spike protein staining and cell viability was measured by MTT assay. The IC50 and CC50 values were determined using the curve regression function of GraphPad Prism 8. Both plots represent mean+SD of three independent experiments performed with three technical replicates. (D) Quantification of viral genomes in supernatant of SARS-CoV-2 infected Caco-2 cells treated with BOT in combination with 2DG or BOT alone. The number of SARS-CoV-2/FFM7 RNA was determined by qRT-PCR of RdRp gene and depicted as RNA copies/ml. The bar plot represents mean+SD of three independent experiments performed with three technical replicates. Statistical significance was determined with a two sided unpaired t-test. ns: not significant; * p≤0.05; ** p≤0.01. (E) Inhibition of viral infection in BOT treated cells in combination with 2DG. Caco-2 cells were pre-treated with different concentration of BOT for 24h. Then the 2DG at concentration 5mM was added and cells were infected with SARS-CoV-2/FFM7 at MOI 0.01. 24h post infection, cells were fixed and stain for spike protein. Percentage of viral inhibition was evaluated by spike protein staining. Bar graph depicts mean+SD of three independent experiments with three technical replicates. Statistical significance was determined with a two sided unpaired t-test. *** p≤0.005 (F) Immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT in combination with 2DG. Representative images of three independent experiments are shown. (G) Simplified scheme of glycolysis and pentose phosphate pathway. The targets for 2DG and BOT are depicted in red. The scheme was created with BioRender.com.
    Figure Legend Snippet: Glycolysis and pentose phosphate pathway as host target for antiviral therapy against SARS-CoV-2 (A) Heatmap of changes in protein abundance of components of glycolysis and pentose phosphate pathway in SARS-CoV-2 infected Caco-2 cells at 24h post infection. A Z score transformation was performed such that red and blue represent high and low protein abundance respectively. The plot was performed using the heatmaps2 function of the gplots package of the R suite. (B) Immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM1 and SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT. Caco-2 cells were pre-treated with different concentration of BOT for 24h. The cells were then infected with two different SARS-CoV-2 strains at MOI 0.01. 24h post infection, cells were fixed and stain for spike protein. Representative images of three independent experiments are shown. (C) Dose-response curves of viral inhibition and cell viability in BOT treated cells. Percentage of viral inhibition was evaluated by spike protein staining and cell viability was measured by MTT assay. The IC50 and CC50 values were determined using the curve regression function of GraphPad Prism 8. Both plots represent mean+SD of three independent experiments performed with three technical replicates. (D) Quantification of viral genomes in supernatant of SARS-CoV-2 infected Caco-2 cells treated with BOT in combination with 2DG or BOT alone. The number of SARS-CoV-2/FFM7 RNA was determined by qRT-PCR of RdRp gene and depicted as RNA copies/ml. The bar plot represents mean+SD of three independent experiments performed with three technical replicates. Statistical significance was determined with a two sided unpaired t-test. ns: not significant; * p≤0.05; ** p≤0.01. (E) Inhibition of viral infection in BOT treated cells in combination with 2DG. Caco-2 cells were pre-treated with different concentration of BOT for 24h. Then the 2DG at concentration 5mM was added and cells were infected with SARS-CoV-2/FFM7 at MOI 0.01. 24h post infection, cells were fixed and stain for spike protein. Percentage of viral inhibition was evaluated by spike protein staining. Bar graph depicts mean+SD of three independent experiments with three technical replicates. Statistical significance was determined with a two sided unpaired t-test. *** p≤0.005 (F) Immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT in combination with 2DG. Representative images of three independent experiments are shown. (G) Simplified scheme of glycolysis and pentose phosphate pathway. The targets for 2DG and BOT are depicted in red. The scheme was created with BioRender.com.

    Techniques Used: Infection, Transformation Assay, Immunohistochemistry, Staining, Concentration Assay, Inhibition, MTT Assay, Quantitative RT-PCR

    16) Product Images from "SARS-CoV and SARS-CoV-2 are transmitted through the air between ferrets over more than one meter distance"

    Article Title: SARS-CoV and SARS-CoV-2 are transmitted through the air between ferrets over more than one meter distance

    Journal: Nature Communications

    doi: 10.1038/s41467-021-21918-6

    Virus RNA shedding in ferrets. A/H1N1 ( a ), SARS-CoV-2 ( b ), and SARS-CoV ( c ) RNA were detected by qRT-PCR in the throat (gray) and nasal (white) swabs collected from a donor (bars) and recipient (circles) ferrets every other day. An individual donor-recipient pair is shown in each panel.
    Figure Legend Snippet: Virus RNA shedding in ferrets. A/H1N1 ( a ), SARS-CoV-2 ( b ), and SARS-CoV ( c ) RNA were detected by qRT-PCR in the throat (gray) and nasal (white) swabs collected from a donor (bars) and recipient (circles) ferrets every other day. An individual donor-recipient pair is shown in each panel.

    Techniques Used: Quantitative RT-PCR

    Antibody responses in donor and recipient ferrets. Sera were collected from donor and recipient ferrets on the indicated days. Antibody responses against A/H1N1 virus ( a ) were measured by hemagglutination inhibition (HI) assay, whereas responses against SARS-CoV-2 ( b ) and SARS-CoV ( c ) were assessed using a nucleoprotein (NP) ELISA. Dotted lines indicate the detection limit of each assay. OD: Optic density.
    Figure Legend Snippet: Antibody responses in donor and recipient ferrets. Sera were collected from donor and recipient ferrets on the indicated days. Antibody responses against A/H1N1 virus ( a ) were measured by hemagglutination inhibition (HI) assay, whereas responses against SARS-CoV-2 ( b ) and SARS-CoV ( c ) were assessed using a nucleoprotein (NP) ELISA. Dotted lines indicate the detection limit of each assay. OD: Optic density.

    Techniques Used: HI Assay, Enzyme-linked Immunosorbent Assay

    Infectious virus shedding in ferrets. A/H1N1 virus ( a ), SARS-CoV-2 ( b ), and SARS-CoV ( c ) titers were detected in the throat (gray) and nasal (white) swabs collected from inoculated donor (bars) and indirect recipient (circles) ferrets. An individual donor-recipient pair is shown in each panel. The dotted line indicates the detection limit.
    Figure Legend Snippet: Infectious virus shedding in ferrets. A/H1N1 virus ( a ), SARS-CoV-2 ( b ), and SARS-CoV ( c ) titers were detected in the throat (gray) and nasal (white) swabs collected from inoculated donor (bars) and indirect recipient (circles) ferrets. An individual donor-recipient pair is shown in each panel. The dotted line indicates the detection limit.

    Techniques Used:

    17) Product Images from "Cross-linking peptide and repurposed drugs inhibit both entry pathways of SARS-CoV-2"

    Article Title: Cross-linking peptide and repurposed drugs inhibit both entry pathways of SARS-CoV-2

    Journal: Nature Communications

    doi: 10.1038/s41467-021-21825-w

    The dual-functional activities of 8P9R. a Cross-linking of SARS-CoV-2 by 8P9R. SARS-CoV-2 was treated by 8P9R, P9R, or P9RS (50 μg ml −1 ). The treated virus was negatively stained for TEM assay. The red triangle indicates the big cluster of cross-linked SARS-CoV-2. Scale bar = 0.5 μm. For quantification, 55 independent viral particles of P9RS-treated virus, 50 independent viral particles of P9R-treated virus, and 13 viral particles (including independent and clustered particles) of 8P9R-treated virus could be accounted in 5 representative microscope fields. The big clustering viral particles in 8P9R-treated samples could be more than 500 nm, which was bigger than the size (~100 nm) of the usual SARS-CoV-2 virion. b H1N1 virus was pre-labelled by green fluorescence dye and then treated by peptides. After 1 h infection in MDCK cells, cells were fixed and stained by cell membrane dye (red) and nuclear dye (blue). White triangles indicate the cross-linked viruses located at cell membrane. Scale bar = 20 μm ( c ). 8P9R could efficiently inhibit endosomal acidification. MDCK cells were treated by 8P9R (25 μg ml −1 ), bafilomycin A1 (BA1, 50 nM), BSA (Mock) and low pH indicator pHrodo TM Red dextran. Red dots indicate the endosomes with low pH. Nuclei were stained with nuclear dye (blue). Live cell images were taken by confocal microscopes. Scale bar = 20 μm. Experiments were repeated twice independently. Source data are provided as a Source Data file.
    Figure Legend Snippet: The dual-functional activities of 8P9R. a Cross-linking of SARS-CoV-2 by 8P9R. SARS-CoV-2 was treated by 8P9R, P9R, or P9RS (50 μg ml −1 ). The treated virus was negatively stained for TEM assay. The red triangle indicates the big cluster of cross-linked SARS-CoV-2. Scale bar = 0.5 μm. For quantification, 55 independent viral particles of P9RS-treated virus, 50 independent viral particles of P9R-treated virus, and 13 viral particles (including independent and clustered particles) of 8P9R-treated virus could be accounted in 5 representative microscope fields. The big clustering viral particles in 8P9R-treated samples could be more than 500 nm, which was bigger than the size (~100 nm) of the usual SARS-CoV-2 virion. b H1N1 virus was pre-labelled by green fluorescence dye and then treated by peptides. After 1 h infection in MDCK cells, cells were fixed and stained by cell membrane dye (red) and nuclear dye (blue). White triangles indicate the cross-linked viruses located at cell membrane. Scale bar = 20 μm ( c ). 8P9R could efficiently inhibit endosomal acidification. MDCK cells were treated by 8P9R (25 μg ml −1 ), bafilomycin A1 (BA1, 50 nM), BSA (Mock) and low pH indicator pHrodo TM Red dextran. Red dots indicate the endosomes with low pH. Nuclei were stained with nuclear dye (blue). Live cell images were taken by confocal microscopes. Scale bar = 20 μm. Experiments were repeated twice independently. Source data are provided as a Source Data file.

    Techniques Used: Functional Assay, Staining, Transmission Electron Microscopy, Microscopy, Fluorescence, Infection

    Drug combination enhanced the antiviral activity against SARS-CoV-2 and SARS-CoV. a Chloroquine (Chl) could significantly enhance the activity of arbidol against SARS-CoV-2 while arbidol alone (0.2 μg ml −1 , Ar-0.2) did not show antiviral activity ( n = 4). SARS-CoV-2 was treated by the indicated Ar-0.2, Chl-3.1 (3.1 μg ml −1 ), or Ar+Chl. P value was compared with Chl-3.1. b Chloroquine (Chl) could significantly enhance the activity of arbidol against SARS-CoV while arbidol alone (0.4 μg ml −1 , Ar-0.4) did not show antiviral activity ( n = 4). SARS-CoV was treated by the indicated Ar-0.4, Chl-6.3 (6.3 μg ml −1 ), or Ar+Chl. Viral RNA copies were measured at 24 h post infection in cell supernatants. The relative RNA copy was compared to mock treated virus. P value was compared with Chl-6.3. c The antiviral activity of indicated drugs or drug combinations against SARS-CoV in mice. Mice were intranasally inoculated with SARS-CoV (5 × 10 3 PFU). 8P9R (intranasal 0.5 mg kg −1 , n = 8), arbidol (Ar, oral 30 mg kg −1 , n = 8), chloroquine (Chl, oral 40 mg kg −1 , n = 6), camostat (Cam, intranasal 0.3 mg kg −1 , n = 5), Ar+Chl ( n = 6), Ar+Cam ( n = 6), Chl+Cam ( n = 6), Ar+Chl+Cam ( n = 5) and mock ( n = 12) were given to mice at 8 h post infection. Two more doses were given to mice in the following day. Viral loads in lung tissues were measured by plaque assay at day 2 post infection. d – e The antiviral activity of 8P9R (12.5 μg ml −1 ), arbidol (12.5 μg ml −1 ), and chloroquine (12.5 μg ml −1 ) in Vero-E6 ( d , n = 4) and Calu-3 ( e , n = 3) cells. Viral RNA copies in cell supernatants were measured by RT-qPCR at 24 h post infection. Relative RNA copy was normalized to mock. f The antiviral activity of indicated drugs or drug combinations against SARS-CoV-2 in hamsters. Hamsters were intranasally inoculated with SARS-CoV-2 (5 × 10 3 PFU). Mock ( n = 9), 8P9R (intranasal 0.5 mg kg −1 , n = 4), Ar+Chl+Cam ( n = 6), Chl+Cam ( n = 6), Ar+Cam (3), Cam (intranasal 0.3 mg kg −1 , n = 5), Ar (oral 30 mg kg −1 , n = 3), and Chl (oral 40 mg kg −1 , n = 4) were given to hamsters at 8 h post infection. Two more doses were given to hamsters in the following day. Viral loads in lung tissues were measured by plaque assay at day 2 post infection. Data are presented as mean ± SD of independent biological samples. P values are calculated by two-tailed student t test when compared with mock. Source data are provided as a Source Data file.
    Figure Legend Snippet: Drug combination enhanced the antiviral activity against SARS-CoV-2 and SARS-CoV. a Chloroquine (Chl) could significantly enhance the activity of arbidol against SARS-CoV-2 while arbidol alone (0.2 μg ml −1 , Ar-0.2) did not show antiviral activity ( n = 4). SARS-CoV-2 was treated by the indicated Ar-0.2, Chl-3.1 (3.1 μg ml −1 ), or Ar+Chl. P value was compared with Chl-3.1. b Chloroquine (Chl) could significantly enhance the activity of arbidol against SARS-CoV while arbidol alone (0.4 μg ml −1 , Ar-0.4) did not show antiviral activity ( n = 4). SARS-CoV was treated by the indicated Ar-0.4, Chl-6.3 (6.3 μg ml −1 ), or Ar+Chl. Viral RNA copies were measured at 24 h post infection in cell supernatants. The relative RNA copy was compared to mock treated virus. P value was compared with Chl-6.3. c The antiviral activity of indicated drugs or drug combinations against SARS-CoV in mice. Mice were intranasally inoculated with SARS-CoV (5 × 10 3 PFU). 8P9R (intranasal 0.5 mg kg −1 , n = 8), arbidol (Ar, oral 30 mg kg −1 , n = 8), chloroquine (Chl, oral 40 mg kg −1 , n = 6), camostat (Cam, intranasal 0.3 mg kg −1 , n = 5), Ar+Chl ( n = 6), Ar+Cam ( n = 6), Chl+Cam ( n = 6), Ar+Chl+Cam ( n = 5) and mock ( n = 12) were given to mice at 8 h post infection. Two more doses were given to mice in the following day. Viral loads in lung tissues were measured by plaque assay at day 2 post infection. d – e The antiviral activity of 8P9R (12.5 μg ml −1 ), arbidol (12.5 μg ml −1 ), and chloroquine (12.5 μg ml −1 ) in Vero-E6 ( d , n = 4) and Calu-3 ( e , n = 3) cells. Viral RNA copies in cell supernatants were measured by RT-qPCR at 24 h post infection. Relative RNA copy was normalized to mock. f The antiviral activity of indicated drugs or drug combinations against SARS-CoV-2 in hamsters. Hamsters were intranasally inoculated with SARS-CoV-2 (5 × 10 3 PFU). Mock ( n = 9), 8P9R (intranasal 0.5 mg kg −1 , n = 4), Ar+Chl+Cam ( n = 6), Chl+Cam ( n = 6), Ar+Cam (3), Cam (intranasal 0.3 mg kg −1 , n = 5), Ar (oral 30 mg kg −1 , n = 3), and Chl (oral 40 mg kg −1 , n = 4) were given to hamsters at 8 h post infection. Two more doses were given to hamsters in the following day. Viral loads in lung tissues were measured by plaque assay at day 2 post infection. Data are presented as mean ± SD of independent biological samples. P values are calculated by two-tailed student t test when compared with mock. Source data are provided as a Source Data file.

    Techniques Used: Activity Assay, Infection, Mouse Assay, Chick Chorioallantoic Membrane Assay, Plaque Assay, Quantitative RT-PCR, Two Tailed Test

    Synergistic mechanism of 8P9R enhancing the antiviral activity of arbidol. a 8P9R could enhance the antiviral activity of arbidol against SARS-CoV-2 in Vero-E6 cells ( n = 5). Virus infected cells at the presence of the indicated concentrations of arbidol (Ar) or Ar+8P9R (1.6 μg ml −1 ) or Ar+8P9R (3.1 μg ml −1 ). b 8P9R could significantly enhance the antiviral activity of arbidol when arbidol alone did not show antiviral activity ( n = 4). SARS-CoV-2 was treated by the indicated Ar-0.2 (0.2 μg m l −1 ), 8P9R-3.1 (3.1 μg ml −1 ), Ar+8P9R, or PBS (Mock). P value was compared with Ar+8P9R. c SARS-CoV-2 (10 6 PFU ml −1 ) were treated by 25 μg ml −1 arbidol, or 8P9R ( n = 3). Then virus was serially diluted to detect the viral titer by plaque assay. d SARS-CoV-2 was treated at the indicated time of post infection by the indicated drugs ( n = 3). Viral titers ( a , b and d ) were measured by RT-qPCR at 24 h post infection. Relative RNA copy was normalized to MOCK. Data in ( a – d ) are presented as mean ± SD from 3–5 independent experiments. P values in ( b – d ) were calculated by two-tailed student t test when compared with mock. e Spike-ACE2 mediated cell-cell fusion could be blocked by arbidol and endosomal acidification inhibitors (bafilomycin A1 and 8P9R). The 293T cells expressed ACE2 or spike+GFP were co-cultured at the presence of indicated 8P9R (125 or 25 μg/ml), arbidol (50 or 25 μg ml −1 ) or bafilomycin A1 (BA1, 50 nM). 8P9R (25 μg ml −1 ) and arbidol (25 μg ml −1 ) did not block cell fusion, of which the fused cells were (2–10)-fold bigger than the non-fused cells. The 293T-GFP cells without spike (-Spike) served as the negative control of cell-cell fusion. Scale bar = 100 μm. The representative pictures were taken at 8 h after co-culture. Experiments were repeated three times independently. Source data are provided as a Source Data file.
    Figure Legend Snippet: Synergistic mechanism of 8P9R enhancing the antiviral activity of arbidol. a 8P9R could enhance the antiviral activity of arbidol against SARS-CoV-2 in Vero-E6 cells ( n = 5). Virus infected cells at the presence of the indicated concentrations of arbidol (Ar) or Ar+8P9R (1.6 μg ml −1 ) or Ar+8P9R (3.1 μg ml −1 ). b 8P9R could significantly enhance the antiviral activity of arbidol when arbidol alone did not show antiviral activity ( n = 4). SARS-CoV-2 was treated by the indicated Ar-0.2 (0.2 μg m l −1 ), 8P9R-3.1 (3.1 μg ml −1 ), Ar+8P9R, or PBS (Mock). P value was compared with Ar+8P9R. c SARS-CoV-2 (10 6 PFU ml −1 ) were treated by 25 μg ml −1 arbidol, or 8P9R ( n = 3). Then virus was serially diluted to detect the viral titer by plaque assay. d SARS-CoV-2 was treated at the indicated time of post infection by the indicated drugs ( n = 3). Viral titers ( a , b and d ) were measured by RT-qPCR at 24 h post infection. Relative RNA copy was normalized to MOCK. Data in ( a – d ) are presented as mean ± SD from 3–5 independent experiments. P values in ( b – d ) were calculated by two-tailed student t test when compared with mock. e Spike-ACE2 mediated cell-cell fusion could be blocked by arbidol and endosomal acidification inhibitors (bafilomycin A1 and 8P9R). The 293T cells expressed ACE2 or spike+GFP were co-cultured at the presence of indicated 8P9R (125 or 25 μg/ml), arbidol (50 or 25 μg ml −1 ) or bafilomycin A1 (BA1, 50 nM). 8P9R (25 μg ml −1 ) and arbidol (25 μg ml −1 ) did not block cell fusion, of which the fused cells were (2–10)-fold bigger than the non-fused cells. The 293T-GFP cells without spike (-Spike) served as the negative control of cell-cell fusion. Scale bar = 100 μm. The representative pictures were taken at 8 h after co-culture. Experiments were repeated three times independently. Source data are provided as a Source Data file.

    Techniques Used: Activity Assay, Infection, Plaque Assay, Quantitative RT-PCR, Two Tailed Test, Cell Culture, Blocking Assay, Negative Control, Co-Culture Assay

    The enhanced antiviral activity of branched P9R (8P9R). a The schematic figure of single P9R binding to single viral particle and branched P9R (8P9R) cross-linking viruses together. b The binding of 8P9R and P9R to SARS-CoV-2 and H1N1 viruses. Peptides coated on ELISA plates could capture virus particles which were then quantified by RT-qPCR. P9RS was the negative control peptide with no viral binding ability. Relative binding and P values were compared to P9R. Data are presented as mean ± SD of three independent experiments. c SARS-CoV-2 was pretreated with the indicated peptides for plaque reduction assay. Data are presented as mean ± SD of four independent experiments. d SARS-CoV-2 was treated by indicated peptides (25 μg ml −1 ) during viral inoculation. Viral RNA copies were detected by RT-qPCR at 24 h post infection in the supernatant of Vero-E6 cells. Data are presented as mean ± SD of three independent experiments. e SARS-CoV-2 was treated by peptides (50 μg ml −1 ) at 6 h post infection. Viral titers were measured at the indicated time by plaque assay. Data are presented as mean ± SD of three independent experiments. P values were compared with BSA. f Hemolysis assay of 8P9R in turkey red blood cells (TRBC). TRBC were treated by the indicated concentration of 8P9R. Hemolysis (%) was normalized to TRBC treated by Triton X-100. Data are presented as mean ± SD three independent experiments. P values are calculated by two-tailed student t test. ** indicates P
    Figure Legend Snippet: The enhanced antiviral activity of branched P9R (8P9R). a The schematic figure of single P9R binding to single viral particle and branched P9R (8P9R) cross-linking viruses together. b The binding of 8P9R and P9R to SARS-CoV-2 and H1N1 viruses. Peptides coated on ELISA plates could capture virus particles which were then quantified by RT-qPCR. P9RS was the negative control peptide with no viral binding ability. Relative binding and P values were compared to P9R. Data are presented as mean ± SD of three independent experiments. c SARS-CoV-2 was pretreated with the indicated peptides for plaque reduction assay. Data are presented as mean ± SD of four independent experiments. d SARS-CoV-2 was treated by indicated peptides (25 μg ml −1 ) during viral inoculation. Viral RNA copies were detected by RT-qPCR at 24 h post infection in the supernatant of Vero-E6 cells. Data are presented as mean ± SD of three independent experiments. e SARS-CoV-2 was treated by peptides (50 μg ml −1 ) at 6 h post infection. Viral titers were measured at the indicated time by plaque assay. Data are presented as mean ± SD of three independent experiments. P values were compared with BSA. f Hemolysis assay of 8P9R in turkey red blood cells (TRBC). TRBC were treated by the indicated concentration of 8P9R. Hemolysis (%) was normalized to TRBC treated by Triton X-100. Data are presented as mean ± SD three independent experiments. P values are calculated by two-tailed student t test. ** indicates P

    Techniques Used: Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Negative Control, Infection, Plaque Assay, Hemolysis Assay, Concentration Assay, Two Tailed Test

    18) Product Images from "High-content screening of Thai medicinal plants reveals Boesenbergia rotunda extract and its component Panduratin A as anti-SARS-CoV-2 agents"

    Article Title: High-content screening of Thai medicinal plants reveals Boesenbergia rotunda extract and its component Panduratin A as anti-SARS-CoV-2 agents

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-77003-3

    Dose-dependent anti-SARS-CoV-2 effects of B. rotunda extract and panduratin A at the pre-entry phase . ( a ) Study design. SARS-CoV-2 at 25TCID 50 were incubated with the extract/compound for 1 h before inoculation into Vero E6 cells. Viral adsorption was allowed for 2 h in the presence of the extract/compound. After washing, the culture was maintained in fresh media for 48 h before harvest. ( b ) Controls. Hydroxychloroquine (HCQ) at the IC 50 (8.07 µM) for pre-entry treatment (details in Supplementary Fig. 1) and the neutralizing serum served as the positive controls (n = 3 biological replicates). ( c , d ) High-content imaging analysis of Boesenbergia rotunda extract ( c ) and Panduratin A ( d ) (the left panel). The percentage of virus inhibition (blue) and cell viability (red) was shown in the right panel) (n = 3 biological replicates). Fluorescent signals: green, anti-SARS-CoV-2 NP mAb; blue, Hoechst. ( e , f ) Plaque reduction assay of B. rotunda extract ( e ) and panduratin A ( f ).
    Figure Legend Snippet: Dose-dependent anti-SARS-CoV-2 effects of B. rotunda extract and panduratin A at the pre-entry phase . ( a ) Study design. SARS-CoV-2 at 25TCID 50 were incubated with the extract/compound for 1 h before inoculation into Vero E6 cells. Viral adsorption was allowed for 2 h in the presence of the extract/compound. After washing, the culture was maintained in fresh media for 48 h before harvest. ( b ) Controls. Hydroxychloroquine (HCQ) at the IC 50 (8.07 µM) for pre-entry treatment (details in Supplementary Fig. 1) and the neutralizing serum served as the positive controls (n = 3 biological replicates). ( c , d ) High-content imaging analysis of Boesenbergia rotunda extract ( c ) and Panduratin A ( d ) (the left panel). The percentage of virus inhibition (blue) and cell viability (red) was shown in the right panel) (n = 3 biological replicates). Fluorescent signals: green, anti-SARS-CoV-2 NP mAb; blue, Hoechst. ( e , f ) Plaque reduction assay of B. rotunda extract ( e ) and panduratin A ( f ).

    Techniques Used: Incubation, Adsorption, Imaging, Inhibition

    Dose-dependent anti-SARS-CoV-2 effects of panduratin A and remdesivir in human airway epithelial cells (Calu-3) at the post-entry phase. ( a ) High-content imaging analysis of panduratin A ( a ) and remdesivir ( b ) (the left panel). The percentage of virus inhibition (blue) and cell viability (red) was shown in the right panel (n = 3 biological replicates). Fluorescent signals: green, anti-SARS-CoV-2 NP mAb; blue, Hoechst. ( c , d ) Plaque reduction assay of panduratin A ( c ) remdesivir ( d ). ( e ) Comparison of IC 50 values of panduration A and remdesivir evaluated by IFA of the high-content imaging technique and plaque assay in two cell types, Vero E6 and Calu-3.
    Figure Legend Snippet: Dose-dependent anti-SARS-CoV-2 effects of panduratin A and remdesivir in human airway epithelial cells (Calu-3) at the post-entry phase. ( a ) High-content imaging analysis of panduratin A ( a ) and remdesivir ( b ) (the left panel). The percentage of virus inhibition (blue) and cell viability (red) was shown in the right panel (n = 3 biological replicates). Fluorescent signals: green, anti-SARS-CoV-2 NP mAb; blue, Hoechst. ( c , d ) Plaque reduction assay of panduratin A ( c ) remdesivir ( d ). ( e ) Comparison of IC 50 values of panduration A and remdesivir evaluated by IFA of the high-content imaging technique and plaque assay in two cell types, Vero E6 and Calu-3.

