sars cov 2  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike RBD rFc Recombinant Protein COVID 19 Spike RBD Research
    Description:
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed with the Fc region of rabbit IgG1 at the C terminus
    Catalog Number:
    40592-v31h
    Product Aliases:
    coronavirus spike Protein 2019-nCoV, cov spike Protein 2019-nCoV, ncov RBD Protein 2019-nCoV, ncov s1 Protein 2019-nCoV, ncov s2 Protein 2019-nCoV, ncov spike Protein 2019-nCoV, NCP-CoV RBD Protein 2019-nCoV, NCP-CoV s1 Protein 2019-nCoV, NCP-CoV s2 Protein 2019-nCoV, NCP-CoV Spike Protein 2019-nCoV, novel coronavirus RBD Protein 2019-nCoV, novel coronavirus s1 Protein 2019-nCoV, novel coronavirus s2 Protein 2019-nCoV, novel coronavirus spike Protein 2019-nCoV, RBD Protein 2019-nCoV, S1 Protein 2019-nCoV, S2 Protein 2019-nCoV, Spike RBD Protein 2019-nCoV
    Price:
    None
    Host:
    HEK293 Cells
    Category:
    recombinant protein
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    Structured Review

    Sino Biological sars cov 2
    Detection of <t>SARS-CoV-2</t> RNA by ISH in FFPE cell pellets. ( A and 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 and 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 and F ) SARS-CoV-2 negative-sense RNA can be detected by ISH using negative-sense RNA probe 1 in infected FFPE cell pellets ( E ), but not in uninfected control FFPE cell pellets ( F ). Nuclei are stained blue (hematoxylin). Scale bars: 50 μm.
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed with the Fc region of rabbit IgG1 at the C terminus
    https://www.bioz.com/result/sars cov 2/product/Sino Biological
    Average 94 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 - by Bioz Stars, 2021-02
    94/100 stars

    Images

    1) 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: JCI Insight

    doi: 10.1172/jci.insight.139042

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

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

    Detection of SARS-CoV-2 antigens by IHC and IFA in FFPE cell pellets. ( A and 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: Uninfected control FFPE cell pellets. Nuclei are stained blue (DAPI). Scale bars: 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 and 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: Uninfected control FFPE cell pellets. Nuclei are stained blue (DAPI). Scale bars: 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 replication in FFPE cells using mFISH. ( A and B ) Compared with 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 bars: 20 μm.
    Figure Legend Snippet: Detection of SARS-CoV-2 replication in FFPE cells using mFISH. ( A and B ) Compared with 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 bars: 20 μm.

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

    Dual staining to detect SARS-CoV-2 antigen and RNA in the same FFPE section. ( A and B ) Compared with 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 bars: 50 μm.
    Figure Legend Snippet: Dual staining to detect SARS-CoV-2 antigen and RNA in the same FFPE section. ( A and B ) Compared with 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 bars: 50 μm.

    Techniques Used: Staining, Formalin-fixed Paraffin-Embedded

    2) Product Images from "Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model"

    Article Title: Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model

    Journal: bioRxiv

    doi: 10.1101/2020.09.24.312165

    JIB-04 exhibits distinct post-entry antiviral mechanisms (A) Drug combination dose-response matrix and VSV-SARS-CoV-2 replication. MA104 cells were treated with JIB-04 and chloroquine or JIB-04 and NTZ for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. (B) Time of compound addition and VSV-SARS-CoV-2 replication. MA104 cells were treated with NTZ or JIB-04 (10 μM) at indicated time points relative to VSV-SARS-CoV-2 infection (MOI=3, 0 hpi). GFP signals at 8 hpi were quantified to calculate the percentage of inhibition. (C) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. S RNA levels were measured by RT-qPCR. (D) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. FL: full-length. S2: cleaved S2 fragment. (* non-specific band) (E) Histone demethylase siRNA knockdown and RV replication. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01). Viral RNA copy numbers at 12 hpi were quantified by RT-qPCR. (F) Volcano plot of differentially expressed transcripts with JIB-04 treatment and RV infection. HEK293 cells were treated with DMSO or JIB-04 (10 μM) for 12 hr, and mock-infected (left panel) or infected with porcine RV (MOI=0.01, right panel) for another 12 hr. Red dots represent upregulated genes and green dots represent downregulated genes in JIB-04 treated cells. (G) Expression of three top genes in (F) with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 12 hr and mock-infected or infected porcine RV (MOI=0.01) for 12 hr. mRNA levels of CYP1A1, CYP1B1, and AHRR at 12 hpi were measured by RT-qPCR. (H) Dose-response analysis of VSV-SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. (I) Dose-response analysis of wild-type SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels at 24 hpi were quantified based on immunofluorescence. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. For all panels except A and I, experiments were repeated at least three times with similar results. Fig. 3A was performed twice. Inhibition assay in Fig. 3I was performed once and cytotoxicity assay was performed in triplicates. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: JIB-04 exhibits distinct post-entry antiviral mechanisms (A) Drug combination dose-response matrix and VSV-SARS-CoV-2 replication. MA104 cells were treated with JIB-04 and chloroquine or JIB-04 and NTZ for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. (B) Time of compound addition and VSV-SARS-CoV-2 replication. MA104 cells were treated with NTZ or JIB-04 (10 μM) at indicated time points relative to VSV-SARS-CoV-2 infection (MOI=3, 0 hpi). GFP signals at 8 hpi were quantified to calculate the percentage of inhibition. (C) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. S RNA levels were measured by RT-qPCR. (D) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. FL: full-length. S2: cleaved S2 fragment. (* non-specific band) (E) Histone demethylase siRNA knockdown and RV replication. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01). Viral RNA copy numbers at 12 hpi were quantified by RT-qPCR. (F) Volcano plot of differentially expressed transcripts with JIB-04 treatment and RV infection. HEK293 cells were treated with DMSO or JIB-04 (10 μM) for 12 hr, and mock-infected (left panel) or infected with porcine RV (MOI=0.01, right panel) for another 12 hr. Red dots represent upregulated genes and green dots represent downregulated genes in JIB-04 treated cells. (G) Expression of three top genes in (F) with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 12 hr and mock-infected or infected porcine RV (MOI=0.01) for 12 hr. mRNA levels of CYP1A1, CYP1B1, and AHRR at 12 hpi were measured by RT-qPCR. (H) Dose-response analysis of VSV-SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. (I) Dose-response analysis of wild-type SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels at 24 hpi were quantified based on immunofluorescence. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. For all panels except A and I, experiments were repeated at least three times with similar results. Fig. 3A was performed twice. Inhibition assay in Fig. 3I was performed once and cytotoxicity assay was performed in triplicates. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Infection, Inhibition, Quantitative RT-PCR, Western Blot, Transfection, Expressing, Immunofluorescence, Cytotoxicity Assay

    Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Chemical structures of NTZ and JIB-04 E-isomer from ChemSpider database. (B) Representative images of Vero E6 cells infected by SARS-CoV-2-mNeonGreen (MOI=0.5) at 24 hpi in Fig. 1A . Experiments were repeated at least three times with similar results.
    Figure Legend Snippet: Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Chemical structures of NTZ and JIB-04 E-isomer from ChemSpider database. (B) Representative images of Vero E6 cells infected by SARS-CoV-2-mNeonGreen (MOI=0.5) at 24 hpi in Fig. 1A . Experiments were repeated at least three times with similar results.

    Techniques Used: Infection

    Nitazoxanide and JIB-04 inhibit the replication of multiple viruses (A) Mean fluorescence intensity of GFP positive cells in Fig. 2B was quantified by flow cytometry. (B) Dose-response analysis of VSV-SARS-CoV-2 replication with NTZ or JIB-04 treatment. MA104 cells were treated with compounds at indicated concentrations for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). At 24 hpi, images of GFP positive infected cells were acquired by the ECHO fluorescence microscope. (C) Same as (B) except that cells were infected with an MOI of 0.1. (D) Dose-response analysis of intracellular viral RNA levels with NTZ, JIB-04, or chloroquine treatment. MA104 cells were treated with compounds at 0.1 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (E) Western blot analysis of RV antigen VP6 levels with JIB-04 treatment. HEK293 cells were treated with JIB-04 at 1, 5, or 10 μM for 6 hr and infected with porcine RV (MOI=0.01) for 12 hr. GAPDH was used as a loading control. All experiments were repeated at least three times with similar results. Data are represented as mean ± SEM.
    Figure Legend Snippet: Nitazoxanide and JIB-04 inhibit the replication of multiple viruses (A) Mean fluorescence intensity of GFP positive cells in Fig. 2B was quantified by flow cytometry. (B) Dose-response analysis of VSV-SARS-CoV-2 replication with NTZ or JIB-04 treatment. MA104 cells were treated with compounds at indicated concentrations for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). At 24 hpi, images of GFP positive infected cells were acquired by the ECHO fluorescence microscope. (C) Same as (B) except that cells were infected with an MOI of 0.1. (D) Dose-response analysis of intracellular viral RNA levels with NTZ, JIB-04, or chloroquine treatment. MA104 cells were treated with compounds at 0.1 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (E) Western blot analysis of RV antigen VP6 levels with JIB-04 treatment. HEK293 cells were treated with JIB-04 at 1, 5, or 10 μM for 6 hr and infected with porcine RV (MOI=0.01) for 12 hr. GAPDH was used as a loading control. All experiments were repeated at least three times with similar results. Data are represented as mean ± SEM.

    Techniques Used: Fluorescence, Flow Cytometry, Infection, Microscopy, Quantitative RT-PCR, Western Blot

    Nitazoxanide and JIB-04 broadly inhibit DNA and RNA viruses in different cell types (A) Dose-response analysis of VSV and VSV-SARS-CoV-2 replication with 15 compounds. MA104 cells were treated with indicated compounds at 0.01 to 30 μM for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. EC 50 values for VSV and VSV-SARS-CoV-2 are shown in each graph in red and blue, respectively. (B) Virus infectivity with NTZ or JIB-04 treatment. Vero E6-TMPRSS2 cells were treated with compounds (10 μM) for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). At 6 hpi, percentages of GFP positive cells were quantified by flow cytometry. (C) Dose-response analysis of VSV-SARS-CoV-2 replication and cytotoxicity with NTZ or JIB-04 treatment. For EC 50 measurement, MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3) for 24 hr. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 3 mM for 25 hr. SI: selectivity index. (D) Intracellular viral RNA levels with NTZ or JIB-04 treatment. MA104 cells were treated with compounds (10 μM) for 1 hr and infected with vaccinia virus (VACV), herpes simplex virus-1 (HSV-1), or rotavirus (RV, RRV and UK strains) (MOI=1). Viral RNA levels at 24 hpi were measured by RT-qPCR for VACV B10R, HSV-1 ICP-27, and RV NSP5, respectively. (E) Viral RNA copy numbers with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 6 hr and infected with porcine rotavirus (MOI=0.01) for 6 hr. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with transmissible gastroenteritis virus (TGEV) (MOI=0.01) for 12 hr. Viral RNA copy numbers were measured by RT-qPCR. (F) TGEV titers in the cell supernatant with JIB-04 treatment. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with TGEV (MOI=0.01). Virus titers at 6 and 12 hpi were measured by plaque assays. (G) Intracellular viral RNA levels with NTZ or JIB-04 treatment in different cell types. HEK293-hACE2, HEK293-hACE2-TMPRSS2, and Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1). VSV RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 2A was performed once. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: Nitazoxanide and JIB-04 broadly inhibit DNA and RNA viruses in different cell types (A) Dose-response analysis of VSV and VSV-SARS-CoV-2 replication with 15 compounds. MA104 cells were treated with indicated compounds at 0.01 to 30 μM for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. EC 50 values for VSV and VSV-SARS-CoV-2 are shown in each graph in red and blue, respectively. (B) Virus infectivity with NTZ or JIB-04 treatment. Vero E6-TMPRSS2 cells were treated with compounds (10 μM) for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). At 6 hpi, percentages of GFP positive cells were quantified by flow cytometry. (C) Dose-response analysis of VSV-SARS-CoV-2 replication and cytotoxicity with NTZ or JIB-04 treatment. For EC 50 measurement, MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3) for 24 hr. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 3 mM for 25 hr. SI: selectivity index. (D) Intracellular viral RNA levels with NTZ or JIB-04 treatment. MA104 cells were treated with compounds (10 μM) for 1 hr and infected with vaccinia virus (VACV), herpes simplex virus-1 (HSV-1), or rotavirus (RV, RRV and UK strains) (MOI=1). Viral RNA levels at 24 hpi were measured by RT-qPCR for VACV B10R, HSV-1 ICP-27, and RV NSP5, respectively. (E) Viral RNA copy numbers with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 6 hr and infected with porcine rotavirus (MOI=0.01) for 6 hr. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with transmissible gastroenteritis virus (TGEV) (MOI=0.01) for 12 hr. Viral RNA copy numbers were measured by RT-qPCR. (F) TGEV titers in the cell supernatant with JIB-04 treatment. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with TGEV (MOI=0.01). Virus titers at 6 and 12 hpi were measured by plaque assays. (G) Intracellular viral RNA levels with NTZ or JIB-04 treatment in different cell types. HEK293-hACE2, HEK293-hACE2-TMPRSS2, and Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1). VSV RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 2A was performed once. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Infection, Inhibition, Flow Cytometry, Quantitative RT-PCR

    Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Small molecule inhibitor screen. Vero E6 cells were treated with individual compounds (listed in Table S1) at 10 μM for 1 hour (hr) and infected with SARS-CoV-2-mNeonGreen (MOI=0.5). At 24 hr post infection (hpi), cells were fixed and nuclei were stained by Hoechst 33342. The intensities of mNeonGreen and Hoechst were quantified by the Typhoon biomolecular imager and Cytation plate reader, respectively. The ratio of mNeonGreen and Hoechst is plotted as percentage of inhibition. (B) Dose-response analysis of wild-type SARS-CoV-2 replication with NTZ or JIB-04 treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels were quantified at 24 hpi based on immunofluorescence. For CC 50 measurement, cells were treated with inhibitors at 0.3 μM to 1 mM for 25 hr. SI: selectivity index. (C) Dose-response analysis of intracellular viral RNA levels with compounds. Vero E6 cells were treated with NTZ (10 μM), JIB-04 (10 μM), chloroquine (10 μM), remdesivir (3 μM), or camostat (10 μM) for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). SARS-CoV-2 RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 1A was performed once with raw data included in Dataset S1. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05).
    Figure Legend Snippet: Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Small molecule inhibitor screen. Vero E6 cells were treated with individual compounds (listed in Table S1) at 10 μM for 1 hour (hr) and infected with SARS-CoV-2-mNeonGreen (MOI=0.5). At 24 hr post infection (hpi), cells were fixed and nuclei were stained by Hoechst 33342. The intensities of mNeonGreen and Hoechst were quantified by the Typhoon biomolecular imager and Cytation plate reader, respectively. The ratio of mNeonGreen and Hoechst is plotted as percentage of inhibition. (B) Dose-response analysis of wild-type SARS-CoV-2 replication with NTZ or JIB-04 treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels were quantified at 24 hpi based on immunofluorescence. For CC 50 measurement, cells were treated with inhibitors at 0.3 μM to 1 mM for 25 hr. SI: selectivity index. (C) Dose-response analysis of intracellular viral RNA levels with compounds. Vero E6 cells were treated with NTZ (10 μM), JIB-04 (10 μM), chloroquine (10 μM), remdesivir (3 μM), or camostat (10 μM) for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). SARS-CoV-2 RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 1A was performed once with raw data included in Dataset S1. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05).

    Techniques Used: Infection, Staining, Inhibition, Immunofluorescence, Quantitative RT-PCR

    Inhibition or knockdown of specific KDM histone demethylases inhibits virus replication (A) Expression of IFN and IFN-stimulated genes with NTZ or JIB-04 treatment. HEK293 cells were treated with NTZ (10 μM), JIB-04 (3 μM), or transfected with low-molecular-weight poly(I:C) (100 ng/ml) for 24 hr. mRNA levels of IFNL3 and CXCL10 were measured by RT-qPCR. (B) Autophagy formation with compound treatment. HEK293 cells were transfected with EGFP-LC3 plasmid for 24 hr and treated with rapamycin (100 nM), NTZ (10 μM), or JIB-04 (3 μM) for another 18 hr. GFP positive punctate structures indicate autophagy activation. Scale bar, 20 μm. (C) Intracellular viral RNA levels with JIB-04 and camostat treatment. Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (D) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. S RNA levels were measured by RT-qPCR. (E) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. FL: full-length. S2: cleaved S2 fragment. (F) siRNA-mediated knockdown of JIB-04 target histone demethylases. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr. mRNA levels of indicated histone demethylases were measured by RT-qPCR. (G) Western blot analysis of RV antigen VP6 levels in cells with histone demethylase siRNA knockdown. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01) for 12 hr. (H) Pathway enrichment analysis of gene expression regulated by JIB-04 treatment. Downregulated genes in Fig. 3F with p values
    Figure Legend Snippet: Inhibition or knockdown of specific KDM histone demethylases inhibits virus replication (A) Expression of IFN and IFN-stimulated genes with NTZ or JIB-04 treatment. HEK293 cells were treated with NTZ (10 μM), JIB-04 (3 μM), or transfected with low-molecular-weight poly(I:C) (100 ng/ml) for 24 hr. mRNA levels of IFNL3 and CXCL10 were measured by RT-qPCR. (B) Autophagy formation with compound treatment. HEK293 cells were transfected with EGFP-LC3 plasmid for 24 hr and treated with rapamycin (100 nM), NTZ (10 μM), or JIB-04 (3 μM) for another 18 hr. GFP positive punctate structures indicate autophagy activation. Scale bar, 20 μm. (C) Intracellular viral RNA levels with JIB-04 and camostat treatment. Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (D) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. S RNA levels were measured by RT-qPCR. (E) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. FL: full-length. S2: cleaved S2 fragment. (F) siRNA-mediated knockdown of JIB-04 target histone demethylases. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr. mRNA levels of indicated histone demethylases were measured by RT-qPCR. (G) Western blot analysis of RV antigen VP6 levels in cells with histone demethylase siRNA knockdown. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01) for 12 hr. (H) Pathway enrichment analysis of gene expression regulated by JIB-04 treatment. Downregulated genes in Fig. 3F with p values

    Techniques Used: Inhibition, Expressing, Transfection, Molecular Weight, Quantitative RT-PCR, Plasmid Preparation, Activation Assay, Infection, Western Blot

    3) Product Images from "SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model"

    Article Title: SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

    Journal: bioRxiv

    doi: 10.1101/2020.08.24.264630

    Gross pathological examination of the lungs of SARS-CoV-2 infected hamsters. Foci (arrowheads) of pulmonary consolidation in untreated SARS-CoV-2 infected animals (A) and animals treated with control MAb (D) or low dose plasma (F). Protection against pulmonary lesions in hamsters treated with MAb 47D11 (C) and high dose plasma (E), similar to mock infected animals (B). Images are from representative animals of each treatment group.
    Figure Legend Snippet: Gross pathological examination of the lungs of SARS-CoV-2 infected hamsters. Foci (arrowheads) of pulmonary consolidation in untreated SARS-CoV-2 infected animals (A) and animals treated with control MAb (D) or low dose plasma (F). Protection against pulmonary lesions in hamsters treated with MAb 47D11 (C) and high dose plasma (E), similar to mock infected animals (B). Images are from representative animals of each treatment group.

    Techniques Used: Infection

    Effect of preventive treatment with MAb or high dose convalescent plasma on severity of pneumonia and level of virus antigen expression in lung parenchyma of hamsters after challenge with SARS-CoV-2. Comparison of extent of histopathological changes (HE) and virus antigen expression (IHC) at four days after SARS-CoV-2 inoculation at low magnification (two left columns) and high magnification (two right columns) in hamsters treated 24 hours before virus inoculation with neutralizing antibodies (second, third and fourth rows) compared to no treatment before SARS-CoV-2 inoculation (first row) and sham inoculation (fifth row).
    Figure Legend Snippet: Effect of preventive treatment with MAb or high dose convalescent plasma on severity of pneumonia and level of virus antigen expression in lung parenchyma of hamsters after challenge with SARS-CoV-2. Comparison of extent of histopathological changes (HE) and virus antigen expression (IHC) at four days after SARS-CoV-2 inoculation at low magnification (two left columns) and high magnification (two right columns) in hamsters treated 24 hours before virus inoculation with neutralizing antibodies (second, third and fourth rows) compared to no treatment before SARS-CoV-2 inoculation (first row) and sham inoculation (fifth row).

    Techniques Used: Expressing, Immunohistochemistry

    Effect of prophylactic neutralizing antibody treatment on weight loss and virus replication following SARS-CoV-2 infection in hamsters. A. Body weights of hamsters treated with antibodies were measured at indicated days after inoculation with SARS-CoV-2. SARS-CoV-2 viral RNA (B, C, E and G) or infectious virus (D, F and H) was detected in throat (B), nasal washes (C and D), lung (E and F) and nasal turbinates (G and H). The mean % of starting weight, the mean copy number or the mean infectious titer is shown, error bars represent the standard error of mean. n = 4. * = P
    Figure Legend Snippet: Effect of prophylactic neutralizing antibody treatment on weight loss and virus replication following SARS-CoV-2 infection in hamsters. A. Body weights of hamsters treated with antibodies were measured at indicated days after inoculation with SARS-CoV-2. SARS-CoV-2 viral RNA (B, C, E and G) or infectious virus (D, F and H) was detected in throat (B), nasal washes (C and D), lung (E and F) and nasal turbinates (G and H). The mean % of starting weight, the mean copy number or the mean infectious titer is shown, error bars represent the standard error of mean. n = 4. * = P

    Techniques Used: Infection

    Quantitative assessment of histopathological changes and virus antigen expression. Percentage of inflamed lung tissue (A) and percentage of lung tissue expressing SARS-CoV-2 antigen (B) estimated by microscopic examination in different groups of hamsters at four days after SARS-CoV-2 inoculation. Individual (symbols) and mean (horizontal lines) percentages are shown. Error bars represent the standard error of mean. n = 4. * = P
    Figure Legend Snippet: Quantitative assessment of histopathological changes and virus antigen expression. Percentage of inflamed lung tissue (A) and percentage of lung tissue expressing SARS-CoV-2 antigen (B) estimated by microscopic examination in different groups of hamsters at four days after SARS-CoV-2 inoculation. Individual (symbols) and mean (horizontal lines) percentages are shown. Error bars represent the standard error of mean. n = 4. * = P

    Techniques Used: Expressing

    Histopathological changes and virus antigen expression in nasal turbinates of hamsters after challenge with SARS-CoV-2. In the nasal turbinate of a sham-inoculated hamster (left column), the nasal cavity is empty and the histology of the olfactory mucosa is normal (A). In a serial section, there is no SARS-CoV-2 antigen expression (C). In the nasal turbinate of a non-treated SARS-CoV-2-inoculated hamster (B and D), the nasal cavity is filled with edema fluid mixed with inflammatory cells and debris and the olfactory mucosa is infiltrated by neutrophils (B). A serial section of this tissue shows SARS-CoV-2 antigen expression in many olfactory mucosal cells, as well as in cells in the lumen (C).
    Figure Legend Snippet: Histopathological changes and virus antigen expression in nasal turbinates of hamsters after challenge with SARS-CoV-2. In the nasal turbinate of a sham-inoculated hamster (left column), the nasal cavity is empty and the histology of the olfactory mucosa is normal (A). In a serial section, there is no SARS-CoV-2 antigen expression (C). In the nasal turbinate of a non-treated SARS-CoV-2-inoculated hamster (B and D), the nasal cavity is filled with edema fluid mixed with inflammatory cells and debris and the olfactory mucosa is infiltrated by neutrophils (B). A serial section of this tissue shows SARS-CoV-2 antigen expression in many olfactory mucosal cells, as well as in cells in the lumen (C).

    Techniques Used: Expressing

    4) Product Images from "Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration"

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

    Journal: eLife

    doi: 10.7554/eLife.61552

    Principal findings and conceptual model. ( A ) ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293 T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. ( B ) SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral entry ( B ) assay are listed. ( C ) Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.
    Figure Legend Snippet: Principal findings and conceptual model. ( A ) ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293 T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. ( B ) SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral entry ( B ) assay are listed. ( C ) Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.

    Techniques Used: Binding Assay, Expressing, Generated

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

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

    Glycan coverage of Spike-ACE2 co-complex. SARS-CoV-2 Spike protein trimer (pink) bound to ACE2 (green). ( A ) Without glycans. ( B ) With N-glycans (red) identified using LC-MS on Spike and ACE2. ( C ) Molecular dynamics simulation analyzed the range of movement of each glycan. The space sampled by glycans is represented by a gray cloud. Glycans cover the Spike-ACE2 interface. They also surround the putative proteolysis site of furin (‘S1-S2’, yellow) and S2’ (blue).
    Figure Legend Snippet: Glycan coverage of Spike-ACE2 co-complex. SARS-CoV-2 Spike protein trimer (pink) bound to ACE2 (green). ( A ) Without glycans. ( B ) With N-glycans (red) identified using LC-MS on Spike and ACE2. ( C ) Molecular dynamics simulation analyzed the range of movement of each glycan. The space sampled by glycans is represented by a gray cloud. Glycans cover the Spike-ACE2 interface. They also surround the putative proteolysis site of furin (‘S1-S2’, yellow) and S2’ (blue).

    Techniques Used: Liquid Chromatography with Mass Spectroscopy

    5) Product Images from "Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells"

    Article Title: Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells

    Journal: Emerging Microbes & Infections

    doi: 10.1080/22221751.2020.1815589

    Comparison of the infection efficiency of pseudotyped viruses in various cell lines. VSVdG viruses bearing the spike protein of SARS-CoV-2 or the G protein of VSV were harvested, and the infectivity of these recombinant viruses were tested in different cell lines, including Vero-E6, BHK21, 293 T and BHK21-hACE2 cells. The fluorescence was detected (A), and the numbers of GFP-positive cells (B) were counted with Opera Phenix 12 h post infection.
    Figure Legend Snippet: Comparison of the infection efficiency of pseudotyped viruses in various cell lines. VSVdG viruses bearing the spike protein of SARS-CoV-2 or the G protein of VSV were harvested, and the infectivity of these recombinant viruses were tested in different cell lines, including Vero-E6, BHK21, 293 T and BHK21-hACE2 cells. The fluorescence was detected (A), and the numbers of GFP-positive cells (B) were counted with Opera Phenix 12 h post infection.