    Techniques Used: Imaging, Inhibition, Immunofluorescence, Plaque Assay

    High-content anti-SARS-CoV-2 compound screening. ( a ) The SARS-CoV-2 (at 25TCID 50 ) infected Vero E6 cells were detected by high-content imaging of the control condition. Fluorescent signals: green, anti-SARS-CoV NP mAb; blue, Hoechst. ( b ) Percentage of the infected Vero E6 of the control conditions. ( c , d ) The high-content images of the infected Vero E6 cells treated with hydroxychloroquine ( c ) and ivermectin ( d ) (the left panel). The percentage of virus inhibition (blue) and cell viability (red) was shown in the right panel (n = 3 biological replicates). ( e , f ) The production of infectious SARS-CoV-2 in Vero E6 cells was evaluated by plaque reduction assay after 48 h of hydroxychloroquine ( e ) and ivermectin ( f ) treatment (n = 2 biological replicates) ( g ) A total of 122 Thai natural products (114 medicinal plant extracts and 8 purified compounds) were screened for anti-SARS-CoV-2 activity (n = 2 technical replicates). ( h ) Percentage of virus inhibition of six selected candidates corresponding to the number-labeled blue dots in ( g ). Full details of the screening results provided in Supplementary Table S1 .
    Figure Legend Snippet: High-content anti-SARS-CoV-2 compound screening. ( a ) The SARS-CoV-2 (at 25TCID 50 ) infected Vero E6 cells were detected by high-content imaging of the control condition. Fluorescent signals: green, anti-SARS-CoV NP mAb; blue, Hoechst. ( b ) Percentage of the infected Vero E6 of the control conditions. ( c , d ) The high-content images of the infected Vero E6 cells treated with hydroxychloroquine ( c ) and ivermectin ( d ) (the left panel). The percentage of virus inhibition (blue) and cell viability (red) was shown in the right panel (n = 3 biological replicates). ( e , f ) The production of infectious SARS-CoV-2 in Vero E6 cells was evaluated by plaque reduction assay after 48 h of hydroxychloroquine ( e ) and ivermectin ( f ) treatment (n = 2 biological replicates) ( g ) A total of 122 Thai natural products (114 medicinal plant extracts and 8 purified compounds) were screened for anti-SARS-CoV-2 activity (n = 2 technical replicates). ( h ) Percentage of virus inhibition of six selected candidates corresponding to the number-labeled blue dots in ( g ). Full details of the screening results provided in Supplementary Table S1 .

    Techniques Used: Infection, Imaging, Inhibition, Purification, Activity Assay, Labeling

    Dose-dependent anti-SARS-CoV-2 effects of six candidates at the post-infectious phase. ( a ) Study design. SARS-CoV-2 infected Vero E6 cells (at 25TCID 50 ) were treated with the extract/compound for 48 h before harvest. ( b ) Controls. Hydroxychloroquine (HCQ) at the IC 50 (5.08 µM) for post-infection treatment (from Fig. 1 c) and the neutralizing serum served as the positive controls. ( c – h ) High-content imaging analysis of Andrographis paniculata extract ( c ), Zingiber officinale extract ( d ), Boesenbergia rotunda extract ( e ), Andrographolide ( f ), 6-Gingerol ( g ), and panduratin A ( h ) was demonstrated in the left panel. The percentage of virus inhibition (blue) and cell viability (red) was shown in the right panel (n = 3 biological replicates). Fluorescent signals: green, anti-SARS-CoV-2 NP mAb; blue, Hoechst. ( i – n ) Plaque reduction assay of six candidates, i.e., A. paniculata extract ( i ), Z. officinale extract ( j ), B. rotunda extract ( k ), Andrographolide ( l ), 6-Gingerol ( m ), and panduratin A ( n ) (n = 2 biological replicates).
    Figure Legend Snippet: Dose-dependent anti-SARS-CoV-2 effects of six candidates at the post-infectious phase. ( a ) Study design. SARS-CoV-2 infected Vero E6 cells (at 25TCID 50 ) were treated with the extract/compound for 48 h before harvest. ( b ) Controls. Hydroxychloroquine (HCQ) at the IC 50 (5.08 µM) for post-infection treatment (from Fig. 1 c) and the neutralizing serum served as the positive controls. ( c – h ) High-content imaging analysis of Andrographis paniculata extract ( c ), Zingiber officinale extract ( d ), Boesenbergia rotunda extract ( e ), Andrographolide ( f ), 6-Gingerol ( g ), and panduratin A ( h ) was demonstrated in the left panel. The percentage of virus inhibition (blue) and cell viability (red) was shown in the right panel (n = 3 biological replicates). Fluorescent signals: green, anti-SARS-CoV-2 NP mAb; blue, Hoechst. ( i – n ) Plaque reduction assay of six candidates, i.e., A. paniculata extract ( i ), Z. officinale extract ( j ), B. rotunda extract ( k ), Andrographolide ( l ), 6-Gingerol ( m ), and panduratin A ( n ) (n = 2 biological replicates).

    Techniques Used: Infection, Imaging, Inhibition

    19) Product Images from "The SARS-CoV-2 multibasic cleavage site facilitates early serine protease-mediated entry into organoid-derived human airway cells"

    Article Title: The SARS-CoV-2 multibasic cleavage site facilitates early serine protease-mediated entry into organoid-derived human airway cells

    Journal: bioRxiv

    doi: 10.1101/2020.09.07.286120

    SARS-CoV-2 entry and replication is dependent on serine proteases in differentiated organoid-derived human airway cells. ( A and B ) Differentiated bronchiolar (A) or bronchial (B) airway spheroid cultures were infected at a MOI of 2. 16 hours ( A ) or 24 hours ( B ) post infection they were fixed and stained for viral nucleoprotein (red). Nuclei were stained with hoechst (blue) and actin was stained using phalloidin (white). AcTub stains ciliated cells (green). Scale bars indicate 200 μm in A and 50 μm in B . Representative images are shown from two independent experiments. ( C to E ) Replication kinetics of SARS-CoV-2 in bronchiolar airway spheroid cultures pretreated with camostat or carrier (DMSO). ( C and D ) TCID50 equivalents (eq.) per ml are shown in culture medium ( C ) and lysed organoids ( D ). ( E ) Live virus titers (TCID50/ml) in lysed organoids. Dotted line indicates limit of detection. ( F ) Replication kinetics of SARS-CoV-2 in 2D tracheal air-liquid interface airway cultures pretreated with camostat or carrier (DMSO). TCID50 eq. per ml in apical washes are shown. Error bars indicate SEM. H p.i. = hours post infection. Two-way ANOVA was performed for statistical analysis. * P
    Figure Legend Snippet: SARS-CoV-2 entry and replication is dependent on serine proteases in differentiated organoid-derived human airway cells. ( A and B ) Differentiated bronchiolar (A) or bronchial (B) airway spheroid cultures were infected at a MOI of 2. 16 hours ( A ) or 24 hours ( B ) post infection they were fixed and stained for viral nucleoprotein (red). Nuclei were stained with hoechst (blue) and actin was stained using phalloidin (white). AcTub stains ciliated cells (green). Scale bars indicate 200 μm in A and 50 μm in B . Representative images are shown from two independent experiments. ( C to E ) Replication kinetics of SARS-CoV-2 in bronchiolar airway spheroid cultures pretreated with camostat or carrier (DMSO). ( C and D ) TCID50 equivalents (eq.) per ml are shown in culture medium ( C ) and lysed organoids ( D ). ( E ) Live virus titers (TCID50/ml) in lysed organoids. Dotted line indicates limit of detection. ( F ) Replication kinetics of SARS-CoV-2 in 2D tracheal air-liquid interface airway cultures pretreated with camostat or carrier (DMSO). TCID50 eq. per ml in apical washes are shown. Error bars indicate SEM. H p.i. = hours post infection. Two-way ANOVA was performed for statistical analysis. * P

    Techniques Used: Derivative Assay, Infection, Staining

    The SARS-CoV-2 multibasic cleavage site increases serine protease usage. ( A and B ) SARS-CoV PP and SARS-CoV-2 PP entry route on VeroE6 cells pretreated with a concentration range of camostat ( A ) or E64D ( B ) to inhibit serine proteases and cathepsins, respectively. ( C and D ) SARS-CoV PP and SARS-CoV-2 PP entry route on VeroE6-TMPRSS2 cells pretreated with a concentration range of camostat ( C ) or E64D ( D ) to inhibit serine proteases and cathepsins, respectively. T-test was performed for statistical analysis at the highest concentration. * P
    Figure Legend Snippet: The SARS-CoV-2 multibasic cleavage site increases serine protease usage. ( A and B ) SARS-CoV PP and SARS-CoV-2 PP entry route on VeroE6 cells pretreated with a concentration range of camostat ( A ) or E64D ( B ) to inhibit serine proteases and cathepsins, respectively. ( C and D ) SARS-CoV PP and SARS-CoV-2 PP entry route on VeroE6-TMPRSS2 cells pretreated with a concentration range of camostat ( C ) or E64D ( D ) to inhibit serine proteases and cathepsins, respectively. T-test was performed for statistical analysis at the highest concentration. * P

    Techniques Used: Concentration Assay

    The SARS-CoV-2 multibasic cleavage site facilitates cell-cell fusion and SARS-CoV-2 is more fusogenic than SARS-CoV on differentiated organoid-derived human airway cells. ( A ) Proteolytic cleavage of SARS-CoV-2 S, SARS-CoV S, and S mutants was assessed by overexpression in HEK-293T cells and subsequent western blots for S1. GAPDH was used as a loading control. ( B and C ) Fusogenicity of SARS-CoV-2 S, SARS-CoV S, and S mutants was assessed after 18 hours by counting the number of nuclei per syncytium ( B ) and by measuring the sum of all GFP+ pixels per well ( C ). Statistical analysis was performed by one-way ANOVA on SARS-CoV or SARS-CoV-2 S-mediated fusion compared with its respective mutants. * P
    Figure Legend Snippet: The SARS-CoV-2 multibasic cleavage site facilitates cell-cell fusion and SARS-CoV-2 is more fusogenic than SARS-CoV on differentiated organoid-derived human airway cells. ( A ) Proteolytic cleavage of SARS-CoV-2 S, SARS-CoV S, and S mutants was assessed by overexpression in HEK-293T cells and subsequent western blots for S1. GAPDH was used as a loading control. ( B and C ) Fusogenicity of SARS-CoV-2 S, SARS-CoV S, and S mutants was assessed after 18 hours by counting the number of nuclei per syncytium ( B ) and by measuring the sum of all GFP+ pixels per well ( C ). Statistical analysis was performed by one-way ANOVA on SARS-CoV or SARS-CoV-2 S-mediated fusion compared with its respective mutants. * P

    Techniques Used: Derivative Assay, Over Expression, Western Blot

    The SARS-CoV-2 S multibasic cleavage site mediates entry into organoid-derived human airway cells. ( A ) Schematic overview of SARS-CoV-2 S protein mutants. Multibasic cleavage site residues are indicated in red; amino acid substitutions are indicated in green. Red arrows indicate cleavage sites. RBD = receptor binding domain, RBM = receptor binding motif. The SARS-CoV-2 S multibasic cleavage site was mutated to either remove the PRRA motif (SARS-2-Del-PRRA) or to substitute the R685 site (SARS-2-R685A and R685H). ( B ) Comparison of S cleavage of SARS-CoV-2 PPs and the multibasic cleavage site mutants. Western blots were performed against S1 with VSV-M silver stains as a production control. ( C and D ) PP infectivity of SARS-CoV-2 S and multibasic cleavage site mutants on VeroE6 ( C ) and Calu-3 ( D ) cells. ( E ) Differentiated airway spheroid cultures were infected with concentrated SARS-CoV-2 PPs containing a GFP reporter, indicated in green. Scale bar indicates 20 μm. ( F ) SARS-CoV-2 PP and multibasic cleavage site mutant infectivity on differentiated bronchiolar airway spheroid cultures. One-way ANOVA was performed for statistical analysis comparing all groups with SARS-CoV-2 PPs. * P
    Figure Legend Snippet: The SARS-CoV-2 S multibasic cleavage site mediates entry into organoid-derived human airway cells. ( A ) Schematic overview of SARS-CoV-2 S protein mutants. Multibasic cleavage site residues are indicated in red; amino acid substitutions are indicated in green. Red arrows indicate cleavage sites. RBD = receptor binding domain, RBM = receptor binding motif. The SARS-CoV-2 S multibasic cleavage site was mutated to either remove the PRRA motif (SARS-2-Del-PRRA) or to substitute the R685 site (SARS-2-R685A and R685H). ( B ) Comparison of S cleavage of SARS-CoV-2 PPs and the multibasic cleavage site mutants. Western blots were performed against S1 with VSV-M silver stains as a production control. ( C and D ) PP infectivity of SARS-CoV-2 S and multibasic cleavage site mutants on VeroE6 ( C ) and Calu-3 ( D ) cells. ( E ) Differentiated airway spheroid cultures were infected with concentrated SARS-CoV-2 PPs containing a GFP reporter, indicated in green. Scale bar indicates 20 μm. ( F ) SARS-CoV-2 PP and multibasic cleavage site mutant infectivity on differentiated bronchiolar airway spheroid cultures. One-way ANOVA was performed for statistical analysis comparing all groups with SARS-CoV-2 PPs. * P

    Techniques Used: Derivative Assay, Binding Assay, Western Blot, Infection, Mutagenesis

    SARS-CoV-2 enters faster on Calu-3 cells than SARS-CoV and entry speed is increased by the multibasic cleavage site. ( A ) SARS-CoV PP and SARS-CoV-2 PP infectivity on VeroE6 and Calu-3 cells. ( B and C ) SARS-CoV PP and SARS-CoV-2 PP entry route on Calu-3 cells. Cells were pretreated with a concentration range of camostat ( B ) or E64D ( C ) to inhibit serine proteases and cathepsins, respectively. T-test was performed for statistical analysis at the highest concentration. * P
    Figure Legend Snippet: SARS-CoV-2 enters faster on Calu-3 cells than SARS-CoV and entry speed is increased by the multibasic cleavage site. ( A ) SARS-CoV PP and SARS-CoV-2 PP infectivity on VeroE6 and Calu-3 cells. ( B and C ) SARS-CoV PP and SARS-CoV-2 PP entry route on Calu-3 cells. Cells were pretreated with a concentration range of camostat ( B ) or E64D ( C ) to inhibit serine proteases and cathepsins, respectively. T-test was performed for statistical analysis at the highest concentration. * P

    Techniques Used: Infection, Concentration Assay

    20) Product Images from "The pulmonary pathology of COVID-19"

    Article Title: The pulmonary pathology of COVID-19

    Journal: Virchows Archiv

    doi: 10.1007/s00428-021-03053-1

    Scheme of the putative pathophysiological mechanisms of SARS-CoV-2 induced acute lung injury, mainly diffuse alveolar damage (background microphotograph (5); H E, ×200), and microangiopathy and immunopathology in lethal (severe) COVID-19. Inserts, except for (1), are visualized by immunoperoxidase, microphotographed at ×400, and represent (1) SARS-CoV-2 in situ hybridization with the 845701 RNAscope probe - V-nCoV2019-S-sense and visualized with the RNAscope 2.5. LS detection kit (brown) from Advanced Cell Diagnostics (Hayward, CA, USA), yielding linear positivity of an alveolar septum; (2) immunohistochemical staining for SARS-CoV-2 Spike protein with the clone 007 from Sino Biological (Wayne, PA, USA), showing protein deposition on the inner side of an alveolar capillary; (3) immunohistochemical staining for fibrin with a polyclonal antibody A0080 from Dako (Glostrup, Denmark), revealing fibrin microthrombi casting the alveolar capillary network; (4) immunohistochemical staining for myeloperoxidase with a polyclonal antibody 760-2659 from Roche/Ventana (Rotkreuz, Switzerland), with neutrophilic granulocytes stuck into an alveolar capillary and displaying microscopic figures suggestive of neutrophilic extracellular traps (NET); (6) immunohistochemical staining for CD105 with the clone EPR10145-10 from Abcam (Cambridge, UK), showing a tight network of newly formed vessels in an alveolar septum; (7) immunohistochemical staining for CD206 with the clone E2L9N from Cell Signaling (Danvers, MA, USA), with significant amounts of intraalveolar M2 macrophages. (8) Kyoto Encyclopedia of Genes and Genomes (KEGG) diagram of disturbed fluid share stress pathways in COVID-19; respective gene expression profiles have been obtained on 25 lethal COVID-19 cases and compared to lungs of 5 patients suffering from arterial hypertension and 5 histopathologically unremarkable autopsy lungs that served as controls, utilizing the HTG EdgeSeq Oncology Biomarker Panel (HTG Molecular Diagnostics, Tucson, AZ, USA). The scheme should be looked at clockwise from 11 to 7. In general, upregulated genes/proteins are outlined in red, downregulated in green, indifferent ones in black
    Figure Legend Snippet: Scheme of the putative pathophysiological mechanisms of SARS-CoV-2 induced acute lung injury, mainly diffuse alveolar damage (background microphotograph (5); H E, ×200), and microangiopathy and immunopathology in lethal (severe) COVID-19. Inserts, except for (1), are visualized by immunoperoxidase, microphotographed at ×400, and represent (1) SARS-CoV-2 in situ hybridization with the 845701 RNAscope probe - V-nCoV2019-S-sense and visualized with the RNAscope 2.5. LS detection kit (brown) from Advanced Cell Diagnostics (Hayward, CA, USA), yielding linear positivity of an alveolar septum; (2) immunohistochemical staining for SARS-CoV-2 Spike protein with the clone 007 from Sino Biological (Wayne, PA, USA), showing protein deposition on the inner side of an alveolar capillary; (3) immunohistochemical staining for fibrin with a polyclonal antibody A0080 from Dako (Glostrup, Denmark), revealing fibrin microthrombi casting the alveolar capillary network; (4) immunohistochemical staining for myeloperoxidase with a polyclonal antibody 760-2659 from Roche/Ventana (Rotkreuz, Switzerland), with neutrophilic granulocytes stuck into an alveolar capillary and displaying microscopic figures suggestive of neutrophilic extracellular traps (NET); (6) immunohistochemical staining for CD105 with the clone EPR10145-10 from Abcam (Cambridge, UK), showing a tight network of newly formed vessels in an alveolar septum; (7) immunohistochemical staining for CD206 with the clone E2L9N from Cell Signaling (Danvers, MA, USA), with significant amounts of intraalveolar M2 macrophages. (8) Kyoto Encyclopedia of Genes and Genomes (KEGG) diagram of disturbed fluid share stress pathways in COVID-19; respective gene expression profiles have been obtained on 25 lethal COVID-19 cases and compared to lungs of 5 patients suffering from arterial hypertension and 5 histopathologically unremarkable autopsy lungs that served as controls, utilizing the HTG EdgeSeq Oncology Biomarker Panel (HTG Molecular Diagnostics, Tucson, AZ, USA). The scheme should be looked at clockwise from 11 to 7. In general, upregulated genes/proteins are outlined in red, downregulated in green, indifferent ones in black

    Techniques Used: In Situ Hybridization, Immunohistochemistry, Staining, Expressing, Biomarker Assay

    21) Product Images from "A single dose of recombinant VSV-ΔG-spike vaccine provides protection against SARS-CoV-2 challenge"

    Article Title: A single dose of recombinant VSV-ΔG-spike vaccine provides protection against SARS-CoV-2 challenge

    Journal: bioRxiv

    doi: 10.1101/2020.06.18.160655

    Histopathology and viral load of rVSV-ΔG-spike vaccinated and infected lungs: General histology (H E) and SARS-CoV-2 Immunolabeling of hamster lungs with and without pre vaccination. Lungs were isolated and processed for paraffin embedding from Naïve (A, E, I), vaccinated + infected 10 6 5dpi (B, F, J; C, G, K), and SARS-CoV-2 5×10 6 5dpi (D, H, L) and groups. Sections (5μm) were taken for H E staining (A-H) and SARS-CoV-2 immunolabeling (I-L, DAPI-Blue, SARS-CoV-2-Green). Pictures A-D: magnification-x1, bar= 100μm; Pictures E-H: magnification-x10, bar= 100μm; Pictures I-L: magnification-x60, bar= 10μm. Black arrows indicate patches of focal inflammation, pleural invaginatio and alveolar collapse. “*”-indicates hemorrhagic areas. “#”-indicates edema and protein rich exudates. Black arrow heads indicate pulmonary mononuclear cells. White arrows indicate CoV-2 positiv immunolabeling. Naïve group: n=4, SARS-CoV-2 5×10 6 pfu/animal 5dpi group: n=1, vaccinated+infecte 10 6 pfu/animal 5dpi group: n=2. (M) Tissue/Air space ratio.
    Figure Legend Snippet: Histopathology and viral load of rVSV-ΔG-spike vaccinated and infected lungs: General histology (H E) and SARS-CoV-2 Immunolabeling of hamster lungs with and without pre vaccination. Lungs were isolated and processed for paraffin embedding from Naïve (A, E, I), vaccinated + infected 10 6 5dpi (B, F, J; C, G, K), and SARS-CoV-2 5×10 6 5dpi (D, H, L) and groups. Sections (5μm) were taken for H E staining (A-H) and SARS-CoV-2 immunolabeling (I-L, DAPI-Blue, SARS-CoV-2-Green). Pictures A-D: magnification-x1, bar= 100μm; Pictures E-H: magnification-x10, bar= 100μm; Pictures I-L: magnification-x60, bar= 10μm. Black arrows indicate patches of focal inflammation, pleural invaginatio and alveolar collapse. “*”-indicates hemorrhagic areas. “#”-indicates edema and protein rich exudates. Black arrow heads indicate pulmonary mononuclear cells. White arrows indicate CoV-2 positiv immunolabeling. Naïve group: n=4, SARS-CoV-2 5×10 6 pfu/animal 5dpi group: n=1, vaccinated+infecte 10 6 pfu/animal 5dpi group: n=2. (M) Tissue/Air space ratio.

    Techniques Used: Histopathology, Infection, Immunolabeling, Isolation, Staining

    Surface antigenic similarity of rVSV-ΔG-spike and SARS-CoV-2: (A) Immunofluorescent images of Vero E6 cells infected with either WT-VSV (left panel), rVSV-ΔG-spike (middle panel), or SARS-CoV-2, stained with serum from COVID-19 human convalescent serum (right panel). (B) Correlation analysis of neutralization of rVSV-ΔG-spike and SARS-CoV-2 by a panel of sera from COVID-19 convalescent patients. For each sera (n=12), NT 50 values were determined for neutralization of rVSV-ΔG-spike, or SARS-CoV-2. The NT 50 values were plotted to determine the correlation between th neutralization assays. R 2 =0.911.
    Figure Legend Snippet: Surface antigenic similarity of rVSV-ΔG-spike and SARS-CoV-2: (A) Immunofluorescent images of Vero E6 cells infected with either WT-VSV (left panel), rVSV-ΔG-spike (middle panel), or SARS-CoV-2, stained with serum from COVID-19 human convalescent serum (right panel). (B) Correlation analysis of neutralization of rVSV-ΔG-spike and SARS-CoV-2 by a panel of sera from COVID-19 convalescent patients. For each sera (n=12), NT 50 values were determined for neutralization of rVSV-ΔG-spike, or SARS-CoV-2. The NT 50 values were plotted to determine the correlation between th neutralization assays. R 2 =0.911.

    Techniques Used: Infection, Staining, Neutralization

    Characterization of rVSV-ΔG-spike: (A) A summary of the genome analysis of several passage of rVSV-ΔG-spike, showing Ct values of VSV-G, VSV-N, and SARS-CoV-2-S indicating the elimination of VSV-G over time, together with increased titer. Also, plaques are formed at late passages. (B) Immunofluorescence images of Vero E6 cells infected with early passage (P5)-rVSV-ΔG-spike, or lat passage (P13)-rVSV-ΔG-spike, stained with a SARS-CoV-2 antibody (green) and DAPI for nuclei stainin (blue). Top and bottom panels show insets at low (10X) and high magnification (25x), respectively. rVSV-ΔG-spike at P5 forms syncytia, whereas P13 show individual infected cells, with no evidence of syncytia. (C) Transmission electron microscopy of rVSV-ΔG-spike (top panel) compared to WT-VSV. Right panel shows immunogold labeling against RBD.
    Figure Legend Snippet: Characterization of rVSV-ΔG-spike: (A) A summary of the genome analysis of several passage of rVSV-ΔG-spike, showing Ct values of VSV-G, VSV-N, and SARS-CoV-2-S indicating the elimination of VSV-G over time, together with increased titer. Also, plaques are formed at late passages. (B) Immunofluorescence images of Vero E6 cells infected with early passage (P5)-rVSV-ΔG-spike, or lat passage (P13)-rVSV-ΔG-spike, stained with a SARS-CoV-2 antibody (green) and DAPI for nuclei stainin (blue). Top and bottom panels show insets at low (10X) and high magnification (25x), respectively. rVSV-ΔG-spike at P5 forms syncytia, whereas P13 show individual infected cells, with no evidence of syncytia. (C) Transmission electron microscopy of rVSV-ΔG-spike (top panel) compared to WT-VSV. Right panel shows immunogold labeling against RBD.

    Techniques Used: Immunofluorescence, Infection, Staining, Transmission Assay, Electron Microscopy, Labeling

    Single-dose rVSV-ΔG-spike vaccine efficacy in hamsters following SARS-CoV-2 challenge. (A) Body weight changes of hamsters infected with SARS-CoV-2 (n=12), and hamsters vaccinated wit rVSV-ΔG-spike and infected with 5×10 6 pfu/hamster (n=10) 25 days post-vaccination, compared to moc hamsters (n=8). p
    Figure Legend Snippet: Single-dose rVSV-ΔG-spike vaccine efficacy in hamsters following SARS-CoV-2 challenge. (A) Body weight changes of hamsters infected with SARS-CoV-2 (n=12), and hamsters vaccinated wit rVSV-ΔG-spike and infected with 5×10 6 pfu/hamster (n=10) 25 days post-vaccination, compared to moc hamsters (n=8). p

    Techniques Used: Infection

    Dose-dependent vaccination of hamsters with rVSV-ΔG-spike. (A) Body weight changes of mock-vaccinated hamsters (n=4), and hamsters vaccinated with rVSV-ΔG-spike ranging from 10 4 to 10 8 pfu/hamster (n=8, n=10, n=10, n=10, n=8, for each vaccinated group, respectively). (B) NT 50 values of neutralization of SARS-CoV-2 by sera from hamsters following i.m. vaccination with rVSV-ΔG-spik ranging from 10 4 to 10 8 pfu/hamster. n=4 for each group. Means and SEM are indicated below the graph.
    Figure Legend Snippet: Dose-dependent vaccination of hamsters with rVSV-ΔG-spike. (A) Body weight changes of mock-vaccinated hamsters (n=4), and hamsters vaccinated with rVSV-ΔG-spike ranging from 10 4 to 10 8 pfu/hamster (n=8, n=10, n=10, n=10, n=8, for each vaccinated group, respectively). (B) NT 50 values of neutralization of SARS-CoV-2 by sera from hamsters following i.m. vaccination with rVSV-ΔG-spik ranging from 10 4 to 10 8 pfu/hamster. n=4 for each group. Means and SEM are indicated below the graph.