    Techniques Used: Infection, Recombinant, Fluorescence

    Validation of the VSVdG-SARS-CoV-2-Sdel18 pseudovirus assay. (A) Specificity of the pseudovirus assay. A negative sample panel including 59 human sera and 58 mouse sera were used to determine the specificity of this assay. (B) Reproducibility of the pseudovirus assay. One COVID-19 convalescent patient serum sample was tested 14 times on individual plates in three independent experiments. The virus titer of VSVdG-SARS-CoV-2-Sdel18 pseudovirus was consistent in these assays (MOI=0.05). (C) The correlation of neutralizing titer measured by the VSVdG-SARS-CoV-2-Sdel18 pseudovirus assay (ID50, log10) and the wild type SARS-CoV-2 neutralization assay (ID100, log10).
    Figure Legend Snippet: Validation of the VSVdG-SARS-CoV-2-Sdel18 pseudovirus assay. (A) Specificity of the pseudovirus assay. A negative sample panel including 59 human sera and 58 mouse sera were used to determine the specificity of this assay. (B) Reproducibility of the pseudovirus assay. One COVID-19 convalescent patient serum sample was tested 14 times on individual plates in three independent experiments. The virus titer of VSVdG-SARS-CoV-2-Sdel18 pseudovirus was consistent in these assays (MOI=0.05). (C) The correlation of neutralizing titer measured by the VSVdG-SARS-CoV-2-Sdel18 pseudovirus assay (ID50, log10) and the wild type SARS-CoV-2 neutralization assay (ID100, log10).

    Techniques Used: Neutralization

    Time course of EGFP expression after VSVdG-SARS-CoV-2-Sdel18 infection. BHK21-hACE2 cells were infected with VSVdG-SARS-CoV-2-Sdel18 virus (MOI=0.05). The fluorescence was detected (A), and the numbers of GFP-positive cells (B) were counted with Opera Phenix at different time points post infection.
    Figure Legend Snippet: Time course of EGFP expression after VSVdG-SARS-CoV-2-Sdel18 infection. BHK21-hACE2 cells were infected with VSVdG-SARS-CoV-2-Sdel18 virus (MOI=0.05). The fluorescence was detected (A), and the numbers of GFP-positive cells (B) were counted with Opera Phenix at different time points post infection.

    Techniques Used: Expressing, Infection, Fluorescence

    VSVdG-SARS-CoV-2-Sdel18-based neutralization assay for screening neutralizing mAbs. (A) Measurement of the neutralizing activity of 35 strains of antibodies. The cultured supernatant of 35 monoclonal hybridoma cells were incubated with VSVdG-SARS-CoV-2-Sdel18 virus (MOI=0.05), and then the mixture was added to BHK21-hACE2 cells. The fluorescence was detected with Opera Phenix 12 h post infection. (B) The numbers of GFP-positive cells were counted to calculate the inhibition rate. (C) The IC50 values of the 7 selected neutralizing antibodies for antiviral activity were also analyzed. The 7 selected neutralizing antibodies were purified and diluted to different concentrations, incubated with VSVdG-SARS-CoV-2-Sdel18 virus (MOI=0.05) for an hour and added to BHK21-hACE2 cells. The numbers of GFP-positive cells were counted with Opera Phenix 12 h post infection to calculate the inhibition ratio. The IC50 was analyzed by nonlinear regression (four-parameter).
    Figure Legend Snippet: VSVdG-SARS-CoV-2-Sdel18-based neutralization assay for screening neutralizing mAbs. (A) Measurement of the neutralizing activity of 35 strains of antibodies. The cultured supernatant of 35 monoclonal hybridoma cells were incubated with VSVdG-SARS-CoV-2-Sdel18 virus (MOI=0.05), and then the mixture was added to BHK21-hACE2 cells. The fluorescence was detected with Opera Phenix 12 h post infection. (B) The numbers of GFP-positive cells were counted to calculate the inhibition rate. (C) The IC50 values of the 7 selected neutralizing antibodies for antiviral activity were also analyzed. The 7 selected neutralizing antibodies were purified and diluted to different concentrations, incubated with VSVdG-SARS-CoV-2-Sdel18 virus (MOI=0.05) for an hour and added to BHK21-hACE2 cells. The numbers of GFP-positive cells were counted with Opera Phenix 12 h post infection to calculate the inhibition ratio. The IC50 was analyzed by nonlinear regression (four-parameter).

    Techniques Used: Neutralization, Activity Assay, Cell Culture, Incubation, Fluorescence, Infection, Inhibition, Purification

    Generation of VSV pseudotyped viruses bearing SARS-CoV-2 spike proteins. (A) The difference between SARS-CoV-2 S protein and SARS-CoV-2-Sdel18. (B) The procedure of producing VSV pseudotyped viruses bearing SARS-CoV-2 spike proteins.
    Figure Legend Snippet: Generation of VSV pseudotyped viruses bearing SARS-CoV-2 spike proteins. (A) The difference between SARS-CoV-2 S protein and SARS-CoV-2-Sdel18. (B) The procedure of producing VSV pseudotyped viruses bearing SARS-CoV-2 spike proteins.

    Techniques Used:

    Comparison of the packaging efficiency of VSVdG-SARS-CoV-2-Sdel18 in various cell lines. Vero-E6, BHK21 and 293 T cells were used to package the VSVdG-SARS-CoV-2-Sdel18 virus. (A) The left picture shows the cells used to package recombinant virus, recorded 48 h post infection with VSVdG-EGFP-G. The right figures show the infectivity of virus produced by three cell lines. The harvested virus was diluted and tested in BHK21-hACE2 cells. The fluorescence was detected (A), and the numbers of GFP-positive cells (B) were counted with Opera Phenix 12 h post infection.
    Figure Legend Snippet: Comparison of the packaging efficiency of VSVdG-SARS-CoV-2-Sdel18 in various cell lines. Vero-E6, BHK21 and 293 T cells were used to package the VSVdG-SARS-CoV-2-Sdel18 virus. (A) The left picture shows the cells used to package recombinant virus, recorded 48 h post infection with VSVdG-EGFP-G. The right figures show the infectivity of virus produced by three cell lines. The harvested virus was diluted and tested in BHK21-hACE2 cells. The fluorescence was detected (A), and the numbers of GFP-positive cells (B) were counted with Opera Phenix 12 h post infection.

    Techniques Used: Recombinant, Infection, Produced, Fluorescence

    6) Product Images from "A novel viral protein translation mechanism reveals mitochondria as a target for antiviral drug development"

    Article Title: A novel viral protein translation mechanism reveals mitochondria as a target for antiviral drug development

    Journal: bioRxiv

    doi: 10.1101/2020.10.19.344713

    The possible effect of Transcription factors binding Leucine-specific tRNAs in viral protein translation (A) Genomic characteristics including histone modification and transcription factor binding status of tRNA TRL-TAA4-1 which matched with rare codon Leu-TTA was analysed using the UCSC database. (B) Overlap analysis of potential TRL-TAA regulatory transcription factors (TFs) and genes that increased in SARS-CoV-2 infection in A549-ACE2 and Calu3 cells using Venny2.1. (C) Heat map demonstrating the expression of 25 TFs in SARS-CoV-2-infected cell lines created using R studio. (D) Western Blot assay to assess the protein level of S after co-transfection with EGR1 or ATF2 in HEK-293 cells. (E). Differentially expressed genes after SARS-CoV-2 infection (GSE147507) were analyzed with R Studio. Pathway enrichment of highly expressed genes was mapped using Metascape (F) Distribution of genes including ACE2, TMPRSS2, ATF2 and identified TFs in human lung cells was created using public single cell sequence data( 18 ).
    Figure Legend Snippet: The possible effect of Transcription factors binding Leucine-specific tRNAs in viral protein translation (A) Genomic characteristics including histone modification and transcription factor binding status of tRNA TRL-TAA4-1 which matched with rare codon Leu-TTA was analysed using the UCSC database. (B) Overlap analysis of potential TRL-TAA regulatory transcription factors (TFs) and genes that increased in SARS-CoV-2 infection in A549-ACE2 and Calu3 cells using Venny2.1. (C) Heat map demonstrating the expression of 25 TFs in SARS-CoV-2-infected cell lines created using R studio. (D) Western Blot assay to assess the protein level of S after co-transfection with EGR1 or ATF2 in HEK-293 cells. (E). Differentially expressed genes after SARS-CoV-2 infection (GSE147507) were analyzed with R Studio. Pathway enrichment of highly expressed genes was mapped using Metascape (F) Distribution of genes including ACE2, TMPRSS2, ATF2 and identified TFs in human lung cells was created using public single cell sequence data( 18 ).

    Techniques Used: Binding Assay, Modification, Infection, Expressing, Western Blot, Cotransfection, Sequencing

    Rare codon bias can prevent translation of SARS-CoV-2 derived sequences (A) Protein expression of plasmids expressing SARS-CoV-2 S using the original or codon-optimized sequence after transfection into human A549 cells. Expression was evaluated using confocal microscopy, and an IRES-RFP sequence was included after the S open reading frame (ORF) for visualisation. (B) Western Blot assay was performed to detect the protein abundance of SARS-CoV-2-derived sequences including the four structural proteins (spike (S), envelope (E), membrane (M) and nucleocapsid (N)) and four accessory proteins (ORF3, ORF6, ORF7 and ORF8). The abbreviation ‘ns’ indicates non-specific. (C) Codon usage variability of selected SARS-CoV-2 ORFs was analysed with GCUA software using the human standard codon table as reference, and the figure was drawn using GraphPad Prism. (D) The number of rare codons (fraction
    Figure Legend Snippet: Rare codon bias can prevent translation of SARS-CoV-2 derived sequences (A) Protein expression of plasmids expressing SARS-CoV-2 S using the original or codon-optimized sequence after transfection into human A549 cells. Expression was evaluated using confocal microscopy, and an IRES-RFP sequence was included after the S open reading frame (ORF) for visualisation. (B) Western Blot assay was performed to detect the protein abundance of SARS-CoV-2-derived sequences including the four structural proteins (spike (S), envelope (E), membrane (M) and nucleocapsid (N)) and four accessory proteins (ORF3, ORF6, ORF7 and ORF8). The abbreviation ‘ns’ indicates non-specific. (C) Codon usage variability of selected SARS-CoV-2 ORFs was analysed with GCUA software using the human standard codon table as reference, and the figure was drawn using GraphPad Prism. (D) The number of rare codons (fraction

    Techniques Used: Derivative Assay, Expressing, Sequencing, Transfection, Confocal Microscopy, Western Blot, Software

    Mitochondrial localization is critical for the translation of SARS-CoV-2 S protein (A) The interaction network of proteins that bound with S was analyzed using Metascape. (B) The frequency of rare codons in SARS-CoV-2, S protein, human nuclear genome and the human mitochondrial genome using the Codon Usage Database. (C) The protein expression of S flanked by SARS-CoV-2 5’- and 3’-UTR sequences in HEK-293 cell lines. (D) The effect of EGR1 and ATF2 on S protein (plus SARS-CoV-2 5’- and 3’ UTS sequences) expression was analysed using Western blot after transfection into HEK-293 cells. Fold change was determined using Image J software. (E) The skeleton of recombinant plasmids encoding S with or without mitochondrial localisation signals (MLS) RnaseP or RMRP. (F) Protein expression of SARS-CoV-2 derived sequences including S, E and ORF8 and variants expressing MLS as analysed by Western blot after transfection into HEK-293 cells. (G) The effect of EGR1 and ATF2 on wild type spike with RMRP binding motif (MRP).
    Figure Legend Snippet: Mitochondrial localization is critical for the translation of SARS-CoV-2 S protein (A) The interaction network of proteins that bound with S was analyzed using Metascape. (B) The frequency of rare codons in SARS-CoV-2, S protein, human nuclear genome and the human mitochondrial genome using the Codon Usage Database. (C) The protein expression of S flanked by SARS-CoV-2 5’- and 3’-UTR sequences in HEK-293 cell lines. (D) The effect of EGR1 and ATF2 on S protein (plus SARS-CoV-2 5’- and 3’ UTS sequences) expression was analysed using Western blot after transfection into HEK-293 cells. Fold change was determined using Image J software. (E) The skeleton of recombinant plasmids encoding S with or without mitochondrial localisation signals (MLS) RnaseP or RMRP. (F) Protein expression of SARS-CoV-2 derived sequences including S, E and ORF8 and variants expressing MLS as analysed by Western blot after transfection into HEK-293 cells. (G) The effect of EGR1 and ATF2 on wild type spike with RMRP binding motif (MRP).

    Techniques Used: Expressing, Western Blot, Transfection, Software, Recombinant, Derivative Assay, Binding Assay

    7) Product Images from "Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion"

    Article Title: Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion

    Journal: bioRxiv

    doi: 10.1101/2020.06.08.141077

    CH25H and 25HC block SARS-CoV-2 S mediated membrane fusion (A) Wild-type (WT) HEK293-hACE2 cells or those stably expressing TMPRSS2 or TMPRSS4 were transfected with mock, IFITM2, IFITM3, or CH25H for 24 hr and infected with VSV-SARS-CoV-2 (MOI=1). At 24 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression. (B) HEK293-hACE2-TMPRSS2 cells with or without CH25H expression were infected with wild-type SARS-CoV-2 (MOI=0.5). At 24 hpi, the mRNA level of SARS-CoV-2 N was measured by RT-qPCR and normalized to GAPDH expression. (C) HEK293-hACE2-TMPRSS2 cells were co-transfected with GFP, either SARS-CoV S or SARS-CoV-2 S, and IFITM2, IFITM3, or CH25H for 24 hr. The red arrows highlight the syncytia formation. Enlarged images of mock condition are highlighted by red boxes and included as insets. Scale bar: 200 µm. (D) HEK293 cells were co-transfected with GFP, Western equine encephalomyelitis virus (WEEV) E1 and E2, VSV G, or reovirus FAST p10, with or without CH25H for 24 hr. The red arrows highlight the syncytia formation. Enlarged images of mock condition are highlighted by red boxes and included as insets. Scale bar: 200 µm. (E) HEK293-hACE2 cells stably expressing TMPRSS2 or TMPRSS4 were co-transfected with SARS-CoV-2 S and GFP with or without 25HC (10 µM) for 24 hr. The red arrows highlight the syncytia formation. Scale bar: 200 µm. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: CH25H and 25HC block SARS-CoV-2 S mediated membrane fusion (A) Wild-type (WT) HEK293-hACE2 cells or those stably expressing TMPRSS2 or TMPRSS4 were transfected with mock, IFITM2, IFITM3, or CH25H for 24 hr and infected with VSV-SARS-CoV-2 (MOI=1). At 24 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression. (B) HEK293-hACE2-TMPRSS2 cells with or without CH25H expression were infected with wild-type SARS-CoV-2 (MOI=0.5). At 24 hpi, the mRNA level of SARS-CoV-2 N was measured by RT-qPCR and normalized to GAPDH expression. (C) HEK293-hACE2-TMPRSS2 cells were co-transfected with GFP, either SARS-CoV S or SARS-CoV-2 S, and IFITM2, IFITM3, or CH25H for 24 hr. The red arrows highlight the syncytia formation. Enlarged images of mock condition are highlighted by red boxes and included as insets. Scale bar: 200 µm. (D) HEK293 cells were co-transfected with GFP, Western equine encephalomyelitis virus (WEEV) E1 and E2, VSV G, or reovirus FAST p10, with or without CH25H for 24 hr. The red arrows highlight the syncytia formation. Enlarged images of mock condition are highlighted by red boxes and included as insets. Scale bar: 200 µm. (E) HEK293-hACE2 cells stably expressing TMPRSS2 or TMPRSS4 were co-transfected with SARS-CoV-2 S and GFP with or without 25HC (10 µM) for 24 hr. The red arrows highlight the syncytia formation. Scale bar: 200 µm. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Blocking Assay, Stable Transfection, Expressing, Transfection, Infection, Quantitative RT-PCR, Western Blot

    CH25H and 25HC do not affect S cleavage or lipid raft organization. (A) VSV-SARS-CoV-2 was incubated with 25HC (10 µM) for 30 min. HEK293-hACE2 cells were treated with 25HC (10 µM) for 1 hr. At 6 hpi, cells were harvested and measured for GFP percentage and intensity by flow cytometry. (B) MA104 cells were treated with 25HC (10 µM) based on the scheme (right panel) and infected with VSV-SARS-CoV-2 (MOI=1). At 24 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression (left panel). (C) HEK293-hACE2 cells were transfected with SARS-CoV-2 for 24 hr. Some cells were also transfected with TMPRSS2 or treated with trypsin (0.5 µg/ml) or 25HC (10 µM). Cells were harvested for western blot and probed for SARS-CoV-2 S1, S2, and GAPDH protein levels. (D) HEK293-hACE2 cells stably expressing indicated ISGs were stained for lipid rafts (cholera toxin B, green) and nucleus (DAPI, blue). Scale bar: 30 µm. (E) HEK293 cells were treated with C4-TopFluor-25HC (10, 1, or 0.1 µM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=0.5) for 24 hr. Scale bar: 500 µm. (F) HEK293-hACE2 cells were transfected GFP-tagged wild-type (WT) or dominant negative (DN) mutants of Rab5 or Rab7 for 24 hr. Cells were harvested for western blot and probed for GFP and GAPDH protein levels. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: CH25H and 25HC do not affect S cleavage or lipid raft organization. (A) VSV-SARS-CoV-2 was incubated with 25HC (10 µM) for 30 min. HEK293-hACE2 cells were treated with 25HC (10 µM) for 1 hr. At 6 hpi, cells were harvested and measured for GFP percentage and intensity by flow cytometry. (B) MA104 cells were treated with 25HC (10 µM) based on the scheme (right panel) and infected with VSV-SARS-CoV-2 (MOI=1). At 24 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression (left panel). (C) HEK293-hACE2 cells were transfected with SARS-CoV-2 for 24 hr. Some cells were also transfected with TMPRSS2 or treated with trypsin (0.5 µg/ml) or 25HC (10 µM). Cells were harvested for western blot and probed for SARS-CoV-2 S1, S2, and GAPDH protein levels. (D) HEK293-hACE2 cells stably expressing indicated ISGs were stained for lipid rafts (cholera toxin B, green) and nucleus (DAPI, blue). Scale bar: 30 µm. (E) HEK293 cells were treated with C4-TopFluor-25HC (10, 1, or 0.1 µM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=0.5) for 24 hr. Scale bar: 500 µm. (F) HEK293-hACE2 cells were transfected GFP-tagged wild-type (WT) or dominant negative (DN) mutants of Rab5 or Rab7 for 24 hr. Cells were harvested for western blot and probed for GFP and GAPDH protein levels. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Incubation, Flow Cytometry, Infection, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Stable Transfection, Staining, Dominant Negative Mutation

    25HC inhibits endosomal cholesterol export to block SARS-CoV-2 fusion (A) HEK293-hACE2-TMPRSS2 cells were treated with or without C4 TopFluor-25HC (F-25HC, 3 µM) and co-cultured at 1:1 ratio with HEK293 cells transfected with SARS-CoV-2 and TdTomato for 24 hr. Note that the fused cells (red) stop at the boundary of 25HC treated cells (green). Scale bar: 200 µm. (B) HEK293 cells were incubated with C4 TopFluor-25HC (F-25HC, 2 µM) for 1 hr, fixed, and stained for early/recycling endosome (Rab4), late endosome (LBPA), lysosome (LAMP1), and nucleus (blue, DAPI). Scale bar: 30 µm. (C) HEK293-hACE2 cells were transfected with wild-type (WT) or dominant negative (DN) mutants of Rab5 or Rab7 for 24 hr and infected with VSV-SARS-CoV-2 (MOI=1) with or without 25HC (10 µM). At 24 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression. (D) HEK293 cells were treated with TopFluor-cholesterol (F-cholesterol, 2 µM) with or without 25HC (20 µM) for 1 hr. Scale bar: 30 µm. (E) MA104 cells were treated with 25HC at indicated concentrations in either complete or serum-free media (SFM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 24 hr. Cells were fixed and scanned with Typhoon. Green signals were quantified by ImageJ. (F) MA104 cells were treated with itraconazole (ICZ) or furin inhibitor (FI) decanoyl-RVKR-CMK at indicated concentrations in either complete or serum-free media for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 24 hr. Cells were fixed and scanned with Typhoon for green signals. (G) HEK293-hACE2-TMPRSS2 cells were treated with 25HC (10 µM) or ICZ (3 µM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 20 hr. Scale bar: 500 µm. (H) HEK293-ACE2-TMPRSS2 cells were transfected with SARS-CoV-2 S and TdTomato plasmids for 24 hr in the presence of chloroquine (10 µM), camostat (10 µM), methyl-β-cyclodextrin (MCBD, 1 mM), ICZ (3 µM), or 25HC (20 µM). Scale bar: 200 µm. (I) Vero-E6 cells were treated with ICZ or 25HC at indicated concentrations for 1 hr and infected with SARS-CoV-2-mNeonGreen (MOI=0.5) for 24 hr. Cells were fixed and green signals were scanned with Typhoon and quantified by ImageJ. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: 25HC inhibits endosomal cholesterol export to block SARS-CoV-2 fusion (A) HEK293-hACE2-TMPRSS2 cells were treated with or without C4 TopFluor-25HC (F-25HC, 3 µM) and co-cultured at 1:1 ratio with HEK293 cells transfected with SARS-CoV-2 and TdTomato for 24 hr. Note that the fused cells (red) stop at the boundary of 25HC treated cells (green). Scale bar: 200 µm. (B) HEK293 cells were incubated with C4 TopFluor-25HC (F-25HC, 2 µM) for 1 hr, fixed, and stained for early/recycling endosome (Rab4), late endosome (LBPA), lysosome (LAMP1), and nucleus (blue, DAPI). Scale bar: 30 µm. (C) HEK293-hACE2 cells were transfected with wild-type (WT) or dominant negative (DN) mutants of Rab5 or Rab7 for 24 hr and infected with VSV-SARS-CoV-2 (MOI=1) with or without 25HC (10 µM). At 24 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression. (D) HEK293 cells were treated with TopFluor-cholesterol (F-cholesterol, 2 µM) with or without 25HC (20 µM) for 1 hr. Scale bar: 30 µm. (E) MA104 cells were treated with 25HC at indicated concentrations in either complete or serum-free media (SFM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 24 hr. Cells were fixed and scanned with Typhoon. Green signals were quantified by ImageJ. (F) MA104 cells were treated with itraconazole (ICZ) or furin inhibitor (FI) decanoyl-RVKR-CMK at indicated concentrations in either complete or serum-free media for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 24 hr. Cells were fixed and scanned with Typhoon for green signals. (G) HEK293-hACE2-TMPRSS2 cells were treated with 25HC (10 µM) or ICZ (3 µM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 20 hr. Scale bar: 500 µm. (H) HEK293-ACE2-TMPRSS2 cells were transfected with SARS-CoV-2 S and TdTomato plasmids for 24 hr in the presence of chloroquine (10 µM), camostat (10 µM), methyl-β-cyclodextrin (MCBD, 1 mM), ICZ (3 µM), or 25HC (20 µM). Scale bar: 200 µm. (I) Vero-E6 cells were treated with ICZ or 25HC at indicated concentrations for 1 hr and infected with SARS-CoV-2-mNeonGreen (MOI=0.5) for 24 hr. Cells were fixed and green signals were scanned with Typhoon and quantified by ImageJ. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Blocking Assay, Cell Culture, Transfection, Incubation, Staining, Dominant Negative Mutation, Infection, Quantitative RT-PCR, Expressing

    CH25H suppresses VSV-SARS-CoV-2 replication in HEK293-hACE2 cells. (A) HEK293-hACE2-mCherry cells were transfected with plasma membrane (PM)-localized GFP and stained for cell surface (green), ACE2 (red), nucleus (DAPI, blue), and actin (white). Scale bar: 30 µm. (B) Wild-type (WT) HEK293 or HEK293-hACE2-mCherry cells were infected with VSV-SARS-CoV-2 (MOI=1) for 8 hr. Scale bar: 200 µm. (C) Same as (B) except that infection was 24 hr and RNA was harvested for RT-qPCR measuring the mRNA level of VSV N compared to GAPDH expression. (D) Same as (B) except that infection was 24 hr and cell lysates were harvested for plaque assays. (E) HEK293-hACE2 cells stably expressing indicated ISGs were harvested for western blot and probed for V5-tagged ISG and GAPDH protein levels. (F) HEK293-hACE2 cells stably expressing indicated ISGs were infected with VSV-SARS-CoV-2 (MOI=1) for 24 hr. Scale bar: 200 µm. (G) HEK293 cells were transfected with mock, IFIH1, or CH25H plasmids for 24 hr or treated with 25HC (10 µM) for 1 hr. RNA was harvested and the mRNA levels of IFN-β (IFNB) and IFN-λ (IFNL3) were measured by RT-qPCR and normalized to GAPDH expression. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: CH25H suppresses VSV-SARS-CoV-2 replication in HEK293-hACE2 cells. (A) HEK293-hACE2-mCherry cells were transfected with plasma membrane (PM)-localized GFP and stained for cell surface (green), ACE2 (red), nucleus (DAPI, blue), and actin (white). Scale bar: 30 µm. (B) Wild-type (WT) HEK293 or HEK293-hACE2-mCherry cells were infected with VSV-SARS-CoV-2 (MOI=1) for 8 hr. Scale bar: 200 µm. (C) Same as (B) except that infection was 24 hr and RNA was harvested for RT-qPCR measuring the mRNA level of VSV N compared to GAPDH expression. (D) Same as (B) except that infection was 24 hr and cell lysates were harvested for plaque assays. (E) HEK293-hACE2 cells stably expressing indicated ISGs were harvested for western blot and probed for V5-tagged ISG and GAPDH protein levels. (F) HEK293-hACE2 cells stably expressing indicated ISGs were infected with VSV-SARS-CoV-2 (MOI=1) for 24 hr. Scale bar: 200 µm. (G) HEK293 cells were transfected with mock, IFIH1, or CH25H plasmids for 24 hr or treated with 25HC (10 µM) for 1 hr. RNA was harvested and the mRNA levels of IFN-β (IFNB) and IFN-λ (IFNL3) were measured by RT-qPCR and normalized to GAPDH expression. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Transfection, Staining, Infection, Quantitative RT-PCR, Expressing, Stable Transfection, Western Blot

    25HC restricts VSV-SARS-CoV-2 replication in MA104 cells. (A) MA104 cells were infected with serially diluted VSV-SARS-CoV-2 (10 5 shown here) with or without 25HC (10 µM). At 3 dpi, GFP signals were scanned with Typhoon. (B) Quantification of plaque sizes in (A). For all figures, experiments were repeated at least three times with similar results. Individual data point is indicated (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: 25HC restricts VSV-SARS-CoV-2 replication in MA104 cells. (A) MA104 cells were infected with serially diluted VSV-SARS-CoV-2 (10 5 shown here) with or without 25HC (10 µM). At 3 dpi, GFP signals were scanned with Typhoon. (B) Quantification of plaque sizes in (A). For all figures, experiments were repeated at least three times with similar results. Individual data point is indicated (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Infection

    25HC inhibits SARS-CoV-2 replication (A) HEK293-hACE2 cells were treated with 7-α, 25-OHC or 25HC at 0.1, 1, or 10 µM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=5). GFP signals were detected at 24 hpi. Scale bar: 200 µm. (B) MA104 cells were treated with 25HC at indicated concentrations for 1 hr and infected with VSV-SARS-CoV-2 (MOI=0.1) for 24 hr. GFP signals were quantified by ImageJ and plotted as percentage of inhibition. (C) HEK293-hACE2 cells were treated with 7-α, 25-OHC or 25HC at 0.1 or 10 µM for 1 hr and infected with SARS-CoV-2 (MOI=0.5). At 24 hpi, the mRNA level of SARS-CoV-2 N was measured by RT-qPCR and normalized to GAPDH expression. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments.
    Figure Legend Snippet: 25HC inhibits SARS-CoV-2 replication (A) HEK293-hACE2 cells were treated with 7-α, 25-OHC or 25HC at 0.1, 1, or 10 µM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=5). GFP signals were detected at 24 hpi. Scale bar: 200 µm. (B) MA104 cells were treated with 25HC at indicated concentrations for 1 hr and infected with VSV-SARS-CoV-2 (MOI=0.1) for 24 hr. GFP signals were quantified by ImageJ and plotted as percentage of inhibition. (C) HEK293-hACE2 cells were treated with 7-α, 25-OHC or 25HC at 0.1 or 10 µM for 1 hr and infected with SARS-CoV-2 (MOI=0.5). At 24 hpi, the mRNA level of SARS-CoV-2 N was measured by RT-qPCR and normalized to GAPDH expression. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments.

    Techniques Used: Infection, Inhibition, Quantitative RT-PCR, Expressing

    ISG screen identifies CH25H as an antiviral host factor that restricts SARS-CoV-2 infection (A) HEK293-hACE2-mCherry cells were transduced with lentiviral vectors encoding individual ISGs for 72 hr and infected with VSV-SARS-CoV or VSV-SARS-CoV-2 (MOI=1) for 24 hr. The percentage of GFP + cells were quantified and plotted. (B) Wild-type (WT) HEK293-hACE2 cells or HEK293-hACE2 cells stably expressing indicated ISGs were infected with VSV-SARS-CoV-2 (MOI=1). At 18 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression. (C) HEK293-hACE2 cells with or without CH25H expression were infected with wild-type VSV, VSV-SARS-CoV or VSV-SARS-CoV-2 (MOI=10) for 6 hr. Cells were harvested and measured for GFP percentage and intensity by flow cytometry. (D) HEK293-hACE2 cells with or without CH25H expression were infected with VSV-SARS-CoV, VSV-SARS-CoV-2, rotavirus RRV strain, or adenovirus serotype 5 (MOI=3) for 24 hr. Viral RNA levels were measured by RT-qPCR and normalized to GAPDH expression. (E) HEK293-hACE2 cells with or without CH25H expression were infected with wild-type SARS-CoV-2 (MOI=0.5). At 24 hpi, the mRNA level of SARS-CoV-2 N was measured by RT-qPCR and normalized to GAPDH expression. For all figures except A, experiments were repeated at least three times with similar results. Fig. 1A was performed twice with average numbers indicated on the graph. Raw data is listed in Dataset S1. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: ISG screen identifies CH25H as an antiviral host factor that restricts SARS-CoV-2 infection (A) HEK293-hACE2-mCherry cells were transduced with lentiviral vectors encoding individual ISGs for 72 hr and infected with VSV-SARS-CoV or VSV-SARS-CoV-2 (MOI=1) for 24 hr. The percentage of GFP + cells were quantified and plotted. (B) Wild-type (WT) HEK293-hACE2 cells or HEK293-hACE2 cells stably expressing indicated ISGs were infected with VSV-SARS-CoV-2 (MOI=1). At 18 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression. (C) HEK293-hACE2 cells with or without CH25H expression were infected with wild-type VSV, VSV-SARS-CoV or VSV-SARS-CoV-2 (MOI=10) for 6 hr. Cells were harvested and measured for GFP percentage and intensity by flow cytometry. (D) HEK293-hACE2 cells with or without CH25H expression were infected with VSV-SARS-CoV, VSV-SARS-CoV-2, rotavirus RRV strain, or adenovirus serotype 5 (MOI=3) for 24 hr. Viral RNA levels were measured by RT-qPCR and normalized to GAPDH expression. (E) HEK293-hACE2 cells with or without CH25H expression were infected with wild-type SARS-CoV-2 (MOI=0.5). At 24 hpi, the mRNA level of SARS-CoV-2 N was measured by RT-qPCR and normalized to GAPDH expression. For all figures except A, experiments were repeated at least three times with similar results. Fig. 1A was performed twice with average numbers indicated on the graph. Raw data is listed in Dataset S1. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Infection, Transduction, Stable Transfection, Expressing, Quantitative RT-PCR, Flow Cytometry

    CH25H and 25HC block SARS-CoV-2 S mediated fusion. (A) HEK293-hACE2-TMPRSS2 cells were infected with wild-type VSV, VSV-SARS-CoV or VSV-SARS-CoV-2 (MOI=10) for 6 hr. Cells were harvested and measured for GFP percentage and intensity by flow cytometry. (B) HEK293-hACE2-TMPRSS2 cells expressing GFP and indicated ISGs or treated with 25HC (10 µM) were mixed at 1:1 ratio and co-cultured with HEK293 cells expressing SARS-CoV-2 S and TdTomato for 24 hr. Note the formation of cell-cell fusion (yellow), highlighted by black arrows. Scale bar: 200 µm. (C) HEK293 cells were co-transfected with GFP, VSV G, or reovirus FAST p10, with or without 25HC (10 µM) for 24 hr. The red arrows highlight the syncytia formation. Scale bar: 200 µm. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM.
    Figure Legend Snippet: CH25H and 25HC block SARS-CoV-2 S mediated fusion. (A) HEK293-hACE2-TMPRSS2 cells were infected with wild-type VSV, VSV-SARS-CoV or VSV-SARS-CoV-2 (MOI=10) for 6 hr. Cells were harvested and measured for GFP percentage and intensity by flow cytometry. (B) HEK293-hACE2-TMPRSS2 cells expressing GFP and indicated ISGs or treated with 25HC (10 µM) were mixed at 1:1 ratio and co-cultured with HEK293 cells expressing SARS-CoV-2 S and TdTomato for 24 hr. Note the formation of cell-cell fusion (yellow), highlighted by black arrows. Scale bar: 200 µm. (C) HEK293 cells were co-transfected with GFP, VSV G, or reovirus FAST p10, with or without 25HC (10 µM) for 24 hr. The red arrows highlight the syncytia formation. Scale bar: 200 µm. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM.

    Techniques Used: Blocking Assay, Infection, Flow Cytometry, Expressing, Cell Culture, Transfection

    8) Product Images from "Native-like SARS-CoV-2 spike glycoprotein expressed by ChAdOx1 nCoV-19/AZD1222 vaccine"

    Article Title: Native-like SARS-CoV-2 spike glycoprotein expressed by ChAdOx1 nCoV-19/AZD1222 vaccine

    Journal: bioRxiv

    doi: 10.1101/2021.01.15.426463

    Site-specific glycan processing of SARS-CoV-2 S upon infection with ChAdOx1 nCoV-19. (A) Western blot analysis of SARS-CoV-2 spike proteins, using anti-S1 and anti-S1+S2 antibodies. Lane 1= Protein pellet from 293F cell lysates infected with ChAdOx1 nCoV-19. Lane 2= Reduced protein pellet from 293F infected with ChAdOx1 nCoV-19. Lane 3=2P-stablilsed SARS-CoV-2 S protein. The white boxes correspond to gel bands that were excised for mass spectrometric analysis. (B) Site-specific N-linked glycosylation of SARS-CoV-2 S0 and S1/S2 glycoproteins. The bar graphs represent the relative quantities of digested glycopeptides possessing the identifiers of oligomannose/hybrid-type glycans (green), complex-type glycans (pink), unoccupied PNGs (grey), or not determined (N.D.) at each N-linked glycan sequon on the S protein, listed from N to C terminus. (C) Glycosylated model of the cleaved (S1/S2) SARS-CoV-2 spike. The pie charts summarise the mass spectrometric analysis of the oligomannose/hybrid (green), complex (pink), or unoccupied (grey) N-linked glycan populations. Representative glycans are modelled onto the prefusion structure of trimeric SARS-CoV-2 S glycoprotein (PDB ID: 6VSB) ( 3 ), with one RBD in the “up” conformation. The modelled glycans are coloured according to oligomannose/hybrid-glycan content with glycan sites labelled in green (80-100%), orange (30-79%), pink (0-29%) or grey (not detected).
    Figure Legend Snippet: Site-specific glycan processing of SARS-CoV-2 S upon infection with ChAdOx1 nCoV-19. (A) Western blot analysis of SARS-CoV-2 spike proteins, using anti-S1 and anti-S1+S2 antibodies. Lane 1= Protein pellet from 293F cell lysates infected with ChAdOx1 nCoV-19. Lane 2= Reduced protein pellet from 293F infected with ChAdOx1 nCoV-19. Lane 3=2P-stablilsed SARS-CoV-2 S protein. The white boxes correspond to gel bands that were excised for mass spectrometric analysis. (B) Site-specific N-linked glycosylation of SARS-CoV-2 S0 and S1/S2 glycoproteins. The bar graphs represent the relative quantities of digested glycopeptides possessing the identifiers of oligomannose/hybrid-type glycans (green), complex-type glycans (pink), unoccupied PNGs (grey), or not determined (N.D.) at each N-linked glycan sequon on the S protein, listed from N to C terminus. (C) Glycosylated model of the cleaved (S1/S2) SARS-CoV-2 spike. The pie charts summarise the mass spectrometric analysis of the oligomannose/hybrid (green), complex (pink), or unoccupied (grey) N-linked glycan populations. Representative glycans are modelled onto the prefusion structure of trimeric SARS-CoV-2 S glycoprotein (PDB ID: 6VSB) ( 3 ), with one RBD in the “up” conformation. The modelled glycans are coloured according to oligomannose/hybrid-glycan content with glycan sites labelled in green (80-100%), orange (30-79%), pink (0-29%) or grey (not detected).

    Techniques Used: Infection, Western Blot

    ChAdOx1 nCoV-19 produces membrane associated SARS-CoV-2 S glycoprotein in native conformations able to bind its host receptor, ACE2. (A) Schematic representation of the vaccine encoded SARS-CoV-2 S protein, showing the position of N-linked glycosylation amino-acid sequons (NXS/T, where X≠P) as branches. Protein domains are illustrated: N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), and transmembrane domain (TM), with the additional tPA secretion signal at the N-terminus. (B) HeLa S3 cells were infected with ChAdOx1 nCoV-19 and incubated with either recombinant ACE2, anti-ChAdOx1 nCoV-19 (derived from vaccinated mice) or a panel of human mAbs (Ab44, Ab45, Ab71 and Ab111, which recognise, S2, RBD, trimeric S and NTD respectively) and compared to non-infected controls, analysed by flow cytometry. (Left). Relative frequency of cells and AlexaFluor 488 fluorescence associated with anti-spike detection is plotted. Left, (blue) anti-ChAdOx1 nCoV-19, middle (red), ACE2 and right (shades of green) human mAbs. In dark grey cells infected with an irrelevant ChAdOx1 vaccine and in light grey non-infected cells are shown as a control. Experimental replicates were performed two times and representative data shown.
    Figure Legend Snippet: ChAdOx1 nCoV-19 produces membrane associated SARS-CoV-2 S glycoprotein in native conformations able to bind its host receptor, ACE2. (A) Schematic representation of the vaccine encoded SARS-CoV-2 S protein, showing the position of N-linked glycosylation amino-acid sequons (NXS/T, where X≠P) as branches. Protein domains are illustrated: N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), and transmembrane domain (TM), with the additional tPA secretion signal at the N-terminus. (B) HeLa S3 cells were infected with ChAdOx1 nCoV-19 and incubated with either recombinant ACE2, anti-ChAdOx1 nCoV-19 (derived from vaccinated mice) or a panel of human mAbs (Ab44, Ab45, Ab71 and Ab111, which recognise, S2, RBD, trimeric S and NTD respectively) and compared to non-infected controls, analysed by flow cytometry. (Left). Relative frequency of cells and AlexaFluor 488 fluorescence associated with anti-spike detection is plotted. Left, (blue) anti-ChAdOx1 nCoV-19, middle (red), ACE2 and right (shades of green) human mAbs. In dark grey cells infected with an irrelevant ChAdOx1 vaccine and in light grey non-infected cells are shown as a control. Experimental replicates were performed two times and representative data shown.

    Techniques Used: Binding Assay, Infection, Incubation, Recombinant, Derivative Assay, Mouse Assay, Flow Cytometry, Fluorescence

    Cryo-ET and subtomogram average of ChAdOx1 nCoV-19 derived spike. (A) Tomographic slice of U2OS cell transduced with ChAdOx1 nCoV-19. The slice is 6.4 Å thick; PM = plasma membrane, scale bar = 100 nm (B) Detailed view of the boxed area marked in (A) . White arrowheads indicate spike proteins on the cell surface; scale bar = 50 nm. (C-E) Subtomogram average of ChAdOx1 nCoV-19 spikes at 11.6 Å resolution as indicated by Fourier-Shell correlation at 0.5 cut-off (C) , shown from side view (D) , and top view (E). SARS-CoV-2 atomic model (PDB 6ZB5) ( 29 ) is fitted for reference.
    Figure Legend Snippet: Cryo-ET and subtomogram average of ChAdOx1 nCoV-19 derived spike. (A) Tomographic slice of U2OS cell transduced with ChAdOx1 nCoV-19. The slice is 6.4 Å thick; PM = plasma membrane, scale bar = 100 nm (B) Detailed view of the boxed area marked in (A) . White arrowheads indicate spike proteins on the cell surface; scale bar = 50 nm. (C-E) Subtomogram average of ChAdOx1 nCoV-19 spikes at 11.6 Å resolution as indicated by Fourier-Shell correlation at 0.5 cut-off (C) , shown from side view (D) , and top view (E). SARS-CoV-2 atomic model (PDB 6ZB5) ( 29 ) is fitted for reference.

    Techniques Used: Derivative Assay, Transduction

    9) Product Images from "Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response"

    Article Title: Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response

    Journal: bioRxiv

    doi: 10.1101/2020.06.27.175166

    Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.
    Figure Legend Snippet: Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.

    Techniques Used: Mouse Assay, Staining, Immunofluorescence, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Binding Assay

    Neutralizing activity against SARS-CoV-2. (A) Percent neutralization of SARS-CoV-2 virus expressing GFP. Serum collected from the naïve animals used as negative controls. (B) Neutralization titer against SARS-CoV-2 virus expressing GFP. (C, D) Correlations between neutralization titer and ELISA binding titer.
    Figure Legend Snippet: Neutralizing activity against SARS-CoV-2. (A) Percent neutralization of SARS-CoV-2 virus expressing GFP. Serum collected from the naïve animals used as negative controls. (B) Neutralization titer against SARS-CoV-2 virus expressing GFP. (C, D) Correlations between neutralization titer and ELISA binding titer.

    Techniques Used: Activity Assay, Neutralization, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay

    Analyzing SARS-CoV-2 RBD and S1 proteins affinities to human ACE2 (hACE2) proteins using biolayer interferometry (BLI). (A) Bio-Layer Interferometry sensograms of the binding of SARS-CoV-2 S1 and RBD proteins to immobilized Fc-human ACE2, after incubation of the analytes at 25°C for 0and 60 minutes. The traces represent BLI response curves for SARS-CoV-2 proteins serially diluted from 800nM to 12.5nM, as indicated. Dotted lines show raw response values, while bold solid lines show the fitted trace. Association and dissociation phases were monitored for 300s and 600s, respectively. The data was globally fit using a 1:1 binding model to estimate binding affinity. (B) Binding affinity specifications of S1 and RBD proteins against hu-ACE2.
    Figure Legend Snippet: Analyzing SARS-CoV-2 RBD and S1 proteins affinities to human ACE2 (hACE2) proteins using biolayer interferometry (BLI). (A) Bio-Layer Interferometry sensograms of the binding of SARS-CoV-2 S1 and RBD proteins to immobilized Fc-human ACE2, after incubation of the analytes at 25°C for 0and 60 minutes. The traces represent BLI response curves for SARS-CoV-2 proteins serially diluted from 800nM to 12.5nM, as indicated. Dotted lines show raw response values, while bold solid lines show the fitted trace. Association and dissociation phases were monitored for 300s and 600s, respectively. The data was globally fit using a 1:1 binding model to estimate binding affinity. (B) Binding affinity specifications of S1 and RBD proteins against hu-ACE2.

    Techniques Used: Binding Assay, Incubation

    Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.
    Figure Legend Snippet: Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.

    Techniques Used: Mouse Assay, Recombinant, Expressing, Binding Assay, Enzyme-linked Immunosorbent Assay, Luminex, Fluorescence

    10) Product Images from "Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients"

    Article Title: Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients

    Journal: bioRxiv

    doi: 10.1101/2020.06.15.153064

    Response of a commercial anti-SARS-CoV-2 rabbit polyclonal antibody (pAb) on the array. (A) array exposed to array exposed to 20% FBS + 10% PNHS; (B) array exposed to 1 μg/mL anti-SARS-CoV-2 pAb in 20% FBS + 10% PNHS. Strong responses to SARS-CoV-2 S1+S2 ECD, S1, and RBD are observed, as well as smaller cross-reactive responses to HCoV-229E, HCoV-OC43, and MERS spike proteins; (C) quantitative data for the titration. Concentrations of pAb are provided at the top of each column in ng/mL; response values at each concentration for each antigen are provided in Angstroms of build. (D) Titration curves for the four SARS-CoV-2 antigens with standard deviation of replicate probe spots at each concentration.
    Figure Legend Snippet: Response of a commercial anti-SARS-CoV-2 rabbit polyclonal antibody (pAb) on the array. (A) array exposed to array exposed to 20% FBS + 10% PNHS; (B) array exposed to 1 μg/mL anti-SARS-CoV-2 pAb in 20% FBS + 10% PNHS. Strong responses to SARS-CoV-2 S1+S2 ECD, S1, and RBD are observed, as well as smaller cross-reactive responses to HCoV-229E, HCoV-OC43, and MERS spike proteins; (C) quantitative data for the titration. Concentrations of pAb are provided at the top of each column in ng/mL; response values at each concentration for each antigen are provided in Angstroms of build. (D) Titration curves for the four SARS-CoV-2 antigens with standard deviation of replicate probe spots at each concentration.

    Techniques Used: Titration, Concentration Assay, Standard Deviation

    11) 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: JCI Insight

    doi: 10.1172/jci.insight.139042

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

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

    Detection of SARS-CoV-2 antigens by IHC and IFA in FFPE cell pellets. ( A and 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: Uninfected control FFPE cell pellets. Nuclei are stained blue (DAPI). Scale bars: 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 and 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: Uninfected control FFPE cell pellets. Nuclei are stained blue (DAPI). Scale bars: 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 replication in FFPE cells using mFISH. ( A and B ) Compared with 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 bars: 20 μm.
    Figure Legend Snippet: Detection of SARS-CoV-2 replication in FFPE cells using mFISH. ( A and B ) Compared with 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 bars: 20 μm.

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

    Dual staining to detect SARS-CoV-2 antigen and RNA in the same FFPE section. ( A and B ) Compared with 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 bars: 50 μm.
    Figure Legend Snippet: Dual staining to detect SARS-CoV-2 antigen and RNA in the same FFPE section. ( A and B ) Compared with 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 bars: 50 μm.

    Techniques Used: Staining, Formalin-fixed Paraffin-Embedded

    12) Product Images from "Neuropilin-1 is a host factor for SARS-CoV-2 infection"

    Article Title: Neuropilin-1 is a host factor for SARS-CoV-2 infection

    Journal: bioRxiv

    doi: 10.1101/2020.06.05.134114

    CendR is required for SARS-CoV-2 infection. ( A ). HeLa NRP1KO + ACE2 cells transiently expressing NRP1-GFP or NRP1(T316R)-GFP were harvested and analysed by western blot. Band intensities were measured from n=3 independent experiments using Odyssey software. GFP bands were quantified from n=3 independent experiments using Odyssey software. The band intensities, normalized to Actin expression, are presented as the average fraction relative to the amount of NRP1 wt-GFP. Two-tailed unpaired t-test; P=0.1167. ACE2 bands were quantified from n=3 independent experiments using Odyssey software. The band intensities, normalized to Actin expression, are presented as the average fraction relative to the amount of ACE2 in HeLa NRP1KO + NRP1 wt-GFP. one-way analysis of variance (ANOVA) and Dunnett’s test; P= 0.5293 (HeLa NRP1KO + GFP), P= 0.9672 (HeLa NRP1KO + NRP1 T316R-GFP) ( B ). Spinning-disk confocal images of HeLa NRP1KO + ACE2 cells expressing NRP1-GFP or NRP1(T316R)-GFP. Hoechst was used to stain nuclei (blue). Maximum projection images of z-stacks acquired with a 20x objective using an automated spinning-disk confocal microscope. Scale bar; 10μm. (C). HeLa NRP1KO + ACE2 cells transfected with GFP, NRP1-GFP or NRP1(T316R)-GFP constructs were infected 24 h later with SARS-CoV-2. At 16 h.p.i. the cells were fixed and stained for SARS-CoV-2-N, and viral infection quantified in the GFP-positive subpopulation of cells. The percentage of infection was normalized to that of GFP-transfected cells. Two-tailed unpaired t-test; p=0.002 (n=3 independent experiments) (D). Inhibition of SARS-CoV-2 infection by treatment with recombinant ACE2 in Caco-2 and Calu-3 cells. Cells were pre-treated with recombinant ACE2 (10 μg/mL) for 1 h prior to SARS-CoV-2 infection. At 16 h.p.i. the cells were fixed and stained for SARS-CoV-2-N, and infection was quantified. Two-tailed unpaired t-test; P
    Figure Legend Snippet: CendR is required for SARS-CoV-2 infection. ( A ). HeLa NRP1KO + ACE2 cells transiently expressing NRP1-GFP or NRP1(T316R)-GFP were harvested and analysed by western blot. Band intensities were measured from n=3 independent experiments using Odyssey software. GFP bands were quantified from n=3 independent experiments using Odyssey software. The band intensities, normalized to Actin expression, are presented as the average fraction relative to the amount of NRP1 wt-GFP. Two-tailed unpaired t-test; P=0.1167. ACE2 bands were quantified from n=3 independent experiments using Odyssey software. The band intensities, normalized to Actin expression, are presented as the average fraction relative to the amount of ACE2 in HeLa NRP1KO + NRP1 wt-GFP. one-way analysis of variance (ANOVA) and Dunnett’s test; P= 0.5293 (HeLa NRP1KO + GFP), P= 0.9672 (HeLa NRP1KO + NRP1 T316R-GFP) ( B ). Spinning-disk confocal images of HeLa NRP1KO + ACE2 cells expressing NRP1-GFP or NRP1(T316R)-GFP. Hoechst was used to stain nuclei (blue). Maximum projection images of z-stacks acquired with a 20x objective using an automated spinning-disk confocal microscope. Scale bar; 10μm. (C). HeLa NRP1KO + ACE2 cells transfected with GFP, NRP1-GFP or NRP1(T316R)-GFP constructs were infected 24 h later with SARS-CoV-2. At 16 h.p.i. the cells were fixed and stained for SARS-CoV-2-N, and viral infection quantified in the GFP-positive subpopulation of cells. The percentage of infection was normalized to that of GFP-transfected cells. Two-tailed unpaired t-test; p=0.002 (n=3 independent experiments) (D). Inhibition of SARS-CoV-2 infection by treatment with recombinant ACE2 in Caco-2 and Calu-3 cells. Cells were pre-treated with recombinant ACE2 (10 μg/mL) for 1 h prior to SARS-CoV-2 infection. At 16 h.p.i. the cells were fixed and stained for SARS-CoV-2-N, and infection was quantified. Two-tailed unpaired t-test; P

    Techniques Used: Infection, Expressing, Western Blot, Software, Two Tailed Test, Staining, Microscopy, Transfection, Construct, Inhibition, Recombinant