    Techniques Used: Neutralization

    Establishment of golden Syrian hamster SARS-CoV-2 model: (A) Body weight changes of hamsters infected with SARS-CoV-2 at either 5×10 4 (n=7), 5×10 5 (n=7), or 5×10 6 (n=10) pfu/hamster, compared to mock-infected hamsters (n=6). p
    Figure Legend Snippet: Establishment of golden Syrian hamster SARS-CoV-2 model: (A) Body weight changes of hamsters infected with SARS-CoV-2 at either 5×10 4 (n=7), 5×10 5 (n=7), or 5×10 6 (n=10) pfu/hamster, compared to mock-infected hamsters (n=6). p

    Techniques Used: Infection

    rVSV-ΔG-spike design and generation strategy: (A) A schematic diagram of the genom organization of WT-VSV genome (top diagram) and rVSV-ΔG-spike (bottom diagram). N: Nucleoprotein, P: Phosphoprotein, M: Matrix, L: Large polymerase, G: Glycoprotein, SPIKE: SARS-CoV-2 spike. (B) pVSV-ΔG-spike map; in red, the inserted S gene. (C) Schematic representation of the generation proces of creating rVSV-ΔG-spike vaccine. Infection of BHK-21 cells with MVA-T7, followed by co-transfecti with pVSV-ΔG-spike, and VSV-system accessory plasmids; Transfection of BHK-21 cells with pCAGGS-VSV-G, followed by infection with the supernatant of the primary transfection to create P1; serial passaging to create rVSV-ΔG-spike.
    Figure Legend Snippet: rVSV-ΔG-spike design and generation strategy: (A) A schematic diagram of the genom organization of WT-VSV genome (top diagram) and rVSV-ΔG-spike (bottom diagram). N: Nucleoprotein, P: Phosphoprotein, M: Matrix, L: Large polymerase, G: Glycoprotein, SPIKE: SARS-CoV-2 spike. (B) pVSV-ΔG-spike map; in red, the inserted S gene. (C) Schematic representation of the generation proces of creating rVSV-ΔG-spike vaccine. Infection of BHK-21 cells with MVA-T7, followed by co-transfecti with pVSV-ΔG-spike, and VSV-system accessory plasmids; Transfection of BHK-21 cells with pCAGGS-VSV-G, followed by infection with the supernatant of the primary transfection to create P1; serial passaging to create rVSV-ΔG-spike.

    Techniques Used: Infection, Transfection, Passaging

    Detection and neutralization of SARS-CoV-2 by sera from hamsters following s.c. vaccinatio with rVSV-ΔG-spike: (A) Immunofluorescence images of Vero E6 cells infected with SARS-CoV-2, stai with sera from either mock-vaccinated hamsters (left panel) or rVSV-ΔG-spike vaccinated-hamsters (right panel). (B) Plaque reduction neutralization test (PRNT) of hamster sera collected from naïve hamster (PBF, n=5) or hamsters vaccinated with rVSV-ΔG-spike 25 days following vaccination (n=5). (C) Bod weight changes of mock-vaccinated hamsters (Mock, n=16), and hamsters vaccinated with rVSV-ΔG-spike (n=8).
    Figure Legend Snippet: Detection and neutralization of SARS-CoV-2 by sera from hamsters following s.c. vaccinatio with rVSV-ΔG-spike: (A) Immunofluorescence images of Vero E6 cells infected with SARS-CoV-2, stai with sera from either mock-vaccinated hamsters (left panel) or rVSV-ΔG-spike vaccinated-hamsters (right panel). (B) Plaque reduction neutralization test (PRNT) of hamster sera collected from naïve hamster (PBF, n=5) or hamsters vaccinated with rVSV-ΔG-spike 25 days following vaccination (n=5). (C) Bod weight changes of mock-vaccinated hamsters (Mock, n=16), and hamsters vaccinated with rVSV-ΔG-spike (n=8).

    Techniques Used: Neutralization, Immunofluorescence, Infection, Plaque Reduction Neutralization Test

    22) Product Images from "BNT162b vaccines are immunogenic and protect non-human primates against SARS-CoV-2"

    Article Title: BNT162b vaccines are immunogenic and protect non-human primates against SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2020.12.11.421008

    BNT162b-elicited antibody responses in mice. BALB/c mice ( n =8) were immunised intramuscularly (IM) with a single dose of each BNT162b vaccine candidate or buffer (control, n =8). Geometric mean of each group (a-c) ± 95% CI (c), Day 28 p-values compared to control (multiple comparison of mixed-effect analysis [ a, b] and one-way ANOVA [ c ], all using Dunnett’s multiple comparisons test) are provided. a, b, RBD- and S1-specific IgG responses in sera obtained 7, 14, 21 and 28 days after immunisation with BNT162b1 ( a ) or BNT162b2 ( b ), determined by ELISA. For day 0 values, a pre-screening of randomly selected mice was performed ( n =4). c, Reciprocal serum endpoint titres of RBD-specific IgG 14 days after immunisation. The horizontal dotted line indicates the lower limit of detection (LLOD). d , Representative surface plasmon resonance sensorgram of the binding kinetics of His-tagged S1 to immobilised mouse IgG from serum drawn 28 days after immunisation with 5 µg BNT162b2. Binding data (in colour) and 1:1 binding model fit to the data (black) are depicted. e, f, Number of infected cells per well in a pseudovirus-based VSV-SARS-CoV-2 50% neutralisation assay conducted with serial dilutions of mouse serum samples drawn 28 days after immunisation with BNT162b1 ( e ) or BNT162b2 ( f ). Lines represent individual sera. Horizontal dotted lines indicate geometric mean ± 95% CI (as grey area) of infected cells in the absence of mouse serum (virus positive control). g , Pearson correlation of pseudovirus-based VSV-SARS-CoV-2 50% neutralisation titres with live SARS-CoV-2 virus neutralisation titres for n = 10 random selected serum samples from mice immunised with BNT162b1 and BNT162b2 each.
    Figure Legend Snippet: BNT162b-elicited antibody responses in mice. BALB/c mice ( n =8) were immunised intramuscularly (IM) with a single dose of each BNT162b vaccine candidate or buffer (control, n =8). Geometric mean of each group (a-c) ± 95% CI (c), Day 28 p-values compared to control (multiple comparison of mixed-effect analysis [ a, b] and one-way ANOVA [ c ], all using Dunnett’s multiple comparisons test) are provided. a, b, RBD- and S1-specific IgG responses in sera obtained 7, 14, 21 and 28 days after immunisation with BNT162b1 ( a ) or BNT162b2 ( b ), determined by ELISA. For day 0 values, a pre-screening of randomly selected mice was performed ( n =4). c, Reciprocal serum endpoint titres of RBD-specific IgG 14 days after immunisation. The horizontal dotted line indicates the lower limit of detection (LLOD). d , Representative surface plasmon resonance sensorgram of the binding kinetics of His-tagged S1 to immobilised mouse IgG from serum drawn 28 days after immunisation with 5 µg BNT162b2. Binding data (in colour) and 1:1 binding model fit to the data (black) are depicted. e, f, Number of infected cells per well in a pseudovirus-based VSV-SARS-CoV-2 50% neutralisation assay conducted with serial dilutions of mouse serum samples drawn 28 days after immunisation with BNT162b1 ( e ) or BNT162b2 ( f ). Lines represent individual sera. Horizontal dotted lines indicate geometric mean ± 95% CI (as grey area) of infected cells in the absence of mouse serum (virus positive control). g , Pearson correlation of pseudovirus-based VSV-SARS-CoV-2 50% neutralisation titres with live SARS-CoV-2 virus neutralisation titres for n = 10 random selected serum samples from mice immunised with BNT162b1 and BNT162b2 each.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, SPR Assay, Binding Assay, Infection, Positive Control

    Virological and serological evidence of protection of rhesus macaques from challenge with infectious SARS-CoV-2. Rhesus macaques immunised with 100 µg of BNT162b1 or BNT162b2 ( n =6 each) or mock immunised with saline challenge (Control, n =9) were challenged with 1.05 × 10 6 total plaque forming units (PFU) of SARS-CoV-2 split equally between the intranasal (IN) and intratracheal (IT) routes. Additional macaques (Sentinel, n =6) were mock-challenged with cell culture medium. Macaque assignments to cohorts and schedules of immunisation, challenge, and sample collection are provided in Extended Data Fig. 6 and Extended Data Table 2 . Viral RNA levels were detected by RT-qPCR. a, Viral RNA in bronchoalveolar lavage (BAL) fluid. b , Viral RNA in nasal swabs. Symbols represent individual animals. Ratios above bars indicate the number of viral RNA positive animals among all animals in a group with evaluable samples. Heights of bars indicate geometric mean viral RNA copies; whiskers indicate geometric standard deviations. Each symbol represents one animal. Dotted lines indicate the lower limit of detection (LLOD). Values below the LLOD were set to ½ the LLOD. The statistical significance by a non-parametric test (Friedman’s test) of differences in viral RNA detection after challenge between 6 BNT162b1-immunised and 6 mock-immunised animals (challenge cohorts 1 and 2) was p = 0.015 for BAL fluid and p = 0.005 for nasal swab; between 6 BNT162b2-immunised animals and 3 mock-immunised animals (challenge cohort 3), the statistical significance was p = 0.001 for BAL fluid and p = 0.262 for nasal swabs. Serum samples were assayed for SARS-CoV-2 50% neutralisation titres (VNT50). c , BNT162b1-immunised macaques and Controls (challenge cohorts 1 and 2). d , BNT162b2-immunised macaques and Controls (challenge cohort 3). Symbols represent individual animal titres. Horizontal dashed lines indicate the LLOQ of 20.
    Figure Legend Snippet: Virological and serological evidence of protection of rhesus macaques from challenge with infectious SARS-CoV-2. Rhesus macaques immunised with 100 µg of BNT162b1 or BNT162b2 ( n =6 each) or mock immunised with saline challenge (Control, n =9) were challenged with 1.05 × 10 6 total plaque forming units (PFU) of SARS-CoV-2 split equally between the intranasal (IN) and intratracheal (IT) routes. Additional macaques (Sentinel, n =6) were mock-challenged with cell culture medium. Macaque assignments to cohorts and schedules of immunisation, challenge, and sample collection are provided in Extended Data Fig. 6 and Extended Data Table 2 . Viral RNA levels were detected by RT-qPCR. a, Viral RNA in bronchoalveolar lavage (BAL) fluid. b , Viral RNA in nasal swabs. Symbols represent individual animals. Ratios above bars indicate the number of viral RNA positive animals among all animals in a group with evaluable samples. Heights of bars indicate geometric mean viral RNA copies; whiskers indicate geometric standard deviations. Each symbol represents one animal. Dotted lines indicate the lower limit of detection (LLOD). Values below the LLOD were set to ½ the LLOD. The statistical significance by a non-parametric test (Friedman’s test) of differences in viral RNA detection after challenge between 6 BNT162b1-immunised and 6 mock-immunised animals (challenge cohorts 1 and 2) was p = 0.015 for BAL fluid and p = 0.005 for nasal swab; between 6 BNT162b2-immunised animals and 3 mock-immunised animals (challenge cohort 3), the statistical significance was p = 0.001 for BAL fluid and p = 0.262 for nasal swabs. Serum samples were assayed for SARS-CoV-2 50% neutralisation titres (VNT50). c , BNT162b1-immunised macaques and Controls (challenge cohorts 1 and 2). d , BNT162b2-immunised macaques and Controls (challenge cohort 3). Symbols represent individual animal titres. Horizontal dashed lines indicate the LLOQ of 20.

    Techniques Used: Cell Culture, Quantitative RT-PCR, RNA Detection

    Vaccine design and characterisation of the expressed antigens. a , Structure of BNT162b RNAs. UTR, untranslated region; SP, signal peptide; RBD, receptor-binding domain; S1 and S2, N-terminal and C-terminal furin cleavage fragments, respectively; S, SARS-CoV-2 S glycoprotein. Proline mutations K986P and V897P are indicated. b , Liquid capillary electropherograms of both in vitro transcribed BNT162b RNAs. c , Representative 2D class averages from EM of negatively stained RBD-foldon trimers. Box edge: 37 nm. d , 2D class average from cryo-EM of the ACE2/B 0 AT1/RBD-foldon trimer complex. Long box edge: 39.2 nm. Peripheral to the relatively well-defined density of each RBD domain bound to ACE2, there is diffuse density attributed to the remainder of the flexibly tethered RBD-foldon trimer. A detergent micelle forms the density at the end of the complex opposite the RBD-foldon. e , Density map of the ACE2/B 0 AT1/RBD-foldon trimer complex at 3.24 Å after focused refinement of the ACE2 extracellular domain bound to a RBD monomer. Surface colour-coding by subunit. The ribbon model refined to the density shows the RBD-ACE2 binding interface, with residues potentially mediating polar interactions labeled. f , 3.29 Å cryo-EM map of P2 S, with fitted and refined atomic model, viewed down the three-fold axis toward the membrane (left) and viewed perpendicular to the three-fold axis (right). Coloured by protomer. g, Mass density map of TwinStrep-tagged P2 S produced by 3D classification of images extracted from cryo-EM micrographs with no symmetry averaging, showing the class in the one RBD ‘up’, two RBD ‘down’ position.
    Figure Legend Snippet: Vaccine design and characterisation of the expressed antigens. a , Structure of BNT162b RNAs. UTR, untranslated region; SP, signal peptide; RBD, receptor-binding domain; S1 and S2, N-terminal and C-terminal furin cleavage fragments, respectively; S, SARS-CoV-2 S glycoprotein. Proline mutations K986P and V897P are indicated. b , Liquid capillary electropherograms of both in vitro transcribed BNT162b RNAs. c , Representative 2D class averages from EM of negatively stained RBD-foldon trimers. Box edge: 37 nm. d , 2D class average from cryo-EM of the ACE2/B 0 AT1/RBD-foldon trimer complex. Long box edge: 39.2 nm. Peripheral to the relatively well-defined density of each RBD domain bound to ACE2, there is diffuse density attributed to the remainder of the flexibly tethered RBD-foldon trimer. A detergent micelle forms the density at the end of the complex opposite the RBD-foldon. e , Density map of the ACE2/B 0 AT1/RBD-foldon trimer complex at 3.24 Å after focused refinement of the ACE2 extracellular domain bound to a RBD monomer. Surface colour-coding by subunit. The ribbon model refined to the density shows the RBD-ACE2 binding interface, with residues potentially mediating polar interactions labeled. f , 3.29 Å cryo-EM map of P2 S, with fitted and refined atomic model, viewed down the three-fold axis toward the membrane (left) and viewed perpendicular to the three-fold axis (right). Coloured by protomer. g, Mass density map of TwinStrep-tagged P2 S produced by 3D classification of images extracted from cryo-EM micrographs with no symmetry averaging, showing the class in the one RBD ‘up’, two RBD ‘down’ position.

    Techniques Used: Binding Assay, In Vitro, Staining, Labeling, Produced

    Schedule of rhesus macaque challenge and necropsy. Timing in days from Dose 2 of vaccine or saline (numbers to the left of the bars) and of necropsy (numbers inside bars) are presented relative to the day of SARS-CoV-2 or mock challenge (Day 0). Numbers of macaques represented by the bars are indicated by red numbers to the right of the bars. Control: macaques challenged but not immunised with BNT162b. Sentinel: macaques mock challenged (cell culture medium only). n/a: macaques not necropsied. Additional details, including timing of sample collections and radiographic examinations, are in Extended Data Table 2 .
    Figure Legend Snippet: Schedule of rhesus macaque challenge and necropsy. Timing in days from Dose 2 of vaccine or saline (numbers to the left of the bars) and of necropsy (numbers inside bars) are presented relative to the day of SARS-CoV-2 or mock challenge (Day 0). Numbers of macaques represented by the bars are indicated by red numbers to the right of the bars. Control: macaques challenged but not immunised with BNT162b. Sentinel: macaques mock challenged (cell culture medium only). n/a: macaques not necropsied. Additional details, including timing of sample collections and radiographic examinations, are in Extended Data Table 2 .

    Techniques Used: Cell Culture

    Gating strategy for rhesus macaque flow cytometry analysis of data shown in Figure 4 e-g . Flow cytometry gating strategy for identification of spike-specific SARS-CoV-2 modRNA vaccine BNT162b2-induced T cells. Starting with events acquired with a constant flow stream and fluorescence intensity, viable cells, lymphocytes and single events were identified and gated (upper row, left to right). Within singlet lymphocytes, CD20 - CD3 + T cells were identified and gated into CD4 + T cells and CD8+ T cells (middle row). Antigen-specific CD4+ T cells were identified by gating on CD154 and cytokine-positive cells, and CD8+ T cells were identified by gating on CD69 and cytokine-positive cells. The antigen-specific cells were used for further analysis (bottom row).
    Figure Legend Snippet: Gating strategy for rhesus macaque flow cytometry analysis of data shown in Figure 4 e-g . Flow cytometry gating strategy for identification of spike-specific SARS-CoV-2 modRNA vaccine BNT162b2-induced T cells. Starting with events acquired with a constant flow stream and fluorescence intensity, viable cells, lymphocytes and single events were identified and gated (upper row, left to right). Within singlet lymphocytes, CD20 - CD3 + T cells were identified and gated into CD4 + T cells and CD8+ T cells (middle row). Antigen-specific CD4+ T cells were identified by gating on CD154 and cytokine-positive cells, and CD8+ T cells were identified by gating on CD69 and cytokine-positive cells. The antigen-specific cells were used for further analysis (bottom row).

    Techniques Used: Flow Cytometry, Fluorescence

    Mouse humoral immunogenicity. BALB/c mice ( n =8) were immunised intramuscularly (IM) with a single dose of each BNT162b vaccine candidate or buffer control. Geometric mean of each group ± 95% confidence interval (CI) (a, b, d). Day 28 p-values compared to control (multiple comparison of mixed-effect analysis [a, d] and OneWay ANOVA [b], all using Dunnett’s multiple comparisons test) are provided. a , RBD-specific IgG levels in sera of mice immunised with 5 µg of BNT162b candidates, determined by ELISA. For day 0 values, a pre-screening of randomly selected animals was performed ( n =4). For IgG levels with lower BNT162b doses and sera testing for detection of S1 see Extended Data Figure 3a, b . b , Reciprocal serum endpoint titres of RBD-specific IgG 28 days after immunisation. The horizontal dotted line indicates the lower limit of detection (LLOD). c , Representative surface plasmon resonance sensorgrams of the binding kinetics of His-tagged RBD to immobilised mouse IgG from serum drawn 28 days after immunisation with 5 µg of each BNT162b. Actual binding (in colour) and the best fit of the data to a 1:1 binding model (black) are depicted. For binding kinetics of same sera to His-tagged S1 see Extended Data Figure 3d . d , Pseudovirus-based VSV-SARS-CoV-2 50% neutralisation titres (pVNT50) in sera of mice immunised with BNT162b vaccine candidates. For number of infected cells per well with serum samples drawn 28 days after immunisation and titre correlation to a SARS-CoV-2 virus neutralisation assay see Extended Data Figure 3e-g .
    Figure Legend Snippet: Mouse humoral immunogenicity. BALB/c mice ( n =8) were immunised intramuscularly (IM) with a single dose of each BNT162b vaccine candidate or buffer control. Geometric mean of each group ± 95% confidence interval (CI) (a, b, d). Day 28 p-values compared to control (multiple comparison of mixed-effect analysis [a, d] and OneWay ANOVA [b], all using Dunnett’s multiple comparisons test) are provided. a , RBD-specific IgG levels in sera of mice immunised with 5 µg of BNT162b candidates, determined by ELISA. For day 0 values, a pre-screening of randomly selected animals was performed ( n =4). For IgG levels with lower BNT162b doses and sera testing for detection of S1 see Extended Data Figure 3a, b . b , Reciprocal serum endpoint titres of RBD-specific IgG 28 days after immunisation. The horizontal dotted line indicates the lower limit of detection (LLOD). c , Representative surface plasmon resonance sensorgrams of the binding kinetics of His-tagged RBD to immobilised mouse IgG from serum drawn 28 days after immunisation with 5 µg of each BNT162b. Actual binding (in colour) and the best fit of the data to a 1:1 binding model (black) are depicted. For binding kinetics of same sera to His-tagged S1 see Extended Data Figure 3d . d , Pseudovirus-based VSV-SARS-CoV-2 50% neutralisation titres (pVNT50) in sera of mice immunised with BNT162b vaccine candidates. For number of infected cells per well with serum samples drawn 28 days after immunisation and titre correlation to a SARS-CoV-2 virus neutralisation assay see Extended Data Figure 3e-g .

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

    Rhesus macaque immunogenicity. Male rhesus macaques, 2-4 years of age, were immunised on Days 0 and 21 (arrows below the x-axis indicate the days of the second immunisation) with 30 µg or 100 µg BNT162b vaccines ( n =6 each). Additional rhesus macaques received saline (C; n =9). Human convalescent sera (HCS) were obtained from SARS-CoV-2-infected patients at least 14 days after PCR-confirmed diagnosis and at a time when acute COVID-19 symptoms had resolved ( n =38). The HCS panel is a benchmark for serology studies in this and other manuscripts. a , Concentrations, in arbitrary units, of IgG binding recombinant SARS-CoV-2 RBD (LLOD = 1.72 U/mL). b , SARS-CoV-2 50% virus neutralisation titres (VNT50, LLOD = 20). c-g , PBMCs collected on Days 0, 14, 28 and 42 were ex vivo re-stimulated with full-length S peptide mix. c, IFNγ ELISpot. d, IL-4 ELISpot. e , S-specific CD4 + T-cell IFNγ, IL-2, or TNFα release by flow cytometry (LLOD = 0.04). f , S-specific CD4 + T-cell IL-4 release by flow cytometry (LLOD = 0.05). g , CD8 + T-cell IFNγ release by flow cytometry (LLOD = 0.03). Heights of bars indicate the geometric (a-b) or arithmetic (c-g) means for each group, with values written above bars (a-b). Whiskers indicate 95% confidence intervals (CI’s; a-b) or standard errors of means (SEMs; c-g). Each symbol represents one animal. Horizontal dashed lines mark LLODs. For serology and ELISpot data (a-d) but not for flow cytometry data (e-g), values below the LLOD were set to ½ the LLOD. Arrows below the x-axis indicate the days of Doses 1 and 2.
    Figure Legend Snippet: Rhesus macaque immunogenicity. Male rhesus macaques, 2-4 years of age, were immunised on Days 0 and 21 (arrows below the x-axis indicate the days of the second immunisation) with 30 µg or 100 µg BNT162b vaccines ( n =6 each). Additional rhesus macaques received saline (C; n =9). Human convalescent sera (HCS) were obtained from SARS-CoV-2-infected patients at least 14 days after PCR-confirmed diagnosis and at a time when acute COVID-19 symptoms had resolved ( n =38). The HCS panel is a benchmark for serology studies in this and other manuscripts. a , Concentrations, in arbitrary units, of IgG binding recombinant SARS-CoV-2 RBD (LLOD = 1.72 U/mL). b , SARS-CoV-2 50% virus neutralisation titres (VNT50, LLOD = 20). c-g , PBMCs collected on Days 0, 14, 28 and 42 were ex vivo re-stimulated with full-length S peptide mix. c, IFNγ ELISpot. d, IL-4 ELISpot. e , S-specific CD4 + T-cell IFNγ, IL-2, or TNFα release by flow cytometry (LLOD = 0.04). f , S-specific CD4 + T-cell IL-4 release by flow cytometry (LLOD = 0.05). g , CD8 + T-cell IFNγ release by flow cytometry (LLOD = 0.03). Heights of bars indicate the geometric (a-b) or arithmetic (c-g) means for each group, with values written above bars (a-b). Whiskers indicate 95% confidence intervals (CI’s; a-b) or standard errors of means (SEMs; c-g). Each symbol represents one animal. Horizontal dashed lines mark LLODs. For serology and ELISpot data (a-d) but not for flow cytometry data (e-g), values below the LLOD were set to ½ the LLOD. Arrows below the x-axis indicate the days of Doses 1 and 2.

    Techniques Used: Infection, Polymerase Chain Reaction, Binding Assay, Recombinant, Ex Vivo, Enzyme-linked Immunospot, Flow Cytometry

    Pulmonary histopathology in rhesus macaques after immunisation with BNT162b1 or BNT162b2 and challenge with infectious SARS-CoV-2. Rhesus macaques were immunised with BNT162b1, BNT162b2, or saline (control) and challenged with SARS-CoV-2. A sentinel group was challenged with cell culture medium. The macaques were necropsied as described in Figs. 4 and 5 and Extended Data Table 2 . Two veterinary pathologists blindly performed microscopic evaluation of formalin fixed, hematoxylin and eosin stained lung tissue sections from each of 7 lobes from each macaque that had been necropsied on Day 7 or 8. Inflammation scores were assigned by consensus between the pathologists on a scale of 1-5 based on the area of involvement. Each dot represents an individual animal and is the mean inflammation area score from the 7 lung lobes.
    Figure Legend Snippet: Pulmonary histopathology in rhesus macaques after immunisation with BNT162b1 or BNT162b2 and challenge with infectious SARS-CoV-2. Rhesus macaques were immunised with BNT162b1, BNT162b2, or saline (control) and challenged with SARS-CoV-2. A sentinel group was challenged with cell culture medium. The macaques were necropsied as described in Figs. 4 and 5 and Extended Data Table 2 . Two veterinary pathologists blindly performed microscopic evaluation of formalin fixed, hematoxylin and eosin stained lung tissue sections from each of 7 lobes from each macaque that had been necropsied on Day 7 or 8. Inflammation scores were assigned by consensus between the pathologists on a scale of 1-5 based on the area of involvement. Each dot represents an individual animal and is the mean inflammation area score from the 7 lung lobes.