    NRP1 interacts with S1 and enhances SARS-CoV-2 infection. ( A ). Alignment of the S protein sequence of SARS-CoV and SARS-CoV-2; SARS-CoV-2 S possesses a furin cleavage site at the S1/S2 boundary. ( B ). Illustration depicting the CendR motif binding to NRPs, the box highlights the similarity between well-established NRP1 ligands and the C-terminal-RRAR motif of SARS-CoV-2 S1. ( C ). Co-immunoprecipitation of the SARS-CoV-2 S protein by GFP-tagged NRP1 in HEK293T cells. HEK293T cells lentivirally transduced to express untagged SARS-CoV-2 S protein were transiently transfected with GFP or NRP1-GFP and subjected to a GFP-trap based immunoprecipitation. ( D ). CendR motif dependent co-immunoprecipitation of the SARS-CoV-2 S1 protein by Nrp1 in HEK293T cells co-transfected to express GFP-tagged S1 or GFP-S1 ΔRRAR and mCherry-tagged Nrp1. Band intensities following mCherry-nanotrap based immunoprecipitation were quantified from n=3 independent experiments using Odyssey software. The band intensities, normalized to mCherry expression, are presented as the average fraction relative to the amount of S1 protein immunoprecipitated by Nrp1-mCherry. Two-tailed unpaired t-test; P= 0.007. (E ). Spinning-disk confocal images (20x objective, 7 z-stacks, 80 fields per well, maximum intensity projected) of HeLa WT + ACE2 and HeLa NRP1KO + ACE2 cells infected with SARS-CoV-2 for 16 hours. Cells were fixed and stained with anti-SARS nucleocapsid (N) and Hoechst to stain nuclei. A blow-up of the dotted square region is shown in the corresponding right-hand panel for each cell line. Images were stitched using CIDRE. ( F ). Expression of NRP1 and ACE2 in HeLa WT , HeLa WT + ACE2, HeLa NRP1KO and HeLa NRP1KO + ACE2 cells. NRP1 bands were quantified from n=3 independent experiments using Odyssey software. The band intensities, normalized to Actin expression, are presented as the average fraction relative to the amount of NRP1 in HeLa WT . Two-tailed unpaired t-test; P= 0.0505. ACE2 bands were quantified from n=3 independent experiments using Odyssey software. The band intensities, normalized to Actin expression, are presented as the average fraction relative to the amount of ACE2 in HeLa WT + ACE2. Two-tailed unpaired t-test; P= 0.1065. ( G ). SARS-CoV-2 infection in HeLa WT , HeLa WT + ACE2, HeLa NRP1KO and HeLa NRP1KO + ACE2 cells. Two-tailed unpaired t-test; P= 0.0002 (n=3 independent experiments). ( H ). The ratio of single-cell and multi-cell (syncytia) infections within the infected-cell population in HeLa WT + ACE2 or HeLa NRP1KO + ACE2 cells were quantified. The bars, error bars and circles represent the mean, s.e.m., and individual data points. Two-tailed unpaired t-test; P
    Figure Legend Snippet: NRP1 interacts with S1 and enhances SARS-CoV-2 infection. ( A ). Alignment of the S protein sequence of SARS-CoV and SARS-CoV-2; SARS-CoV-2 S possesses a furin cleavage site at the S1/S2 boundary. ( B ). Illustration depicting the CendR motif binding to NRPs, the box highlights the similarity between well-established NRP1 ligands and the C-terminal-RRAR motif of SARS-CoV-2 S1. ( C ). Co-immunoprecipitation of the SARS-CoV-2 S protein by GFP-tagged NRP1 in HEK293T cells. HEK293T cells lentivirally transduced to express untagged SARS-CoV-2 S protein were transiently transfected with GFP or NRP1-GFP and subjected to a GFP-trap based immunoprecipitation. ( D ). CendR motif dependent co-immunoprecipitation of the SARS-CoV-2 S1 protein by Nrp1 in HEK293T cells co-transfected to express GFP-tagged S1 or GFP-S1 ΔRRAR and mCherry-tagged Nrp1. Band intensities following mCherry-nanotrap based immunoprecipitation were quantified from n=3 independent experiments using Odyssey software. The band intensities, normalized to mCherry expression, are presented as the average fraction relative to the amount of S1 protein immunoprecipitated by Nrp1-mCherry. Two-tailed unpaired t-test; P= 0.007. (E ). Spinning-disk confocal images (20x objective, 7 z-stacks, 80 fields per well, maximum intensity projected) of HeLa WT + ACE2 and HeLa NRP1KO + ACE2 cells infected with SARS-CoV-2 for 16 hours. Cells were fixed and stained with anti-SARS nucleocapsid (N) and Hoechst to stain nuclei. A blow-up of the dotted square region is shown in the corresponding right-hand panel for each cell line. Images were stitched using CIDRE. ( F ). Expression of NRP1 and ACE2 in HeLa WT , HeLa WT + ACE2, HeLa NRP1KO and HeLa NRP1KO + ACE2 cells. NRP1 bands were quantified from n=3 independent experiments using Odyssey software. The band intensities, normalized to Actin expression, are presented as the average fraction relative to the amount of NRP1 in HeLa WT . Two-tailed unpaired t-test; P= 0.0505. ACE2 bands were quantified from n=3 independent experiments using Odyssey software. The band intensities, normalized to Actin expression, are presented as the average fraction relative to the amount of ACE2 in HeLa WT + ACE2. Two-tailed unpaired t-test; P= 0.1065. ( G ). SARS-CoV-2 infection in HeLa WT , HeLa WT + ACE2, HeLa NRP1KO and HeLa NRP1KO + ACE2 cells. Two-tailed unpaired t-test; P= 0.0002 (n=3 independent experiments). ( H ). The ratio of single-cell and multi-cell (syncytia) infections within the infected-cell population in HeLa WT + ACE2 or HeLa NRP1KO + ACE2 cells were quantified. The bars, error bars and circles represent the mean, s.e.m., and individual data points. Two-tailed unpaired t-test; P

    Techniques Used: Infection, Sequencing, Binding Assay, Immunoprecipitation, Transfection, Software, Expressing, Two Tailed Test, Staining

    13) Product Images from "An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2"

    Article Title: An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2

    Journal: bioRxiv

    doi: 10.1101/2020.08.19.258244

    The immunogenicity, efficacy and safety of C-Vac for SARS-CoV-2 infection. (A) Flow cytometry-based detection of MHC(HLA-A2)-peptide complex binding affinity in T2 cells. (B) The expression of antigens in 293T-based C-Vac is confirmed by Western Blot. N, 293T cells transfected with the plasmid expressing a full length N gene. (C D) Pseudovirus neutralization titers of hamster serum at day 7 after the first immunization and day 21 (boosted at day 14) after vaccination with 293T-based C-Vac, MiT C-Vac: Mitomycin C-treated C-Vac, Lys C-Vac: Lysed C-Vac. (E) Histological characteristics of hamster lung at day 7 after the first vaccination. Original magnification 200× (F) Allograft volume of transformed fibroblasts expressing RBD-truncated N protein in the Syrian hamsters immunized with different regime. 5×10 6 BHK21 cells expressing C-Vac antigen (RBD-Ntap) were subcutaneously injected into immunized hamsters for challenge at day 45 after boost, and the volume of allografts were measured at 14 days after inoculation of the BHK21 cells expressing RBD-Ntap into the immunized hamsters.
    Figure Legend Snippet: The immunogenicity, efficacy and safety of C-Vac for SARS-CoV-2 infection. (A) Flow cytometry-based detection of MHC(HLA-A2)-peptide complex binding affinity in T2 cells. (B) The expression of antigens in 293T-based C-Vac is confirmed by Western Blot. N, 293T cells transfected with the plasmid expressing a full length N gene. (C D) Pseudovirus neutralization titers of hamster serum at day 7 after the first immunization and day 21 (boosted at day 14) after vaccination with 293T-based C-Vac, MiT C-Vac: Mitomycin C-treated C-Vac, Lys C-Vac: Lysed C-Vac. (E) Histological characteristics of hamster lung at day 7 after the first vaccination. Original magnification 200× (F) Allograft volume of transformed fibroblasts expressing RBD-truncated N protein in the Syrian hamsters immunized with different regime. 5×10 6 BHK21 cells expressing C-Vac antigen (RBD-Ntap) were subcutaneously injected into immunized hamsters for challenge at day 45 after boost, and the volume of allografts were measured at 14 days after inoculation of the BHK21 cells expressing RBD-Ntap into the immunized hamsters.

    Techniques Used: Infection, Flow Cytometry, Binding Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Neutralization, Transformation Assay, Injection

    The RBD domain of Spike is crucial for the SARS-CoV2 Vaccine. (A) The functional domain of SARS-CoV-2 spike protein. (B) Potential B cell antigen of RBD domain from SARS-CoV2 is predicted by Discotope software based on their 3D structure. (C) Potential linear B cell epitopes of SARS-CoV-2 full S protein are analysed with the IEDB database. (D) The location of potential antigens in RBD domain (SARS-CoV-2:red, SARS-CoV: Purple) and interaction model between RBD and ACE2 receptor (interface is marked yellow) are marked with Discovery Studio. (E) The expression of Spike and nucleocapsid with wild-type sequence in 293T cells are detected by Western Blot assay. (F) The expression of Spike and its derivatives with codon optimization (opt).
    Figure Legend Snippet: The RBD domain of Spike is crucial for the SARS-CoV2 Vaccine. (A) The functional domain of SARS-CoV-2 spike protein. (B) Potential B cell antigen of RBD domain from SARS-CoV2 is predicted by Discotope software based on their 3D structure. (C) Potential linear B cell epitopes of SARS-CoV-2 full S protein are analysed with the IEDB database. (D) The location of potential antigens in RBD domain (SARS-CoV-2:red, SARS-CoV: Purple) and interaction model between RBD and ACE2 receptor (interface is marked yellow) are marked with Discovery Studio. (E) The expression of Spike and nucleocapsid with wild-type sequence in 293T cells are detected by Western Blot assay. (F) The expression of Spike and its derivatives with codon optimization (opt).

    Techniques Used: Functional Assay, Software, Expressing, Sequencing, Western Blot

    Construction of chimeric vaccine for SARS-CoV-2. (A) Potential B-cell epitopes of N protein is predicted by IEDB database. (B) Potential MHCI-binding peptides of N. (C) Functional domain of SARS-CoV N protein (Upper) and its antibody epitope map reported in previous study. (D) The skeleton of Chimeric Vaccine for SARS-CoV-2, RBD: spike RBD domain (306-541 aa), Ntap: T-cell-associated peptide of N (211-339 aa). (E) Characterization of SARS-CoV-2-derived protein and C-Vac antigen by SARS-CoV-2 antisera and commercial antibodies against SARS-CoV2 spike RBD or Nucleocapsid.
    Figure Legend Snippet: Construction of chimeric vaccine for SARS-CoV-2. (A) Potential B-cell epitopes of N protein is predicted by IEDB database. (B) Potential MHCI-binding peptides of N. (C) Functional domain of SARS-CoV N protein (Upper) and its antibody epitope map reported in previous study. (D) The skeleton of Chimeric Vaccine for SARS-CoV-2, RBD: spike RBD domain (306-541 aa), Ntap: T-cell-associated peptide of N (211-339 aa). (E) Characterization of SARS-CoV-2-derived protein and C-Vac antigen by SARS-CoV-2 antisera and commercial antibodies against SARS-CoV2 spike RBD or Nucleocapsid.

    Techniques Used: Binding Assay, Functional Assay, Derivative Assay

    14) Product Images from "Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response"

    Article Title: Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response

    Journal: bioRxiv

    doi: 10.1101/2020.06.27.175166

    Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.
    Figure Legend Snippet: Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.

    Techniques Used: Mouse Assay, Staining, Immunofluorescence, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Binding Assay

    Neutralizing activity against SARS-CoV-2. (A) Percent neutralization of SARS-CoV-2 virus expressing GFP. Serum collected from the naïve animals used as negative controls. (B) Neutralization titer against SARS-CoV-2 virus expressing GFP. (C, D) Correlations between neutralization titer and ELISA binding titer.
    Figure Legend Snippet: Neutralizing activity against SARS-CoV-2. (A) Percent neutralization of SARS-CoV-2 virus expressing GFP. Serum collected from the naïve animals used as negative controls. (B) Neutralization titer against SARS-CoV-2 virus expressing GFP. (C, D) Correlations between neutralization titer and ELISA binding titer.

    Techniques Used: Activity Assay, Neutralization, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay

    Analyzing SARS-CoV-2 RBD and S1 proteins affinities to human ACE2 (hACE2) proteins using biolayer interferometry (BLI). (A) Bio-Layer Interferometry sensograms of the binding of SARS-CoV-2 S1 and RBD proteins to immobilized Fc-human ACE2, after incubation of the analytes at 25°C for 0and 60 minutes. The traces represent BLI response curves for SARS-CoV-2 proteins serially diluted from 800nM to 12.5nM, as indicated. Dotted lines show raw response values, while bold solid lines show the fitted trace. Association and dissociation phases were monitored for 300s and 600s, respectively. The data was globally fit using a 1:1 binding model to estimate binding affinity. (B) Binding affinity specifications of S1 and RBD proteins against hu-ACE2.
    Figure Legend Snippet: Analyzing SARS-CoV-2 RBD and S1 proteins affinities to human ACE2 (hACE2) proteins using biolayer interferometry (BLI). (A) Bio-Layer Interferometry sensograms of the binding of SARS-CoV-2 S1 and RBD proteins to immobilized Fc-human ACE2, after incubation of the analytes at 25°C for 0and 60 minutes. The traces represent BLI response curves for SARS-CoV-2 proteins serially diluted from 800nM to 12.5nM, as indicated. Dotted lines show raw response values, while bold solid lines show the fitted trace. Association and dissociation phases were monitored for 300s and 600s, respectively. The data was globally fit using a 1:1 binding model to estimate binding affinity. (B) Binding affinity specifications of S1 and RBD proteins against hu-ACE2.

    Techniques Used: Binding Assay, Incubation

    Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.
    Figure Legend Snippet: Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.

    Techniques Used: Mouse Assay, Recombinant, Expressing, Binding Assay, Enzyme-linked Immunosorbent Assay, Luminex, Fluorescence

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

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

    Journal: JCI Insight

    doi: 10.1172/jci.insight.142386

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

    Techniques Used: Enzyme-linked Immunosorbent Assay

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

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

    Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.
    Figure Legend Snippet: Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.

    Techniques Used: Neutralization, Activity Assay, Binding Assay, Standard Deviation

    16) Product Images from "SARS-CoV-2 causes severe alveolar inflammation and barrier dysfunction"

    Article Title: SARS-CoV-2 causes severe alveolar inflammation and barrier dysfunction

    Journal: bioRxiv

    doi: 10.1101/2020.08.31.276725

    SARS-CoV-2 infection results in induction of antiviral and proinflammatory mRNA synthesis. Calu-3 cells were left uninfected (mock) or were infected with a SARS-CoV-2 patient isolate (5159, 5587, 5588) (MOI=1). RNA-lysates were performed 24h p.i. Levels of IFNα, IFNβ, IFNλ1, IFNλ2,3, IL6, IL8, IP10, TNFα, cIAP2, TRAIL, and RIPK1 Mrna were measured of three patient isolate (5159, 5587, 5588) and two technical samples in 3 independent experiments. Means ± SD of three independent experiments are shown. Levels of mock-treated samples were arbitrarily set as 1. After normalization, two-tailed unpaired t-tests were performed for comparison of mock-treated and SARS-CoV-2-infected and samples. (*p
    Figure Legend Snippet: SARS-CoV-2 infection results in induction of antiviral and proinflammatory mRNA synthesis. Calu-3 cells were left uninfected (mock) or were infected with a SARS-CoV-2 patient isolate (5159, 5587, 5588) (MOI=1). RNA-lysates were performed 24h p.i. Levels of IFNα, IFNβ, IFNλ1, IFNλ2,3, IL6, IL8, IP10, TNFα, cIAP2, TRAIL, and RIPK1 Mrna were measured of three patient isolate (5159, 5587, 5588) and two technical samples in 3 independent experiments. Means ± SD of three independent experiments are shown. Levels of mock-treated samples were arbitrarily set as 1. After normalization, two-tailed unpaired t-tests were performed for comparison of mock-treated and SARS-CoV-2-infected and samples. (*p

    Techniques Used: Infection, Two Tailed Test

    Infection with SARS-CoV-2 results in the disruption of the epithelial- and endothelial barrier. The epithelial side of the alveolus-on-a-chip model was left uninfected (mock) or infected with three different SARS-CoV-2 patient isolates (5159, 5587, 5588) (MOI=1). Immunofluorescence staining was performed 40h p.i., (A) The E-cadherin of the epithelial layer and the (B) VE-cadherin of the endothelial layer were visualized by an anti-E-Cadherin-specific antibody or an anti-VE-Cadherin antiserum, respectively, and a Cy5 goat anti-rabbit IgG (red). (A, B) The SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Scale bars represent 100 μm.
    Figure Legend Snippet: Infection with SARS-CoV-2 results in the disruption of the epithelial- and endothelial barrier. The epithelial side of the alveolus-on-a-chip model was left uninfected (mock) or infected with three different SARS-CoV-2 patient isolates (5159, 5587, 5588) (MOI=1). Immunofluorescence staining was performed 40h p.i., (A) The E-cadherin of the epithelial layer and the (B) VE-cadherin of the endothelial layer were visualized by an anti-E-Cadherin-specific antibody or an anti-VE-Cadherin antiserum, respectively, and a Cy5 goat anti-rabbit IgG (red). (A, B) The SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Scale bars represent 100 μm.

    Techniques Used: Infection, Chromatin Immunoprecipitation, Immunofluorescence, Staining

    Phylogenetic tree of SARS-CoV-2. (A) Phylogenetic analysis revealed a close relationship of SARS-CoV-2 to the SARS-related coronaviruses RaTG13, bat-SL-CoVZXC21 and bat-SL-CoVZC45. Sequences of strains 5587 and 5588 exhibit two base substitutions T8,782C ( nsp1ab: synonymous) and C28,144T ( nsp8: S84L). (B) Accordingly, 5587 and 5588 clustered with lineage L/lineage B strains in the phylogenetic analysis. Both strains exhibit deletion of nsp1ab D448 and two synonymous substitutions (T514C, C5512T). Beside the nsp8 S84L substitution, strain 5159 has accumulated three additional amino acid substitutions ( S: D614G, nsp1ab: P4715L and N: R203K/G204R) which place this virus in lineage B.1.1.
    Figure Legend Snippet: Phylogenetic tree of SARS-CoV-2. (A) Phylogenetic analysis revealed a close relationship of SARS-CoV-2 to the SARS-related coronaviruses RaTG13, bat-SL-CoVZXC21 and bat-SL-CoVZC45. Sequences of strains 5587 and 5588 exhibit two base substitutions T8,782C ( nsp1ab: synonymous) and C28,144T ( nsp8: S84L). (B) Accordingly, 5587 and 5588 clustered with lineage L/lineage B strains in the phylogenetic analysis. Both strains exhibit deletion of nsp1ab D448 and two synonymous substitutions (T514C, C5512T). Beside the nsp8 S84L substitution, strain 5159 has accumulated three additional amino acid substitutions ( S: D614G, nsp1ab: P4715L and N: R203K/G204R) which place this virus in lineage B.1.1.

    Techniques Used:

    SARS-CoV-2 infects epithelial cells productively. (A) Vero-76, Calu-3, and HUVECs were infected with a SARS-CoV-2 patient isolate (5159, 5587, 5588) (MOI=1). RNA-lysates were performed 24h p.i. and copies of viral RNA (E-gene) were determined by r-biopharm qRT-PCR. Means ± SD of three independent experiments are shown. (B) HUVECs were infected with a SARS-CoV-2 patient isolate (5159) (MOI=1) for 4h, 8h, and 24h. SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Immunofluorescence (IF) microscopy was acquired by use of the Axio Observer.Z1 (Zeiss) with a 200×magnification.
    Figure Legend Snippet: SARS-CoV-2 infects epithelial cells productively. (A) Vero-76, Calu-3, and HUVECs were infected with a SARS-CoV-2 patient isolate (5159, 5587, 5588) (MOI=1). RNA-lysates were performed 24h p.i. and copies of viral RNA (E-gene) were determined by r-biopharm qRT-PCR. Means ± SD of three independent experiments are shown. (B) HUVECs were infected with a SARS-CoV-2 patient isolate (5159) (MOI=1) for 4h, 8h, and 24h. SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Immunofluorescence (IF) microscopy was acquired by use of the Axio Observer.Z1 (Zeiss) with a 200×magnification.

    Techniques Used: Infection, Quantitative RT-PCR, Staining, Immunofluorescence, Microscopy

    SARS-CoV-2 efficiently infects epithelial cells of the human-alveolus-on-a chip model and provokes type I and III interferon production. (A-C) The epithelial chamber of the alveolus-on-a-chip model was left uninfected (mock) or infected with three different SARS-CoV-2 patient isolates (5159, 5587, 5588) (MOI=1). (A, B) Immunofluorescence staining was performed 28h p.i. and analyzed by immunofluorescence microscopy (Axio Observer.Z1 (Zeiss)). (A) The E-cadherin of the epithelial layer and the (B) VE-cadherin of the endothelial layer were visualized by an anti-E-Cadherin-specific antibody or an anti-VE-Cadherin antiserum, respectively, and a Cy5 goat anti-rabbit IgG (red). (A, B) The SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Scale bars represent 100 μm. (C) Production of antiviral cytokines derived from the epithelial side was determined by use of Legendplex Panel (Biolegend, CA, USA). SARS-CoV-2 induced IFNβ, IFNλ1 and IFNλ2,3 release (pg/ml) was measured. Means ± SD of three independent experiments each infected with another patient isolate (5159, 5587, 5588) are shown. Levels of mock-treated samples were arbitrarily set as 1. After normalization, two-tailed unpaired t-tests were performed for comparison of mock-treated and SARS-CoV-2-infected and samples. (**p
    Figure Legend Snippet: SARS-CoV-2 efficiently infects epithelial cells of the human-alveolus-on-a chip model and provokes type I and III interferon production. (A-C) The epithelial chamber of the alveolus-on-a-chip model was left uninfected (mock) or infected with three different SARS-CoV-2 patient isolates (5159, 5587, 5588) (MOI=1). (A, B) Immunofluorescence staining was performed 28h p.i. and analyzed by immunofluorescence microscopy (Axio Observer.Z1 (Zeiss)). (A) The E-cadherin of the epithelial layer and the (B) VE-cadherin of the endothelial layer were visualized by an anti-E-Cadherin-specific antibody or an anti-VE-Cadherin antiserum, respectively, and a Cy5 goat anti-rabbit IgG (red). (A, B) The SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Scale bars represent 100 μm. (C) Production of antiviral cytokines derived from the epithelial side was determined by use of Legendplex Panel (Biolegend, CA, USA). SARS-CoV-2 induced IFNβ, IFNλ1 and IFNλ2,3 release (pg/ml) was measured. Means ± SD of three independent experiments each infected with another patient isolate (5159, 5587, 5588) are shown. Levels of mock-treated samples were arbitrarily set as 1. After normalization, two-tailed unpaired t-tests were performed for comparison of mock-treated and SARS-CoV-2-infected and samples. (**p

    Techniques Used: Chromatin Immunoprecipitation, Infection, Immunofluorescence, Staining, Microscopy, Derivative Assay, Two Tailed Test

    SARS-CoV-2 infection results in the disruption of barrier integrity in the human-alveolus-on-a chip model. The epithelial side of the alveolus-on-a-chip model was left uninfected (mock) or infected with the SARS-CoV-2 patient isolate (5159) (MOI=1) for 28h. An overview (upper panel) of the (A) epithelial layer and (B) endothelial layer are depicted. Dead cells (middle panel) are focused. The surface of dead cells (lower panel) shows particles (arrows) attached to the plasma membranes of the epithelial cells only. Scale bars represent 50 μm (200×magnification), 5 μm (2.000×magnification) and 200 nm (60.000×magnification). (C) Barrier function of the human alveolus-on-a-chip model was analyzed by a permeability assay of mock-infected and SARS-CoV-2-infected human alveolus-on-a-chip model using FITC-dextran at 28h p.i., FITC-dextran was measured via the fluorescence intensity (exc. 488nm; em. 518 nm) and depicted as the permeability coefficient ( P app ), calculated according to P app (cm s -1 ) = (dQ/d t ) (1/AC o ). Results show significant higher barrier permeability after SARS-CoV-2 infection. (D) Supernatants of the epithelial- and endothelial side of SARS-CoV-2 infected human alveolus-on-a-chip models were used to perform LDH-assays indicating cell membrane rupture at 28h and 40h p.i.. (E) Progeny virus titers were analyzed in the supernatants of the epithelial- and endothelial layer by standard plaque assay. Shown are means (±SD) of (C) three independent experiments each infected with another patient isolate (5159, 5587, 5588), (D) LDH release, and (E) plaque forming units (PFU/ml). Statistical significance was analyzed by unpaired, two-tailed t-test (*p
    Figure Legend Snippet: SARS-CoV-2 infection results in the disruption of barrier integrity in the human-alveolus-on-a chip model. The epithelial side of the alveolus-on-a-chip model was left uninfected (mock) or infected with the SARS-CoV-2 patient isolate (5159) (MOI=1) for 28h. An overview (upper panel) of the (A) epithelial layer and (B) endothelial layer are depicted. Dead cells (middle panel) are focused. The surface of dead cells (lower panel) shows particles (arrows) attached to the plasma membranes of the epithelial cells only. Scale bars represent 50 μm (200×magnification), 5 μm (2.000×magnification) and 200 nm (60.000×magnification). (C) Barrier function of the human alveolus-on-a-chip model was analyzed by a permeability assay of mock-infected and SARS-CoV-2-infected human alveolus-on-a-chip model using FITC-dextran at 28h p.i., FITC-dextran was measured via the fluorescence intensity (exc. 488nm; em. 518 nm) and depicted as the permeability coefficient ( P app ), calculated according to P app (cm s -1 ) = (dQ/d t ) (1/AC o ). Results show significant higher barrier permeability after SARS-CoV-2 infection. (D) Supernatants of the epithelial- and endothelial side of SARS-CoV-2 infected human alveolus-on-a-chip models were used to perform LDH-assays indicating cell membrane rupture at 28h and 40h p.i.. (E) Progeny virus titers were analyzed in the supernatants of the epithelial- and endothelial layer by standard plaque assay. Shown are means (±SD) of (C) three independent experiments each infected with another patient isolate (5159, 5587, 5588), (D) LDH release, and (E) plaque forming units (PFU/ml). Statistical significance was analyzed by unpaired, two-tailed t-test (*p

    Techniques Used: Infection, Chromatin Immunoprecipitation, Permeability, Fluorescence, Plaque Assay, Two Tailed Test

    SARS-CoV-2 replicates in Vero-76 and Calu-3 cells. Vero-76 (A-D) and Calu-3 (B-D) cells were left uninfected (mock) (B-D) or were infected (A-D) with a SARS-CoV-2 patient isolate (5159) (MOI=1). (A) Transmission electron microscopy was performed 24h post infection (p.i.): (upper panel, scale bar: 5 μm) overview of 3 SARS-CoV-2-infected Vero-76 cells; (lower left panel, scale bar: 200 nm) generation of double membrane vesicles; (lower middle panel, scale bar: 200 nm) virion assembly in the ER–Golgi-intermediate compartment (ERGIC); (lower right panel, scale bar: 200 nm) viral release. (B) SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Immunofluorescence (IF) microscopy was acquired by use of the Axio Observer.Z1 (Zeiss) with a 200×magnification. (C) Total cell lysates were harvested at the times indicated and expression of the spike protein was analyzed by western-blot assay. ERK2 served as loading control. (D) Progeny virus particles were measured in the supernatant by standard plaque assay at the indicated times post infection. Shown are means (±SD) of plaque forming units (PFU) ml -1 of three independent experiments including two biological samples. Statistical significance was analyzed by unpaired, two-tailed t-test (***p
    Figure Legend Snippet: SARS-CoV-2 replicates in Vero-76 and Calu-3 cells. Vero-76 (A-D) and Calu-3 (B-D) cells were left uninfected (mock) (B-D) or were infected (A-D) with a SARS-CoV-2 patient isolate (5159) (MOI=1). (A) Transmission electron microscopy was performed 24h post infection (p.i.): (upper panel, scale bar: 5 μm) overview of 3 SARS-CoV-2-infected Vero-76 cells; (lower left panel, scale bar: 200 nm) generation of double membrane vesicles; (lower middle panel, scale bar: 200 nm) virion assembly in the ER–Golgi-intermediate compartment (ERGIC); (lower right panel, scale bar: 200 nm) viral release. (B) SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Immunofluorescence (IF) microscopy was acquired by use of the Axio Observer.Z1 (Zeiss) with a 200×magnification. (C) Total cell lysates were harvested at the times indicated and expression of the spike protein was analyzed by western-blot assay. ERK2 served as loading control. (D) Progeny virus particles were measured in the supernatant by standard plaque assay at the indicated times post infection. Shown are means (±SD) of plaque forming units (PFU) ml -1 of three independent experiments including two biological samples. Statistical significance was analyzed by unpaired, two-tailed t-test (***p

    Techniques Used: Infection, Transmission Assay, Electron Microscopy, Staining, Immunofluorescence, Microscopy, Expressing, Western Blot, Plaque Assay, Two Tailed Test

    17) Product Images from "Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mice and non-human primates"

    Article Title: Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mice and non-human primates

    Journal: bioRxiv

    doi: 10.1101/2021.01.05.422952

    Quantitative assessment of humoral responses in two mouse strains. (A-B) Monthly monitoring of SARS-CoV-2 RBD-binding IgG titers in 6-10 week-old BALB/c (A) and C57BL/6 (B) mice injected IM with two doses (10 10 gc and 10 11 gc) of AC1 or AC3, n=20 (10 females and 10 males). Mean geometric titers (MGT) shown above each group. (C-D) Pseudovirus neutralizing titers of a subset of BALB/c (C) and C57BL/6 (D) animals (6 females and 6 males per group) from the studies described in A and B. TheGMT are shown above each group. (E-F) Correlation of pseudovirus neutralizing titers and RBD-binding IgG titers in BALB/c (E) and C57BL/6 (F). (G) Live SARS-CoV-2 neutralizing titers measured on a PRNT assay on week 4 samples harvested from BALB/c animals (n≥8, both genders). The GMT is shown above each group. (H) Correlation of SARS-CoV-2 neutralizing and pseudovirus neutralizing titers. (I) Titer of binding antibodies against SARS-CoV-2 RBD (SARS2 RBD), SARS-CoV-2 Spike ectodomain (SARS2 Ecto) and SARS-CoV RBD (SARS RBD) in female BALB/c sera 28 days after AC1 or AC3 injection. (J) RBD-binding antibody titers in BALB/c male animals (n=5) vaccinated with 10 11 gc of AC1 or AAV1-S (same genomic sequence packaged in different capsids), which were naïve (0 mg IVIG) or passively pre-immunized with 15 mg of human IVIG 24h and 2h prior to the vaccination. Ctr: unvaccinated control. (A-J) Data are represented as mean ± SD. For (A-D and G) groups were compared by one-way ANOVA and Tukey’s post-test. * p
    Figure Legend Snippet: Quantitative assessment of humoral responses in two mouse strains. (A-B) Monthly monitoring of SARS-CoV-2 RBD-binding IgG titers in 6-10 week-old BALB/c (A) and C57BL/6 (B) mice injected IM with two doses (10 10 gc and 10 11 gc) of AC1 or AC3, n=20 (10 females and 10 males). Mean geometric titers (MGT) shown above each group. (C-D) Pseudovirus neutralizing titers of a subset of BALB/c (C) and C57BL/6 (D) animals (6 females and 6 males per group) from the studies described in A and B. TheGMT are shown above each group. (E-F) Correlation of pseudovirus neutralizing titers and RBD-binding IgG titers in BALB/c (E) and C57BL/6 (F). (G) Live SARS-CoV-2 neutralizing titers measured on a PRNT assay on week 4 samples harvested from BALB/c animals (n≥8, both genders). The GMT is shown above each group. (H) Correlation of SARS-CoV-2 neutralizing and pseudovirus neutralizing titers. (I) Titer of binding antibodies against SARS-CoV-2 RBD (SARS2 RBD), SARS-CoV-2 Spike ectodomain (SARS2 Ecto) and SARS-CoV RBD (SARS RBD) in female BALB/c sera 28 days after AC1 or AC3 injection. (J) RBD-binding antibody titers in BALB/c male animals (n=5) vaccinated with 10 11 gc of AC1 or AAV1-S (same genomic sequence packaged in different capsids), which were naïve (0 mg IVIG) or passively pre-immunized with 15 mg of human IVIG 24h and 2h prior to the vaccination. Ctr: unvaccinated control. (A-J) Data are represented as mean ± SD. For (A-D and G) groups were compared by one-way ANOVA and Tukey’s post-test. * p

    Techniques Used: Binding Assay, Mouse Assay, Injection, Plaque Reduction Neutralization Test, Sequencing

    Characterization of humoral immune responses in NHP. (A) SARS-CoV-2 RBD-binding IgG titers 20-week follow up in Rhesus macaques (n=2, 1 female and 1 male) treated IM with 10 12 gc of AC1 or AC3. (B) Pseudovirus neutralizing antibody titers in NHPs for 20 weeks (left) and 60 convalescent human plasma samples of patients with different disease severity and NIBSC 20/130 (red dot) reference plasma (right). The Geometric Mean Titer (GMT) is shown for each cohort of convalescent plasma. (C) Live SARS-CoV-2 neutralizing titers. (D) Identification of RBD-binding B cells with a memory phenotype (CD27+ or CD27-IgD-) in peripheral blood of a representative macaque at multiple dates post-vaccination. (E) Frequency of RBD-binding B cells in memory B cell compartment. (F) Frequency of RBD-binding memory B cells with isotype-switched (IgD-IgM-) phenotype. (G) Quantification of S1 subunit concentration (pg/mL) in sera of animals treated with AC3 during the first month after vaccination. (H) Titration of binding antibodies against SARS-CoV-2 RBD (SARS2 RBD), SARS-CoV-2 Spike ectodomain (SARS2 Ecto) and SARS-CoV RBD (SARS RBD) 9 weeks after vaccination. (I) Ratio between RBD-binding IgG1 and IgG4 isotypes 8 weeks post-vaccination.
    Figure Legend Snippet: Characterization of humoral immune responses in NHP. (A) SARS-CoV-2 RBD-binding IgG titers 20-week follow up in Rhesus macaques (n=2, 1 female and 1 male) treated IM with 10 12 gc of AC1 or AC3. (B) Pseudovirus neutralizing antibody titers in NHPs for 20 weeks (left) and 60 convalescent human plasma samples of patients with different disease severity and NIBSC 20/130 (red dot) reference plasma (right). The Geometric Mean Titer (GMT) is shown for each cohort of convalescent plasma. (C) Live SARS-CoV-2 neutralizing titers. (D) Identification of RBD-binding B cells with a memory phenotype (CD27+ or CD27-IgD-) in peripheral blood of a representative macaque at multiple dates post-vaccination. (E) Frequency of RBD-binding B cells in memory B cell compartment. (F) Frequency of RBD-binding memory B cells with isotype-switched (IgD-IgM-) phenotype. (G) Quantification of S1 subunit concentration (pg/mL) in sera of animals treated with AC3 during the first month after vaccination. (H) Titration of binding antibodies against SARS-CoV-2 RBD (SARS2 RBD), SARS-CoV-2 Spike ectodomain (SARS2 Ecto) and SARS-CoV RBD (SARS RBD) 9 weeks after vaccination. (I) Ratio between RBD-binding IgG1 and IgG4 isotypes 8 weeks post-vaccination.

    Techniques Used: Binding Assay, Concentration Assay, Titration

    Quality of the host response to AAVCOVID. (A) Several RBD-binding antibody isotype titers (IgG, IgG1, IgG2a, IgG2b, IgG3, IgA and IgM) measured weekly in 6-10 week-old BALB/c (n=10, 5 females and 5 males) treated IM with two doses of AC1 and AC3. (B) Ratio of RBD-binding IgG2a and IgG1 antibody titers in serum samples harvested 28 days after vaccination of BALB/c mice as described in A. The Geometric Mean Titer (GMT) is shown above each group. (C and F) Cytokine concentration (pg/mL) in supernatants harvested from splenocytes stimulated for 48h with peptides spanning SARS-CoV-2 Spike protein. Splenocytes were extracted from BALB/c (C) and C57BL/6 (F) animals 4 and 6 weeks, respectively, after vaccination with 10 11 gc of AC1 or AC3. (D-E) Spot forming units (SFU) detected by IFN-γ (D) or IL-4 (E) ELISpot in splenocytes extracted from BALB/c animals 4 weeks after vaccination with 10 11 gc of AC1 or AC3 and stimulated with peptides spanning SARS-CoV-2 Spike protein for 48h. (G-H) Spot forming units (SFU) detected by IFN-γ (G) or IL-4 (H) ELISpot in splenocytes extracted from C57BL/6 animals 6 weeks after vaccination with 10 10 gc of AC1 or AC3 and stimulated with peptides spanning SARS-CoV-2 Spike protein for 48h. For (B-H) data are represented as mean ± SD and groups were compared by Kruskal Wallis and Dunn’s post-test.
    Figure Legend Snippet: Quality of the host response to AAVCOVID. (A) Several RBD-binding antibody isotype titers (IgG, IgG1, IgG2a, IgG2b, IgG3, IgA and IgM) measured weekly in 6-10 week-old BALB/c (n=10, 5 females and 5 males) treated IM with two doses of AC1 and AC3. (B) Ratio of RBD-binding IgG2a and IgG1 antibody titers in serum samples harvested 28 days after vaccination of BALB/c mice as described in A. The Geometric Mean Titer (GMT) is shown above each group. (C and F) Cytokine concentration (pg/mL) in supernatants harvested from splenocytes stimulated for 48h with peptides spanning SARS-CoV-2 Spike protein. Splenocytes were extracted from BALB/c (C) and C57BL/6 (F) animals 4 and 6 weeks, respectively, after vaccination with 10 11 gc of AC1 or AC3. (D-E) Spot forming units (SFU) detected by IFN-γ (D) or IL-4 (E) ELISpot in splenocytes extracted from BALB/c animals 4 weeks after vaccination with 10 11 gc of AC1 or AC3 and stimulated with peptides spanning SARS-CoV-2 Spike protein for 48h. (G-H) Spot forming units (SFU) detected by IFN-γ (G) or IL-4 (H) ELISpot in splenocytes extracted from C57BL/6 animals 6 weeks after vaccination with 10 10 gc of AC1 or AC3 and stimulated with peptides spanning SARS-CoV-2 Spike protein for 48h. For (B-H) data are represented as mean ± SD and groups were compared by Kruskal Wallis and Dunn’s post-test.

    Techniques Used: Binding Assay, Mouse Assay, Concentration Assay, Enzyme-linked Immunospot

    Composition and characterization of AAVCOVID vaccine candidates. (A) Schematic representation of the recombinant genome of AAVCOVID19-1 (AC1) and AAVCOVID19-3 (AC3) vaccine candidates. SV40: Simian virus 40 promoter. RBD: receptor binding domain. S1: SARS-CoV-2 Spike subunit 1. S2: SARS-CoV-2 Spike subunit 2. CMV: cytomegalovirus promoter. tPA-SP: tissue plasminogen activator signal peptide. WPRE: woodchuck hepatitis virus posttranscriptional regulatory element. bGH: bovine growth hormone. ITR: inverted terminal repeat. (B) Phylogenetic tree of several AAV clades and percentage of sequence identity with AAVrh32.33. (C) Percentage of seropositivity of neutralizing antibodies and titer range against AAV2, AAV8 and AAVrh32.33 among 50 donor plasma samples. (D) Productivity of several AC1 and AC3 (vector genome copies produced per producer cell or Gc/cell) compared to various AAV serotypes carrying a CMV-EGFP-WPRE transgene in small scale production and purification. Data are represented as mean ± SD. One-way ANOVA and Tukey’s tests were used to compare groups between them. * p
    Figure Legend Snippet: Composition and characterization of AAVCOVID vaccine candidates. (A) Schematic representation of the recombinant genome of AAVCOVID19-1 (AC1) and AAVCOVID19-3 (AC3) vaccine candidates. SV40: Simian virus 40 promoter. RBD: receptor binding domain. S1: SARS-CoV-2 Spike subunit 1. S2: SARS-CoV-2 Spike subunit 2. CMV: cytomegalovirus promoter. tPA-SP: tissue plasminogen activator signal peptide. WPRE: woodchuck hepatitis virus posttranscriptional regulatory element. bGH: bovine growth hormone. ITR: inverted terminal repeat. (B) Phylogenetic tree of several AAV clades and percentage of sequence identity with AAVrh32.33. (C) Percentage of seropositivity of neutralizing antibodies and titer range against AAV2, AAV8 and AAVrh32.33 among 50 donor plasma samples. (D) Productivity of several AC1 and AC3 (vector genome copies produced per producer cell or Gc/cell) compared to various AAV serotypes carrying a CMV-EGFP-WPRE transgene in small scale production and purification. Data are represented as mean ± SD. One-way ANOVA and Tukey’s tests were used to compare groups between them. * p

    Techniques Used: Recombinant, Binding Assay, Sequencing, Plasmid Preparation, Produced, Purification

    18) Product Images from "Prunella vulgaris extract and suramin block SARS-coronavirus 2 virus Spike protein D614 and G614 variants mediated receptor association and virus entry in cell culture system"

    Article Title: Prunella vulgaris extract and suramin block SARS-coronavirus 2 virus Spike protein D614 and G614 variants mediated receptor association and virus entry in cell culture system

    Journal: bioRxiv

    doi: 10.1101/2020.08.28.270306

    SARS-CoV-2 SP-PVs’s infection in different cell lines and SARS-CoV-2 SP G614 variant exhibited stronger virus entry. A) 293T, 293T ACE2 and Vero-E6 cells were infected by equal amounts of SARS-CoV-2SP-, SARS-CoV-2SPΔC-pseudotyped viruses. At 48 hrs pi, the Gluc activity in supernatants was measured. B) the expression of SARS-CoV-2SP receptor, ACE2, in 293T, 293T ACE2 and Vero-E6 cells detected by WB with anti-ACE2 antibodies. C) The SPΔC G614 -GFP + PVs were produced with 293T cells and used to infect 293T ACE2 cells in 96-well plate After 48 hrs pi, GFP-positive cells (per well) were counted and photographed by fluorescence microscope (on the top of the panel). D) Detection of SARS-CoV-2 SPΔC, SPΔC G614 and HIV p24 protein expression in transfected 293T cells and viral particles by WB. E) Infectivity comparison of SPΔC-PVs and SPΔC G614 -PVs in 293T ACE2 cells. Equal amounts of SPΔC D614 -PVs and SPΔC G614 -PVs virions (adjusted by p24 level) were used to infect 293T ACE2 cells. At different days post-infection (pi), Gluc activity in supernatants was measured.
    Figure Legend Snippet: SARS-CoV-2 SP-PVs’s infection in different cell lines and SARS-CoV-2 SP G614 variant exhibited stronger virus entry. A) 293T, 293T ACE2 and Vero-E6 cells were infected by equal amounts of SARS-CoV-2SP-, SARS-CoV-2SPΔC-pseudotyped viruses. At 48 hrs pi, the Gluc activity in supernatants was measured. B) the expression of SARS-CoV-2SP receptor, ACE2, in 293T, 293T ACE2 and Vero-E6 cells detected by WB with anti-ACE2 antibodies. C) The SPΔC G614 -GFP + PVs were produced with 293T cells and used to infect 293T ACE2 cells in 96-well plate After 48 hrs pi, GFP-positive cells (per well) were counted and photographed by fluorescence microscope (on the top of the panel). D) Detection of SARS-CoV-2 SPΔC, SPΔC G614 and HIV p24 protein expression in transfected 293T cells and viral particles by WB. E) Infectivity comparison of SPΔC-PVs and SPΔC G614 -PVs in 293T ACE2 cells. Equal amounts of SPΔC D614 -PVs and SPΔC G614 -PVs virions (adjusted by p24 level) were used to infect 293T ACE2 cells. At different days post-infection (pi), Gluc activity in supernatants was measured.

    Techniques Used: Infection, Variant Assay, Activity Assay, Expressing, Western Blot, Produced, Fluorescence, Microscopy, Transfection

    SARS-CoV-2-SP-PV’s infection was efficiently blocked by CHPV and suramin. A) Images of the dried Prunella Vulgaris flowers and its water extract (CHPV). B) Dose -response anti-SARS-CoV-2 analysis by Gluc activity for CHPV or suramin. 293T ACE2 cells were infected by equal amounts of SARS-CoV-2SPΔC-pseudotyped viruses in the presence of different dose of CHPV or suramin. At 48 hrs pi, the Gluc activity in supernatants was measured. (% inhibition = 100 ⨯ [1 - (Gluc value in presence of drug)/(Gluc value in absence of drug)). C) Infection inhibition of CHPV or suramin on SARS-CoV-2-SPΔC G614 -PVs in 293T ACE2 cells. Equal amounts of SCoV-2-SPΔC G614 -PVs (adjusted by p24 level) were used to infect 293T ACE2 cells in presence of different concentrations of CHPV or suramin, in indicated at bottom of the panel. At 48 hrs pi, Gluc activity in supernatants was measured and present as % inhibition. Means ± SD were calculated from duplicate experiments. D) 293T ACE2 cells in 96-well plate were infected with SPΔC G614 -GFP + PVs. After 48 hrs pi, GFP-positive cells (per well) were counted (left panel) and photographed by fluorescence microscope (right panel, a. Without drugs; b. Without infection; c. In the presence of CHPV (100 μg/ml ) ; d. In the presence of suramin (100 μg/ml ) .
    Figure Legend Snippet: SARS-CoV-2-SP-PV’s infection was efficiently blocked by CHPV and suramin. A) Images of the dried Prunella Vulgaris flowers and its water extract (CHPV). B) Dose -response anti-SARS-CoV-2 analysis by Gluc activity for CHPV or suramin. 293T ACE2 cells were infected by equal amounts of SARS-CoV-2SPΔC-pseudotyped viruses in the presence of different dose of CHPV or suramin. At 48 hrs pi, the Gluc activity in supernatants was measured. (% inhibition = 100 ⨯ [1 - (Gluc value in presence of drug)/(Gluc value in absence of drug)). C) Infection inhibition of CHPV or suramin on SARS-CoV-2-SPΔC G614 -PVs in 293T ACE2 cells. Equal amounts of SCoV-2-SPΔC G614 -PVs (adjusted by p24 level) were used to infect 293T ACE2 cells in presence of different concentrations of CHPV or suramin, in indicated at bottom of the panel. At 48 hrs pi, Gluc activity in supernatants was measured and present as % inhibition. Means ± SD were calculated from duplicate experiments. D) 293T ACE2 cells in 96-well plate were infected with SPΔC G614 -GFP + PVs. After 48 hrs pi, GFP-positive cells (per well) were counted (left panel) and photographed by fluorescence microscope (right panel, a. Without drugs; b. Without infection; c. In the presence of CHPV (100 μg/ml ) ; d. In the presence of suramin (100 μg/ml ) .

    Techniques Used: Infection, Activity Assay, Inhibition, Fluorescence, Microscopy

    Characterization of the mechanisms of CHPV and suramin for their anti-SARS-COV-2-SP action. A) Time-dependent inhibition of SPΔC G614 -PVs infection mediated by CHPV or suramin. CHPV (100 μg/mL) or suramin (100 μg/mL) was added at 1 hr prior to infection, during infection (0 hr), and at 1 hr, and 3 hr pi. The positive controls (PC) were 293T ACE2 cells infected with SPΔC G614 -PVs in the absence of compounds. At 3 hrs pi, all of the cell cultures were replaced with fresh DMEM and cultured for 48 hrs. Then, the Gluc activity was monitored in the supernatant, and the data are shown as a percentage of inhibition (%). B) inhibitory effect of CHPV or suramin on SARS-CoV2-SP/ACE2 binding by ELISA as described in materials and methods. nAB: anti-COVID-19 neutralizing antibody (SAD-S35). The results are the mean ± SD of duplicate samples, and the data are representative of results obtained in two independent experiments.
    Figure Legend Snippet: Characterization of the mechanisms of CHPV and suramin for their anti-SARS-COV-2-SP action. A) Time-dependent inhibition of SPΔC G614 -PVs infection mediated by CHPV or suramin. CHPV (100 μg/mL) or suramin (100 μg/mL) was added at 1 hr prior to infection, during infection (0 hr), and at 1 hr, and 3 hr pi. The positive controls (PC) were 293T ACE2 cells infected with SPΔC G614 -PVs in the absence of compounds. At 3 hrs pi, all of the cell cultures were replaced with fresh DMEM and cultured for 48 hrs. Then, the Gluc activity was monitored in the supernatant, and the data are shown as a percentage of inhibition (%). B) inhibitory effect of CHPV or suramin on SARS-CoV2-SP/ACE2 binding by ELISA as described in materials and methods. nAB: anti-COVID-19 neutralizing antibody (SAD-S35). The results are the mean ± SD of duplicate samples, and the data are representative of results obtained in two independent experiments.

    Techniques Used: Inhibition, Infection, Cell Culture, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

    Generation of a SARS-COV2-SP-pseudotyped lentiviruse particles (SCoV-2-SP-PVs). A) Schematic representation of SARS-CoV-2SP, SARS-CoV-2SPΔC, and SARS-CoV-2SP G614 ΔC expressing plasmids. B) Schematic representation of plasmids and and procedures for production of SARS-COV2-SP-pseudotyped lentivirus particles (SCoV-2-SP-PVs). C) Detection of SARS-CoV-2 SPs and HIV p24 protein expression in transfected 293T cells and viral particles by Western blot (WB) with anti-SP or anti-p24 antibodies. D) Different amounts of SCoV-2-SP-PVs and SCoV-2-SPΔC-PVs virions (adjusted by p24) were used to infect 293T ACE2 cells. At different time intervels, the Gaussia Luciferase activity (Gluc) (left panel) and PVs-associated p24 (at 72 hrs) in supernatants was measured.
    Figure Legend Snippet: Generation of a SARS-COV2-SP-pseudotyped lentiviruse particles (SCoV-2-SP-PVs). A) Schematic representation of SARS-CoV-2SP, SARS-CoV-2SPΔC, and SARS-CoV-2SP G614 ΔC expressing plasmids. B) Schematic representation of plasmids and and procedures for production of SARS-COV2-SP-pseudotyped lentivirus particles (SCoV-2-SP-PVs). C) Detection of SARS-CoV-2 SPs and HIV p24 protein expression in transfected 293T cells and viral particles by Western blot (WB) with anti-SP or anti-p24 antibodies. D) Different amounts of SCoV-2-SP-PVs and SCoV-2-SPΔC-PVs virions (adjusted by p24) were used to infect 293T ACE2 cells. At different time intervels, the Gaussia Luciferase activity (Gluc) (left panel) and PVs-associated p24 (at 72 hrs) in supernatants was measured.

    Techniques Used: Expressing, Transfection, Western Blot, Luciferase, Activity Assay

    Inhibitory effect of CHPV and Suramin on SARS-CoV-2 infection-induced cytopathic effects. Vero cells were infected with a wild type SARS-CoV-2 virus (hCoV-19/Canada/ON-VIDO-01/2020) in the presence or absence of different concentrations of CHPV and Suramin. After 72 hrs pi., the SARS-CoV-2 infection-induced cytopathic effects in Vero cells were monitored. Error bars represent variation between triplicate samples, and the data of (A) and (B) are representative of results obtained in two independent experiments.
    Figure Legend Snippet: Inhibitory effect of CHPV and Suramin on SARS-CoV-2 infection-induced cytopathic effects. Vero cells were infected with a wild type SARS-CoV-2 virus (hCoV-19/Canada/ON-VIDO-01/2020) in the presence or absence of different concentrations of CHPV and Suramin. After 72 hrs pi., the SARS-CoV-2 infection-induced cytopathic effects in Vero cells were monitored. Error bars represent variation between triplicate samples, and the data of (A) and (B) are representative of results obtained in two independent experiments.

    Techniques Used: Infection

    19) Product Images from "An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2"

    Article Title: An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2

    Journal: bioRxiv

    doi: 10.1101/2020.08.19.258244

    The immunogenicity, efficacy and safety of C-Vac for SARS-CoV-2 infection. (A) Flow cytometry-based detection of MHC(HLA-A2)-peptide complex binding affinity in T2 cells. (B) The expression of antigens in 293T-based C-Vac is confirmed by Western Blot. N, 293T cells transfected with the plasmid expressing a full length N gene. (C D) Pseudovirus neutralization titers of hamster serum at day 7 after the first immunization and day 21 (boosted at day 14) after vaccination with 293T-based C-Vac, MiT C-Vac: Mitomycin C-treated C-Vac, Lys C-Vac: Lysed C-Vac. (E) Histological characteristics of hamster lung at day 7 after the first vaccination. Original magnification 200× (F) Allograft volume of transformed fibroblasts expressing RBD-truncated N protein in the Syrian hamsters immunized with different regime. 5×10 6 BHK21 cells expressing C-Vac antigen (RBD-Ntap) were subcutaneously injected into immunized hamsters for challenge at day 45 after boost, and the volume of allografts were measured at 14 days after inoculation of the BHK21 cells expressing RBD-Ntap into the immunized hamsters.
    Figure Legend Snippet: The immunogenicity, efficacy and safety of C-Vac for SARS-CoV-2 infection. (A) Flow cytometry-based detection of MHC(HLA-A2)-peptide complex binding affinity in T2 cells. (B) The expression of antigens in 293T-based C-Vac is confirmed by Western Blot. N, 293T cells transfected with the plasmid expressing a full length N gene. (C D) Pseudovirus neutralization titers of hamster serum at day 7 after the first immunization and day 21 (boosted at day 14) after vaccination with 293T-based C-Vac, MiT C-Vac: Mitomycin C-treated C-Vac, Lys C-Vac: Lysed C-Vac. (E) Histological characteristics of hamster lung at day 7 after the first vaccination. Original magnification 200× (F) Allograft volume of transformed fibroblasts expressing RBD-truncated N protein in the Syrian hamsters immunized with different regime. 5×10 6 BHK21 cells expressing C-Vac antigen (RBD-Ntap) were subcutaneously injected into immunized hamsters for challenge at day 45 after boost, and the volume of allografts were measured at 14 days after inoculation of the BHK21 cells expressing RBD-Ntap into the immunized hamsters.