    Techniques Used: Histopathology, Cell Culture, Staining

    Vaccine antigen expression and receptor affinity. a , Detection of BNT162b1-encoded RBD-foldon and BNT162b2-encoded P2 S in HEK293T cells by S1-specific antibody staining and flow cytometry. HEK293T cells analysed by flow cytometry were incubated with: no RNA (control), BNT162b RNAs formulated as LNPs (BNT162b1, BNT162b2) or BNT162b RNAs mixed with a transfection reagent (BNT162b1 RNA, BNT162b2 RNA). b , Localisation of BNT162b1 RNA-encoded RBD-foldon or BNT162b2 RNA-encoded P2 S in HEK293T cells transfected as in panel a, determined by immunofluorescence staining. Endoplasmic reticulum and Golgi (ER/Golgi, red), S1 (green) and DNA (blue). Scale bar: 10 µm. c , Western blot of denatured and non-denatured samples of size exclusion chromatography (SEC) fractions (chromatogram in Supplementary Fig. 1 ) of concentrated medium from HEK293T cells transfected with BNT162b1 RNA. The RBD-foldon was detected with a rabbit monoclonal antibody against the S1 fragment of SARS-CoV-2 S. Protein controls (ctrl): purified, recombinant RBD and S. d, Biolayer interferometry sensorgram demonstrating the binding kinetics of the purified RBD-foldon trimer, expressed from DNA, to immobilised human ACE2-PD. e , f Biolayer inferometry sensorgrams showing binding of a DNA-expressed P2 S preparation from a size exclusion chromatography peak (not shown) that contains intact P2 S and dissociated S1 and S2 to immobilised ( e ) human ACE2-PD and ( f ) B38 monoclonal antibody. Binding data are in colour; 1:1 binding models fit to the data are in black.
    Figure Legend Snippet: Vaccine antigen expression and receptor affinity. a , Detection of BNT162b1-encoded RBD-foldon and BNT162b2-encoded P2 S in HEK293T cells by S1-specific antibody staining and flow cytometry. HEK293T cells analysed by flow cytometry were incubated with: no RNA (control), BNT162b RNAs formulated as LNPs (BNT162b1, BNT162b2) or BNT162b RNAs mixed with a transfection reagent (BNT162b1 RNA, BNT162b2 RNA). b , Localisation of BNT162b1 RNA-encoded RBD-foldon or BNT162b2 RNA-encoded P2 S in HEK293T cells transfected as in panel a, determined by immunofluorescence staining. Endoplasmic reticulum and Golgi (ER/Golgi, red), S1 (green) and DNA (blue). Scale bar: 10 µm. c , Western blot of denatured and non-denatured samples of size exclusion chromatography (SEC) fractions (chromatogram in Supplementary Fig. 1 ) of concentrated medium from HEK293T cells transfected with BNT162b1 RNA. The RBD-foldon was detected with a rabbit monoclonal antibody against the S1 fragment of SARS-CoV-2 S. Protein controls (ctrl): purified, recombinant RBD and S. d, Biolayer interferometry sensorgram demonstrating the binding kinetics of the purified RBD-foldon trimer, expressed from DNA, to immobilised human ACE2-PD. e , f Biolayer inferometry sensorgrams showing binding of a DNA-expressed P2 S preparation from a size exclusion chromatography peak (not shown) that contains intact P2 S and dissociated S1 and S2 to immobilised ( e ) human ACE2-PD and ( f ) B38 monoclonal antibody. Binding data are in colour; 1:1 binding models fit to the data are in black.

    Techniques Used: Expressing, Staining, Flow Cytometry, Incubation, Transfection, Immunofluorescence, Western Blot, Size-exclusion Chromatography, Purification, Recombinant, Binding Assay

    Viral RNA detection in oropharyngeal (OP) and rectal swabs from rhesus macaques after BNT162b immunisation and challenge with infectious SARS-CoV-2. Rhesus macaques immunised with 100 µg of BNT162b1 or BNT162b2 ( n =6 each) and macaques immunised with saline or not immunised (Control, n =9), as described in Fig. 4 , Extended Data Fig. 6 , and Extended Data Table 2 , were challenged with 1.05 × 10 6 total plaque forming units (PFU) of SARS-CoV-2 split equally between the intranasal (IN) and intratracheal (IT) routes. Additional macaques (sentinel, n =6) were mock-challenged with cell culture medium. Viral RNA levels were detected by RT-qPCR. a , Viral RNA in OP swabs. b , Viral RNA in rectal swabs. Ratios above data points indicate the number of viral RNA positive animals among all animals providing evaluable samples in a group. Heights of bars indicate geometric mean of viral RNA copies; whiskers indicate geometric standard deviations. Every symbol represents one animal. Dotted lines indicate the lower limits of detection (LLODs). Values below the LLOD were set to ½ the LLOD. The statistical significance by Friedman’s non-parametric test of differences in viral RNA detection between 6 BNT162b1-immunised and 6 contemporaneously control-immunised animals (challenge cohorts 1 and 2) after challenge was p
    Figure Legend Snippet: Viral RNA detection in oropharyngeal (OP) and rectal swabs from rhesus macaques after BNT162b immunisation and challenge with infectious SARS-CoV-2. Rhesus macaques immunised with 100 µg of BNT162b1 or BNT162b2 ( n =6 each) and macaques immunised with saline or not immunised (Control, n =9), as described in Fig. 4 , Extended Data Fig. 6 , and Extended Data Table 2 , were challenged with 1.05 × 10 6 total plaque forming units (PFU) of SARS-CoV-2 split equally between the intranasal (IN) and intratracheal (IT) routes. Additional macaques (sentinel, n =6) were mock-challenged with cell culture medium. Viral RNA levels were detected by RT-qPCR. a , Viral RNA in OP swabs. b , Viral RNA in rectal swabs. Ratios above data points indicate the number of viral RNA positive animals among all animals providing evaluable samples in a group. Heights of bars indicate geometric mean of viral RNA copies; whiskers indicate geometric standard deviations. Every symbol represents one animal. Dotted lines indicate the lower limits of detection (LLODs). Values below the LLOD were set to ½ the LLOD. The statistical significance by Friedman’s non-parametric test of differences in viral RNA detection between 6 BNT162b1-immunised and 6 contemporaneously control-immunised animals (challenge cohorts 1 and 2) after challenge was p

    Techniques Used: RNA Detection, Cell Culture, Quantitative RT-PCR

    Radiographic signs in rhesus macaques after immunisation with BNT162b1 or BNT162b2 and challenge with SARS-CoV-2. Rhesus macaques were immunised with BNT162b1, BNT162b2, or saline (control) and challenged with SARS-CoV-2. A sentinel group was challenged with cell culture medium and imaged as described in Figs. 4 and 5 and Extended Data Table 2 . Three-view thoracic radiographs (ventrodorsal, right and left lateral) and lung field CT images were obtained prior to challenge (pre), and post-challenge at the indicated time points. The animals were anesthetised and intubated to perform end inspiratory breath-hold. Images were interpreted by two board-certified veterinary radiologists blinded to treatment groups. Scores were assigned to 7 lung regions on a severity scale of 0-3 per region, with a maximum severity score of 21. Pulmonary lesions evident prior to challenge or those which could not be unequivocally attributed to the viral challenge (such as atelectasis secondary to recumbency and anesthesia) received a score of “0”. a, Thoracic radiograph scores. b, Lung field CT scores.
    Figure Legend Snippet: Radiographic signs in rhesus macaques after immunisation with BNT162b1 or BNT162b2 and challenge with SARS-CoV-2. Rhesus macaques were immunised with BNT162b1, BNT162b2, or saline (control) and challenged with SARS-CoV-2. A sentinel group was challenged with cell culture medium and imaged as described in Figs. 4 and 5 and Extended Data Table 2 . Three-view thoracic radiographs (ventrodorsal, right and left lateral) and lung field CT images were obtained prior to challenge (pre), and post-challenge at the indicated time points. The animals were anesthetised and intubated to perform end inspiratory breath-hold. Images were interpreted by two board-certified veterinary radiologists blinded to treatment groups. Scores were assigned to 7 lung regions on a severity scale of 0-3 per region, with a maximum severity score of 21. Pulmonary lesions evident prior to challenge or those which could not be unequivocally attributed to the viral challenge (such as atelectasis secondary to recumbency and anesthesia) received a score of “0”. a, Thoracic radiograph scores. b, Lung field CT scores.

    Techniques Used: Cell Culture

    Clinical signs in BNT162b vaccine-immunised rhesus macaques after challenge with infectious SARS-CoV-2. Rhesus macaques were immunised with BNT162b vaccine candidates ( n =6 per group) or saline (control; n =9) and challenged with SARS-CoV-2. A sentinel group was challenged with cell culture medium ( n =6) as described in Figs. 4 and 5 and Extended Data Table 2 . Vital signs were recorded. a, Body weight change. b , Temperature change. c , Heart rate. d , Oxygen saturation.
    Figure Legend Snippet: Clinical signs in BNT162b vaccine-immunised rhesus macaques after challenge with infectious SARS-CoV-2. Rhesus macaques were immunised with BNT162b vaccine candidates ( n =6 per group) or saline (control; n =9) and challenged with SARS-CoV-2. A sentinel group was challenged with cell culture medium ( n =6) as described in Figs. 4 and 5 and Extended Data Table 2 . Vital signs were recorded. a, Body weight change. b , Temperature change. c , Heart rate. d , Oxygen saturation.

    Techniques Used: Cell Culture

    23) Product Images from "Collapsing Glomerulopathy in a Patient With Coronavirus Disease 2019 (COVID-19)"

    Article Title: Collapsing Glomerulopathy in a Patient With Coronavirus Disease 2019 (COVID-19)

    Journal: Kidney International Reports

    doi: 10.1016/j.ekir.2020.04.002

    In situ hybridization for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (a) Tissue quality was evaluated by performing RNAscope analysis for mRNA of the housekeeping gene peptidylprolyl isomerase B ( PPIB ). Positive cytoplasmic staining confirms adequate quality. Signal was detected using 3,3′-diaminobenzidine (DAB) (brown) chromogen. (periodic acid–Schiff counter stain; original magnification ×400). (b) RNAscope using probes directed against SARS-CoV-2 shows absence of signal in the patient's kidney parenchyma (periodic acid–Schiff counter stain; original magnification ×400).
    Figure Legend Snippet: In situ hybridization for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (a) Tissue quality was evaluated by performing RNAscope analysis for mRNA of the housekeeping gene peptidylprolyl isomerase B ( PPIB ). Positive cytoplasmic staining confirms adequate quality. Signal was detected using 3,3′-diaminobenzidine (DAB) (brown) chromogen. (periodic acid–Schiff counter stain; original magnification ×400). (b) RNAscope using probes directed against SARS-CoV-2 shows absence of signal in the patient's kidney parenchyma (periodic acid–Schiff counter stain; original magnification ×400).

    Techniques Used: In Situ Hybridization, Staining

    24) Product Images from "Molecular Detection of SARS-CoV-2 in Formalin Fixed Paraffin Embedded Specimens"

    Article Title: Molecular Detection of SARS-CoV-2 in Formalin Fixed Paraffin Embedded Specimens

    Journal: bioRxiv

    doi: 10.1101/2020.04.21.042911

    Dual staining to detect SARS-CoV-2 antigen and RNA in the same FFPE section. ( A–B ) Compared to uninfected control FFPE cell pellets ( A ), SARS-CoV-2 S (brown) and positive-sense RNA (red) were detected in the same section ( B ). Nuclei are stained blue (hematoxylin). Scale bar, 50 μm in ( A – B ).
    Figure Legend Snippet: Dual staining to detect SARS-CoV-2 antigen and RNA in the same FFPE section. ( A–B ) Compared to uninfected control FFPE cell pellets ( A ), SARS-CoV-2 S (brown) and positive-sense RNA (red) were detected in the same section ( B ). Nuclei are stained blue (hematoxylin). Scale bar, 50 μm in ( A – B ).

    Techniques Used: Staining, Formalin-fixed Paraffin-Embedded

    Detection of SARS-CoV-2 replication in FFPE cells using multiplex fluorescence ISH. ( A–B ) Compared to uninfected control ( A ), SARS-CoV-2 negative-sense RNA (green), a replicative intermediate that indicates viral replication, can be detected in infected FFPE cell pellets in addition to positive-sense (red) RNA ( B ). Nuclei are stained blue (DAPI). Scale bar, 20 μm in ( A – B ).
    Figure Legend Snippet: Detection of SARS-CoV-2 replication in FFPE cells using multiplex fluorescence ISH. ( A–B ) Compared to uninfected control ( A ), SARS-CoV-2 negative-sense RNA (green), a replicative intermediate that indicates viral replication, can be detected in infected FFPE cell pellets in addition to positive-sense (red) RNA ( B ). Nuclei are stained blue (DAPI). Scale bar, 20 μm in ( A – B ).

    Techniques Used: Formalin-fixed Paraffin-Embedded, Multiplex Assay, Fluorescence, In Situ Hybridization, Infection, Staining

    Detection of SARS-CoV-2 antigens by IHC and IFA in FFPE cell pellets. ( A–B ) In comparison to uninfected control FFPE cell pellets ( A and C ), SARS-CoV-2 S (brown, B ) and SARS-CoV-2 NP (brown, D ) can be detected in FFPE SARS-CoV-2-infected cell pellets. Nuclei are stained blue (hematoxylin). ( E ) Immunofluorescence staining to detect SARS-CoV-2 S (green) and NP (red) in FFPE SARS-CoV-2-infected cell pellets. Inset of ( E ) is uninfected control FFPE cell pellets. Nuclei are stained blue (DAPI). Scale bar, 50 μm in ( A – D ), 20 μm in inset of ( E) , and 10 μm in ( E) .
    Figure Legend Snippet: Detection of SARS-CoV-2 antigens by IHC and IFA in FFPE cell pellets. ( A–B ) In comparison to uninfected control FFPE cell pellets ( A and C ), SARS-CoV-2 S (brown, B ) and SARS-CoV-2 NP (brown, D ) can be detected in FFPE SARS-CoV-2-infected cell pellets. Nuclei are stained blue (hematoxylin). ( E ) Immunofluorescence staining to detect SARS-CoV-2 S (green) and NP (red) in FFPE SARS-CoV-2-infected cell pellets. Inset of ( E ) is uninfected control FFPE cell pellets. Nuclei are stained blue (DAPI). Scale bar, 50 μm in ( A – D ), 20 μm in inset of ( E) , and 10 μm in ( E) .

    Techniques Used: Immunohistochemistry, Immunofluorescence, Formalin-fixed Paraffin-Embedded, Infection, Staining

    Detection of SARS-CoV-2 RNA by ISH in FFPE cell pellets. ( A–B ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 1 in infected FFPE cell pellets ( B ), but not in uninfected control FFPE cell pellets ( A ). ( C–D ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 2 in infected FFPE cell pellets ( D ), but not in uninfected control FFPE cell pellets ( C ). ( E–F ) SARS-CoV-2 negative-sense RNA can be detected by ISH using negative-sense RNA probe 1in infected FFPE cell pellets ( E ), but not in uninfected control FFPE cell pellets ( F ). Nuclei are stained blue (hematoxylin). Scale bar, 50 μm in ( A – F ).
    Figure Legend Snippet: Detection of SARS-CoV-2 RNA by ISH in FFPE cell pellets. ( A–B ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 1 in infected FFPE cell pellets ( B ), but not in uninfected control FFPE cell pellets ( A ). ( C–D ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 2 in infected FFPE cell pellets ( D ), but not in uninfected control FFPE cell pellets ( C ). ( E–F ) SARS-CoV-2 negative-sense RNA can be detected by ISH using negative-sense RNA probe 1in infected FFPE cell pellets ( E ), but not in uninfected control FFPE cell pellets ( F ). Nuclei are stained blue (hematoxylin). Scale bar, 50 μm in ( A – F ).

    Techniques Used: In Situ Hybridization, Formalin-fixed Paraffin-Embedded, Infection, Staining

    25) Product Images from "A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates"

    Article Title: A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates

    Journal: bioRxiv

    doi: 10.1101/2020.09.08.280818

    Mouse immunogenicity. BALB/c mice (n=8 per group unless otherwise specified) were immunised intramuscularly (IM) with a single dose of with 0.2, 1 or 5 μg BNT162b2 or buffer. Geometric mean of each group ± 95% CI, P-values compare Day 28 to non-immunised (0 μg; n=8) baseline sera (multiple comparison of mixed-effect analysis using Dunnett’s multiple comparisons test) (a, b). a , S1-binding IgG responses in sera obtained 7, 14, 21 and 28 days after immunisation with 0, 0.2, 1, or 5 μg BNT162b2, determined by ELISA. For day 0 values, a pre-screening of randomly selected animals was performed (n=4). b, VSV-SARS-CoV-2 pseudovirus 50% serum neutralising titers (pVNT 50 ) of sera from (a). c-f, Splenocytes of BALB/c mice immunised IM with BNT162b2 or buffer (control) were ex vivo re-stimulated with full-length S peptide mix or negative controls ([c], [e], [f]: no peptide; [d]: irrelevant peptide). Individual values and mean of each group, P-values were determined by a two-tailed paired t-test. c , IFNγ ELISpot of splenocytes collected 12 days after immunisation with 5 μg BNT162b2. d , IFNγ ELISpot of isolated splenic CD4 + or CD8 + T cells collected 28 days after immunisation with 1 μg BNT162b2. e , CD8 + T-cell specific cytokine release by splenocytes collected 12 days after immunisation with 5 μg BNT162b2 or buffer (Control), determined by flow cytometry. S-peptide specific responses are corrected for background (no peptide). f , Cytokine production by splenocytes collected 28 days after immunisation with 1 μg BNT162b2, determined by bead-based multiplex analysis (n=7 for IL-4, IL-5 and IL-13, one outlier removed via routs test [Q=1%] for the S peptide stimulated samples).
    Figure Legend Snippet: Mouse immunogenicity. BALB/c mice (n=8 per group unless otherwise specified) were immunised intramuscularly (IM) with a single dose of with 0.2, 1 or 5 μg BNT162b2 or buffer. Geometric mean of each group ± 95% CI, P-values compare Day 28 to non-immunised (0 μg; n=8) baseline sera (multiple comparison of mixed-effect analysis using Dunnett’s multiple comparisons test) (a, b). a , S1-binding IgG responses in sera obtained 7, 14, 21 and 28 days after immunisation with 0, 0.2, 1, or 5 μg BNT162b2, determined by ELISA. For day 0 values, a pre-screening of randomly selected animals was performed (n=4). b, VSV-SARS-CoV-2 pseudovirus 50% serum neutralising titers (pVNT 50 ) of sera from (a). c-f, Splenocytes of BALB/c mice immunised IM with BNT162b2 or buffer (control) were ex vivo re-stimulated with full-length S peptide mix or negative controls ([c], [e], [f]: no peptide; [d]: irrelevant peptide). Individual values and mean of each group, P-values were determined by a two-tailed paired t-test. c , IFNγ ELISpot of splenocytes collected 12 days after immunisation with 5 μg BNT162b2. d , IFNγ ELISpot of isolated splenic CD4 + or CD8 + T cells collected 28 days after immunisation with 1 μg BNT162b2. e , CD8 + T-cell specific cytokine release by splenocytes collected 12 days after immunisation with 5 μg BNT162b2 or buffer (Control), determined by flow cytometry. S-peptide specific responses are corrected for background (no peptide). f , Cytokine production by splenocytes collected 28 days after immunisation with 1 μg BNT162b2, determined by bead-based multiplex analysis (n=7 for IL-4, IL-5 and IL-13, one outlier removed via routs test [Q=1%] for the S peptide stimulated samples).

    Techniques Used: Mouse Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Ex Vivo, Two Tailed Test, Enzyme-linked Immunospot, Isolation, Flow Cytometry, Multiplex Assay

    Rhesus macaque immunogenicity. Rhesus macaques (n=6 per group) were immunised on Days 0 and 21 with 30 μg or 100 μg BNT162b2 or buffer. Sera and PBMCs were collected at the times indicated. Human convalescent sera (HCS) were obtained from SARS-CoV-2-infected patients at least 14 days after PCR-confirmed diagnosis and at a time when acute COVID-19 symptoms had resolved (n=38). a , Concentration, in arbitrary units, of IgG binding recombinant SARS-CoV-2 S1. b , SARS-CoV-2 50% virus neutralisation titers (VNT 50 ). c-g , PBMCs collected on Day 42 were ex vivo re-stimulated with full-length S peptide mix. c, IFNγ, and d, IL-4 ELISpot. e , f , CD4 + T-cell specific, and g , CD8 + T-cell specific cytokine release, determined by flow cytometry. Heights of bars indicate the geometric (a-b) or arithmetic (c-g) means for each group. Whiskers indicate 95% confidence intervals (CI’s; a-b) or standard errors of means (SEMs; c-g). Every symbol represents one animal. Horizontal dotted lines mark the LLODs. Values below the LLOD set to ½ the LLOD. Asterisks below the x-axis indicate the day of Dose 2.
    Figure Legend Snippet: Rhesus macaque immunogenicity. Rhesus macaques (n=6 per group) were immunised on Days 0 and 21 with 30 μg or 100 μg BNT162b2 or buffer. Sera and PBMCs were collected at the times indicated. Human convalescent sera (HCS) were obtained from SARS-CoV-2-infected patients at least 14 days after PCR-confirmed diagnosis and at a time when acute COVID-19 symptoms had resolved (n=38). a , Concentration, in arbitrary units, of IgG binding recombinant SARS-CoV-2 S1. b , SARS-CoV-2 50% virus neutralisation titers (VNT 50 ). c-g , PBMCs collected on Day 42 were ex vivo re-stimulated with full-length S peptide mix. c, IFNγ, and d, IL-4 ELISpot. e , f , CD4 + T-cell specific, and g , CD8 + T-cell specific cytokine release, determined by flow cytometry. Heights of bars indicate the geometric (a-b) or arithmetic (c-g) means for each group. Whiskers indicate 95% confidence intervals (CI’s; a-b) or standard errors of means (SEMs; c-g). Every symbol represents one animal. Horizontal dotted lines mark the LLODs. Values below the LLOD set to ½ the LLOD. Asterisks below the x-axis indicate the day of Dose 2.

    Techniques Used: Infection, Polymerase Chain Reaction, Concentration Assay, Binding Assay, Recombinant, Ex Vivo, Enzyme-linked Immunospot, Flow Cytometry

    Protection of rhesus macaques from challenge with infectious SARS-CoV-2. Fifty-five days after the Dose 2 of 100 μg BNT162b2 (n=6) or saline control (n=3), rhesus macaques were challenged with 1.05 × 10 6 total pfu of SARS-CoV-2 split equally between the IN and IT routes. Non-immunised rhesus macaques (n=3) were mock-challenged with cell culture medium (sentinel). Viral RNA levels were detected by RT-qPCR. BAL was performed and nasal and oropharyngeal (OP) swabs obtained at the indicated time points. Final collection of samples was on Day 10 relative to challenge for the sentinel and control groups and at the end of protocol (EOP) on Day 7 or 8 for the BNT162b2-immunised group. Ratios above data points indicate the number of viral RNA positive animals among all animals per group. Heights of bars indicate geometric means. Whiskers indicate geometric standard deviations. Every symbol represents one animal. Dotted lines indicate the lower limits of detection (LLOD). Values below the LLOD were set to ½ the LLOD. a , Viral RNA in bronchoalveolar lavage (BAL) fluid. b , Viral RNA in nasal swabs. c , Viral RNA in OP swabs. The statistical significance by a non-parametric test (Friedman’s test) of differences in viral RNA detection between control-immunised and BNT162b2-immunised animals after challenge was p=0.0014 for BAL fluid, p=0.2622 for nasal swabs, and p=0.0007 for OP swabs. n/a – not available.
    Figure Legend Snippet: Protection of rhesus macaques from challenge with infectious SARS-CoV-2. Fifty-five days after the Dose 2 of 100 μg BNT162b2 (n=6) or saline control (n=3), rhesus macaques were challenged with 1.05 × 10 6 total pfu of SARS-CoV-2 split equally between the IN and IT routes. Non-immunised rhesus macaques (n=3) were mock-challenged with cell culture medium (sentinel). Viral RNA levels were detected by RT-qPCR. BAL was performed and nasal and oropharyngeal (OP) swabs obtained at the indicated time points. Final collection of samples was on Day 10 relative to challenge for the sentinel and control groups and at the end of protocol (EOP) on Day 7 or 8 for the BNT162b2-immunised group. Ratios above data points indicate the number of viral RNA positive animals among all animals per group. Heights of bars indicate geometric means. Whiskers indicate geometric standard deviations. Every symbol represents one animal. Dotted lines indicate the lower limits of detection (LLOD). Values below the LLOD were set to ½ the LLOD. a , Viral RNA in bronchoalveolar lavage (BAL) fluid. b , Viral RNA in nasal swabs. c , Viral RNA in OP swabs. The statistical significance by a non-parametric test (Friedman’s test) of differences in viral RNA detection between control-immunised and BNT162b2-immunised animals after challenge was p=0.0014 for BAL fluid, p=0.2622 for nasal swabs, and p=0.0007 for OP swabs. n/a – not available.

    Techniques Used: Cell Culture, Quantitative RT-PCR, RNA Detection

    Vaccine design and characterisation of the expressed antigen. a , BNT162b2 RNA structure. UTR, untranslated region; S, SARS-CoV-2 S glycoprotein; S1, N-terminal furin cleavage fragment; S2, C-terminal furin cleavage fragment; RBD, receptor-binding domain. Positions of the P2 mutation (K986P and V897P) are indicated. b , Liquid capillary electropherogram of in vitro transcribed BNT162b2 RNA. c , A 3.29 Å cryoEM map of P2 S, with fitted and refined atomic model, viewed down the three-fold axis toward the membrane. d, Cryo-EM map and model of (d) viewed perpendicular to the three-fold axis. e, Mass density map of TwinStrep-tagged P2 S produced by 3D classification of images extracted from cryo-EM micrographs with no symmetry averaging. This class, in the one-RBD ‘up’, two RBD ‘down’ positioning, represents 20.4% of the population.
    Figure Legend Snippet: Vaccine design and characterisation of the expressed antigen. a , BNT162b2 RNA structure. UTR, untranslated region; S, SARS-CoV-2 S glycoprotein; S1, N-terminal furin cleavage fragment; S2, C-terminal furin cleavage fragment; RBD, receptor-binding domain. Positions of the P2 mutation (K986P and V897P) are indicated. b , Liquid capillary electropherogram of in vitro transcribed BNT162b2 RNA. c , A 3.29 Å cryoEM map of P2 S, with fitted and refined atomic model, viewed down the three-fold axis toward the membrane. d, Cryo-EM map and model of (d) viewed perpendicular to the three-fold axis. e, Mass density map of TwinStrep-tagged P2 S produced by 3D classification of images extracted from cryo-EM micrographs with no symmetry averaging. This class, in the one-RBD ‘up’, two RBD ‘down’ positioning, represents 20.4% of the population.