    Techniques Used: Infection, Flow Cytometry, Binding Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Neutralization, Transformation Assay, Injection

    The RBD domain of Spike is crucial for the SARS-CoV2 Vaccine. (A) The functional domain of SARS-CoV-2 spike protein. (B) Potential B cell antigen of RBD domain from SARS-CoV2 is predicted by Discotope software based on their 3D structure. (C) Potential linear B cell epitopes of SARS-CoV-2 full S protein are analysed with the IEDB database. (D) The location of potential antigens in RBD domain (SARS-CoV-2:red, SARS-CoV: Purple) and interaction model between RBD and ACE2 receptor (interface is marked yellow) are marked with Discovery Studio. (E) The expression of Spike and nucleocapsid with wild-type sequence in 293T cells are detected by Western Blot assay. (F) The expression of Spike and its derivatives with codon optimization (opt).
    Figure Legend Snippet: The RBD domain of Spike is crucial for the SARS-CoV2 Vaccine. (A) The functional domain of SARS-CoV-2 spike protein. (B) Potential B cell antigen of RBD domain from SARS-CoV2 is predicted by Discotope software based on their 3D structure. (C) Potential linear B cell epitopes of SARS-CoV-2 full S protein are analysed with the IEDB database. (D) The location of potential antigens in RBD domain (SARS-CoV-2:red, SARS-CoV: Purple) and interaction model between RBD and ACE2 receptor (interface is marked yellow) are marked with Discovery Studio. (E) The expression of Spike and nucleocapsid with wild-type sequence in 293T cells are detected by Western Blot assay. (F) The expression of Spike and its derivatives with codon optimization (opt).

    Techniques Used: Functional Assay, Software, Expressing, Sequencing, Western Blot

    Construction of chimeric vaccine for SARS-CoV-2. (A) Potential B-cell epitopes of N protein is predicted by IEDB database. (B) Potential MHCI-binding peptides of N. (C) Functional domain of SARS-CoV N protein (Upper) and its antibody epitope map reported in previous study. (D) The skeleton of Chimeric Vaccine for SARS-CoV-2, RBD: spike RBD domain (306-541 aa), Ntap: T-cell-associated peptide of N (211-339 aa). (E) Characterization of SARS-CoV-2-derived protein and C-Vac antigen by SARS-CoV-2 antisera and commercial antibodies against SARS-CoV2 spike RBD or Nucleocapsid.
    Figure Legend Snippet: Construction of chimeric vaccine for SARS-CoV-2. (A) Potential B-cell epitopes of N protein is predicted by IEDB database. (B) Potential MHCI-binding peptides of N. (C) Functional domain of SARS-CoV N protein (Upper) and its antibody epitope map reported in previous study. (D) The skeleton of Chimeric Vaccine for SARS-CoV-2, RBD: spike RBD domain (306-541 aa), Ntap: T-cell-associated peptide of N (211-339 aa). (E) Characterization of SARS-CoV-2-derived protein and C-Vac antigen by SARS-CoV-2 antisera and commercial antibodies against SARS-CoV2 spike RBD or Nucleocapsid.

    Techniques Used: Binding Assay, Functional Assay, Derivative Assay

    20) Product Images from "Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii"

    Article Title: Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii

    Journal: bioRxiv

    doi: 10.1101/2021.01.29.428890

    (A) Diagram of the SARS-CoV-2 viral particle with crystal structure of the spike (S) protein highlighted with Subunits 1 and 2 indicated (S1, S2, respectively). The Receptor Binding Domain (RBD) is located in the more variable subunit 1. (B) Vector design and construction. The peptide structure of the spike protein indicating the N-Terminal Domain (NTD), Receptor Binding Domain (RBD), Fusion Peptide (FP, Homology region 1 and 2 (HR1, HR2), Transmembrane association domain (TA) and Intracellular Terminal (IT). A C. reinhardtii nuclear codon optimized version of the RBD-coding sequence was cloned in to a vector containing the AR1 promoter (P AR1 ) driving a transcriptional fusion of the Bleomycin resistance gene (BleR), FMDV Foot-and-mouth disease virus 2A (F2A) ribosomal-skip motif and 5’ mClover green fluorescent protein tag. A separate Beta-tubulin2 promoter driving Hygromycin resistance was used for secondary selection. Three different versions of the RBD were generated. A chloroplast-directed version through N-terminal fusion of the PsaE chloroplast transit sequence, a secreted version by the addition of the PHC2 secretion signal peptide, and an ER-Golgi system retained version by the subsequent addition of a C-terminal KDEL Golgi retention sequence. (C) Schematic summarizing transformation process and timeline including drug selection, clone down selection through 96-well microtiter plates, and then flask-scale characterization of candidate RBD-expressing lines.
    Figure Legend Snippet: (A) Diagram of the SARS-CoV-2 viral particle with crystal structure of the spike (S) protein highlighted with Subunits 1 and 2 indicated (S1, S2, respectively). The Receptor Binding Domain (RBD) is located in the more variable subunit 1. (B) Vector design and construction. The peptide structure of the spike protein indicating the N-Terminal Domain (NTD), Receptor Binding Domain (RBD), Fusion Peptide (FP, Homology region 1 and 2 (HR1, HR2), Transmembrane association domain (TA) and Intracellular Terminal (IT). A C. reinhardtii nuclear codon optimized version of the RBD-coding sequence was cloned in to a vector containing the AR1 promoter (P AR1 ) driving a transcriptional fusion of the Bleomycin resistance gene (BleR), FMDV Foot-and-mouth disease virus 2A (F2A) ribosomal-skip motif and 5’ mClover green fluorescent protein tag. A separate Beta-tubulin2 promoter driving Hygromycin resistance was used for secondary selection. Three different versions of the RBD were generated. A chloroplast-directed version through N-terminal fusion of the PsaE chloroplast transit sequence, a secreted version by the addition of the PHC2 secretion signal peptide, and an ER-Golgi system retained version by the subsequent addition of a C-terminal KDEL Golgi retention sequence. (C) Schematic summarizing transformation process and timeline including drug selection, clone down selection through 96-well microtiter plates, and then flask-scale characterization of candidate RBD-expressing lines.

    Techniques Used: Binding Assay, Plasmid Preparation, Sequencing, Clone Assay, Selection, Generated, Transformation Assay, Expressing

    21) Product Images from "Immune response to vaccine candidates based on different types of nanoscaffolded RBD domain of the SARS-CoV-2 spike protein"

    Article Title: Immune response to vaccine candidates based on different types of nanoscaffolded RBD domain of the SARS-CoV-2 spike protein

    Journal: bioRxiv

    doi: 10.1101/2020.08.28.244269

    Neutralization of binding of viral RBD to the ACE2 receptor and inhibition of pseudoviral infection of cells by mouse antisera. A) Sera of mice immunized with DNA vaccines comprising scaffolded RBD were diluted and pre-incubated with Spike protein. Afterwards, Spike that bound to ACE2 was detected using streptactin-HRP. Mean and SEM of 6 (RBD-AaLs) or 5 (all others) biological replicates are shown (A) Sera of mice immunized with DNA vaccines comprising scaffolded RBD were diluted 50-fold and Spike-pseudotyped virus infection of ACE2 and TMPRSS2 –transfected HEK293 cells was followed by luminescence. Mean and SEM of 6 (RBD-AaLs) or 5 (RBD-bann, RBD-foldon-RBD, RBD-ferritin) or 4 (empty pcDNA3 vector, RBD) biological replicates are shown (B). *P
    Figure Legend Snippet: Neutralization of binding of viral RBD to the ACE2 receptor and inhibition of pseudoviral infection of cells by mouse antisera. A) Sera of mice immunized with DNA vaccines comprising scaffolded RBD were diluted and pre-incubated with Spike protein. Afterwards, Spike that bound to ACE2 was detected using streptactin-HRP. Mean and SEM of 6 (RBD-AaLs) or 5 (all others) biological replicates are shown (A) Sera of mice immunized with DNA vaccines comprising scaffolded RBD were diluted 50-fold and Spike-pseudotyped virus infection of ACE2 and TMPRSS2 –transfected HEK293 cells was followed by luminescence. Mean and SEM of 6 (RBD-AaLs) or 5 (RBD-bann, RBD-foldon-RBD, RBD-ferritin) or 4 (empty pcDNA3 vector, RBD) biological replicates are shown (B). *P

    Techniques Used: Neutralization, Binding Assay, Inhibition, Infection, Mouse Assay, Incubation, Transfection, Plasmid Preparation

    Total IgG in mice that underwent switch immunization. Mice were immunized with combinations of differently scaffolded RBD plasmid DNA (β-annulus and foldon) for prime and boost immunization. Titers of antibodies against RBD after prime and boost (A,B) and against Spike protein (C, D) were determined via ELISA. Graphs represent mean of EPT of group of mice (n=6 per group). Each dot represents an individual animal. To determine NS, Mann-Whitney test was performed.
    Figure Legend Snippet: Total IgG in mice that underwent switch immunization. Mice were immunized with combinations of differently scaffolded RBD plasmid DNA (β-annulus and foldon) for prime and boost immunization. Titers of antibodies against RBD after prime and boost (A,B) and against Spike protein (C, D) were determined via ELISA. Graphs represent mean of EPT of group of mice (n=6 per group). Each dot represents an individual animal. To determine NS, Mann-Whitney test was performed.

    Techniques Used: Mouse Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Analysis of different classes of antibodies against RBD for different scaffolded RBDs and immunization by the scaffold. Mice were immunized with different combination of RBD plasmid DNA. End point titers 6 weeks after the first immunization of IgA (A), IgM (B), IgG1 (C), IgG2b (D) and IgG3 (E) against RBD protein were determined by ELISA. Graphs represent mean of EPT of group of mice (n=5 per group). Each dot represents an individual animal.
    Figure Legend Snippet: Analysis of different classes of antibodies against RBD for different scaffolded RBDs and immunization by the scaffold. Mice were immunized with different combination of RBD plasmid DNA. End point titers 6 weeks after the first immunization of IgA (A), IgM (B), IgG1 (C), IgG2b (D) and IgG3 (E) against RBD protein were determined by ELISA. Graphs represent mean of EPT of group of mice (n=5 per group). Each dot represents an individual animal.

    Techniques Used: Mouse Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

    Total IgG against scaffold in mice that underwent switch immunization. Mice were immunized with combinations of differently scaffolded RBD plasmid DNA (β-annulus and foldon) for prime and boost immunization and vice versa. Titers of antibodies against scaffold (depicted in blue) after prime and boost were determined via ELISA. Graphs represent mean of EPT of group of mice (n=6 per group). Each dot represents an individual animal.
    Figure Legend Snippet: Total IgG against scaffold in mice that underwent switch immunization. Mice were immunized with combinations of differently scaffolded RBD plasmid DNA (β-annulus and foldon) for prime and boost immunization and vice versa. Titers of antibodies against scaffold (depicted in blue) after prime and boost were determined via ELISA. Graphs represent mean of EPT of group of mice (n=6 per group). Each dot represents an individual animal.

    Techniques Used: Mouse Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

    Protection of pseudoviral infection by DNA plasmid immunization in a mouse model. Mice were immunized by two injections of plasmids separated by two weeks. After one month hACE2 and TMPRRS was introduced by intranasal plasmid transfection followed by intranasal infection with SARS_CoV-2 S-typed virus (PV). Luminescence based on pseudovirus intranasal infection was measured after 24 hrs (A). Bioluminescence imaging revealing the protective state of immunized animals against pseudovirus infection in animals. Subsequent quantification of bioluminescence average radiance was carried out (B, C). Dashed line represent merging of pictures of mice from the same test group taken separately. Each dot represents an individual animal (pcDNA3 n=4; RBD and RBD-bann n=5). **P
    Figure Legend Snippet: Protection of pseudoviral infection by DNA plasmid immunization in a mouse model. Mice were immunized by two injections of plasmids separated by two weeks. After one month hACE2 and TMPRRS was introduced by intranasal plasmid transfection followed by intranasal infection with SARS_CoV-2 S-typed virus (PV). Luminescence based on pseudovirus intranasal infection was measured after 24 hrs (A). Bioluminescence imaging revealing the protective state of immunized animals against pseudovirus infection in animals. Subsequent quantification of bioluminescence average radiance was carried out (B, C). Dashed line represent merging of pictures of mice from the same test group taken separately. Each dot represents an individual animal (pcDNA3 n=4; RBD and RBD-bann n=5). **P

    Techniques Used: Infection, Plasmid Preparation, Mouse Assay, Transfection, Imaging

    Titer of total IgG antibodies against the RBD and Spike protein for immunization with plasmids for different scaffolded RBDs and scaffold alone. Mice were immunized with different combination of RBD plasmid DNA, complexed with jetPEI- in vivo transfection reagent, according to immunization protocol (A). End point titer (EPT) for total IgG against RBD (B-D) and against Spike protein (E-G). Graphs represent mean of EPT of group of mice (n=5 per group). Each dot represents an individual animal. *P
    Figure Legend Snippet: Titer of total IgG antibodies against the RBD and Spike protein for immunization with plasmids for different scaffolded RBDs and scaffold alone. Mice were immunized with different combination of RBD plasmid DNA, complexed with jetPEI- in vivo transfection reagent, according to immunization protocol (A). End point titer (EPT) for total IgG against RBD (B-D) and against Spike protein (E-G). Graphs represent mean of EPT of group of mice (n=5 per group). Each dot represents an individual animal. *P

    Techniques Used: Mouse Assay, Plasmid Preparation, In Vivo, Transfection

    DNA plasmid immunization with naked DNA. Mice were immunized with 20 μg per animal of naked DNA (empty vector, RBD, RBD-bann), dissolved in 150 mM NaCl. End point titer (EPT) for total IgG against RBD (A) and against Spike protein (B) were determined by ELISA. Graphs represent mean of EPT of group of mice (n=6 per group). Each dot represents an individual animal. *P
    Figure Legend Snippet: DNA plasmid immunization with naked DNA. Mice were immunized with 20 μg per animal of naked DNA (empty vector, RBD, RBD-bann), dissolved in 150 mM NaCl. End point titer (EPT) for total IgG against RBD (A) and against Spike protein (B) were determined by ELISA. Graphs represent mean of EPT of group of mice (n=6 per group). Each dot represents an individual animal. *P

    Techniques Used: Plasmid Preparation, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Secretion of RDB protein domains fused to different scaffolding proteins produced in plasmid-transfected mammalian cells and size analysis of the isolated RBD-bann protein. Supernatant of HEK293 cells transfected with indicated construct was harvested 3 days post transfection and the presence of differently scaffolded RBD domain variants was detected with anti RBD antibodies (A). Size analysis of the purified RBD-bann by DLS confirms the presence of particles around 500 nm (B).
    Figure Legend Snippet: Secretion of RDB protein domains fused to different scaffolding proteins produced in plasmid-transfected mammalian cells and size analysis of the isolated RBD-bann protein. Supernatant of HEK293 cells transfected with indicated construct was harvested 3 days post transfection and the presence of differently scaffolded RBD domain variants was detected with anti RBD antibodies (A). Size analysis of the purified RBD-bann by DLS confirms the presence of particles around 500 nm (B).

    Techniques Used: Scaffolding, Produced, Plasmid Preparation, Transfection, Isolation, Construct, Purification

    22) Product Images from "Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response"

    Article Title: Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response

    Journal: bioRxiv

    doi: 10.1101/2020.06.27.175166

    Construction and characterization of MVA/S and MVA/S1 recombinants. (A) Schematic representation of MVA/S and MVA/S1. Recombinant inserts were cloned in the essential region in between 18R and G1L under mH5 promoter. (B) Representative flow plots showing the expression of membrane anchored spike on the surface and S1 intracellularly. (C) Westernblotting analysis of expressed proteins in supernatants and lysates of MVA infected cells. (D) Size-exclusion chromatography analysis of S1 protein expressed by MVA/S1. (E) Binding of hACE2 to MVA/S expressing cells.
    Figure Legend Snippet: Construction and characterization of MVA/S and MVA/S1 recombinants. (A) Schematic representation of MVA/S and MVA/S1. Recombinant inserts were cloned in the essential region in between 18R and G1L under mH5 promoter. (B) Representative flow plots showing the expression of membrane anchored spike on the surface and S1 intracellularly. (C) Westernblotting analysis of expressed proteins in supernatants and lysates of MVA infected cells. (D) Size-exclusion chromatography analysis of S1 protein expressed by MVA/S1. (E) Binding of hACE2 to MVA/S expressing cells.

    Techniques Used: Recombinant, Clone Assay, Expressing, Infection, Size-exclusion Chromatography, Binding Assay

    23) Product Images from "mRNA vaccine CVnCoV protects non-human primates from SARS-CoV-2 challenge infection"

    Article Title: mRNA vaccine CVnCoV protects non-human primates from SARS-CoV-2 challenge infection

    Journal: bioRxiv

    doi: 10.1101/2020.12.23.424138

    CVnCoV induces humoral response in non-human primates. (A) Schematic drawing of study setup. Rhesus macaques (n=6; 3 male, 3 female/group) were vaccinated IM on day 0 and day 28 with 0.5 μg or 8 μg of CVnCoV or remained unvaccinated. All animals were challenge with 5.0 x 106 PFU of SARS-CoV-2 on d56. Two animals of each group were terminated on d62, d63 and d64, respectively (B) Trimeric Spike protein or (C) RBD specific binding IgG antibodies, displayed as endpoint titres at different time points as indicated (C) Virus neutralising antibodies determined via focus reduction neutralisation test at different time points as indicated. All values are displayed as median with range. Square symbols represent male, round symbols female animals. Dotted lines represent vaccinations and challenge infection, respectively. RBD receptor binding domain; VNT virus neutralising titre
    Figure Legend Snippet: CVnCoV induces humoral response in non-human primates. (A) Schematic drawing of study setup. Rhesus macaques (n=6; 3 male, 3 female/group) were vaccinated IM on day 0 and day 28 with 0.5 μg or 8 μg of CVnCoV or remained unvaccinated. All animals were challenge with 5.0 x 106 PFU of SARS-CoV-2 on d56. Two animals of each group were terminated on d62, d63 and d64, respectively (B) Trimeric Spike protein or (C) RBD specific binding IgG antibodies, displayed as endpoint titres at different time points as indicated (C) Virus neutralising antibodies determined via focus reduction neutralisation test at different time points as indicated. All values are displayed as median with range. Square symbols represent male, round symbols female animals. Dotted lines represent vaccinations and challenge infection, respectively. RBD receptor binding domain; VNT virus neutralising titre

    Techniques Used: Binding Assay, Infection

    Exemplary sections showing histopathology (H E) and SARS-CoV-2 in situ hybridisation (ISH). A. Alveolar necrosis and inflammatory exudates (*) in the alveolar spaces and type II pneumocyte hyperplasia (arrows). B) Mild perivascular cuffing (arrow). C) Inflammatory cell infiltration in the alveolar spaces and the interalveolar septa (*) and type II pneumocyte hyperplasia (arrows). D) SARS-CoV-2 ISH staining in abundant cell within inflammatory foci (arrows). E. SARS-CoV-2 ISH staining in a single cell within an interalveolar septum (arrow). F. Abundant foci of SARS-CoV-2 ISH stained cells within the alveolar lining and the interalveolar septa (arrows). Bar = 100μm. ISH in situ hybridisation
    Figure Legend Snippet: Exemplary sections showing histopathology (H E) and SARS-CoV-2 in situ hybridisation (ISH). A. Alveolar necrosis and inflammatory exudates (*) in the alveolar spaces and type II pneumocyte hyperplasia (arrows). B) Mild perivascular cuffing (arrow). C) Inflammatory cell infiltration in the alveolar spaces and the interalveolar septa (*) and type II pneumocyte hyperplasia (arrows). D) SARS-CoV-2 ISH staining in abundant cell within inflammatory foci (arrows). E. SARS-CoV-2 ISH staining in a single cell within an interalveolar septum (arrow). F. Abundant foci of SARS-CoV-2 ISH stained cells within the alveolar lining and the interalveolar septa (arrows). Bar = 100μm. ISH in situ hybridisation

    Techniques Used: Histopathology, In Situ, Hybridization, In Situ Hybridization, Staining

    24) Product Images from "Single-dose intranasal administration of AdCOVID elicits systemic and mucosal immunity against SARS-CoV-2 in mice"

    Article Title: Single-dose intranasal administration of AdCOVID elicits systemic and mucosal immunity against SARS-CoV-2 in mice

    Journal: bioRxiv

    doi: 10.1101/2020.10.10.331348

    SARS-CoV-2 neutralizing antibody responses in serum following single intranasal administration of the S1 and RBD vectors. (A) Neutralizing antibody response by C57BL/6J or CD-1 mice vaccinated 28 days earlier with the mid or high dose of the S1 or RBD vector as indicated. Results are expressed as the reciprocal of the dilution of serum samples required to achieve 50% neutralization (FRNT 50 ) of wild-type SARS-CoV-2 infection of permissive Vero E6 cells. Line represent the group median value. (B-C) Correlation between neutralizing antibody response and Spike-specific IgG response in serum of vaccinated animals. Correlation analysis was performed with a two-tailed Spearman test.
    Figure Legend Snippet: SARS-CoV-2 neutralizing antibody responses in serum following single intranasal administration of the S1 and RBD vectors. (A) Neutralizing antibody response by C57BL/6J or CD-1 mice vaccinated 28 days earlier with the mid or high dose of the S1 or RBD vector as indicated. Results are expressed as the reciprocal of the dilution of serum samples required to achieve 50% neutralization (FRNT 50 ) of wild-type SARS-CoV-2 infection of permissive Vero E6 cells. Line represent the group median value. (B-C) Correlation between neutralizing antibody response and Spike-specific IgG response in serum of vaccinated animals. Correlation analysis was performed with a two-tailed Spearman test.

    Techniques Used: Mouse Assay, Plasmid Preparation, Neutralization, Infection, Two Tailed Test

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

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

    Journal: JCI Insight

    doi: 10.1172/jci.insight.142386

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

    Techniques Used: Enzyme-linked Immunosorbent Assay

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

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

    Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.
    Figure Legend Snippet: Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.

    Techniques Used: Neutralization, Activity Assay, Binding Assay, Standard Deviation

    26) Product Images from "Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response"

    Article Title: Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response

    Journal: bioRxiv

    doi: 10.1101/2020.06.27.175166

    Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.
    Figure Legend Snippet: Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.

    Techniques Used: Mouse Assay, Staining, Immunofluorescence, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Binding Assay

    Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.
    Figure Legend Snippet: Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.

    Techniques Used: Mouse Assay, Recombinant, Expressing, Binding Assay, Enzyme-linked Immunosorbent Assay, Luminex, Fluorescence

    Construction and characterization of MVA/S and MVA/S1 recombinants. (A) Schematic representation of MVA/S and MVA/S1. Recombinant inserts were cloned in the essential region in between 18R and G1L under mH5 promoter. (B) Representative flow plots showing the expression of membrane anchored spike on the surface and S1 intracellularly. (C) Westernblotting analysis of expressed proteins in supernatants and lysates of MVA infected cells. (D) Size-exclusion chromatography analysis of S1 protein expressed by MVA/S1. (E) Binding of hACE2 to MVA/S expressing cells.
    Figure Legend Snippet: Construction and characterization of MVA/S and MVA/S1 recombinants. (A) Schematic representation of MVA/S and MVA/S1. Recombinant inserts were cloned in the essential region in between 18R and G1L under mH5 promoter. (B) Representative flow plots showing the expression of membrane anchored spike on the surface and S1 intracellularly. (C) Westernblotting analysis of expressed proteins in supernatants and lysates of MVA infected cells. (D) Size-exclusion chromatography analysis of S1 protein expressed by MVA/S1. (E) Binding of hACE2 to MVA/S expressing cells.

    Techniques Used: Recombinant, Clone Assay, Expressing, Infection, Size-exclusion Chromatography, Binding Assay

    27) 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

    28) Product Images from "Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response"

    Article Title: Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response

    Journal: bioRxiv

    doi: 10.1101/2020.06.27.175166

    Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.
    Figure Legend Snippet: Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.

    Techniques Used: Mouse Assay, Staining, Immunofluorescence, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Binding Assay

    Neutralizing activity against SARS-CoV-2. (A) Percent neutralization of SARS-CoV-2 virus expressing GFP. Serum collected from the naïve animals used as negative controls. (B) Neutralization titer against SARS-CoV-2 virus expressing GFP. (C, D) Correlations between neutralization titer and ELISA binding titer.
    Figure Legend Snippet: Neutralizing activity against SARS-CoV-2. (A) Percent neutralization of SARS-CoV-2 virus expressing GFP. Serum collected from the naïve animals used as negative controls. (B) Neutralization titer against SARS-CoV-2 virus expressing GFP. (C, D) Correlations between neutralization titer and ELISA binding titer.

    Techniques Used: Activity Assay, Neutralization, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay

    Analyzing SARS-CoV-2 RBD and S1 proteins affinities to human ACE2 (hACE2) proteins using biolayer interferometry (BLI). (A) Bio-Layer Interferometry sensograms of the binding of SARS-CoV-2 S1 and RBD proteins to immobilized Fc-human ACE2, after incubation of the analytes at 25°C for 0and 60 minutes. The traces represent BLI response curves for SARS-CoV-2 proteins serially diluted from 800nM to 12.5nM, as indicated. Dotted lines show raw response values, while bold solid lines show the fitted trace. Association and dissociation phases were monitored for 300s and 600s, respectively. The data was globally fit using a 1:1 binding model to estimate binding affinity. (B) Binding affinity specifications of S1 and RBD proteins against hu-ACE2.
    Figure Legend Snippet: Analyzing SARS-CoV-2 RBD and S1 proteins affinities to human ACE2 (hACE2) proteins using biolayer interferometry (BLI). (A) Bio-Layer Interferometry sensograms of the binding of SARS-CoV-2 S1 and RBD proteins to immobilized Fc-human ACE2, after incubation of the analytes at 25°C for 0and 60 minutes. The traces represent BLI response curves for SARS-CoV-2 proteins serially diluted from 800nM to 12.5nM, as indicated. Dotted lines show raw response values, while bold solid lines show the fitted trace. Association and dissociation phases were monitored for 300s and 600s, respectively. The data was globally fit using a 1:1 binding model to estimate binding affinity. (B) Binding affinity specifications of S1 and RBD proteins against hu-ACE2.