    Techniques Used: Binding Assay, Mutagenesis, In Vitro, Produced

    26) Product Images from "CoVaccine HT™ adjuvant potentiates robust immune responses to recombinant SARS-CoV-2 Spike S1 immunisation"

    Article Title: CoVaccine HT™ adjuvant potentiates robust immune responses to recombinant SARS-CoV-2 Spike S1 immunisation

    Journal: bioRxiv

    doi: 10.1101/2020.07.24.220715

    Detection of IFN-γ secreting cells from mice immunised with SARS-CoV-2 vaccines. The splenocytes were obtained from mice (2 to 3 per group) immunised with SARS-CoV-2 S1 protein, adjuvanted with CoVaccine HT™ or Alum, or S1 protein alone on day 28 (one-week after booster immunisations). Pooled splenocytes obtained from two naïve mice were used as controls. The cells were incubated for 40 hours with PepTivator® SARS-CoV-2 Prot_S1 peptide pools at 0.2 μg/mL or 0.5 μg/mL per peptide or medium. IFN-γ secreting cells were enumerated by FluoroSpot as detailed in the methods section. The results are expressed as the number of spot forming cells (SFC)/106 splenocytes after subtraction of the number of spots formed by cells in medium only wells to correct for background activity. *** p ≤ 0.001, **** p ≤ 0.0001.
    Figure Legend Snippet: Detection of IFN-γ secreting cells from mice immunised with SARS-CoV-2 vaccines. The splenocytes were obtained from mice (2 to 3 per group) immunised with SARS-CoV-2 S1 protein, adjuvanted with CoVaccine HT™ or Alum, or S1 protein alone on day 28 (one-week after booster immunisations). Pooled splenocytes obtained from two naïve mice were used as controls. The cells were incubated for 40 hours with PepTivator® SARS-CoV-2 Prot_S1 peptide pools at 0.2 μg/mL or 0.5 μg/mL per peptide or medium. IFN-γ secreting cells were enumerated by FluoroSpot as detailed in the methods section. The results are expressed as the number of spot forming cells (SFC)/106 splenocytes after subtraction of the number of spots formed by cells in medium only wells to correct for background activity. *** p ≤ 0.001, **** p ≤ 0.0001.

    Techniques Used: Mouse Assay, Incubation, Activity Assay

    Immunogenicity and specificity to SARS-CoV-2 S1 immunisation. A Timeline schematic of BALB/c immunisations and bleeds with a table detailing the study design. B Median fluorescence intensity (MFI) of serum antibodies from each group binding to custom magnetic beads coupled with Spike S1 proteins from either SARS-CoV-2 (SARS-2), SARS-CoV (SARS), or MERS-CoV (MERS) on day 14 and 35. C Antibody reactivity to SARS-2, SARS, and MERS antigens throughout the study. Graphs in panels (B) and (C) are on a logarithmic scale representing geometric mean MFI responses with 95% confidence interval (CI). The dashed lines represent assay cut-off values determined by the mean plus three standard deviations of the negative control (BSA coupled beads).
    Figure Legend Snippet: Immunogenicity and specificity to SARS-CoV-2 S1 immunisation. A Timeline schematic of BALB/c immunisations and bleeds with a table detailing the study design. B Median fluorescence intensity (MFI) of serum antibodies from each group binding to custom magnetic beads coupled with Spike S1 proteins from either SARS-CoV-2 (SARS-2), SARS-CoV (SARS), or MERS-CoV (MERS) on day 14 and 35. C Antibody reactivity to SARS-2, SARS, and MERS antigens throughout the study. Graphs in panels (B) and (C) are on a logarithmic scale representing geometric mean MFI responses with 95% confidence interval (CI). The dashed lines represent assay cut-off values determined by the mean plus three standard deviations of the negative control (BSA coupled beads).

    Techniques Used: Fluorescence, Binding Assay, Magnetic Beads, Negative Control

    27) Product Images from "Fast isolation of sub-nanomolar affinity alpaca nanobody against the Spike RBD of SARS-CoV-2 by combining bacterial display and a simple single-step density gradient selection"

    Article Title: Fast isolation of sub-nanomolar affinity alpaca nanobody against the Spike RBD of SARS-CoV-2 by combining bacterial display and a simple single-step density gradient selection

    Journal: bioRxiv

    doi: 10.1101/2020.06.09.137935

    Immunization of the Spike of SARS-CoV-2 and a simple density gradient method for the selection of Nanobodies A) SDS-Page to ensure protein integrity of full-length Spike of SARS-CoV-2 before immunization. B) Adult alpaca immunized with Spike. C) Evaluation of the alpaca’s immune response by Dot blot. Image shows the reaction to decreasing amounts of Spike-1 and Bovine serum albumin (negative control) using a pre-immunization control, and after one immunization (1 week), or two immunizations (3 weeks) with full-length SARS-CoV-2 Spike, using alpaca serums as a primary antibody source followed by an anti-camelid IgG-HRP secondary antibody. D) ELISA assay before and after the second immunization (3 weeks). E) Schematic representation of novel protocol for isolation of Nanobodies using density gradient separation. The bacterial display library expressing the Nanobodies on the surface of bacteria is briefly incubated with conventional Sepharose beads coated with the epitope of interest. Directly after the mixture is deposited on a Ficoll gradient conic tube and centrifuged at 200 x g for 1 min, the beads drive through the gradient to the bottom of the tube with the bacteria expressing specific Nanobodies, while the unbound bacteria remain on the surface of the gradient. The beads are then resuspended, and bacterial clones are isolated for biochemical binding confirmation.
    Figure Legend Snippet: Immunization of the Spike of SARS-CoV-2 and a simple density gradient method for the selection of Nanobodies A) SDS-Page to ensure protein integrity of full-length Spike of SARS-CoV-2 before immunization. B) Adult alpaca immunized with Spike. C) Evaluation of the alpaca’s immune response by Dot blot. Image shows the reaction to decreasing amounts of Spike-1 and Bovine serum albumin (negative control) using a pre-immunization control, and after one immunization (1 week), or two immunizations (3 weeks) with full-length SARS-CoV-2 Spike, using alpaca serums as a primary antibody source followed by an anti-camelid IgG-HRP secondary antibody. D) ELISA assay before and after the second immunization (3 weeks). E) Schematic representation of novel protocol for isolation of Nanobodies using density gradient separation. The bacterial display library expressing the Nanobodies on the surface of bacteria is briefly incubated with conventional Sepharose beads coated with the epitope of interest. Directly after the mixture is deposited on a Ficoll gradient conic tube and centrifuged at 200 x g for 1 min, the beads drive through the gradient to the bottom of the tube with the bacteria expressing specific Nanobodies, while the unbound bacteria remain on the surface of the gradient. The beads are then resuspended, and bacterial clones are isolated for biochemical binding confirmation.

    Techniques Used: Selection, SDS Page, Dot Blot, Negative Control, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Incubation, Clone Assay, Binding Assay

    Binding Characterization of W25UACh to SARS-CoV-2 Spike. A) Pulldown of the W25UACh nanobody, a recombinant Spike RBD domain of the SARS-CoV-2 Spike protein or control BSA protein was covalently bound to NHS-Sepharose beads. Further, the W25UACh nanobody was incubated with control and Spike RBD beads, washed, and further eluted in LSD lysis buffer (Invitrogen). B) HeLa cells were transiently transfected with full-length Spike-GFP and immunofluorescence was performed with the pure W25UACh nanobody, showing increased resolution of Spike visualized on a Celldiscoverer7 microscope. C) Unfolding profiles of 2 μM SARS-CoV-2 S1, Spike RBD in the absence (black) and presence (red) of 2 μM W25UACh, measured with Tycho NT.6. Binding of W25UACh to Spike RBD leads to strong stabilization and shifts the inflection unfolding temperature (T i ) from 52.1°C to 66.3°C. D) MST binding curve for the titration of 1 nM fluorescently labeled W25UACh into a 16-point serial dilution of SARS-CoV-2 S1, Spike RBD (250 nM – 7.6 pM). W25UACh binds Spike RBD with sub-nanomolar affinity (K d = 295 ± 84 pM). Error bars show the SD calculated from experiments performed in triplicate.
    Figure Legend Snippet: Binding Characterization of W25UACh to SARS-CoV-2 Spike. A) Pulldown of the W25UACh nanobody, a recombinant Spike RBD domain of the SARS-CoV-2 Spike protein or control BSA protein was covalently bound to NHS-Sepharose beads. Further, the W25UACh nanobody was incubated with control and Spike RBD beads, washed, and further eluted in LSD lysis buffer (Invitrogen). B) HeLa cells were transiently transfected with full-length Spike-GFP and immunofluorescence was performed with the pure W25UACh nanobody, showing increased resolution of Spike visualized on a Celldiscoverer7 microscope. C) Unfolding profiles of 2 μM SARS-CoV-2 S1, Spike RBD in the absence (black) and presence (red) of 2 μM W25UACh, measured with Tycho NT.6. Binding of W25UACh to Spike RBD leads to strong stabilization and shifts the inflection unfolding temperature (T i ) from 52.1°C to 66.3°C. D) MST binding curve for the titration of 1 nM fluorescently labeled W25UACh into a 16-point serial dilution of SARS-CoV-2 S1, Spike RBD (250 nM – 7.6 pM). W25UACh binds Spike RBD with sub-nanomolar affinity (K d = 295 ± 84 pM). Error bars show the SD calculated from experiments performed in triplicate.

    Techniques Used: Binding Assay, Recombinant, Incubation, Lysis, Transfection, Immunofluorescence, Microscopy, Titration, Labeling, Serial Dilution

    Dual biochemical and microscopy-based selection fo Nanobodies A) Dot blot immunodetection of full-length SARS-CoV-2 Spike using direct total protein extracts of clones W25UACh and W23UACh as the primary antibody. Mouse anti-Myc 1:3000 followed by anti-Mouse-HRP were used for detection. Protein extract from E. coli (BL21 strain) was used as a negative control. B) Immunodetection of Spike-GFP transiently transfected in HeLa cells using total protein extract selected clones as the primary antibody, followed by Mouse anti-Myc 1:3000 and anti-Mouse-Alexa 647. The image shows two positive clones W25UACh and W23UACh, and an example of a negative nanobody. C) Sequence alignment of W25UACh and W23UACh predicted aminoacidic sequence. CDR sequences are marked with a black line.
    Figure Legend Snippet: Dual biochemical and microscopy-based selection fo Nanobodies A) Dot blot immunodetection of full-length SARS-CoV-2 Spike using direct total protein extracts of clones W25UACh and W23UACh as the primary antibody. Mouse anti-Myc 1:3000 followed by anti-Mouse-HRP were used for detection. Protein extract from E. coli (BL21 strain) was used as a negative control. B) Immunodetection of Spike-GFP transiently transfected in HeLa cells using total protein extract selected clones as the primary antibody, followed by Mouse anti-Myc 1:3000 and anti-Mouse-Alexa 647. The image shows two positive clones W25UACh and W23UACh, and an example of a negative nanobody. C) Sequence alignment of W25UACh and W23UACh predicted aminoacidic sequence. CDR sequences are marked with a black line.

    Techniques Used: Microscopy, Selection, Dot Blot, Immunodetection, Clone Assay, Negative Control, Transfection, Sequencing

    28) Product Images from "Double Lock of a Potent Human Monoclonal Antibody against SARS-CoV-2"

    Article Title: Double Lock of a Potent Human Monoclonal Antibody against SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2020.11.24.393629

    Schematic diagram of SARS-CoV-2 S and the secondary structure of the RBD. Related to Figure 7 . (A) Overall topology of SARS-CoV-2 S. NTD: N-terminal domain; RBD: receptor-binding domain; RBM: receptor-binding motif; SD1: subdomain 1; SD2: subdomain 2; FP: fusion peptide; HR1: heptad repeat 1; HR2: heptad repeat 2; TM: transmembrane region; IC: intracellular domain. (B) Protein sequence and the secondary structure of SARS-CoV-2 RBD. The red three-pointed stars and blue rectangles mark the residues in SARS-CoV-2 S RBD that interact with HB27 and ACE2, respectively.
    Figure Legend Snippet: Schematic diagram of SARS-CoV-2 S and the secondary structure of the RBD. Related to Figure 7 . (A) Overall topology of SARS-CoV-2 S. NTD: N-terminal domain; RBD: receptor-binding domain; RBM: receptor-binding motif; SD1: subdomain 1; SD2: subdomain 2; FP: fusion peptide; HR1: heptad repeat 1; HR2: heptad repeat 2; TM: transmembrane region; IC: intracellular domain. (B) Protein sequence and the secondary structure of SARS-CoV-2 RBD. The red three-pointed stars and blue rectangles mark the residues in SARS-CoV-2 S RBD that interact with HB27 and ACE2, respectively.

    Techniques Used: Binding Assay, Sequencing

    HB27 potently binds and neutralizes SARS-CoV-2 wide type and mutant strain D614G. Related to Figure 7 . (A) The spike proteins of WT and D614G were transient expressed in 293T cells which were then examined for binding to HB27 by flow cytometry. (B) Neutralizing activities of HB27 against SARS-CoV-2 WT and D614G pseudoviruses (PSV).
    Figure Legend Snippet: HB27 potently binds and neutralizes SARS-CoV-2 wide type and mutant strain D614G. Related to Figure 7 . (A) The spike proteins of WT and D614G were transient expressed in 293T cells which were then examined for binding to HB27 by flow cytometry. (B) Neutralizing activities of HB27 against SARS-CoV-2 WT and D614G pseudoviruses (PSV).

    Techniques Used: Mutagenesis, Binding Assay, Flow Cytometry

    Prophylactic and therapeutic efficacy of HB27 in two SARS-CoV-2 susceptible mice models (A) Experimental design for therapeutic and prophylactic evaluations of HB27 in two SARS-CoV-2 susceptible mice models. Group of 6-to-8 week-old hACE2 mice and BALB/c mice were infected intranasally with 5×10 4 PFU of SARS-CoV-2 BetaCoV/Beijing/IME-BJ01/2020 or 1.6×10 4 PFU of MASCp6 as described previously, respectively. A dose of 20 mg/kg HB27 was injected intraperitoneally at 12 hours before infection (the prophylactic group, P) or at 2 hours after infection (the therapeutic group, T). PBS injections were used as control group. Then, the lung tissues of mice were collected at 3 and 5 dpi for virus titer, H E and Immunostaining. (B) and (C) Virus titers of lung and trachea tissues at 3 or 5 dpi in mouse model based on a SARS-CoV-2 mouse adapted strain MASCp6. The viral loads of the tissues were determined by qRT-PCR (*P
    Figure Legend Snippet: Prophylactic and therapeutic efficacy of HB27 in two SARS-CoV-2 susceptible mice models (A) Experimental design for therapeutic and prophylactic evaluations of HB27 in two SARS-CoV-2 susceptible mice models. Group of 6-to-8 week-old hACE2 mice and BALB/c mice were infected intranasally with 5×10 4 PFU of SARS-CoV-2 BetaCoV/Beijing/IME-BJ01/2020 or 1.6×10 4 PFU of MASCp6 as described previously, respectively. A dose of 20 mg/kg HB27 was injected intraperitoneally at 12 hours before infection (the prophylactic group, P) or at 2 hours after infection (the therapeutic group, T). PBS injections were used as control group. Then, the lung tissues of mice were collected at 3 and 5 dpi for virus titer, H E and Immunostaining. (B) and (C) Virus titers of lung and trachea tissues at 3 or 5 dpi in mouse model based on a SARS-CoV-2 mouse adapted strain MASCp6. The viral loads of the tissues were determined by qRT-PCR (*P

    Techniques Used: Mouse Assay, Infection, Injection, Immunostaining, Quantitative RT-PCR

    HB27 strongly binds various SARS-CoV-2 RBD mutants. Related to Figure 6 . (A) Sequence alignments of the mutated RBDs of circulating SARS-CoV-2 strains used in (A) and SARS-CoV. The genome sequences used in the alignments were downloaded from NCBI and GISAID with accession numbers: NC_045512.2 , EPI_ISL_406596, EPI_ISL_406595, EPI_ISL_413602, EPI_ISL_415605, EPI_ISL_408511, EPI_ISL_413522, EPI_ISL_415655, EPI_ISL_418055, EPI_ISL_416507, EPI_ISL_407071 and AY429078.1, respectively. The alignments were analyzed by Clustal W and BioEdit. (B) ELISA binding assays of HB27 with selected SARS-CoV-2 RBD mutants. SARS-CoV-2 RBD proteins with previously reported site mutations were examined for their binding abilities to HB27.
    Figure Legend Snippet: HB27 strongly binds various SARS-CoV-2 RBD mutants. Related to Figure 6 . (A) Sequence alignments of the mutated RBDs of circulating SARS-CoV-2 strains used in (A) and SARS-CoV. The genome sequences used in the alignments were downloaded from NCBI and GISAID with accession numbers: NC_045512.2 , EPI_ISL_406596, EPI_ISL_406595, EPI_ISL_413602, EPI_ISL_415605, EPI_ISL_408511, EPI_ISL_413522, EPI_ISL_415655, EPI_ISL_418055, EPI_ISL_416507, EPI_ISL_407071 and AY429078.1, respectively. The alignments were analyzed by Clustal W and BioEdit. (B) ELISA binding assays of HB27 with selected SARS-CoV-2 RBD mutants. SARS-CoV-2 RBD proteins with previously reported site mutations were examined for their binding abilities to HB27.

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

    Structural basis for neutralization of SARS-CoV-2 by HB27 (A) Orthogonal views of the clashes between HB27 Fabs and ACE2 upon binding to SARS-CoV-2 S trimer. The SARS-CoV-2 S trimer is presented as ribbon diagrams and translucent molecular surfaces with three monomers colored in cyan, yellow and violet, respectively. The three copies of HB27 Fabs are rendered as molecular surfaces colored the same as in Figure 6 . The superposed ACE2 is presented as green ribbon diagrams as well as translucent molecular surface. Insets are close-up views of the clashes between ACE2 and HB27 upon binding to SARS-CoV-2 RBD. (B) Orthogonal views of the structure of HB27 Fab-A, Fab-B and Fab-C complexed with SARS-CoV-2 RBD. The S1 subunits of SARS-CoV-2 S trimer are rendered as cyan, yellow and violet surfaces and the S2 subunits are rendered as gray surfaces. See also Figures S6 and S7 .
    Figure Legend Snippet: Structural basis for neutralization of SARS-CoV-2 by HB27 (A) Orthogonal views of the clashes between HB27 Fabs and ACE2 upon binding to SARS-CoV-2 S trimer. The SARS-CoV-2 S trimer is presented as ribbon diagrams and translucent molecular surfaces with three monomers colored in cyan, yellow and violet, respectively. The three copies of HB27 Fabs are rendered as molecular surfaces colored the same as in Figure 6 . The superposed ACE2 is presented as green ribbon diagrams as well as translucent molecular surface. Insets are close-up views of the clashes between ACE2 and HB27 upon binding to SARS-CoV-2 RBD. (B) Orthogonal views of the structure of HB27 Fab-A, Fab-B and Fab-C complexed with SARS-CoV-2 RBD. The S1 subunits of SARS-CoV-2 S trimer are rendered as cyan, yellow and violet surfaces and the S2 subunits are rendered as gray surfaces. See also Figures S6 and S7 .

    Techniques Used: Neutralization, Binding Assay

    Characterization of SARS-CoV-2 and HB27, and cryo-EM maps and atomic models of SARS-CoV-2 S and HB27 complex. Related to Figure 6 . (A) Gel filtration of SARS-CoV-2 S trimer. (B) SDS-PAGE analysis of the SARS-CoV-2 S trimer, the HB27 IgG and the Fab fragment. (C) The gold-standard Fourier Shell Correlation (FSC) curves of the final cryo-EM maps of the SARS-CoV-2 S trimer-HB27 Fabs complex and of the binding interface. (D) Local resolution evaluations of the cryo-EM maps of SARS-CoV-2 S trimer complexed with three HB27 Fabs and the binding interface using ResMap ( Kucukelbir et al., 2014 ) are shown. (E) Cryo-EM map of SARS-CoV-2 S trimer complexed with three HB27 Fabs. (F) Cryo-EM map of the binding interface between SARS-CoV-2 RBD and one HB27 Fab. The color scheme is the same as in Figure 6 . The magnified panels illustrate both maps (mesh) and related atomic models. Residues are shown as sticks,
    Figure Legend Snippet: Characterization of SARS-CoV-2 and HB27, and cryo-EM maps and atomic models of SARS-CoV-2 S and HB27 complex. Related to Figure 6 . (A) Gel filtration of SARS-CoV-2 S trimer. (B) SDS-PAGE analysis of the SARS-CoV-2 S trimer, the HB27 IgG and the Fab fragment. (C) The gold-standard Fourier Shell Correlation (FSC) curves of the final cryo-EM maps of the SARS-CoV-2 S trimer-HB27 Fabs complex and of the binding interface. (D) Local resolution evaluations of the cryo-EM maps of SARS-CoV-2 S trimer complexed with three HB27 Fabs and the binding interface using ResMap ( Kucukelbir et al., 2014 ) are shown. (E) Cryo-EM map of SARS-CoV-2 S trimer complexed with three HB27 Fabs. (F) Cryo-EM map of the binding interface between SARS-CoV-2 RBD and one HB27 Fab. The color scheme is the same as in Figure 6 . The magnified panels illustrate both maps (mesh) and related atomic models. Residues are shown as sticks,

    Techniques Used: Filtration, SDS Page, Binding Assay

    HB27 potently competes with ACE2 for binding to SARS-CoV-2 RBD. Related to Figure 4 . (A) HB27 was demonstrated to compete with recombinant ACE2 for binding to SARS-CoV-2 RBD with an EC 50 value of 0.5 nM by the enzyme-linked immunosorbent assay (ELISA). (B) BIAcore SPR kinetic profile of SARS-CoV-2 S trimer and HB27. The binding affinity K D (equilibrium dissociation constant, K D = Kd/Ka, where Kd and Ka represent the dissociation rate constant and association rate constant, respectively) values were obtained using a series of HB27 concentrations and fitted in a global mode in each sensorgram. (C) BIAcore SPR kinetic profiles of SARS-CoV-2 RBD (left panel) and S trimer (right panel) with ACE2. The binding affinity K D (equilibrium dissociation constant, K D = Kd/Ka, where Kd and Ka represent the dissociation rate constant and association rate constant, respectively) values were obtained using a series of HB27 concentrations and fitted in a global mode in each sensorgram. (D) Competition of HB27 for SARS-CoV-2 RBD binding to 293T cells expressing GFP-tagged ACE2 as detected by immunofluorescence assay, scale bar, 100 μm. Anti-H7N9 mAb was used as an isotype control. (E) Competition of HB27 for ACE2-Fc-Apc binding to 293T cells expressing GFP-tagged SARS-CoV-2-Spike as detected by immunofluorescence assay. Anti-H7N9 mAb was used as an isotype control.
    Figure Legend Snippet: HB27 potently competes with ACE2 for binding to SARS-CoV-2 RBD. Related to Figure 4 . (A) HB27 was demonstrated to compete with recombinant ACE2 for binding to SARS-CoV-2 RBD with an EC 50 value of 0.5 nM by the enzyme-linked immunosorbent assay (ELISA). (B) BIAcore SPR kinetic profile of SARS-CoV-2 S trimer and HB27. The binding affinity K D (equilibrium dissociation constant, K D = Kd/Ka, where Kd and Ka represent the dissociation rate constant and association rate constant, respectively) values were obtained using a series of HB27 concentrations and fitted in a global mode in each sensorgram. (C) BIAcore SPR kinetic profiles of SARS-CoV-2 RBD (left panel) and S trimer (right panel) with ACE2. The binding affinity K D (equilibrium dissociation constant, K D = Kd/Ka, where Kd and Ka represent the dissociation rate constant and association rate constant, respectively) values were obtained using a series of HB27 concentrations and fitted in a global mode in each sensorgram. (D) Competition of HB27 for SARS-CoV-2 RBD binding to 293T cells expressing GFP-tagged ACE2 as detected by immunofluorescence assay, scale bar, 100 μm. Anti-H7N9 mAb was used as an isotype control. (E) Competition of HB27 for ACE2-Fc-Apc binding to 293T cells expressing GFP-tagged SARS-CoV-2-Spike as detected by immunofluorescence assay. Anti-H7N9 mAb was used as an isotype control.

    Techniques Used: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, SPR Assay, Expressing, Immunofluorescence

    HB27 blocks the interactions of SARS-CoV-2 with ACE2 (A) BIAcore SPR kinetics showing the competitive binding of HB27 and ACE2 to SARS-CoV-2 S trimer. For both panels, SARS-CoV-2 S protein was immobilized onto the sensor chips. In the upper panel, HB27 was first injected, followed by ACE2, whereas in the lower panel, ACE2 was injected first and then HB27. The control groups are as shown by the curves. (B) Blocking of SARS-CoV-2 RBD binding to 293T-ACE2 cells by HB27 (upper panel). Recombinant SARS-CoV-2 RBD protein and serially diluted HB27 were incubated with ACE2 expressing 293T cells (293T-ACE2) and tested for binding of HB27 to 293T-ACE2 cells. Competitive binding of HB27 and ACE2 to SARS-CoV-2-S cells (lower panel). Recombinant ACE2 and serially diluted HB27 were incubated with 293T cells expressing SARS-CoV-2 S (SARS-CoV-2-S) and tested for binding of HB27 to SARS-CoV-2-S cells. BSA was used as a negative control (NC). (C) Amount of virus on the cell surface, as detected by RT-PCR, when exposed to HB27 prior to (upper panel) and after (lower panel) the virus was allowed to attach to cells. Values are mean ± SD. Experiments were repeated in triplicate. See also Figure S2 .
    Figure Legend Snippet: HB27 blocks the interactions of SARS-CoV-2 with ACE2 (A) BIAcore SPR kinetics showing the competitive binding of HB27 and ACE2 to SARS-CoV-2 S trimer. For both panels, SARS-CoV-2 S protein was immobilized onto the sensor chips. In the upper panel, HB27 was first injected, followed by ACE2, whereas in the lower panel, ACE2 was injected first and then HB27. The control groups are as shown by the curves. (B) Blocking of SARS-CoV-2 RBD binding to 293T-ACE2 cells by HB27 (upper panel). Recombinant SARS-CoV-2 RBD protein and serially diluted HB27 were incubated with ACE2 expressing 293T cells (293T-ACE2) and tested for binding of HB27 to 293T-ACE2 cells. Competitive binding of HB27 and ACE2 to SARS-CoV-2-S cells (lower panel). Recombinant ACE2 and serially diluted HB27 were incubated with 293T cells expressing SARS-CoV-2 S (SARS-CoV-2-S) and tested for binding of HB27 to SARS-CoV-2-S cells. BSA was used as a negative control (NC). (C) Amount of virus on the cell surface, as detected by RT-PCR, when exposed to HB27 prior to (upper panel) and after (lower panel) the virus was allowed to attach to cells. Values are mean ± SD. Experiments were repeated in triplicate. See also Figure S2 .

    Techniques Used: SPR Assay, Binding Assay, Injection, Blocking Assay, Recombinant, Incubation, Expressing, Negative Control, Reverse Transcription Polymerase Chain Reaction

    HB27 is a SARS-CoV-2-specific antibody of high potency (A) Analysis of affinity of HB27 (left panel) and HB27 Fab fragments (right panel) for SARS-CoV-2 RBD. Biotinylated SARS-CoV-2 RBD protein was loaded on Octet SA sensor and tested for real-time association and dissociation from HB27 IgG and HB27 Fab fragments, respectively. (B) and (C) Analysis of affinity of HB27 for SARS-CoV RBD and MERS-CoV RBD, respectively. (D) and (E) Neutralizing activity of HB27 against SARS-CoV-2 and SARS-CoV pseudoviruses (PSV), respectively. Serially diluted HB27 titres were added to test neutralizing activity against SARS-CoV-2 and SARS-CoV PSV. (F) In vitro neutralization activity of HB27 against SARS-CoV-2 by plaque reduction neutralization test (PRNT) in Vero cells. Neutralizing activities are represented as mean ± SD. Experiments were performed in duplicates See also Figure S1 .
    Figure Legend Snippet: HB27 is a SARS-CoV-2-specific antibody of high potency (A) Analysis of affinity of HB27 (left panel) and HB27 Fab fragments (right panel) for SARS-CoV-2 RBD. Biotinylated SARS-CoV-2 RBD protein was loaded on Octet SA sensor and tested for real-time association and dissociation from HB27 IgG and HB27 Fab fragments, respectively. (B) and (C) Analysis of affinity of HB27 for SARS-CoV RBD and MERS-CoV RBD, respectively. (D) and (E) Neutralizing activity of HB27 against SARS-CoV-2 and SARS-CoV pseudoviruses (PSV), respectively. Serially diluted HB27 titres were added to test neutralizing activity against SARS-CoV-2 and SARS-CoV PSV. (F) In vitro neutralization activity of HB27 against SARS-CoV-2 by plaque reduction neutralization test (PRNT) in Vero cells. Neutralizing activities are represented as mean ± SD. Experiments were performed in duplicates See also Figure S1 .

    Techniques Used: Activity Assay, In Vitro, Neutralization, Plaque Reduction Neutralization Test

    Murine antibody mhB27 strongly binds SARS-CoV-2 RBD and neutralizes SARS-CoV-2 PSV. Related to Figure 1 . (A) Binding assay of mhB27 to SARS-CoV-2 RBD. mhB27 was serial diluted and tested its ability to bind to SARS-CoV-2 RBD by ELISA. (B) Neutralizing activities of mhB27 against SARS-CoV-2 pseudoviruses (PSV).
    Figure Legend Snippet: Murine antibody mhB27 strongly binds SARS-CoV-2 RBD and neutralizes SARS-CoV-2 PSV. Related to Figure 1 . (A) Binding assay of mhB27 to SARS-CoV-2 RBD. mhB27 was serial diluted and tested its ability to bind to SARS-CoV-2 RBD by ELISA. (B) Neutralizing activities of mhB27 against SARS-CoV-2 pseudoviruses (PSV).

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

    HB27 inhibits SARS-CoV-2 membrane fusion. (A) HB27 had potent neutralization activities when exposed to virus before or after attachment to Huh7 cells. Values are mean ± SD. Experiments were repeated in triplicate. (B) HB27 inhibits S protein-mediated cell-cell fusion. 293T cells were transfected with SARS-CoV-2 S-GFP protein, co-cultured with Vero E6 cells in the absence or presence of 100 μg/mL H014 or HB27 or anti-influenza H7N9 antibody (isotype control). No Ab: in the absence of antibodies. Images were taken after 48 h. Cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min and stained for nuclei with 4,6-diamidino-2-phenylindole (DAPI). (C) HB27 inhibits SARS-CoV-2-mediated cell-cell fusion. Huh7 cells were infected with 100 PFU of SARS-CoV-2 for 1 h at 4°C and washed for 3 times. After that cells were further cultured in the presence of a series of concentrations (0, 4, 20 and 100 nM) of HB27, or 100 nM of H014 at 37 °C for 48 h. Images were taken after 48 h. Cells were fixed with 4% (w/v) PFA for 20 min and incubated with anti-SARS-CoV-2 S protein antibody and stained for nuclei with DAPI. Scale bar equals 200 μm. (D) HB27 blocks receptor-mediated fusion of SARS-CoV-2 with liposomes. Liposomes were loaded with self-quenching concentrations of the fluorescent dye calcein. Perturbation of the bilayer causes the release of calcein resulting in dilution and a consequent increase in its fluorescence. Fusion of SARS-CoV-2 with liposomes occurred in the presence of both ACE2 and trypsin and a series of HB27 concentrations were used to inhibit the fusion. 10% Triton X-100 treatment was used to achieve 100% calcein leakage. All data shown are representative of three independent experiments.
    Figure Legend Snippet: HB27 inhibits SARS-CoV-2 membrane fusion. (A) HB27 had potent neutralization activities when exposed to virus before or after attachment to Huh7 cells. Values are mean ± SD. Experiments were repeated in triplicate. (B) HB27 inhibits S protein-mediated cell-cell fusion. 293T cells were transfected with SARS-CoV-2 S-GFP protein, co-cultured with Vero E6 cells in the absence or presence of 100 μg/mL H014 or HB27 or anti-influenza H7N9 antibody (isotype control). No Ab: in the absence of antibodies. Images were taken after 48 h. Cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min and stained for nuclei with 4,6-diamidino-2-phenylindole (DAPI). (C) HB27 inhibits SARS-CoV-2-mediated cell-cell fusion. Huh7 cells were infected with 100 PFU of SARS-CoV-2 for 1 h at 4°C and washed for 3 times. After that cells were further cultured in the presence of a series of concentrations (0, 4, 20 and 100 nM) of HB27, or 100 nM of H014 at 37 °C for 48 h. Images were taken after 48 h. Cells were fixed with 4% (w/v) PFA for 20 min and incubated with anti-SARS-CoV-2 S protein antibody and stained for nuclei with DAPI. Scale bar equals 200 μm. (D) HB27 blocks receptor-mediated fusion of SARS-CoV-2 with liposomes. Liposomes were loaded with self-quenching concentrations of the fluorescent dye calcein. Perturbation of the bilayer causes the release of calcein resulting in dilution and a consequent increase in its fluorescence. Fusion of SARS-CoV-2 with liposomes occurred in the presence of both ACE2 and trypsin and a series of HB27 concentrations were used to inhibit the fusion. 10% Triton X-100 treatment was used to achieve 100% calcein leakage. All data shown are representative of three independent experiments.

    Techniques Used: Neutralization, Transfection, Cell Culture, Staining, Infection, Incubation, Fluorescence

    Flowchart of Cryo-EM data processing of SARS-CoV-2 S trimer and HB27 complex. Related to Figure 6 .
    Figure Legend Snippet: Flowchart of Cryo-EM data processing of SARS-CoV-2 S trimer and HB27 complex. Related to Figure 6 .

    Techniques Used:

    Structure and interaction of the SARS-CoV-2 S trimer with HB27. (A) Orthogonal views of SARS-CoV-2 S trimer in complex with three copies of HB27 Fab. (B) Individual views of the three monomers each complexed with one HB27 Fab. (A) and (B) The S trimer and HB27 are rendered as molecular surfaces. Three monomers of the S trimer are colored in yellow, cyan and violet, respectively. The HB27 light and heavy chains are colored in hotpink and purpleblue, respectively. RBD: receptor binding domain. NTD: N-terminal domain. S2: the S2 subunit. (C) S1 subunits of the three monomers from SARS-CoV-2 S trimer complexed with HB27 are superposed; HB27 Fabs are not shown. All domains are presented as ribbon diagrams. Three RBD domains are colored in yellow, cyan and violet, respectively. SD1: subdomain 1. SD2: subdomain 2. (D) Cartoon representations of the structure of SARS-CoV-2 RBD in complex with HB27. The RBD is cyan, and the light and heavy chains of HB27 are hotpink and purpleblue, respectively. Residues constituting the HB27 epitope and the RBM are drawn as spheres and colored in green and blue, respectively. The overlapped residues between the HB27 epitope and the RBM are colored in red. The CDRs involved in the interactions with the RBD are labelled. CDR: complementary determining region. RBM: receptor binding motif. (E) Residues in SARS-CoV-2 RBD comprising the HB27 epitope and RBM are labeled. The RBD is rendered as cyan surface. Blue, green and red mark the HB27 epitope, the RBM and overlapped residues of them both, respectively. (F) Hydrophobic interactions between SARS-CoV-2 RBD and HB27. The RBD is shown as cyan ribbon diagrams, and the residues of which involved in hydrophobic interactions with HB27 are shown as side chains and labeled, the four dark orange circles mark the positions of four glycine residues. The HB27 light and heavy chain are rendered as light pink and pale blue molecular surfaces, respectively, of which the residues involved in the hydrophobic interactions with the RBD are highlighted in hotpink and purpleblue and labeled. (G) A few key interactions between SARS-CoV-2 RBD and the HB27 heavy (left) and light chain (right). Hydrogen bonds are presented as dashed lines. See also Figures S3 , S4 and S5 . Tables S2 and S3 .
    Figure Legend Snippet: Structure and interaction of the SARS-CoV-2 S trimer with HB27. (A) Orthogonal views of SARS-CoV-2 S trimer in complex with three copies of HB27 Fab. (B) Individual views of the three monomers each complexed with one HB27 Fab. (A) and (B) The S trimer and HB27 are rendered as molecular surfaces. Three monomers of the S trimer are colored in yellow, cyan and violet, respectively. The HB27 light and heavy chains are colored in hotpink and purpleblue, respectively. RBD: receptor binding domain. NTD: N-terminal domain. S2: the S2 subunit. (C) S1 subunits of the three monomers from SARS-CoV-2 S trimer complexed with HB27 are superposed; HB27 Fabs are not shown. All domains are presented as ribbon diagrams. Three RBD domains are colored in yellow, cyan and violet, respectively. SD1: subdomain 1. SD2: subdomain 2. (D) Cartoon representations of the structure of SARS-CoV-2 RBD in complex with HB27. The RBD is cyan, and the light and heavy chains of HB27 are hotpink and purpleblue, respectively. Residues constituting the HB27 epitope and the RBM are drawn as spheres and colored in green and blue, respectively. The overlapped residues between the HB27 epitope and the RBM are colored in red. The CDRs involved in the interactions with the RBD are labelled. CDR: complementary determining region. RBM: receptor binding motif. (E) Residues in SARS-CoV-2 RBD comprising the HB27 epitope and RBM are labeled. The RBD is rendered as cyan surface. Blue, green and red mark the HB27 epitope, the RBM and overlapped residues of them both, respectively. (F) Hydrophobic interactions between SARS-CoV-2 RBD and HB27. The RBD is shown as cyan ribbon diagrams, and the residues of which involved in hydrophobic interactions with HB27 are shown as side chains and labeled, the four dark orange circles mark the positions of four glycine residues. The HB27 light and heavy chain are rendered as light pink and pale blue molecular surfaces, respectively, of which the residues involved in the hydrophobic interactions with the RBD are highlighted in hotpink and purpleblue and labeled. (G) A few key interactions between SARS-CoV-2 RBD and the HB27 heavy (left) and light chain (right). Hydrogen bonds are presented as dashed lines. See also Figures S3 , S4 and S5 . Tables S2 and S3 .

    Techniques Used: Binding Assay, Labeling

    29) Product Images from "SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface"

    Article Title: SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

    Journal: Nature Communications

    doi: 10.1038/s41467-021-21609-2

    Binding to RBD mutants, epitopes, and structure models. A ELISA using STE73-2E9, -9G3, and -2G8 on S1-His with different RBD mutations. B Overview of the binding of STE73-2E9, -9G3, and -2G8 to different RBD mutations analyzed by ELISA, SPR, and protein array. Sequence SARS-CoV-2 (Gene bank QHD43416). ELISA experiments were performed in duplicate and mean values are given. C The three antibodies STE73-2E9, -9G3, and -2G8 are binding to the ACE–RBD interface (docking models based on epitope data from binding to RBD mutations). Experimentally validated computational models of the variable regions of the antibodies (colored cartoons) binding to the RBD (white surface, same orientation in all images) are shown. The cartoon representation of ACE2 is also shown for comparison.
    Figure Legend Snippet: Binding to RBD mutants, epitopes, and structure models. A ELISA using STE73-2E9, -9G3, and -2G8 on S1-His with different RBD mutations. B Overview of the binding of STE73-2E9, -9G3, and -2G8 to different RBD mutations analyzed by ELISA, SPR, and protein array. Sequence SARS-CoV-2 (Gene bank QHD43416). ELISA experiments were performed in duplicate and mean values are given. C The three antibodies STE73-2E9, -9G3, and -2G8 are binding to the ACE–RBD interface (docking models based on epitope data from binding to RBD mutations). Experimentally validated computational models of the variable regions of the antibodies (colored cartoons) binding to the RBD (white surface, same orientation in all images) are shown. The cartoon representation of ACE2 is also shown for comparison.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay, Protein Array, Sequencing

    Characterization of the neutralizing antibody STE73-2E9 in IgG format. A Neutralization of 20–30 pfu SARS-CoV-2 by STE73-2E9, -9G3, and -2G8. Palivizumab was used as isotype control. B Validation of neutralization potency of STE73-2E9 using 100 pfu. Neutralization assays were performed in triplicates, mean ± s.e.m. are given. C Titration ELISA on the indicated antigens. ELISA shows single titration of two representative experiments (see also Supplementary Fig. 7 ). D Cross-reactivity to other coronavirus spike proteins analzyed by ELISA. S1-HIS SARS-CoV-2 Hi5 was produced in house. S1-HIS SARS-CoV-2 HEK and all other coronavirus S1 domain proteins were obtained commercially. ELISA experiments were performed in duplicate and the mean values are given. E , F Kinetic parameter determination through single-cycle kinetic titration SPR of STE73-2E9 IgG on HEK cell produced RBD-SD1 and S1-S2, respectively (concentrations: 200, 100, 50, 25, 12.5, 6.25 nM).
    Figure Legend Snippet: Characterization of the neutralizing antibody STE73-2E9 in IgG format. A Neutralization of 20–30 pfu SARS-CoV-2 by STE73-2E9, -9G3, and -2G8. Palivizumab was used as isotype control. B Validation of neutralization potency of STE73-2E9 using 100 pfu. Neutralization assays were performed in triplicates, mean ± s.e.m. are given. C Titration ELISA on the indicated antigens. ELISA shows single titration of two representative experiments (see also Supplementary Fig. 7 ). D Cross-reactivity to other coronavirus spike proteins analzyed by ELISA. S1-HIS SARS-CoV-2 Hi5 was produced in house. S1-HIS SARS-CoV-2 HEK and all other coronavirus S1 domain proteins were obtained commercially. ELISA experiments were performed in duplicate and the mean values are given. E , F Kinetic parameter determination through single-cycle kinetic titration SPR of STE73-2E9 IgG on HEK cell produced RBD-SD1 and S1-S2, respectively (concentrations: 200, 100, 50, 25, 12.5, 6.25 nM).

    Techniques Used: Neutralization, Titration, Enzyme-linked Immunosorbent Assay, Produced, SPR Assay

    Use of V region genes in human anti-SARS-CoV-2 antibodies. Comparison of the distribution of V region gene subfamilies in the universal HAL9/10 library 50 , the in vivo distribution of subfamilies 82 , and the distribution of antibodies against S1 selected from HAL9/10. A Abundance of VH, B Vκ, and C Vλ.
    Figure Legend Snippet: Use of V region genes in human anti-SARS-CoV-2 antibodies. Comparison of the distribution of V region gene subfamilies in the universal HAL9/10 library 50 , the in vivo distribution of subfamilies 82 , and the distribution of antibodies against S1 selected from HAL9/10. A Abundance of VH, B Vκ, and C Vλ.

    Techniques Used: In Vivo

    Inhibition of SARS-CoV-2 spike protein binding to cell (flow cytometry). A Inhibition prescreen of 109 scFv-Fc antibodies on ACE2-positive cells using 1500 nM antibody and 50 nM spike protein (30:1 ratio). The antibodies selected for detailed analysis are marked in colors. Data show single measurements. B IC50 determination by flow cytometry using 50 nM S1-S2 trimer and 4.7–1500 nM scFv-Fc. C IC50 determination by flow cytometry using 10 nM RBD and 0.03–1000 nM scFv-Fc. The inhibition assays were made as single titrations. Logistic5 fit of Origin was used to determine the IC50.
    Figure Legend Snippet: Inhibition of SARS-CoV-2 spike protein binding to cell (flow cytometry). A Inhibition prescreen of 109 scFv-Fc antibodies on ACE2-positive cells using 1500 nM antibody and 50 nM spike protein (30:1 ratio). The antibodies selected for detailed analysis are marked in colors. Data show single measurements. B IC50 determination by flow cytometry using 50 nM S1-S2 trimer and 4.7–1500 nM scFv-Fc. C IC50 determination by flow cytometry using 10 nM RBD and 0.03–1000 nM scFv-Fc. The inhibition assays were made as single titrations. Logistic5 fit of Origin was used to determine the IC50.

    Techniques Used: Inhibition, Protein Binding, Flow Cytometry

    SARS-CoV-2 neutralization in the scFv-Fc format. Neutralization analysis using 250 pfu of SARS-CoV-2 in a CPE-based neutralization assay. A Cell monolayer occupancy at 4 days post infection in the absence of neutralizing antibodies was compared to uninfected control cells and median values were normalized as 0 and 100% occupancy, respectively. Histograms indicate medians of normalized monolayer occupancy in a neutralization assay using 1 µg/mL (~10 nM) antibody for each of the 17 tested antibodies. Data show the median from 4 or 6 replicates, the black dots indicate monolayer occupancy in individual assays, and the range is given for the maximum and minimum measurements. B Representative phase-contrast microscopic pictures of uninfected cells, cells infected in the absence of antibodies, in the presence of a poorly neutralizing scFv-Fc (STE73-2C2), or of a highly neutralizing scFv-Fc (STE73-6C8).
    Figure Legend Snippet: SARS-CoV-2 neutralization in the scFv-Fc format. Neutralization analysis using 250 pfu of SARS-CoV-2 in a CPE-based neutralization assay. A Cell monolayer occupancy at 4 days post infection in the absence of neutralizing antibodies was compared to uninfected control cells and median values were normalized as 0 and 100% occupancy, respectively. Histograms indicate medians of normalized monolayer occupancy in a neutralization assay using 1 µg/mL (~10 nM) antibody for each of the 17 tested antibodies. Data show the median from 4 or 6 replicates, the black dots indicate monolayer occupancy in individual assays, and the range is given for the maximum and minimum measurements. B Representative phase-contrast microscopic pictures of uninfected cells, cells infected in the absence of antibodies, in the presence of a poorly neutralizing scFv-Fc (STE73-2C2), or of a highly neutralizing scFv-Fc (STE73-6C8).

    Techniques Used: Neutralization, Infection

    Determination of EC50 on RBD. Binding in titration ELISA of the 17 best inhibiting scFv-Fc on RBD (fusion protein with murine Fc part), S1 (fusion protein with murine Fc part), or S1-S2 (fusion protein with His tag). Sequence SARS-CoV-2 (Gene bank QHD43416). An unrelated antibody with murine Fc part (TUN219-2C1), human HEK293 cell lysate, BSA, or lysozyme were used as controls. Experiments were performed in duplicate and mean values are given. EC50 were calculated with GraphPad Prism Version 6.1, fitting to a four-parameter logistic curve.
    Figure Legend Snippet: Determination of EC50 on RBD. Binding in titration ELISA of the 17 best inhibiting scFv-Fc on RBD (fusion protein with murine Fc part), S1 (fusion protein with murine Fc part), or S1-S2 (fusion protein with His tag). Sequence SARS-CoV-2 (Gene bank QHD43416). An unrelated antibody with murine Fc part (TUN219-2C1), human HEK293 cell lysate, BSA, or lysozyme were used as controls. Experiments were performed in duplicate and mean values are given. EC50 were calculated with GraphPad Prism Version 6.1, fitting to a four-parameter logistic curve.

    Techniques Used: Binding Assay, Titration, Enzyme-linked Immunosorbent Assay, Sequencing

    30) Product Images from "SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface"

    Article Title: SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

    Journal: Nature Communications

    doi: 10.1038/s41467-021-21609-2

    Binding to RBD mutants, epitopes, and structure models. A ELISA using STE73-2E9, -9G3, and -2G8 on S1-His with different RBD mutations. B Overview of the binding of STE73-2E9, -9G3, and -2G8 to different RBD mutations analyzed by ELISA, SPR, and protein array. Sequence SARS-CoV-2 (Gene bank QHD43416). ELISA experiments were performed in duplicate and mean values are given. C The three antibodies STE73-2E9, -9G3, and -2G8 are binding to the ACE–RBD interface (docking models based on epitope data from binding to RBD mutations). Experimentally validated computational models of the variable regions of the antibodies (colored cartoons) binding to the RBD (white surface, same orientation in all images) are shown. The cartoon representation of ACE2 is also shown for comparison.
    Figure Legend Snippet: Binding to RBD mutants, epitopes, and structure models. A ELISA using STE73-2E9, -9G3, and -2G8 on S1-His with different RBD mutations. B Overview of the binding of STE73-2E9, -9G3, and -2G8 to different RBD mutations analyzed by ELISA, SPR, and protein array. Sequence SARS-CoV-2 (Gene bank QHD43416). ELISA experiments were performed in duplicate and mean values are given. C The three antibodies STE73-2E9, -9G3, and -2G8 are binding to the ACE–RBD interface (docking models based on epitope data from binding to RBD mutations). Experimentally validated computational models of the variable regions of the antibodies (colored cartoons) binding to the RBD (white surface, same orientation in all images) are shown. The cartoon representation of ACE2 is also shown for comparison.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay, Protein Array, Sequencing

    Characterization of the neutralizing antibody STE73-2E9 in IgG format. A Neutralization of 20–30 pfu SARS-CoV-2 by STE73-2E9, -9G3, and -2G8. Palivizumab was used as isotype control. B Validation of neutralization potency of STE73-2E9 using 100 pfu. Neutralization assays were performed in triplicates, mean ± s.e.m. are given. C Titration ELISA on the indicated antigens. ELISA shows single titration of two representative experiments (see also Supplementary Fig. 7 ). D Cross-reactivity to other coronavirus spike proteins analzyed by ELISA. S1-HIS SARS-CoV-2 Hi5 was produced in house. S1-HIS SARS-CoV-2 HEK and all other coronavirus S1 domain proteins were obtained commercially. ELISA experiments were performed in duplicate and the mean values are given. E , F Kinetic parameter determination through single-cycle kinetic titration SPR of STE73-2E9 IgG on HEK cell produced RBD-SD1 and S1-S2, respectively (concentrations: 200, 100, 50, 25, 12.5, 6.25 nM).
    Figure Legend Snippet: Characterization of the neutralizing antibody STE73-2E9 in IgG format. A Neutralization of 20–30 pfu SARS-CoV-2 by STE73-2E9, -9G3, and -2G8. Palivizumab was used as isotype control. B Validation of neutralization potency of STE73-2E9 using 100 pfu. Neutralization assays were performed in triplicates, mean ± s.e.m. are given. C Titration ELISA on the indicated antigens. ELISA shows single titration of two representative experiments (see also Supplementary Fig. 7 ). D Cross-reactivity to other coronavirus spike proteins analzyed by ELISA. S1-HIS SARS-CoV-2 Hi5 was produced in house. S1-HIS SARS-CoV-2 HEK and all other coronavirus S1 domain proteins were obtained commercially. ELISA experiments were performed in duplicate and the mean values are given. E , F Kinetic parameter determination through single-cycle kinetic titration SPR of STE73-2E9 IgG on HEK cell produced RBD-SD1 and S1-S2, respectively (concentrations: 200, 100, 50, 25, 12.5, 6.25 nM).

    Techniques Used: Neutralization, Titration, Enzyme-linked Immunosorbent Assay, Produced, SPR Assay

    Use of V region genes in human anti-SARS-CoV-2 antibodies. Comparison of the distribution of V region gene subfamilies in the universal HAL9/10 library 50 , the in vivo distribution of subfamilies 82 , and the distribution of antibodies against S1 selected from HAL9/10. A Abundance of VH, B Vκ, and C Vλ.
    Figure Legend Snippet: Use of V region genes in human anti-SARS-CoV-2 antibodies. Comparison of the distribution of V region gene subfamilies in the universal HAL9/10 library 50 , the in vivo distribution of subfamilies 82 , and the distribution of antibodies against S1 selected from HAL9/10. A Abundance of VH, B Vκ, and C Vλ.

    Techniques Used: In Vivo

    Inhibition of SARS-CoV-2 spike protein binding to cell (flow cytometry). A Inhibition prescreen of 109 scFv-Fc antibodies on ACE2-positive cells using 1500 nM antibody and 50 nM spike protein (30:1 ratio). The antibodies selected for detailed analysis are marked in colors. Data show single measurements. B IC50 determination by flow cytometry using 50 nM S1-S2 trimer and 4.7–1500 nM scFv-Fc. C IC50 determination by flow cytometry using 10 nM RBD and 0.03–1000 nM scFv-Fc. The inhibition assays were made as single titrations. Logistic5 fit of Origin was used to determine the IC50.
    Figure Legend Snippet: Inhibition of SARS-CoV-2 spike protein binding to cell (flow cytometry). A Inhibition prescreen of 109 scFv-Fc antibodies on ACE2-positive cells using 1500 nM antibody and 50 nM spike protein (30:1 ratio). The antibodies selected for detailed analysis are marked in colors. Data show single measurements. B IC50 determination by flow cytometry using 50 nM S1-S2 trimer and 4.7–1500 nM scFv-Fc. C IC50 determination by flow cytometry using 10 nM RBD and 0.03–1000 nM scFv-Fc. The inhibition assays were made as single titrations. Logistic5 fit of Origin was used to determine the IC50.