    Techniques Used: Binding Assay, Incubation

    Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.
    Figure Legend Snippet: Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.

    Techniques Used: Mouse Assay, Recombinant, Expressing, Binding Assay, Enzyme-linked Immunosorbent Assay, Luminex, Fluorescence

    29) Product Images from "Inhibition of SARS-CoV-2 viral entry in vitro upon blocking N- and O-glycan elaboration"

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

    Journal: bioRxiv

    doi: 10.1101/2020.10.15.339838

    Mannosidase-I blockade using kifunensine inhibits SARS-CoV-2 pseudovirus entry into 293T/ACE2 cells. A . VSVG, Spike-WT and Spike-mutant pseudovirus were produced in the presence of 15µM kifunensine or vehicle control. The 6 viruses were added to 293T/ACE2 at equal titer. B-D . Microscopy (panel B) and cytometry (panel C, D) show ∼90% loss of viral infection in the case of Spike-WT and Spike-mutant virus upon kifunensine treatment (* P
    Figure Legend Snippet: Mannosidase-I blockade using kifunensine inhibits SARS-CoV-2 pseudovirus entry into 293T/ACE2 cells. A . VSVG, Spike-WT and Spike-mutant pseudovirus were produced in the presence of 15µM kifunensine or vehicle control. The 6 viruses were added to 293T/ACE2 at equal titer. B-D . Microscopy (panel B) and cytometry (panel C, D) show ∼90% loss of viral infection in the case of Spike-WT and Spike-mutant virus upon kifunensine treatment (* P

    Techniques Used: Mutagenesis, Produced, Microscopy, Cytometry, Infection

    Principal findings and conceptual model: A . ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. B . SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral-entry ( B ) assay are listed. C . Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.
    Figure Legend Snippet: Principal findings and conceptual model: A . ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. B . SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral-entry ( B ) assay are listed. C . Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.

    Techniques Used: Binding Assay, Expressing, Generated

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

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

    Glycan coverage of Spike-ACE2 co-complex: SARS-CoV-2 spike protein trimer (pink) bound to ACE2 (green). A . without glycans. B . with N-glycans (red) identified using LC-MS on Spike and ACE2. C . Molecular dynamics simulation analyzed the range of movement of each glycan. The space sampled by glycans is represented by a gray cloud. Glycans cover the Spike-ACE2 interface. They also surround the putative proteolysis site of furin (“S1-S2”, yellow) and S2’ (blue).
    Figure Legend Snippet: Glycan coverage of Spike-ACE2 co-complex: SARS-CoV-2 spike protein trimer (pink) bound to ACE2 (green). A . without glycans. B . with N-glycans (red) identified using LC-MS on Spike and ACE2. C . Molecular dynamics simulation analyzed the range of movement of each glycan. The space sampled by glycans is represented by a gray cloud. Glycans cover the Spike-ACE2 interface. They also surround the putative proteolysis site of furin (“S1-S2”, yellow) and S2’ (blue).

    Techniques Used: Liquid Chromatography with Mass Spectroscopy

    30) Product Images from "A Next Generation Bivalent Human Ad5 COVID-19 Vaccine Delivering Both Spike and Nucleocapsid Antigens Elicits Th1 Dominant CD4+, CD8+ T-cell and Neutralizing Antibody Responses"

    Article Title: A Next Generation Bivalent Human Ad5 COVID-19 Vaccine Delivering Both Spike and Nucleocapsid Antigens Elicits Th1 Dominant CD4+, CD8+ T-cell and Neutralizing Antibody Responses

    Journal: bioRxiv

    doi: 10.1101/2020.07.29.227595

    cPass and Vero E6 cell SARS-CoV-2 confirm neutralization by antibodies. (a) In the cPass assay, inhibition of S RBD interaction with ACE2 was significant at both 1:20 and 1:60 dilutions of serum from hAd5 S-Fusion + N-ETSD vaccinated mice. (b) The results in the Vero E6 cell SARS-CoV-2 viral infection for mice that showed S-specific antibodies by ELISA also showed high neutralization for mice and very high neutralization for pooled sera (G4 pool, blue line) even compared to COVID-19 convalescent serum. G4 pool – mice with S-specific antibodies; M1, M2, M3, M4 – mouse ID; +C – convalescent serum; and media – media only negative control.
    Figure Legend Snippet: cPass and Vero E6 cell SARS-CoV-2 confirm neutralization by antibodies. (a) In the cPass assay, inhibition of S RBD interaction with ACE2 was significant at both 1:20 and 1:60 dilutions of serum from hAd5 S-Fusion + N-ETSD vaccinated mice. (b) The results in the Vero E6 cell SARS-CoV-2 viral infection for mice that showed S-specific antibodies by ELISA also showed high neutralization for mice and very high neutralization for pooled sera (G4 pool, blue line) even compared to COVID-19 convalescent serum. G4 pool – mice with S-specific antibodies; M1, M2, M3, M4 – mouse ID; +C – convalescent serum; and media – media only negative control.

    Techniques Used: Neutralization, Inhibition, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Negative Control

    The SARS-CoV-2 virus, spike, the hAd5 [E1-, E2b-, E3-] vector and vaccine candidate constructs. (a) Trimeric spike (S) protein (▾) is displayed on the viral surface; the nucleocapsid (N) protein (○) is associated with the viral RNA. (b) The Receptor Binding Domain (RBD) is within the S1 region, followed by other functional regions, the transmembrane domain (TM) and the C-terminus (CT), which is within the virus. (c) The second-generation human adenovirus serotype 5 (hAd5) vector used has the E1, E2b, and E3 regions deleted. Constructs are shown for (d) S wild type (S-WT), (e) S-RBD with the Enhanced T-cell Stimulation Domain (S RBD-ETSD), (f) S-Fusion, (g) N-ETSD, and (h) bivalent hAd5 S-Fusion + N-ETSD; LP – Leader peptide.
    Figure Legend Snippet: The SARS-CoV-2 virus, spike, the hAd5 [E1-, E2b-, E3-] vector and vaccine candidate constructs. (a) Trimeric spike (S) protein (▾) is displayed on the viral surface; the nucleocapsid (N) protein (○) is associated with the viral RNA. (b) The Receptor Binding Domain (RBD) is within the S1 region, followed by other functional regions, the transmembrane domain (TM) and the C-terminus (CT), which is within the virus. (c) The second-generation human adenovirus serotype 5 (hAd5) vector used has the E1, E2b, and E3 regions deleted. Constructs are shown for (d) S wild type (S-WT), (e) S-RBD with the Enhanced T-cell Stimulation Domain (S RBD-ETSD), (f) S-Fusion, (g) N-ETSD, and (h) bivalent hAd5 S-Fusion + N-ETSD; LP – Leader peptide.

    Techniques Used: Plasmid Preparation, Construct, Binding Assay, Functional Assay, Cell Stimulation

    Transfection of HEK293T cells with hAd5 S-Fusion + ETSD results in enhanced surface expression of the spike receptor binding domain (RBD). Flow cytometric analysis of an anti-RBD antibody with construct-transfected cells reveals no detectable surface expression of RBD in either (a) S-WT or (b) S-WT + N-ETSD transfected cells. Surface RBD expression was high for S RBD-ETSD and S RBD-ETSD + N-ETSD (c, d). Expression was low in (e) S-Fusion transfected cells. Cell surface expression of the RBD was high in (f) S-Fusion + N-ETSD transfected cells, particularly at day 1 and 2. (g) No expression was detected the N-ETSD negative control. Y-axis scale is normalized to mode (NM).
    Figure Legend Snippet: Transfection of HEK293T cells with hAd5 S-Fusion + ETSD results in enhanced surface expression of the spike receptor binding domain (RBD). Flow cytometric analysis of an anti-RBD antibody with construct-transfected cells reveals no detectable surface expression of RBD in either (a) S-WT or (b) S-WT + N-ETSD transfected cells. Surface RBD expression was high for S RBD-ETSD and S RBD-ETSD + N-ETSD (c, d). Expression was low in (e) S-Fusion transfected cells. Cell surface expression of the RBD was high in (f) S-Fusion + N-ETSD transfected cells, particularly at day 1 and 2. (g) No expression was detected the N-ETSD negative control. Y-axis scale is normalized to mode (NM).

    Techniques Used: Transfection, Expressing, Binding Assay, Construct, Negative Control

    Binding of recombinant ACE2-Fc HEK293T cell-surface expressed RBD after transfection confirms native protein folding. Flow cytometric analysis of binding between recombinant ACE2-Fc, with which the spike RBD interacts in vivo to initiate infection, and cell-surface antigens expressed after transfection of HEK293T cells with (a) hAd5 S-WT, (b) hAd5 S-Fusion, (c) hAd5 S-Fusion + N-ETSD, (d) hAd5 S RBD-ETSD, or (e) hAd5 S RBD-ETSD + N-ETSD constructs reveals the highest binding is seen for both ACE-Fc and an anti-RBD specific antibody (f-j) after transfection with the bivalent S-Fusion + N-ETSD. Both S RBD-ETSD-containing constructs also showed binding. Y-axis scale is normalized to mode (NM).
    Figure Legend Snippet: Binding of recombinant ACE2-Fc HEK293T cell-surface expressed RBD after transfection confirms native protein folding. Flow cytometric analysis of binding between recombinant ACE2-Fc, with which the spike RBD interacts in vivo to initiate infection, and cell-surface antigens expressed after transfection of HEK293T cells with (a) hAd5 S-WT, (b) hAd5 S-Fusion, (c) hAd5 S-Fusion + N-ETSD, (d) hAd5 S RBD-ETSD, or (e) hAd5 S RBD-ETSD + N-ETSD constructs reveals the highest binding is seen for both ACE-Fc and an anti-RBD specific antibody (f-j) after transfection with the bivalent S-Fusion + N-ETSD. Both S RBD-ETSD-containing constructs also showed binding. Y-axis scale is normalized to mode (NM).

    Techniques Used: Binding Assay, Recombinant, Transfection, In Vivo, Infection, Construct

    Immunoblot analysis of S expression. Cell surface RBD expression with (a) hAd5 S-WT, (b) S-Fusion, and (c) S-Fusion + N-ETSD in HEK 293T cells shows high correlation with (d) expression of S in immunoblots of HEK 293T cell lysates probed using anti-full length (S2) antibody. Y-axis scale is normalized to mode (NM).
    Figure Legend Snippet: Immunoblot analysis of S expression. Cell surface RBD expression with (a) hAd5 S-WT, (b) S-Fusion, and (c) S-Fusion + N-ETSD in HEK 293T cells shows high correlation with (d) expression of S in immunoblots of HEK 293T cell lysates probed using anti-full length (S2) antibody. Y-axis scale is normalized to mode (NM).

    Techniques Used: Expressing, Western Blot

    31) Product Images from "Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells"

    Article Title: Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

    Journal: bioRxiv

    doi: 10.1101/2020.04.08.026948

    VSVdG-SARS-CoV-2-Sdel18 based system for screening of neutralizing mAbs. A Neutralizing antibodies were screened from 35 antibodies. The culture supernatant of 35 monoclonal hybridoma cells were incubated with VSVdG-SARS-CoV-2-Sdel18 virus and then added the mixture to BHK21-hACE2 cell. The fluorescence was detected using Opera Phenix 12 h post infection. The GFP positive cell number was also counted to calculate the inhibition rate (B). C The IC50 of 7 selected neutralizing antibodies for antiviral activity were also analyzed. The 7 selected neutralizing antibodies were purified and diluted to different concentration, then incubated with VSVdG-SARS-CoV-2-Sdel18 virus for an hour and added to BHK21-hACE2 cell. The GFP positive cell number was counted using Opera Phenix 12 h post infection to calculate the inhibit ratio. The IC50 was analysed by nonlinear regression.
    Figure Legend Snippet: VSVdG-SARS-CoV-2-Sdel18 based system for screening of neutralizing mAbs. A Neutralizing antibodies were screened from 35 antibodies. The culture supernatant of 35 monoclonal hybridoma cells were incubated with VSVdG-SARS-CoV-2-Sdel18 virus and then added the mixture to BHK21-hACE2 cell. The fluorescence was detected using Opera Phenix 12 h post infection. The GFP positive cell number was also counted to calculate the inhibition rate (B). C The IC50 of 7 selected neutralizing antibodies for antiviral activity were also analyzed. The 7 selected neutralizing antibodies were purified and diluted to different concentration, then incubated with VSVdG-SARS-CoV-2-Sdel18 virus for an hour and added to BHK21-hACE2 cell. The GFP positive cell number was counted using Opera Phenix 12 h post infection to calculate the inhibit ratio. The IC50 was analysed by nonlinear regression.

    Techniques Used: Incubation, Fluorescence, Infection, Inhibition, Activity Assay, Purification, Concentration Assay

    Generation of VSV pseudotypes bearing SARS-CoV-2 spike proteins. (A) The difference between SARS-CoV-2-S and SARS-CoV-2-Sde18. (B) The procedure of producing VSV pseudotypes bearing SARS-CoV-2 spike proteins.
    Figure Legend Snippet: Generation of VSV pseudotypes bearing SARS-CoV-2 spike proteins. (A) The difference between SARS-CoV-2-S and SARS-CoV-2-Sde18. (B) The procedure of producing VSV pseudotypes bearing SARS-CoV-2 spike proteins.

    Techniques Used:

    Time course of EGFP expression after VSVdG-SARS-CoV-2-Sdel18 infection. BHK21-hACE2 cell was infected with VSVdG-SARS-CoV-2-Sdel18 virus. The fluorescence was detected (A) and GFP positive cell number (B) was counted using Opera Phenix at different time point post infection.
    Figure Legend Snippet: Time course of EGFP expression after VSVdG-SARS-CoV-2-Sdel18 infection. BHK21-hACE2 cell was infected with VSVdG-SARS-CoV-2-Sdel18 virus. The fluorescence was detected (A) and GFP positive cell number (B) was counted using Opera Phenix at different time point post infection.

    Techniques Used: Expressing, Infection, Fluorescence

    Verify the correlation between VSVdG-SARS-CoV-2-Sdel18 based system and live SARS-CoV-2 system. A Analyze the neutralizing antibodies in the serum of 18 convalescent patients using VSVdG-SARS-CoV-2-Sdel18 based system. ID50 was used to indicate the neutralizing activity of sera. B Analyze the neutralizing antibodies in the serum of 18 convalescent patients using live SARS-CoV-2 system. ID50 was used to indicate the neutralizing activity of sera. C The correlation between VSVdG-SARS-CoV-2-Sdel18 based system and live SARS-CoV-2 system.
    Figure Legend Snippet: Verify the correlation between VSVdG-SARS-CoV-2-Sdel18 based system and live SARS-CoV-2 system. A Analyze the neutralizing antibodies in the serum of 18 convalescent patients using VSVdG-SARS-CoV-2-Sdel18 based system. ID50 was used to indicate the neutralizing activity of sera. B Analyze the neutralizing antibodies in the serum of 18 convalescent patients using live SARS-CoV-2 system. ID50 was used to indicate the neutralizing activity of sera. C The correlation between VSVdG-SARS-CoV-2-Sdel18 based system and live SARS-CoV-2 system.

    Techniques Used: Activity Assay

    Comparison of the packaging efficiency of VSVdG-SARS-CoV-2-Sdel18 in various cell lines. Vero-E6, BHK21 and 293T cells were used to package VSVdG-SARS-CoV-2-Sdel18 virus. The left picture shows the cell used to package recombinant virus, recorded 48h post infection of VSVdG-EGFP-G. The right figures show the infectivity of virus produced by three cell lines. The harvested virus was diluted and tested in BHK21-hACE2 cell. The fluorescence was detected (A) and GFP positive cell number (B) was counted using Opera Phenix 12 h post infection.
    Figure Legend Snippet: Comparison of the packaging efficiency of VSVdG-SARS-CoV-2-Sdel18 in various cell lines. Vero-E6, BHK21 and 293T cells were used to package VSVdG-SARS-CoV-2-Sdel18 virus. The left picture shows the cell used to package recombinant virus, recorded 48h post infection of VSVdG-EGFP-G. The right figures show the infectivity of virus produced by three cell lines. The harvested virus was diluted and tested in BHK21-hACE2 cell. The fluorescence was detected (A) and GFP positive cell number (B) was counted using Opera Phenix 12 h post infection.

    Techniques Used: Recombinant, Infection, Produced, Fluorescence

    Comparison of the infectious efficiency of pseudotypes in various cell lines. VSVdG virus bearing spike protein of SARS-CoV-2 or G protein of VSV were harvested and the infectivity of these recombinant virus were tested in different cell lines, including Vero-E6, BHK21, 293T and BHK21-hACE2. The fluorescence was detected (A) and GFP positive cell number (B) was counted using Opera Phenix 12 h post infection.
    Figure Legend Snippet: Comparison of the infectious efficiency of pseudotypes in various cell lines. VSVdG virus bearing spike protein of SARS-CoV-2 or G protein of VSV were harvested and the infectivity of these recombinant virus were tested in different cell lines, including Vero-E6, BHK21, 293T and BHK21-hACE2. The fluorescence was detected (A) and GFP positive cell number (B) was counted using Opera Phenix 12 h post infection.

    Techniques Used: Infection, Recombinant, Fluorescence

    32) Product Images from "Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients"

    Article Title: Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients

    Journal: bioRxiv

    doi: 10.1101/2020.06.15.153064

    Results from the Adarza Ziva system for pre-COVID-19 serum samples and single-donor samples from convalescent COVID-19 (PCR-positive) subjects. Pre-COVID-19 single-donor results were averaged (blue bars). Black bars indicate threshold positive values, calculated as two standard deviations above the average negative (pre-COVID-19) signal. Red bars indicate PCR+ individuals yielding signals below the threshold on all SARS-CoV-2 antigens, while green bars indicate signals from single-donor convalescent COVID-19 samples with at least one SARS-CoV-2 antigen response above threshold.
    Figure Legend Snippet: Results from the Adarza Ziva system for pre-COVID-19 serum samples and single-donor samples from convalescent COVID-19 (PCR-positive) subjects. Pre-COVID-19 single-donor results were averaged (blue bars). Black bars indicate threshold positive values, calculated as two standard deviations above the average negative (pre-COVID-19) signal. Red bars indicate PCR+ individuals yielding signals below the threshold on all SARS-CoV-2 antigens, while green bars indicate signals from single-donor convalescent COVID-19 samples with at least one SARS-CoV-2 antigen response above threshold.

    Techniques Used: Polymerase Chain Reaction

    AIR assay for antibodies to respiratory viruses. For each antigen, six replicate spots are printed in two different locations on the chip. Each group of six spots is surrounded by negative control reference spots (anti-FITC). Blank (background) areas are included as additional negative controls. Key: 1: human coronavirus (HKU isolate) spike glycoprotein, aa 1-760; 2: MERS-CoV spike glycoprotein, S1 domain; 3: MERS-CoV spike glycoprotein, receptor binding domain (RBD); 4: SARS-CoV spike glycoprotein, S1 domain; 5: SARS-CoV spike glycoprotein, RBD; 6: SARS-CoV-2 spike glycoprotein, S1+S2 ECD; 7: SARS-CoV-2 spike glycoprotein, S2 ECD; 8: SARS-CoV-2 spike glycoprotein, S1 domain; 9: SARS-CoV-2 spike glycoprotein, RBD; 10: human coronavirus (HCoV-229E isolate) spike glycoprotein, S1+S2 ECD; 11: human coronavirus (HCoV-OC43 isolate) spike glycoprotein, S1+S2 ECD; 12: influenza B/Brisbane/2008 hemagglutinin; 13: influenza A/California/2009 (H1N1) hemagglutinin; 14: influenza A/Wisconsin/2005 (H3N2) influenza. F1 , F2 , and F3 are derived from spotting three different dilutions of anti-FITC. The image at right is a representative array exposed to Pooled Normal Human Serum (PNHS) at a 1:4 dilution.
    Figure Legend Snippet: AIR assay for antibodies to respiratory viruses. For each antigen, six replicate spots are printed in two different locations on the chip. Each group of six spots is surrounded by negative control reference spots (anti-FITC). Blank (background) areas are included as additional negative controls. Key: 1: human coronavirus (HKU isolate) spike glycoprotein, aa 1-760; 2: MERS-CoV spike glycoprotein, S1 domain; 3: MERS-CoV spike glycoprotein, receptor binding domain (RBD); 4: SARS-CoV spike glycoprotein, S1 domain; 5: SARS-CoV spike glycoprotein, RBD; 6: SARS-CoV-2 spike glycoprotein, S1+S2 ECD; 7: SARS-CoV-2 spike glycoprotein, S2 ECD; 8: SARS-CoV-2 spike glycoprotein, S1 domain; 9: SARS-CoV-2 spike glycoprotein, RBD; 10: human coronavirus (HCoV-229E isolate) spike glycoprotein, S1+S2 ECD; 11: human coronavirus (HCoV-OC43 isolate) spike glycoprotein, S1+S2 ECD; 12: influenza B/Brisbane/2008 hemagglutinin; 13: influenza A/California/2009 (H1N1) hemagglutinin; 14: influenza A/Wisconsin/2005 (H3N2) influenza. F1 , F2 , and F3 are derived from spotting three different dilutions of anti-FITC. The image at right is a representative array exposed to Pooled Normal Human Serum (PNHS) at a 1:4 dilution.

    Techniques Used: Chromatin Immunoprecipitation, Negative Control, Binding Assay, Derivative Assay

    Correlation of AIR and ELISA data for SARS-CoV-2 S1+S2 ECD (left) and RBD (right). Exponential trend lines and associated R 2 values are indicated.
    Figure Legend Snippet: Correlation of AIR and ELISA data for SARS-CoV-2 S1+S2 ECD (left) and RBD (right). Exponential trend lines and associated R 2 values are indicated.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Representative AIR array images (100 ms exposures) of (A) 5% FBS; (B) 10% PNHS; (C) a negative single-donor sample, and (D) one convalescent serum sample. Strong responses to SARS-CoV-2 antigens are readily observed in (D), but not in (A), (B), or (C). In each case, samples were diluted 1:20 in Adarza diluent, and incubated with the arrays overnight at 4 °C. See Figure 1 for key to the array. All arrays in this figure were imaged at an exposure of 100 ms.
    Figure Legend Snippet: Representative AIR array images (100 ms exposures) of (A) 5% FBS; (B) 10% PNHS; (C) a negative single-donor sample, and (D) one convalescent serum sample. Strong responses to SARS-CoV-2 antigens are readily observed in (D), but not in (A), (B), or (C). In each case, samples were diluted 1:20 in Adarza diluent, and incubated with the arrays overnight at 4 °C. See Figure 1 for key to the array. All arrays in this figure were imaged at an exposure of 100 ms.

    Techniques Used: Incubation

    Response of a commercial anti-SARS-CoV-2 rabbit polyclonal antibody (pAb) on the array. (A) array exposed to array exposed to 20% FBS + 10% PNHS; (B) array exposed to 1 μg/mL anti-SARS-CoV-2 pAb in 20% FBS + 10% PNHS. Strong responses to SARS-CoV-2 S1+S2 ECD, S1, and RBD are observed, as well as smaller cross-reactive responses to HCoV-229E, HCoV-OC43, and MERS spike proteins; (C) quantitative data for the titration. Concentrations of pAb are provided at the top of each column in ng/mL; response values at each concentration for each antigen are provided in Angstroms of build. (D) Titration curves for the four SARS-CoV-2 antigens with standard deviation of replicate probe spots at each concentration.
    Figure Legend Snippet: Response of a commercial anti-SARS-CoV-2 rabbit polyclonal antibody (pAb) on the array. (A) array exposed to array exposed to 20% FBS + 10% PNHS; (B) array exposed to 1 μg/mL anti-SARS-CoV-2 pAb in 20% FBS + 10% PNHS. Strong responses to SARS-CoV-2 S1+S2 ECD, S1, and RBD are observed, as well as smaller cross-reactive responses to HCoV-229E, HCoV-OC43, and MERS spike proteins; (C) quantitative data for the titration. Concentrations of pAb are provided at the top of each column in ng/mL; response values at each concentration for each antigen are provided in Angstroms of build. (D) Titration curves for the four SARS-CoV-2 antigens with standard deviation of replicate probe spots at each concentration.

    Techniques Used: Titration, Concentration Assay, Standard Deviation

    33) Product Images from "The Rhinolophus affinis bat ACE2 and multiple animal orthologs are functional receptors for bat coronavirus RaTG13 and SARS-CoV-2"

    Article Title: The Rhinolophus affinis bat ACE2 and multiple animal orthologs are functional receptors for bat coronavirus RaTG13 and SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2020.11.16.385849

    Entry mediated by the S protein of RaTG13, SARS-CoV-2, and SARS-CoV on cells expressing different ACE2 proteins. HEK-293 cells transiently expressing different ACE2 proteins were transduced with RaTG13 S pseudovirions (A), SARS-CoV-2 S pseudovirions (B), SARS-CoV S pseudovirions (C), and VSV-G pseudovirions (D). Experiments were done in triplicate and repeated at least three times. One representative is shown with error bars indicating SEM.
    Figure Legend Snippet: Entry mediated by the S protein of RaTG13, SARS-CoV-2, and SARS-CoV on cells expressing different ACE2 proteins. HEK-293 cells transiently expressing different ACE2 proteins were transduced with RaTG13 S pseudovirions (A), SARS-CoV-2 S pseudovirions (B), SARS-CoV S pseudovirions (C), and VSV-G pseudovirions (D). Experiments were done in triplicate and repeated at least three times. One representative is shown with error bars indicating SEM.