    Techniques Used: Inhibition, Protein Binding, Flow Cytometry

    SARS-CoV-2 neutralization in the scFv-Fc format. Neutralization analysis using 250 pfu of SARS-CoV-2 in a CPE-based neutralization assay. A Cell monolayer occupancy at 4 days post infection in the absence of neutralizing antibodies was compared to uninfected control cells and median values were normalized as 0 and 100% occupancy, respectively. Histograms indicate medians of normalized monolayer occupancy in a neutralization assay using 1 µg/mL (~10 nM) antibody for each of the 17 tested antibodies. Data show the median from 4 or 6 replicates, the black dots indicate monolayer occupancy in individual assays, and the range is given for the maximum and minimum measurements. B Representative phase-contrast microscopic pictures of uninfected cells, cells infected in the absence of antibodies, in the presence of a poorly neutralizing scFv-Fc (STE73-2C2), or of a highly neutralizing scFv-Fc (STE73-6C8).
    Figure Legend Snippet: SARS-CoV-2 neutralization in the scFv-Fc format. Neutralization analysis using 250 pfu of SARS-CoV-2 in a CPE-based neutralization assay. A Cell monolayer occupancy at 4 days post infection in the absence of neutralizing antibodies was compared to uninfected control cells and median values were normalized as 0 and 100% occupancy, respectively. Histograms indicate medians of normalized monolayer occupancy in a neutralization assay using 1 µg/mL (~10 nM) antibody for each of the 17 tested antibodies. Data show the median from 4 or 6 replicates, the black dots indicate monolayer occupancy in individual assays, and the range is given for the maximum and minimum measurements. B Representative phase-contrast microscopic pictures of uninfected cells, cells infected in the absence of antibodies, in the presence of a poorly neutralizing scFv-Fc (STE73-2C2), or of a highly neutralizing scFv-Fc (STE73-6C8).

    Techniques Used: Neutralization, Infection

    Determination of EC50 on RBD. Binding in titration ELISA of the 17 best inhibiting scFv-Fc on RBD (fusion protein with murine Fc part), S1 (fusion protein with murine Fc part), or S1-S2 (fusion protein with His tag). Sequence SARS-CoV-2 (Gene bank QHD43416). An unrelated antibody with murine Fc part (TUN219-2C1), human HEK293 cell lysate, BSA, or lysozyme were used as controls. Experiments were performed in duplicate and mean values are given. EC50 were calculated with GraphPad Prism Version 6.1, fitting to a four-parameter logistic curve.
    Figure Legend Snippet: Determination of EC50 on RBD. Binding in titration ELISA of the 17 best inhibiting scFv-Fc on RBD (fusion protein with murine Fc part), S1 (fusion protein with murine Fc part), or S1-S2 (fusion protein with His tag). Sequence SARS-CoV-2 (Gene bank QHD43416). An unrelated antibody with murine Fc part (TUN219-2C1), human HEK293 cell lysate, BSA, or lysozyme were used as controls. Experiments were performed in duplicate and mean values are given. EC50 were calculated with GraphPad Prism Version 6.1, fitting to a four-parameter logistic curve.

    Techniques Used: Binding Assay, Titration, Enzyme-linked Immunosorbent Assay, Sequencing

    31) Product Images from "Multi-level proteomics reveals host-perturbation strategies of SARS-CoV-2 and SARS-CoV"

    Article Title: Multi-level proteomics reveals host-perturbation strategies of SARS-CoV-2 and SARS-CoV

    Journal: bioRxiv

    doi: 10.1101/2020.06.17.156455

    SARS-CoV-2 uses a multi-pronged approach to perturb host-pathways at multiple levels. (a) The host subnetwork perturbed by SARS-CoV-2 ORF7a, as predicted by the network diffusion approach. (b) Selection of the optimal cutting threshold for the network diffusion graph of SARS-CoV-2 ORF7a-induced proteome changes. The plot shows the correlation between the minimal allowed edge weight (X axis), and the average path length from the regulated proteins to the host targets of the viral protein along the edges of the filtered subnetwork (Y axis). The red curve represents the path length for the network diffusion analysis of the actual data. The grey band shows 50% confidence interval, and dashed lines correspond to 95% confidence interval for the path lengths of 1000 randomised datasets. Optimal edge weight threshold that maximises the difference between the median path length in randomised data and the path length in the real data is highlighted by the blue vertical line. (c) Overview of perturbations to host-cell innate immunity related pathways, induced by distinct proteins of SARS-CoV-2, derived from the network diffusion model and overlaid with transcriptional, protein abundance, ubiquitination and phosphorylation changes upon SARS-CoV-2 infection. (d) Heatmap showing effects of the indicated SARS-CoV-2 proteins on type-I IFN expression levels, ISRE and GAS promoter activation in HEK293-R1. Accumulation of type-I IFN in the supernatant was evaluated by testing supernatants of PPP-RNA (IVT4) stimulated cells on MX1-luciferase reporter cells, ISRE promoter activation - by luciferase assay after IFN-α stimulation and GAS promoter activation - by luciferase assay after INF-γ stimulation in cells expressing SARS-CoV-2 proteins as compared to the controls (ZIKV NS5 and SMN1). Average of three independent experiments is shown. (e) Overview of perturbations to host-cell Integrin-TGFβ-EGFR-RTK signaling, induced by distinct proteins of SARS-CoV-2, derived from the network diffusion model and overlaid with transcriptional, protein abundance, ubiquitination and phosphorylation changes upon SARS-CoV-2 infection. (f) Profile plots showing intensities of indicated phosphosites and total protein levels of EGFR, EPHA2 and AKAP12 in SARS-CoV-2 infected A549-ACE2 cells at the indicated time points post infection. Points are normalized intensities of individual replicates, solid line is median, filled area corresponds to 25–75 percentiles, dashed lines mark 2.5–97.5 percentiles of the posterior distribution. n = 3 independent experiments; Bayesian statistical modelling. (g) Western blot showing phosphospecies and total protein levels of p38 (T180/Y182, MAPK14) and JNK (T183/Y185, MAPK8) in SARS-CoV-2 infected A549-ACE2 cells. (h) Profile plots of total protein levels of ITGA3, SERPINE1 and FN1 in SARS-CoV-2 infected A549-ACE2 cells at 6 and 24 hours post infection with indicated median and confidence intervals. n = 4 independent experiments.
    Figure Legend Snippet: SARS-CoV-2 uses a multi-pronged approach to perturb host-pathways at multiple levels. (a) The host subnetwork perturbed by SARS-CoV-2 ORF7a, as predicted by the network diffusion approach. (b) Selection of the optimal cutting threshold for the network diffusion graph of SARS-CoV-2 ORF7a-induced proteome changes. The plot shows the correlation between the minimal allowed edge weight (X axis), and the average path length from the regulated proteins to the host targets of the viral protein along the edges of the filtered subnetwork (Y axis). The red curve represents the path length for the network diffusion analysis of the actual data. The grey band shows 50% confidence interval, and dashed lines correspond to 95% confidence interval for the path lengths of 1000 randomised datasets. Optimal edge weight threshold that maximises the difference between the median path length in randomised data and the path length in the real data is highlighted by the blue vertical line. (c) Overview of perturbations to host-cell innate immunity related pathways, induced by distinct proteins of SARS-CoV-2, derived from the network diffusion model and overlaid with transcriptional, protein abundance, ubiquitination and phosphorylation changes upon SARS-CoV-2 infection. (d) Heatmap showing effects of the indicated SARS-CoV-2 proteins on type-I IFN expression levels, ISRE and GAS promoter activation in HEK293-R1. Accumulation of type-I IFN in the supernatant was evaluated by testing supernatants of PPP-RNA (IVT4) stimulated cells on MX1-luciferase reporter cells, ISRE promoter activation - by luciferase assay after IFN-α stimulation and GAS promoter activation - by luciferase assay after INF-γ stimulation in cells expressing SARS-CoV-2 proteins as compared to the controls (ZIKV NS5 and SMN1). Average of three independent experiments is shown. (e) Overview of perturbations to host-cell Integrin-TGFβ-EGFR-RTK signaling, induced by distinct proteins of SARS-CoV-2, derived from the network diffusion model and overlaid with transcriptional, protein abundance, ubiquitination and phosphorylation changes upon SARS-CoV-2 infection. (f) Profile plots showing intensities of indicated phosphosites and total protein levels of EGFR, EPHA2 and AKAP12 in SARS-CoV-2 infected A549-ACE2 cells at the indicated time points post infection. Points are normalized intensities of individual replicates, solid line is median, filled area corresponds to 25–75 percentiles, dashed lines mark 2.5–97.5 percentiles of the posterior distribution. n = 3 independent experiments; Bayesian statistical modelling. (g) Western blot showing phosphospecies and total protein levels of p38 (T180/Y182, MAPK14) and JNK (T183/Y185, MAPK8) in SARS-CoV-2 infected A549-ACE2 cells. (h) Profile plots of total protein levels of ITGA3, SERPINE1 and FN1 in SARS-CoV-2 infected A549-ACE2 cells at 6 and 24 hours post infection with indicated median and confidence intervals. n = 4 independent experiments.

    Techniques Used: Diffusion-based Assay, Selection, Derivative Assay, Infection, Expressing, Activation Assay, Luciferase, Western Blot

    SARS-CoV-2-targeted pathways, revealed by multi-omics profiling approach, allow systematic testing of novel antiviral therapies. (a) A549-ACE2 cells, exposed for 6h to the specified concentrations of interferon alpha and infected with SARS-CoV-2-GFP reporter virus (MOI 3). GFP signal and cell confluency were analysed by live-cell imaging for 48h. Line diagrams show virus growth over time of GFP-positive vs total cell area with indicated mean of four biological replicates. (b) A549-ACE2 cells were treated with the indicated drugs 6h prior to infection with SARS-CoV-2-GFP (MOI 3). Scatter plot represents GFP vs total cell area signal (y-axis) versus cell confluency in uninfected control treatments (x-axis) at 48h after infection. A confluence cutoff of −0.2 log2 fold change was applied to remove cytotoxic compounds. (c-e) as (a) but line diagrams showing virus replication after (c) Prinomastat, (d) Ipatasertib and (e) Gilteritinib pre-treatment. Asterisks indicate significance to control treatment (Wilcoxon test; p-value ≤ 0.01).
    Figure Legend Snippet: SARS-CoV-2-targeted pathways, revealed by multi-omics profiling approach, allow systematic testing of novel antiviral therapies. (a) A549-ACE2 cells, exposed for 6h to the specified concentrations of interferon alpha and infected with SARS-CoV-2-GFP reporter virus (MOI 3). GFP signal and cell confluency were analysed by live-cell imaging for 48h. Line diagrams show virus growth over time of GFP-positive vs total cell area with indicated mean of four biological replicates. (b) A549-ACE2 cells were treated with the indicated drugs 6h prior to infection with SARS-CoV-2-GFP (MOI 3). Scatter plot represents GFP vs total cell area signal (y-axis) versus cell confluency in uninfected control treatments (x-axis) at 48h after infection. A confluence cutoff of −0.2 log2 fold change was applied to remove cytotoxic compounds. (c-e) as (a) but line diagrams showing virus replication after (c) Prinomastat, (d) Ipatasertib and (e) Gilteritinib pre-treatment. Asterisks indicate significance to control treatment (Wilcoxon test; p-value ≤ 0.01).

    Techniques Used: Infection, Live Cell Imaging

    Drug screen, focusing on pathways perturbed by SARS-CoV-2 on several levels, reveals potential candidates for use in antiviral therapy. (a) A549-ACE2 cells were pre-treated for 6h or treated at the time of infection with SARS-CoV-2-GFP reporter virus (MOI 3). GFP signal and cell growth were evaluated for 48h by live cell imaging using an Incucyte S3 platform. Heatmap show the cell growth rate over time in uninfected conditions, and GFP signal vs total cell confluency and normalized to the signal measured in control treatment (water, DMSO), over time. Only treatments with significant effects on SARS-CoV-2-GFP are shown. Asterisks indicate significance to control treatment (Wilcoxon test; p-value ≤ 0.05).
    Figure Legend Snippet: Drug screen, focusing on pathways perturbed by SARS-CoV-2 on several levels, reveals potential candidates for use in antiviral therapy. (a) A549-ACE2 cells were pre-treated for 6h or treated at the time of infection with SARS-CoV-2-GFP reporter virus (MOI 3). GFP signal and cell growth were evaluated for 48h by live cell imaging using an Incucyte S3 platform. Heatmap show the cell growth rate over time in uninfected conditions, and GFP signal vs total cell confluency and normalized to the signal measured in control treatment (water, DMSO), over time. Only treatments with significant effects on SARS-CoV-2-GFP are shown. Asterisks indicate significance to control treatment (Wilcoxon test; p-value ≤ 0.05).

    Techniques Used: Infection, Live Cell Imaging

    Orthogonal profiling of SARS-CoV-2 infection. (a) Time-resolved profiling of SARS-CoV-2 infection by multiple-omics methods. The plot shows normalized MS intensities of three SARS-CoV-2 viral proteins over time. (b) Numbers of distinct transcripts, proteins, ubiquitination and phosphorylation sites, up- or down-regulated at the indicated time points after infection, as identified using data independent (DIA) or dependent (DDA) acquisition methods. (c) Volcano plot showing ubiquitination sites regulated at 24h after SARS-CoV-2 infection. Viral proteins are marked in orange. Selected significant ubiquitination sites (Student’s t-test, two-tailed, permutation-based FDR
    Figure Legend Snippet: Orthogonal profiling of SARS-CoV-2 infection. (a) Time-resolved profiling of SARS-CoV-2 infection by multiple-omics methods. The plot shows normalized MS intensities of three SARS-CoV-2 viral proteins over time. (b) Numbers of distinct transcripts, proteins, ubiquitination and phosphorylation sites, up- or down-regulated at the indicated time points after infection, as identified using data independent (DIA) or dependent (DDA) acquisition methods. (c) Volcano plot showing ubiquitination sites regulated at 24h after SARS-CoV-2 infection. Viral proteins are marked in orange. Selected significant ubiquitination sites (Student’s t-test, two-tailed, permutation-based FDR

    Techniques Used: Infection, Two Tailed Test

    Expression of viral proteins in transduced A549 cells induces changes to the host proteome. (a) Expression of HA-tagged viral proteins, in stably transduced A549 cells, used in AP-MS and proteome expression measurements. (b) The extended version of the virus-host protein-protein interaction network with 24 SARS-CoV-2 and 27 SARS-CoV proteins, as well as ORF3 of HCoV-NL63 and ORF4 and 4a of HCoV-229E, used as baits. Host targets regulated upon viral protein overexpression or SARS-CoV-2 infection (based on the analysis of all data of this study) are highlighted (see the in-plot legend). (c-f) Co-precipitation experiments in HEK 293T cells showing a specific enrichment of (c) endogenous MAVS co-precipitated with c-term HA-tagged ORF7b of SARS-CoV-2 and SARS-CoV (negative controls: SARS-CoV-2 ORF6-HA, ORF7a-HA), (d) ORF7b-HA of SARS-CoV-2 and SARS-CoV co-precipitated with SII-HA-UNC93B1 (control precipitation: SII-HA-RSAD2), (e) endogenous HSPA1A co-precipitated with N-HA of SARS-CoV-2 and SARS-CoV (control: SARS-CoV-2 ORF6-HA) and (f) endogenous TGFβ with ORF8-HA of SARS-CoV-2 vs ORF8-HA, ORF8a-HA, ORF8b-HA of SARS-CoV or ORF9b-HA of SARS-CoV-2. (g, h) Differential enrichment of proteins in (g) NSP2 and (h) ORF8 of SARS-CoV-2 (x-axis) vs SARS-CoV (y-axis) AP-MS experiments.
    Figure Legend Snippet: Expression of viral proteins in transduced A549 cells induces changes to the host proteome. (a) Expression of HA-tagged viral proteins, in stably transduced A549 cells, used in AP-MS and proteome expression measurements. (b) The extended version of the virus-host protein-protein interaction network with 24 SARS-CoV-2 and 27 SARS-CoV proteins, as well as ORF3 of HCoV-NL63 and ORF4 and 4a of HCoV-229E, used as baits. Host targets regulated upon viral protein overexpression or SARS-CoV-2 infection (based on the analysis of all data of this study) are highlighted (see the in-plot legend). (c-f) Co-precipitation experiments in HEK 293T cells showing a specific enrichment of (c) endogenous MAVS co-precipitated with c-term HA-tagged ORF7b of SARS-CoV-2 and SARS-CoV (negative controls: SARS-CoV-2 ORF6-HA, ORF7a-HA), (d) ORF7b-HA of SARS-CoV-2 and SARS-CoV co-precipitated with SII-HA-UNC93B1 (control precipitation: SII-HA-RSAD2), (e) endogenous HSPA1A co-precipitated with N-HA of SARS-CoV-2 and SARS-CoV (control: SARS-CoV-2 ORF6-HA) and (f) endogenous TGFβ with ORF8-HA of SARS-CoV-2 vs ORF8-HA, ORF8a-HA, ORF8b-HA of SARS-CoV or ORF9b-HA of SARS-CoV-2. (g, h) Differential enrichment of proteins in (g) NSP2 and (h) ORF8 of SARS-CoV-2 (x-axis) vs SARS-CoV (y-axis) AP-MS experiments.

    Techniques Used: Expressing, Stable Transfection, Over Expression, Infection

    Validation of the in vitro SARS-CoV-2 infection model and tracking of virus-specific changes at the multi-omics level. (a) Western blot showing ACE2-HA expression levels in A549 cells untransduced (wt) or transduced with ACE2-HA encoding lentivirus. (b) mRNA expression levels of SARS-CoV-2 N relative to RPLP0 as measured by qRT-PCR upon infection of wt A549 and A549-ACE2 cells at the indicated MOIs. Mean +/- standard deviation of three biological replicates are shown. (c) Volcano plot of mRNA expression changes of A549-ACE2 cells, infected with SARS-CoV-2 at an MOI of 3, shown as a fold change versus mock at 24 h.p.i.. Selected significant hits are marked in black (Wald test, n=3). (d) Expression levels, as measured by qRT-PCR, of SARS-CoV-2 N and host transcripts relative to RPLP0 after infection of A549-ACE2 cells at MOI of 3 at indicated time points after infection with indicated mean +/- standard deviation (n=3). ND: not detectable. (e) Transcription factor enrichment analysis of up- (red arrow) and down- (blue arrow) regulated genes in A549-ACE2 cells infected with SARS-CoV-2 for indicated time periods (Fisher’s exact test, unadjusted). (f) Volcano plot of protein abundance changes at 24 h.p.i. in comparison to mock measured by proteome profiling (DDA MS). Viral proteins are highlighted in orange, selected significant hits are marked in black (Student’s t-test, two-tailed, permutation-based FDR
    Figure Legend Snippet: Validation of the in vitro SARS-CoV-2 infection model and tracking of virus-specific changes at the multi-omics level. (a) Western blot showing ACE2-HA expression levels in A549 cells untransduced (wt) or transduced with ACE2-HA encoding lentivirus. (b) mRNA expression levels of SARS-CoV-2 N relative to RPLP0 as measured by qRT-PCR upon infection of wt A549 and A549-ACE2 cells at the indicated MOIs. Mean +/- standard deviation of three biological replicates are shown. (c) Volcano plot of mRNA expression changes of A549-ACE2 cells, infected with SARS-CoV-2 at an MOI of 3, shown as a fold change versus mock at 24 h.p.i.. Selected significant hits are marked in black (Wald test, n=3). (d) Expression levels, as measured by qRT-PCR, of SARS-CoV-2 N and host transcripts relative to RPLP0 after infection of A549-ACE2 cells at MOI of 3 at indicated time points after infection with indicated mean +/- standard deviation (n=3). ND: not detectable. (e) Transcription factor enrichment analysis of up- (red arrow) and down- (blue arrow) regulated genes in A549-ACE2 cells infected with SARS-CoV-2 for indicated time periods (Fisher’s exact test, unadjusted). (f) Volcano plot of protein abundance changes at 24 h.p.i. in comparison to mock measured by proteome profiling (DDA MS). Viral proteins are highlighted in orange, selected significant hits are marked in black (Student’s t-test, two-tailed, permutation-based FDR

    Techniques Used: In Vitro, Infection, Western Blot, Expressing, Transduction, Quantitative RT-PCR, Standard Deviation, Two Tailed Test

    Network diffusion approach identifies molecular pathways linking protein-protein interactions with downstream changes in the host proteome. (a) Network diffusion approach to identify functional connections between the host targets of a viral protein and downstream proteome changes followed by the integration of RNA expression, protein abundance, ubiquitination and phosphorylation changes upon SARS-CoV-2 infection to streamline the identification of affected host pathways. (b-d) Subnetworks of the network diffusion predictions linking host targets of (b) SARS-CoV-2 ORF3 to the accumulation of factors involved in autophagy, (c) ORF7b to the factors involved in innate immunity and (d) ORF8 to the factors involved in TGFβ signaling. (e) Overview of perturbations to host-cell autophagy, induced by distinct proteins of SARS-CoV-2, derived from the network diffusion model and overlaid with the changes in protein levels, ubiquitination and phosphorylation induced by SARS-CoV-2 infection. (f-i) Western blot of autophagy-associated factors MAP1LC3B-II and SQSTM1 accumulation upon SARS-CoV-2 ORF3 expression in (f) HEK293R1 and (g-i) SARS-CoV-2 infection of A549-ACE2 cells. (h) Profile plot of SQSTM1 MS intensity and (i) line diagram showing SQSTM1 mRNA level relative to RPLP0 tested by qRT-PCR upon SARS-CoV-2 infection.
    Figure Legend Snippet: Network diffusion approach identifies molecular pathways linking protein-protein interactions with downstream changes in the host proteome. (a) Network diffusion approach to identify functional connections between the host targets of a viral protein and downstream proteome changes followed by the integration of RNA expression, protein abundance, ubiquitination and phosphorylation changes upon SARS-CoV-2 infection to streamline the identification of affected host pathways. (b-d) Subnetworks of the network diffusion predictions linking host targets of (b) SARS-CoV-2 ORF3 to the accumulation of factors involved in autophagy, (c) ORF7b to the factors involved in innate immunity and (d) ORF8 to the factors involved in TGFβ signaling. (e) Overview of perturbations to host-cell autophagy, induced by distinct proteins of SARS-CoV-2, derived from the network diffusion model and overlaid with the changes in protein levels, ubiquitination and phosphorylation induced by SARS-CoV-2 infection. (f-i) Western blot of autophagy-associated factors MAP1LC3B-II and SQSTM1 accumulation upon SARS-CoV-2 ORF3 expression in (f) HEK293R1 and (g-i) SARS-CoV-2 infection of A549-ACE2 cells. (h) Profile plot of SQSTM1 MS intensity and (i) line diagram showing SQSTM1 mRNA level relative to RPLP0 tested by qRT-PCR upon SARS-CoV-2 infection.

    Techniques Used: Diffusion-based Assay, Functional Assay, RNA Expression, Infection, Derivative Assay, Western Blot, Expressing, Quantitative RT-PCR

    Joint analysis of SARS-CoV-2 and SARS-CoV protein-protein virus-host interactomes. (a) Experimental design to systematically compare the AP-MS interactomes and induced host proteome changes of the homologous SARS-CoV-2 and SARS-CoV viral proteins, with ORF3 homologs of HCoV-NL63 and HCoV-229E as reference for pan coronavirus specificity. (b) Combined virus-host protein interaction network of SARS-CoV-2 and SARS-CoV measured by affinity-purification coupled to mass spectrometry. Homolog viral proteins are displayed as one node. Shared and virus-specific interactions are denoted by the edge color. (c) The numbers of unique and shared host interactions between the homologous proteins of SARS-CoV-2 and SARS-CoV. (d) Gene Ontology Biological Processes enriched among the cellular proteins that are up- (red arrow) or down- (blue arrow) regulated upon overexpression of individual viral proteins.
    Figure Legend Snippet: Joint analysis of SARS-CoV-2 and SARS-CoV protein-protein virus-host interactomes. (a) Experimental design to systematically compare the AP-MS interactomes and induced host proteome changes of the homologous SARS-CoV-2 and SARS-CoV viral proteins, with ORF3 homologs of HCoV-NL63 and HCoV-229E as reference for pan coronavirus specificity. (b) Combined virus-host protein interaction network of SARS-CoV-2 and SARS-CoV measured by affinity-purification coupled to mass spectrometry. Homolog viral proteins are displayed as one node. Shared and virus-specific interactions are denoted by the edge color. (c) The numbers of unique and shared host interactions between the homologous proteins of SARS-CoV-2 and SARS-CoV. (d) Gene Ontology Biological Processes enriched among the cellular proteins that are up- (red arrow) or down- (blue arrow) regulated upon overexpression of individual viral proteins.

    Techniques Used: Affinity Purification, Mass Spectrometry, Over Expression

    32) Product Images from "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"

    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

    Journal: Small Methods

    doi: 10.1002/smtd.202001031

    Evaluation of neutralization potential of human plasmas from convalescent COVID‐19 patients by CSBT and CRBT assays. A) Comparisons of cMFI inhibitions on CSBT and CRBT assays between plasma samples from convalescent COVID‐19 patients and healthy control (HC) subjects. The cMFI inhibition (%) at 1:20 dilution was plotted at the left Y ‐axis. The cutoff values for CSBT and CRBT were inhibition of 25% (median HC value +3.3 × SD) on cMFI at 1:20 dilution. B) Heatmaps showing CSBT and CRBT effects of two‐fold serial dilutions of 32 plasmas from convalescent COVID‐19 patients. C) Distributions of the levels of TAb, IgM, IgG, CSBT, CRBT, and LVppNAT of convalescent plasma samples. The numbers indicated the average titers at log 10 . The titers of Ab, IgM, and IgG were expressed as relative S/CO values determined by serial dilution measurements of each sample (maximum reactive dilution fold multiplied by S/CO). The CRBT and CSBT titers were expresses at ID25, whereas the LVppNAT was expressed as ID50. D) Correlation analyses between the CSBT titer and the CRBT efficiency (at 1:20 dilution), the TAb titer, the IgM titer, the IgG titer, the LVppNAT and the NAT against authentic SARS‐CoV‐2 virus among convalescent plasmas. The correlation of CSBT titer and neutralization activity against authentic SARS‐CoV‐2 virus in 12 representative samples (included 11 convalescent COVID‐19 plasmas and 1 control sample).
    Figure Legend Snippet: Evaluation of neutralization potential of human plasmas from convalescent COVID‐19 patients by CSBT and CRBT assays. A) Comparisons of cMFI inhibitions on CSBT and CRBT assays between plasma samples from convalescent COVID‐19 patients and healthy control (HC) subjects. The cMFI inhibition (%) at 1:20 dilution was plotted at the left Y ‐axis. The cutoff values for CSBT and CRBT were inhibition of 25% (median HC value +3.3 × SD) on cMFI at 1:20 dilution. B) Heatmaps showing CSBT and CRBT effects of two‐fold serial dilutions of 32 plasmas from convalescent COVID‐19 patients. C) Distributions of the levels of TAb, IgM, IgG, CSBT, CRBT, and LVppNAT of convalescent plasma samples. The numbers indicated the average titers at log 10 . The titers of Ab, IgM, and IgG were expressed as relative S/CO values determined by serial dilution measurements of each sample (maximum reactive dilution fold multiplied by S/CO). The CRBT and CSBT titers were expresses at ID25, whereas the LVppNAT was expressed as ID50. D) Correlation analyses between the CSBT titer and the CRBT efficiency (at 1:20 dilution), the TAb titer, the IgM titer, the IgG titer, the LVppNAT and the NAT against authentic SARS‐CoV‐2 virus among convalescent plasmas. The correlation of CSBT titer and neutralization activity against authentic SARS‐CoV‐2 virus in 12 representative samples (included 11 convalescent COVID‐19 plasmas and 1 control sample).

    Techniques Used: Neutralization, Inhibition, Serial Dilution, Activity Assay

    The 83H7 mAb inhibits SARS‐CoV‐2 via the intracellular neutralization pathway. The 293T‐ACE2iRb3 cells were incubated with 20 × 10 −9 m of dylight633‐labeled mAbs (Ab633) of 36H6, 53G2, 83H7, and 8H6 and an irrelevant control antibody (ctrAb), in the presence or absence of STG (2.5 × 10 −9 m ). Live‐cell fluorescence image dynamically tracked using a 63× water immersion objective. Five replicate wells were measured for each group, and 16 fields of each well were imaged. Time‐series (at 10 min, 1 h, 2 h, 3 h, 5 h, 7 h, 9 h, 11 h, and 13 h) analyses of the A) STG‐IVNs, B) STG‐IVpMFI, C) Ab633‐IVNs, and D) Ab633‐IVpMFI and E) the percentage of STG/Ab633 colocalized vesicles to total internalized STG vesicles. IVNs, average internalized vesicle numbers; IVpMFI, the average peak MFI of internalized vesicles. F) Comparisons of the STG‐IVA of the internalized STG vesicles among groups co‐incubated with various mAbs at 5 h postincubation. ** indicates p
    Figure Legend Snippet: The 83H7 mAb inhibits SARS‐CoV‐2 via the intracellular neutralization pathway. The 293T‐ACE2iRb3 cells were incubated with 20 × 10 −9 m of dylight633‐labeled mAbs (Ab633) of 36H6, 53G2, 83H7, and 8H6 and an irrelevant control antibody (ctrAb), in the presence or absence of STG (2.5 × 10 −9 m ). Live‐cell fluorescence image dynamically tracked using a 63× water immersion objective. Five replicate wells were measured for each group, and 16 fields of each well were imaged. Time‐series (at 10 min, 1 h, 2 h, 3 h, 5 h, 7 h, 9 h, 11 h, and 13 h) analyses of the A) STG‐IVNs, B) STG‐IVpMFI, C) Ab633‐IVNs, and D) Ab633‐IVpMFI and E) the percentage of STG/Ab633 colocalized vesicles to total internalized STG vesicles. IVNs, average internalized vesicle numbers; IVpMFI, the average peak MFI of internalized vesicles. F) Comparisons of the STG‐IVA of the internalized STG vesicles among groups co‐incubated with various mAbs at 5 h postincubation. ** indicates p

    Techniques Used: Neutralization, Incubation, Labeling, Fluorescence

    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.
    Figure Legend 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.

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

    Detection of compound‐induced influence on SARS‐CoV‐2 S‐mediated cellular entry. A) Schematic summary of the possible mechanisms of 11 compound inhibitors involved in the study. CytD, cytochalasin D; MDC, dansylcadaverine; Baf.A1, bafilomycin A1; vRNA, viral RNA. B) Dose‐dependent inhibitions of 11 compounds against SARS‐CoV‐2 LVpp infection on H1299‐ACE2hR cells. All compounds were tested in a 2‐fold dilution series, and the initial drug concentrations were begun at their maximal noncytotoxic concentrations. The initial concentrations were 200 × 10 −6 m for amiloride, MDC and DMSO (as a solvent control); 100 × 10 −6 m for dynasore; 10 × 10 −6 m for filipin, APY0201, YM201636, and tetrandrine; 4 × 10 −6 m for nystatin; 100 × 10 −9 m for Baf.A1 and apilimod. ND, not detected. C) Confocal images of STG (green channel), ACE2‐mRuby3 (red channel), and nucleus (blue channel) in 293T‐ACE2iRb3 cells at 5 h post STG incubation. The cells were pretreated with compounds for 1 h before STG loading. These pictures were obtained by using Leica gSTED confocal microscopy on cells treated with compounds at their respective initial concentrations as above‐mentioned. Scale bar, 10 µm. D) Quantitative analysis of the influence of entry inhibitors on STG internalization. Dose‐dependent influence of various compounds on STG internalization characteristics on 293T‐ACE2iRb3 cells at 1 h (left panels) and 5 h (right panels) after incubation. All compounds were tested in a 4‐fold dilution series (4 gradients for DMSO control, and 5 gradients for others), and the initial drug concentrations were identical with as (B). Three replicate wells were measured for each group, and 16 fields of each well were imaged. For each compound, 5 colored bars from left‐to‐right orderly displayed the values measured from cells treated with 4‐fold serial high‐to‐low concentrations of compounds. STG‐IFR, internalized STG fluorescence intensity ratio; STG‐IVA, average area (µm 2 ) of internalized STG vesicles; STG‐IVNs, average numbers of internalized STG vesicles per cell; * p
    Figure Legend Snippet: Detection of compound‐induced influence on SARS‐CoV‐2 S‐mediated cellular entry. A) Schematic summary of the possible mechanisms of 11 compound inhibitors involved in the study. CytD, cytochalasin D; MDC, dansylcadaverine; Baf.A1, bafilomycin A1; vRNA, viral RNA. B) Dose‐dependent inhibitions of 11 compounds against SARS‐CoV‐2 LVpp infection on H1299‐ACE2hR cells. All compounds were tested in a 2‐fold dilution series, and the initial drug concentrations were begun at their maximal noncytotoxic concentrations. The initial concentrations were 200 × 10 −6 m for amiloride, MDC and DMSO (as a solvent control); 100 × 10 −6 m for dynasore; 10 × 10 −6 m for filipin, APY0201, YM201636, and tetrandrine; 4 × 10 −6 m for nystatin; 100 × 10 −9 m for Baf.A1 and apilimod. ND, not detected. C) Confocal images of STG (green channel), ACE2‐mRuby3 (red channel), and nucleus (blue channel) in 293T‐ACE2iRb3 cells at 5 h post STG incubation. The cells were pretreated with compounds for 1 h before STG loading. These pictures were obtained by using Leica gSTED confocal microscopy on cells treated with compounds at their respective initial concentrations as above‐mentioned. Scale bar, 10 µm. D) Quantitative analysis of the influence of entry inhibitors on STG internalization. Dose‐dependent influence of various compounds on STG internalization characteristics on 293T‐ACE2iRb3 cells at 1 h (left panels) and 5 h (right panels) after incubation. All compounds were tested in a 4‐fold dilution series (4 gradients for DMSO control, and 5 gradients for others), and the initial drug concentrations were identical with as (B). Three replicate wells were measured for each group, and 16 fields of each well were imaged. For each compound, 5 colored bars from left‐to‐right orderly displayed the values measured from cells treated with 4‐fold serial high‐to‐low concentrations of compounds. STG‐IFR, internalized STG fluorescence intensity ratio; STG‐IVA, average area (µm 2 ) of internalized STG vesicles; STG‐IVNs, average numbers of internalized STG vesicles per cell; * p

    Techniques Used: Infection, Incubation, Confocal Microscopy, Fluorescence

    Generation and characterization of FP‐fused SARS‐CoV‐2 S proteins. A) Schematics of STG and RBG constructs. Functional domains are colored. NTD, N‐terminal domain; RBD, receptor binding domain; FP, fusion peptide; HR1/2, heptad repeat 1/2; CH, central helix; TM, transmembrane domain; cyt, cytoplasmic tail; TFd, T4 fibritin trimerization motif; mGam, monomeric Gamillus; mNG, mNeonGreen. B) SDS‐PAGE and fluorescence analyses for purified ST‐based and RBD‐based SARS‐CoV‐2 S proteins. C) Size‐exclusion chromatogram (SEC) of the purified SARS‐CoV2‐ST, SARS‐CoV2‐STG, and SARS‐CoV2‐STN. Data from UV280 detector (upper panel) and fluorescence detector (lower panel) from a G3000 HPLC Column were showed. The molecular weight of SARS‐CoV2‐STG (or SARS‐CoV2‐STN) was about 808 kd, which was calculated according to its elution time in referring to the standard curve of determining the molecular weight as shown in Figure S2A,B (Supporting Information). D) SPR sensorgrams showing the binding kinetics for SARS‐CoV2‐STG (upper panel) or SARS‐CoV2‐RBG (lower panel) with immobilized rACE2 (human). Colored lines represented a global fit of the data using a 1:1 binding model.
    Figure Legend Snippet: Generation and characterization of FP‐fused SARS‐CoV‐2 S proteins. A) Schematics of STG and RBG constructs. Functional domains are colored. NTD, N‐terminal domain; RBD, receptor binding domain; FP, fusion peptide; HR1/2, heptad repeat 1/2; CH, central helix; TM, transmembrane domain; cyt, cytoplasmic tail; TFd, T4 fibritin trimerization motif; mGam, monomeric Gamillus; mNG, mNeonGreen. B) SDS‐PAGE and fluorescence analyses for purified ST‐based and RBD‐based SARS‐CoV‐2 S proteins. C) Size‐exclusion chromatogram (SEC) of the purified SARS‐CoV2‐ST, SARS‐CoV2‐STG, and SARS‐CoV2‐STN. Data from UV280 detector (upper panel) and fluorescence detector (lower panel) from a G3000 HPLC Column were showed. The molecular weight of SARS‐CoV2‐STG (or SARS‐CoV2‐STN) was about 808 kd, which was calculated according to its elution time in referring to the standard curve of determining the molecular weight as shown in Figure S2A,B (Supporting Information). D) SPR sensorgrams showing the binding kinetics for SARS‐CoV2‐STG (upper panel) or SARS‐CoV2‐RBG (lower panel) with immobilized rACE2 (human). Colored lines represented a global fit of the data using a 1:1 binding model.

    Techniques Used: Construct, Functional Assay, Binding Assay, SDS Page, Fluorescence, Purification, High Performance Liquid Chromatography, Molecular Weight, SPR Assay

    33) Product Images from "Low ozone concentration and negative ions for rapid SARS-CoV-2 inactivation"

    Article Title: Low ozone concentration and negative ions for rapid SARS-CoV-2 inactivation

    Journal: bioRxiv

    doi: 10.1101/2021.03.11.434968

    A low concentration of ozone inactivates SARS-CoV-2 in different size droplets ICON3 was used to expose SARS-CoV-2 to ozone/ions by keeping the ozone concentration between 5.44 - 1.47 ppm (mean 3.18 ppm) for 20 minutes (orange bars), compared to control (exposed to air, blue bars) and by varying the size of the droplets from 10 to 3 to 0.5 µL in Figures a, b and c, respectively. Replicative capacity is indicated as percentage viral nucleoprotein compared to control.
    Figure Legend Snippet: A low concentration of ozone inactivates SARS-CoV-2 in different size droplets ICON3 was used to expose SARS-CoV-2 to ozone/ions by keeping the ozone concentration between 5.44 - 1.47 ppm (mean 3.18 ppm) for 20 minutes (orange bars), compared to control (exposed to air, blue bars) and by varying the size of the droplets from 10 to 3 to 0.5 µL in Figures a, b and c, respectively. Replicative capacity is indicated as percentage viral nucleoprotein compared to control.

    Techniques Used: Concentration Assay

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    Article Snippet: The purified antibodies were used in following binding and neutralization analyses. .. ELISA binding assay and competitive ELISA2 μg/ml the recombinant S or RBD proteins derived from SARS-CoV-2, SARS-CoV, or MERS-CoV (Sino Biological, Beijing) were coated on 384-well plates (Corning) at 4°C overnight. ..

    Article Title: Potent human neutralizing antibodies elicited by SARS-CoV-2 infection
    Article Snippet: Secreted RBD and trimeric Spike were harvested from the supernatant and purified by gel filtration chromatography as previously reported , , - . .. ELISA analysis of plasma and antibody binding to RBD, trimeric Spike, and NP proteinsThe recombinant RBDs and trimeric Spike derived from SARS-CoV-2, SARS-CoV and MERS-CoV and the SARS-CoV-2 NP protein (Sino Biological, Beijing) were diluted to final concentrations of 0.5 μg/ml or 2μg/ml, then coated onto 96-well plates and incubated at 4°C overnight. .. Samples were washed with PBS-T (PBS containing 0.05% Tween 20) and blocked with blocking buffer (PBS containing 5% skim milk and 2% BSA) at RT for 1h.

    Binding Assay:

    Article Title: A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD
    Article Snippet: The purified antibodies were used in following binding and neutralization analyses. .. ELISA binding assay and competitive ELISA2 μg/ml the recombinant S or RBD proteins derived from SARS-CoV-2, SARS-CoV, or MERS-CoV (Sino Biological, Beijing) were coated on 384-well plates (Corning) at 4°C overnight. ..

    Article Title: Potent human neutralizing antibodies elicited by SARS-CoV-2 infection
    Article Snippet: Secreted RBD and trimeric Spike were harvested from the supernatant and purified by gel filtration chromatography as previously reported , , - . .. ELISA analysis of plasma and antibody binding to RBD, trimeric Spike, and NP proteinsThe recombinant RBDs and trimeric Spike derived from SARS-CoV-2, SARS-CoV and MERS-CoV and the SARS-CoV-2 NP protein (Sino Biological, Beijing) were diluted to final concentrations of 0.5 μg/ml or 2μg/ml, then coated onto 96-well plates and incubated at 4°C overnight. .. Samples were washed with PBS-T (PBS containing 0.05% Tween 20) and blocked with blocking buffer (PBS containing 5% skim milk and 2% BSA) at RT for 1h.

    Recombinant:

    Article Title: A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD
    Article Snippet: The purified antibodies were used in following binding and neutralization analyses. .. ELISA binding assay and competitive ELISA2 μg/ml the recombinant S or RBD proteins derived from SARS-CoV-2, SARS-CoV, or MERS-CoV (Sino Biological, Beijing) were coated on 384-well plates (Corning) at 4°C overnight. ..

    Article Title: Potent human neutralizing antibodies elicited by SARS-CoV-2 infection
    Article Snippet: Secreted RBD and trimeric Spike were harvested from the supernatant and purified by gel filtration chromatography as previously reported , , - . .. ELISA analysis of plasma and antibody binding to RBD, trimeric Spike, and NP proteinsThe recombinant RBDs and trimeric Spike derived from SARS-CoV-2, SARS-CoV and MERS-CoV and the SARS-CoV-2 NP protein (Sino Biological, Beijing) were diluted to final concentrations of 0.5 μg/ml or 2μg/ml, then coated onto 96-well plates and incubated at 4°C overnight. .. Samples were washed with PBS-T (PBS containing 0.05% Tween 20) and blocked with blocking buffer (PBS containing 5% skim milk and 2% BSA) at RT for 1h.

    Derivative Assay:

    Article Title: A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD
    Article Snippet: The purified antibodies were used in following binding and neutralization analyses. .. ELISA binding assay and competitive ELISA2 μg/ml the recombinant S or RBD proteins derived from SARS-CoV-2, SARS-CoV, or MERS-CoV (Sino Biological, Beijing) were coated on 384-well plates (Corning) at 4°C overnight. ..

    Article Title: Potent human neutralizing antibodies elicited by SARS-CoV-2 infection
    Article Snippet: Secreted RBD and trimeric Spike were harvested from the supernatant and purified by gel filtration chromatography as previously reported , , - . .. ELISA analysis of plasma and antibody binding to RBD, trimeric Spike, and NP proteinsThe recombinant RBDs and trimeric Spike derived from SARS-CoV-2, SARS-CoV and MERS-CoV and the SARS-CoV-2 NP protein (Sino Biological, Beijing) were diluted to final concentrations of 0.5 μg/ml or 2μg/ml, then coated onto 96-well plates and incubated at 4°C overnight. .. Samples were washed with PBS-T (PBS containing 0.05% Tween 20) and blocked with blocking buffer (PBS containing 5% skim milk and 2% BSA) at RT for 1h.

    Infection:

    Article Title: Cell entry of SARS-CoV-2 conferred by angiotensin-converting enzyme 2 (ACE2) of different species
    Article Snippet: An HA-Alexa Fluor 488 monoclonal antibody (Thermo Fisher Scientific, USA) was used to stain ACE2 with an HA tag. .. The nucleoprotein (N) of SARS-CoV was detected for infection and replication of the virus using an N-specific polyclonal antibody (Sinobiological, China), and a donkey anti-rabbit IgG (H+L) labeled with Cy3 (Jacksion, USA) was used as the secondary antibody. .. All the cells were stained with DAPI (Sigma, USA) for nuclear visualization.

    Labeling:

    Article Title: Cell entry of SARS-CoV-2 conferred by angiotensin-converting enzyme 2 (ACE2) of different species
    Article Snippet: An HA-Alexa Fluor 488 monoclonal antibody (Thermo Fisher Scientific, USA) was used to stain ACE2 with an HA tag. .. The nucleoprotein (N) of SARS-CoV was detected for infection and replication of the virus using an N-specific polyclonal antibody (Sinobiological, China), and a donkey anti-rabbit IgG (H+L) labeled with Cy3 (Jacksion, USA) was used as the secondary antibody. .. All the cells were stained with DAPI (Sigma, USA) for nuclear visualization.

    Incubation:

    Article Title: Potent human neutralizing antibodies elicited by SARS-CoV-2 infection
    Article Snippet: Secreted RBD and trimeric Spike were harvested from the supernatant and purified by gel filtration chromatography as previously reported , , - . .. ELISA analysis of plasma and antibody binding to RBD, trimeric Spike, and NP proteinsThe recombinant RBDs and trimeric Spike derived from SARS-CoV-2, SARS-CoV and MERS-CoV and the SARS-CoV-2 NP protein (Sino Biological, Beijing) were diluted to final concentrations of 0.5 μg/ml or 2μg/ml, then coated onto 96-well plates and incubated at 4°C overnight. .. Samples were washed with PBS-T (PBS containing 0.05% Tween 20) and blocked with blocking buffer (PBS containing 5% skim milk and 2% BSA) at RT for 1h.

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  • 95
    Sino Biological sars cov 2 s antigen
    Flow cytometric analysis of total T cell (CD4 + ) populations producing IL-2 on mouse splenocyte upon <t>SARS-CoV-2</t> S protein stimulation. Cells were gated in an orderly manner, like singlets were gated, followed by lymphocytes, CD45 + , CD45 + CD4 + and CD45 + CD4 + IL2 + (A, B, C) 3 control panels where 0.26%, 0.26% and 0.13% CD45 + CD4 + IL2 + cells were identified respectively, (D, E, F) 3 treatment panels where 0.73%, 0.69% and 0.63% CD45 + CD4 + IL2 + cells were identified respectively.
    Sars Cov 2 S Antigen, 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
    https://www.bioz.com/result/sars cov 2 s antigen/product/Sino Biological
    Average 95 stars, based on 1 article reviews
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    sars cov 2 s antigen - by Bioz Stars, 2021-04
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    96
    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
    https://www.bioz.com/result/sars cov 2 rbd protein/product/Sino Biological
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 rbd protein - by Bioz Stars, 2021-04
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    95
    Sino Biological sars cov 2
    Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from <t>SARS-CoV-2.</t> (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.
    Sars Cov 2, 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
    https://www.bioz.com/result/sars cov 2/product/Sino Biological
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 - by Bioz Stars, 2021-04
    95/100 stars
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    Flow cytometric analysis of total T cell (CD4 + ) populations producing IL-2 on mouse splenocyte upon SARS-CoV-2 S protein stimulation. Cells were gated in an orderly manner, like singlets were gated, followed by lymphocytes, CD45 + , CD45 + CD4 + and CD45 + CD4 + IL2 + (A, B, C) 3 control panels where 0.26%, 0.26% and 0.13% CD45 + CD4 + IL2 + cells were identified respectively, (D, E, F) 3 treatment panels where 0.73%, 0.69% and 0.63% CD45 + CD4 + IL2 + cells were identified respectively.

    Journal: bioRxiv

    Article Title: BANCOVID, the first D614G variant mRNA-based vaccine candidate against SARS-CoV-2 elicits neutralizing antibody and balanced cellular immune response

    doi: 10.1101/2020.09.29.319061

    Figure Lengend Snippet: Flow cytometric analysis of total T cell (CD4 + ) populations producing IL-2 on mouse splenocyte upon SARS-CoV-2 S protein stimulation. Cells were gated in an orderly manner, like singlets were gated, followed by lymphocytes, CD45 + , CD45 + CD4 + and CD45 + CD4 + IL2 + (A, B, C) 3 control panels where 0.26%, 0.26% and 0.13% CD45 + CD4 + IL2 + cells were identified respectively, (D, E, F) 3 treatment panels where 0.73%, 0.69% and 0.63% CD45 + CD4 + IL2 + cells were identified respectively.

    Article Snippet: The reactivity of the sera from each group of mice immunized with BANCOVID was measured against SARS-CoV-2 S antigen (SinoBiologicals, China).

    Techniques:

    Flow cytometric analysis of total T cell (CD4 + ) populations producing TFN alpha on mouse splenocyte upon SARS-CoV-2 S protein stimulation. Cells were gated in an orderly manner, like singlets were gated, followed by lymphocytes, CD45 + , CD45 + CD4 + and CD45 + CD4 + TFNalpha + (A, B, C) 3 control panels where 0.48%, 0.43% and 0.37% CD45 + CD4 + TFNalpha + cells were identified respectively, (D, E, F) 3 treatment panels where 0.99%, 0.95% and 0.81% CD45 + CD4 + TFNalpha + cells were identified respectively.

    Journal: bioRxiv

    Article Title: BANCOVID, the first D614G variant mRNA-based vaccine candidate against SARS-CoV-2 elicits neutralizing antibody and balanced cellular immune response

    doi: 10.1101/2020.09.29.319061

    Figure Lengend Snippet: Flow cytometric analysis of total T cell (CD4 + ) populations producing TFN alpha on mouse splenocyte upon SARS-CoV-2 S protein stimulation. Cells were gated in an orderly manner, like singlets were gated, followed by lymphocytes, CD45 + , CD45 + CD4 + and CD45 + CD4 + TFNalpha + (A, B, C) 3 control panels where 0.48%, 0.43% and 0.37% CD45 + CD4 + TFNalpha + cells were identified respectively, (D, E, F) 3 treatment panels where 0.99%, 0.95% and 0.81% CD45 + CD4 + TFNalpha + cells were identified respectively.

    Article Snippet: The reactivity of the sera from each group of mice immunized with BANCOVID was measured against SARS-CoV-2 S antigen (SinoBiologicals, China).

    Techniques:

    SARS-CoV-2 S protein mapping via LC-MS/MS.

    Journal: bioRxiv

    Article Title: BANCOVID, the first D614G variant mRNA-based vaccine candidate against SARS-CoV-2 elicits neutralizing antibody and balanced cellular immune response

    doi: 10.1101/2020.09.29.319061

    Figure Lengend Snippet: SARS-CoV-2 S protein mapping via LC-MS/MS.

    Article Snippet: The reactivity of the sera from each group of mice immunized with BANCOVID was measured against SARS-CoV-2 S antigen (SinoBiologicals, China).

    Techniques: Liquid Chromatography with Mass Spectroscopy

    SARS-CoV-2 S protein mapping via ExPASy PeptideMass.

    Journal: bioRxiv

    Article Title: BANCOVID, the first D614G variant mRNA-based vaccine candidate against SARS-CoV-2 elicits neutralizing antibody and balanced cellular immune response

    doi: 10.1101/2020.09.29.319061

    Figure Lengend Snippet: SARS-CoV-2 S protein mapping via ExPASy PeptideMass.

    Article Snippet: The reactivity of the sera from each group of mice immunized with BANCOVID was measured against SARS-CoV-2 S antigen (SinoBiologicals, China).

    Techniques:

    Flow cytometric analysis of total T cell (CD4 + ) populations producing IL-6 on mouse splenocyte upon SARS-CoV-2 S protein stimulation. Cells were gated in an orderly manner, like singlets were gated, followed by lymphocytes, CD45 + , CD45 + CD4 + and CD45 + CD4 + IL6 + (A, B, C) 3 control panels where 0.32%, 0.27% and 0.22% CD45 + CD4 + IL6 + cells were identified respectively, (D, E, F) 3 treatment panels where 0.47%, 0.39% and 0.34% CD45 + CD4 + IL6 + cells were identified respectively.

    Journal: bioRxiv

    Article Title: BANCOVID, the first D614G variant mRNA-based vaccine candidate against SARS-CoV-2 elicits neutralizing antibody and balanced cellular immune response

    doi: 10.1101/2020.09.29.319061

    Figure Lengend Snippet: Flow cytometric analysis of total T cell (CD4 + ) populations producing IL-6 on mouse splenocyte upon SARS-CoV-2 S protein stimulation. Cells were gated in an orderly manner, like singlets were gated, followed by lymphocytes, CD45 + , CD45 + CD4 + and CD45 + CD4 + IL6 + (A, B, C) 3 control panels where 0.32%, 0.27% and 0.22% CD45 + CD4 + IL6 + cells were identified respectively, (D, E, F) 3 treatment panels where 0.47%, 0.39% and 0.34% CD45 + CD4 + IL6 + cells were identified respectively.

    Article Snippet: The reactivity of the sera from each group of mice immunized with BANCOVID was measured against SARS-CoV-2 S antigen (SinoBiologicals, China).

    Techniques:

    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

    Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.

    Journal: Biosensors & Bioelectronics

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

    doi: 10.1016/j.bios.2020.112572

    Figure Lengend Snippet: Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.

    Article Snippet: As shown in (A), these four antibodies have very different affinities towards the S1 protein of SARS-CoV-2.

    Techniques: Enzyme-linked Immunosorbent Assay

    SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL

    Journal: Biosensors & Bioelectronics

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

    doi: 10.1016/j.bios.2020.112572

    Figure Lengend Snippet: SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL

    Article Snippet: As shown in (A), these four antibodies have very different affinities towards the S1 protein of SARS-CoV-2.

    Techniques: Standard Deviation

    Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Journal: Biosensors & Bioelectronics

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

    doi: 10.1016/j.bios.2020.112572

    Figure Lengend Snippet: Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Article Snippet: As shown in (A), these four antibodies have very different affinities towards the S1 protein of SARS-CoV-2.

    Techniques: Serial Dilution, Standard Deviation, Generated

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

    Journal: Biosensors & Bioelectronics

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

    doi: 10.1016/j.bios.2020.112572

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

    Article Snippet: As shown in (A), these four antibodies have very different affinities towards the S1 protein of SARS-CoV-2.

    Techniques: Chemiluminescent ELISA