    Techniques Used: Expressing, Transduction

    Binding of different ACE2 proteins by RBDs of bat SL-CoV RaTG13, SARS-CoV-2, and SARS-CoV. HEK293 cells transiently expressing different ACE2 cells were incubated with either RaTG13 (A), SARS-CoV-2 (B), or SARS-CoV (C) RBDs, followed by rabbit anti-His tag antibodies and Alexa-488 conjugated goat anti rabbit IgG, and analyzed by flow cytometry. The experiments were done at least three times. The results of percentage of positive cells from hACE2 binding were set to 100%, the rest was calculated as percentage of hACE2 binding according to results in flow cytometry analysis. Data are shown as the means ± standard deviations.
    Figure Legend Snippet: Binding of different ACE2 proteins by RBDs of bat SL-CoV RaTG13, SARS-CoV-2, and SARS-CoV. HEK293 cells transiently expressing different ACE2 cells were incubated with either RaTG13 (A), SARS-CoV-2 (B), or SARS-CoV (C) RBDs, followed by rabbit anti-His tag antibodies and Alexa-488 conjugated goat anti rabbit IgG, and analyzed by flow cytometry. The experiments were done at least three times. The results of percentage of positive cells from hACE2 binding were set to 100%, the rest was calculated as percentage of hACE2 binding according to results in flow cytometry analysis. Data are shown as the means ± standard deviations.

    Techniques Used: Binding Assay, Expressing, Incubation, Flow Cytometry

    Cell–cell fusion mediated by RaTG13, SARS-CoV, and SARS-CoV-2 spike proteins. HEK293T cells transiently expressing eGFP and spike proteins of either RaTG13, SARS-CoV, or SARS-CoV-2 were detached with trypsin, and overlaid on different ACE2 expressing HEK293 cells. After 4 hrs of incubation, images were taken. (A) Representative images of syncytia for hACE2; (B-D) Percentage of nuclei in syncytia induced by RaTG13 S (B), SARS-CoV-2 S (C), and SARS-CoV S (D). Syncytium formation for each image was quantified by counting the total nuclei in syncytia and total nuclei in the image and calculated as the percentage of nuclei in syncytia, and three images were selected for each sample. Experiments were done three times, and one representative is shown with error bars indicating SEM. The scale bar indicates 250 μm.
    Figure Legend Snippet: Cell–cell fusion mediated by RaTG13, SARS-CoV, and SARS-CoV-2 spike proteins. HEK293T cells transiently expressing eGFP and spike proteins of either RaTG13, SARS-CoV, or SARS-CoV-2 were detached with trypsin, and overlaid on different ACE2 expressing HEK293 cells. After 4 hrs of incubation, images were taken. (A) Representative images of syncytia for hACE2; (B-D) Percentage of nuclei in syncytia induced by RaTG13 S (B), SARS-CoV-2 S (C), and SARS-CoV S (D). Syncytium formation for each image was quantified by counting the total nuclei in syncytia and total nuclei in the image and calculated as the percentage of nuclei in syncytia, and three images were selected for each sample. Experiments were done three times, and one representative is shown with error bars indicating SEM. The scale bar indicates 250 μm.

    Techniques Used: Expressing, Incubation

    Bat SL-CoV RaTG13 uses hACE2 and RaACE2 for virus entry. (A) Schematic diagram of the full length of different CoV S proteins and the amino acid sequence identities of each region are shown in corresponding places. S1, receptor binding subunit; S2, membrane fusion subunit; TM, transmembrane domain. (B) Detection of the S proteins of SARS-CoV, SARS-CoV-2, Bat SL-CoV RaTG13 and ZC45 in cells lysates and pseudovirions by western blot. HEK293T cells transfected with either empty vector or plasmids encoding the indicated CoV S proteins were lysed at 40 hrs post transfection. The S proteins in cell lysates and pseudovirions were subjected to WB analysis by blotting with mouse monoclonal anti-FLAG M2 antibody. Actin and gag-p24 served as loading controls (cell lysate, top panel, pseudovirions, bottom panel). The full length S protein is about 180 kDa, while cleaved S protein is about 90 kDa. Experiments were done three times and the representative was shown. (C) Entry by RaTG13 S pseudovirons on different CoV receptors. Cells were spin-inoculated with indicated pseudovirions. At 48 hrs post inoculation, transduction efficiency was determined by measurement of luciferase activities. HEK293 cells (grey), HEK293/hACE2 (red), HEK293 cells stably expressing hACE2; 293/mCEACAM (green), HEK293 cells stably expressing mCEACAM, the MHV receptor; 293/hDPP4 (blue), HEK293 cells stably expressing hDPP4, the MERS-CoV receptor. BHK/hAPN(purple), BHK cells stably expressing hAPN, the hCoV-229E receptor; Experiments were done triplicate and repeated at least three times. One representative is shown with error bars indicate SEM. (D) Expression of Rhinolophus affinis ACE2 protein in HEK 293 cells. HEK 293 cells transiently transfected with the plasmids encoding either FLAG-tagged hACE2 or Rhinolophus affinis ACE2 (RaACE2) proteins were lysed at 40 hrs post-transfection. Expression of ACE2 proteins were detected by mouse monoclonal anti-FLAG M2 antibody. (E) Binding of hACE2 and RaACE2 by SARS-CoV-2 and RaTG13 RBDs. HEK 293 cells transiently expressing hACE2 or RaACE2 proteins were incubated with either SARS-CoV-2 RBD or RaTG13 RBD on ice, followed by rabbit anti-his tag antibodies and alexa-488 conjugated goat anti rabbit IgG, and analyzed by flow cytometry. The experiments were done three times, and one representative is shown. (F) Mean fluorescence intensities of the gated cells positive for SARS-CoV-2 RBD binding to 293/hACE2 and 293/RaACE2 cells in (E). (G) Entry of SARS-CoV, SARS-CoV-2, and RaTG13 S protein pseudovirions on 293/RaACE2 cells. Experiments were done three times, and one representative is shown with error bars indicating SEM. *P
    Figure Legend Snippet: Bat SL-CoV RaTG13 uses hACE2 and RaACE2 for virus entry. (A) Schematic diagram of the full length of different CoV S proteins and the amino acid sequence identities of each region are shown in corresponding places. S1, receptor binding subunit; S2, membrane fusion subunit; TM, transmembrane domain. (B) Detection of the S proteins of SARS-CoV, SARS-CoV-2, Bat SL-CoV RaTG13 and ZC45 in cells lysates and pseudovirions by western blot. HEK293T cells transfected with either empty vector or plasmids encoding the indicated CoV S proteins were lysed at 40 hrs post transfection. The S proteins in cell lysates and pseudovirions were subjected to WB analysis by blotting with mouse monoclonal anti-FLAG M2 antibody. Actin and gag-p24 served as loading controls (cell lysate, top panel, pseudovirions, bottom panel). The full length S protein is about 180 kDa, while cleaved S protein is about 90 kDa. Experiments were done three times and the representative was shown. (C) Entry by RaTG13 S pseudovirons on different CoV receptors. Cells were spin-inoculated with indicated pseudovirions. At 48 hrs post inoculation, transduction efficiency was determined by measurement of luciferase activities. HEK293 cells (grey), HEK293/hACE2 (red), HEK293 cells stably expressing hACE2; 293/mCEACAM (green), HEK293 cells stably expressing mCEACAM, the MHV receptor; 293/hDPP4 (blue), HEK293 cells stably expressing hDPP4, the MERS-CoV receptor. BHK/hAPN(purple), BHK cells stably expressing hAPN, the hCoV-229E receptor; Experiments were done triplicate and repeated at least three times. One representative is shown with error bars indicate SEM. (D) Expression of Rhinolophus affinis ACE2 protein in HEK 293 cells. HEK 293 cells transiently transfected with the plasmids encoding either FLAG-tagged hACE2 or Rhinolophus affinis ACE2 (RaACE2) proteins were lysed at 40 hrs post-transfection. Expression of ACE2 proteins were detected by mouse monoclonal anti-FLAG M2 antibody. (E) Binding of hACE2 and RaACE2 by SARS-CoV-2 and RaTG13 RBDs. HEK 293 cells transiently expressing hACE2 or RaACE2 proteins were incubated with either SARS-CoV-2 RBD or RaTG13 RBD on ice, followed by rabbit anti-his tag antibodies and alexa-488 conjugated goat anti rabbit IgG, and analyzed by flow cytometry. The experiments were done three times, and one representative is shown. (F) Mean fluorescence intensities of the gated cells positive for SARS-CoV-2 RBD binding to 293/hACE2 and 293/RaACE2 cells in (E). (G) Entry of SARS-CoV, SARS-CoV-2, and RaTG13 S protein pseudovirions on 293/RaACE2 cells. Experiments were done three times, and one representative is shown with error bars indicating SEM. *P

    Techniques Used: Sequencing, Binding Assay, Western Blot, Transfection, Plasmid Preparation, Transduction, Luciferase, Stable Transfection, Expressing, Incubation, Flow Cytometry, Fluorescence

    Entry of lentiviral pseudovirions with mutant RaTG13 S and SARS-CoV-2 S proteins on 293/hACE2, 293/mouse ACE2, and 293/pangolin ACE2 cells. (A) Alignment of partial amino acid sequences of RaTG13 and SARS-CoV-2 S proteins. Residues 449, 484, 493, and 498 are labeled in red. Detection of mutant S proteins in cells lysates and pseudovirions by western blotting using a mouse monoclonal anti-FLAG M2 antibody. (B) RaTG13 S. (C) SARS-CoV-2 S. Top panel, cell lysate; bottom panel, pseudovirions; β-actin and HIV p24 were used as loading controls. (D)(E) Entry of pseudovirons with mutant RaTG13 (D) and SARS-CoV-2 (E) S proteins on 293/hACE2 cells. Pseudovirions carrying mutant S proteins were inoculated on 293/hACE2 cells. After 40 hrs incubation, transduction efficiency was determined by measuring the luciferase activities in cell lysate. Transduction from WT pseudovirions was set as 100%. Experiments were done in quadruplicate and repeated at least three times, and one representative was shown with SEM. (F) Entry of pseudovirons with mutant RaTG13 S proteins on 293 cells expressing mouse (blue) and pangolin (red) ACE2 proteins. Transduction from WT pseudovirions on mouse ACE2 cells was set as 100%. (G) Entry of pseudovirons with mutant SARS-CoV-2 S proteins on 293 cells expressing mouse (blue) and pangolin (red) ACE2 proteins. Transduction from WT pseudovirions on pangolin ACE2 cells was set as 100%. The experiments were performed in quadruplicate with at least three replications and the representative data are shown with SEM. *P
    Figure Legend Snippet: Entry of lentiviral pseudovirions with mutant RaTG13 S and SARS-CoV-2 S proteins on 293/hACE2, 293/mouse ACE2, and 293/pangolin ACE2 cells. (A) Alignment of partial amino acid sequences of RaTG13 and SARS-CoV-2 S proteins. Residues 449, 484, 493, and 498 are labeled in red. Detection of mutant S proteins in cells lysates and pseudovirions by western blotting using a mouse monoclonal anti-FLAG M2 antibody. (B) RaTG13 S. (C) SARS-CoV-2 S. Top panel, cell lysate; bottom panel, pseudovirions; β-actin and HIV p24 were used as loading controls. (D)(E) Entry of pseudovirons with mutant RaTG13 (D) and SARS-CoV-2 (E) S proteins on 293/hACE2 cells. Pseudovirions carrying mutant S proteins were inoculated on 293/hACE2 cells. After 40 hrs incubation, transduction efficiency was determined by measuring the luciferase activities in cell lysate. Transduction from WT pseudovirions was set as 100%. Experiments were done in quadruplicate and repeated at least three times, and one representative was shown with SEM. (F) Entry of pseudovirons with mutant RaTG13 S proteins on 293 cells expressing mouse (blue) and pangolin (red) ACE2 proteins. Transduction from WT pseudovirions on mouse ACE2 cells was set as 100%. (G) Entry of pseudovirons with mutant SARS-CoV-2 S proteins on 293 cells expressing mouse (blue) and pangolin (red) ACE2 proteins. Transduction from WT pseudovirions on pangolin ACE2 cells was set as 100%. The experiments were performed in quadruplicate with at least three replications and the representative data are shown with SEM. *P

    Techniques Used: Mutagenesis, Labeling, Western Blot, Incubation, Transduction, Luciferase, Expressing

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    Sino Biological sars cov 2 2019 ncov spike rbd antibody rabbit pab
    JIB-04 exhibits distinct post-entry antiviral mechanisms (A) Drug combination dose-response matrix and <t>VSV-SARS-CoV-2</t> replication. MA104 cells were treated with JIB-04 and chloroquine or JIB-04 and NTZ for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. (B) Time of compound addition and VSV-SARS-CoV-2 replication. MA104 cells were treated with NTZ or JIB-04 (10 μM) at indicated time points relative to VSV-SARS-CoV-2 infection (MOI=3, 0 hpi). GFP signals at 8 hpi were quantified to calculate the percentage of inhibition. (C) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. S RNA levels were measured by RT-qPCR. (D) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. FL: full-length. S2: cleaved S2 fragment. (* non-specific band) (E) Histone demethylase siRNA knockdown and RV replication. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01). Viral RNA copy numbers at 12 hpi were quantified by RT-qPCR. (F) Volcano plot of differentially expressed transcripts with JIB-04 treatment and RV infection. HEK293 cells were treated with DMSO or JIB-04 (10 μM) for 12 hr, and mock-infected (left panel) or infected with porcine RV (MOI=0.01, right panel) for another 12 hr. Red dots represent upregulated genes and green dots represent downregulated genes in JIB-04 treated cells. (G) Expression of three top genes in (F) with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 12 hr and mock-infected or infected porcine RV (MOI=0.01) for 12 hr. mRNA levels of CYP1A1, CYP1B1, and AHRR at 12 hpi were measured by RT-qPCR. (H) Dose-response analysis of VSV-SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. (I) Dose-response analysis of wild-type SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels at 24 hpi were quantified based on immunofluorescence. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. For all panels except A and I, experiments were repeated at least three times with similar results. Fig. 3A was performed twice. Inhibition assay in Fig. 3I was performed once and cytotoxicity assay was performed in triplicates. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Sars Cov 2 2019 Ncov Spike Rbd Antibody Rabbit Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Sino Biological sars cov 2 s protein
    <t>SARS-CoV-2</t> Spike Ectodomain Protein Binding to Cells Is Differentially Affected by HS from Different Organs and Potently Inhibited by Heparinoids (A) LC-MS/MS disaccharide analysis of HS isolated from human kidney, liver, tonsil, and lung tissue. (B) Inhibition of binding of recombinant SARS-CoV-2 S RBD protein to H1299 cells, using tissue HS. Analysis by flow cytometry. (C) Inhibition of recombinant trimeric SARS-CoV-2 protein (20 μg/mL) binding to H1299 cells, using CHO HS, heparin, MST heparin, and split-glycol heparin. Analysis by flow cytometry. (D) Similar analysis of A549 cells. Curve fitting was performed using non-linear regression and the inhibitor versus response least-squares fit algorithm. IC 50 values are listed in Table 1 . Graphs show representative experiments performed in technical duplicates or triplicates. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).
    Sars Cov 2 S Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sars cov 2 s protein/product/Sino Biological
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 s protein - by Bioz Stars, 2021-02
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    93
    Sino Biological sars cov 2 antigen
    Gross pathological examination of the lungs of <t>SARS-CoV-2</t> infected hamsters. Foci (arrowheads) of pulmonary consolidation in untreated SARS-CoV-2 infected animals (A) and animals treated with control MAb (D) or low dose plasma (F). Protection against pulmonary lesions in hamsters treated with MAb 47D11 (C) and high dose plasma (E), similar to mock infected animals (B). Images are from representative animals of each treatment group.
    Sars Cov 2 Antigen, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sars cov 2 antigen/product/Sino Biological
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    sars cov 2 antigen - by Bioz Stars, 2021-02
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    Image Search Results


    JIB-04 exhibits distinct post-entry antiviral mechanisms (A) Drug combination dose-response matrix and VSV-SARS-CoV-2 replication. MA104 cells were treated with JIB-04 and chloroquine or JIB-04 and NTZ for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. (B) Time of compound addition and VSV-SARS-CoV-2 replication. MA104 cells were treated with NTZ or JIB-04 (10 μM) at indicated time points relative to VSV-SARS-CoV-2 infection (MOI=3, 0 hpi). GFP signals at 8 hpi were quantified to calculate the percentage of inhibition. (C) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. S RNA levels were measured by RT-qPCR. (D) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. FL: full-length. S2: cleaved S2 fragment. (* non-specific band) (E) Histone demethylase siRNA knockdown and RV replication. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01). Viral RNA copy numbers at 12 hpi were quantified by RT-qPCR. (F) Volcano plot of differentially expressed transcripts with JIB-04 treatment and RV infection. HEK293 cells were treated with DMSO or JIB-04 (10 μM) for 12 hr, and mock-infected (left panel) or infected with porcine RV (MOI=0.01, right panel) for another 12 hr. Red dots represent upregulated genes and green dots represent downregulated genes in JIB-04 treated cells. (G) Expression of three top genes in (F) with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 12 hr and mock-infected or infected porcine RV (MOI=0.01) for 12 hr. mRNA levels of CYP1A1, CYP1B1, and AHRR at 12 hpi were measured by RT-qPCR. (H) Dose-response analysis of VSV-SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. (I) Dose-response analysis of wild-type SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels at 24 hpi were quantified based on immunofluorescence. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. For all panels except A and I, experiments were repeated at least three times with similar results. Fig. 3A was performed twice. Inhibition assay in Fig. 3I was performed once and cytotoxicity assay was performed in triplicates. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Journal: bioRxiv

    Article Title: Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model

    doi: 10.1101/2020.09.24.312165

    Figure Lengend Snippet: JIB-04 exhibits distinct post-entry antiviral mechanisms (A) Drug combination dose-response matrix and VSV-SARS-CoV-2 replication. MA104 cells were treated with JIB-04 and chloroquine or JIB-04 and NTZ for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. (B) Time of compound addition and VSV-SARS-CoV-2 replication. MA104 cells were treated with NTZ or JIB-04 (10 μM) at indicated time points relative to VSV-SARS-CoV-2 infection (MOI=3, 0 hpi). GFP signals at 8 hpi were quantified to calculate the percentage of inhibition. (C) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. S RNA levels were measured by RT-qPCR. (D) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. FL: full-length. S2: cleaved S2 fragment. (* non-specific band) (E) Histone demethylase siRNA knockdown and RV replication. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01). Viral RNA copy numbers at 12 hpi were quantified by RT-qPCR. (F) Volcano plot of differentially expressed transcripts with JIB-04 treatment and RV infection. HEK293 cells were treated with DMSO or JIB-04 (10 μM) for 12 hr, and mock-infected (left panel) or infected with porcine RV (MOI=0.01, right panel) for another 12 hr. Red dots represent upregulated genes and green dots represent downregulated genes in JIB-04 treated cells. (G) Expression of three top genes in (F) with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 12 hr and mock-infected or infected porcine RV (MOI=0.01) for 12 hr. mRNA levels of CYP1A1, CYP1B1, and AHRR at 12 hpi were measured by RT-qPCR. (H) Dose-response analysis of VSV-SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. (I) Dose-response analysis of wild-type SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels at 24 hpi were quantified based on immunofluorescence. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. For all panels except A and I, experiments were repeated at least three times with similar results. Fig. 3A was performed twice. Inhibition assay in Fig. 3I was performed once and cytotoxicity assay was performed in triplicates. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Article Snippet: Proteins were resolved in SDS-PAGE and detected as describedusing the following antibodies: GAPDH (631402, Biolegend), rotavirus VP6 (rabbit polyclonal, ABclonal technology), and SARS-CoV-2 S2 (40592-T62, Sino Biological).

    Techniques: Infection, Inhibition, Quantitative RT-PCR, Western Blot, Transfection, Expressing, Immunofluorescence, Cytotoxicity Assay

    Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Chemical structures of NTZ and JIB-04 E-isomer from ChemSpider database. (B) Representative images of Vero E6 cells infected by SARS-CoV-2-mNeonGreen (MOI=0.5) at 24 hpi in Fig. 1A . Experiments were repeated at least three times with similar results.

    Journal: bioRxiv

    Article Title: Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model

    doi: 10.1101/2020.09.24.312165

    Figure Lengend Snippet: Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Chemical structures of NTZ and JIB-04 E-isomer from ChemSpider database. (B) Representative images of Vero E6 cells infected by SARS-CoV-2-mNeonGreen (MOI=0.5) at 24 hpi in Fig. 1A . Experiments were repeated at least three times with similar results.

    Article Snippet: Proteins were resolved in SDS-PAGE and detected as describedusing the following antibodies: GAPDH (631402, Biolegend), rotavirus VP6 (rabbit polyclonal, ABclonal technology), and SARS-CoV-2 S2 (40592-T62, Sino Biological).

    Techniques: Infection

    Nitazoxanide and JIB-04 inhibit the replication of multiple viruses (A) Mean fluorescence intensity of GFP positive cells in Fig. 2B was quantified by flow cytometry. (B) Dose-response analysis of VSV-SARS-CoV-2 replication with NTZ or JIB-04 treatment. MA104 cells were treated with compounds at indicated concentrations for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). At 24 hpi, images of GFP positive infected cells were acquired by the ECHO fluorescence microscope. (C) Same as (B) except that cells were infected with an MOI of 0.1. (D) Dose-response analysis of intracellular viral RNA levels with NTZ, JIB-04, or chloroquine treatment. MA104 cells were treated with compounds at 0.1 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (E) Western blot analysis of RV antigen VP6 levels with JIB-04 treatment. HEK293 cells were treated with JIB-04 at 1, 5, or 10 μM for 6 hr and infected with porcine RV (MOI=0.01) for 12 hr. GAPDH was used as a loading control. All experiments were repeated at least three times with similar results. Data are represented as mean ± SEM.

    Journal: bioRxiv

    Article Title: Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model

    doi: 10.1101/2020.09.24.312165

    Figure Lengend Snippet: Nitazoxanide and JIB-04 inhibit the replication of multiple viruses (A) Mean fluorescence intensity of GFP positive cells in Fig. 2B was quantified by flow cytometry. (B) Dose-response analysis of VSV-SARS-CoV-2 replication with NTZ or JIB-04 treatment. MA104 cells were treated with compounds at indicated concentrations for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). At 24 hpi, images of GFP positive infected cells were acquired by the ECHO fluorescence microscope. (C) Same as (B) except that cells were infected with an MOI of 0.1. (D) Dose-response analysis of intracellular viral RNA levels with NTZ, JIB-04, or chloroquine treatment. MA104 cells were treated with compounds at 0.1 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (E) Western blot analysis of RV antigen VP6 levels with JIB-04 treatment. HEK293 cells were treated with JIB-04 at 1, 5, or 10 μM for 6 hr and infected with porcine RV (MOI=0.01) for 12 hr. GAPDH was used as a loading control. All experiments were repeated at least three times with similar results. Data are represented as mean ± SEM.

    Article Snippet: Proteins were resolved in SDS-PAGE and detected as describedusing the following antibodies: GAPDH (631402, Biolegend), rotavirus VP6 (rabbit polyclonal, ABclonal technology), and SARS-CoV-2 S2 (40592-T62, Sino Biological).

    Techniques: Fluorescence, Flow Cytometry, Infection, Microscopy, Quantitative RT-PCR, Western Blot

    Nitazoxanide and JIB-04 broadly inhibit DNA and RNA viruses in different cell types (A) Dose-response analysis of VSV and VSV-SARS-CoV-2 replication with 15 compounds. MA104 cells were treated with indicated compounds at 0.01 to 30 μM for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. EC 50 values for VSV and VSV-SARS-CoV-2 are shown in each graph in red and blue, respectively. (B) Virus infectivity with NTZ or JIB-04 treatment. Vero E6-TMPRSS2 cells were treated with compounds (10 μM) for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). At 6 hpi, percentages of GFP positive cells were quantified by flow cytometry. (C) Dose-response analysis of VSV-SARS-CoV-2 replication and cytotoxicity with NTZ or JIB-04 treatment. For EC 50 measurement, MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3) for 24 hr. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 3 mM for 25 hr. SI: selectivity index. (D) Intracellular viral RNA levels with NTZ or JIB-04 treatment. MA104 cells were treated with compounds (10 μM) for 1 hr and infected with vaccinia virus (VACV), herpes simplex virus-1 (HSV-1), or rotavirus (RV, RRV and UK strains) (MOI=1). Viral RNA levels at 24 hpi were measured by RT-qPCR for VACV B10R, HSV-1 ICP-27, and RV NSP5, respectively. (E) Viral RNA copy numbers with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 6 hr and infected with porcine rotavirus (MOI=0.01) for 6 hr. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with transmissible gastroenteritis virus (TGEV) (MOI=0.01) for 12 hr. Viral RNA copy numbers were measured by RT-qPCR. (F) TGEV titers in the cell supernatant with JIB-04 treatment. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with TGEV (MOI=0.01). Virus titers at 6 and 12 hpi were measured by plaque assays. (G) Intracellular viral RNA levels with NTZ or JIB-04 treatment in different cell types. HEK293-hACE2, HEK293-hACE2-TMPRSS2, and Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1). VSV RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 2A was performed once. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Journal: bioRxiv

    Article Title: Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model

    doi: 10.1101/2020.09.24.312165

    Figure Lengend Snippet: Nitazoxanide and JIB-04 broadly inhibit DNA and RNA viruses in different cell types (A) Dose-response analysis of VSV and VSV-SARS-CoV-2 replication with 15 compounds. MA104 cells were treated with indicated compounds at 0.01 to 30 μM for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. EC 50 values for VSV and VSV-SARS-CoV-2 are shown in each graph in red and blue, respectively. (B) Virus infectivity with NTZ or JIB-04 treatment. Vero E6-TMPRSS2 cells were treated with compounds (10 μM) for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). At 6 hpi, percentages of GFP positive cells were quantified by flow cytometry. (C) Dose-response analysis of VSV-SARS-CoV-2 replication and cytotoxicity with NTZ or JIB-04 treatment. For EC 50 measurement, MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3) for 24 hr. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 3 mM for 25 hr. SI: selectivity index. (D) Intracellular viral RNA levels with NTZ or JIB-04 treatment. MA104 cells were treated with compounds (10 μM) for 1 hr and infected with vaccinia virus (VACV), herpes simplex virus-1 (HSV-1), or rotavirus (RV, RRV and UK strains) (MOI=1). Viral RNA levels at 24 hpi were measured by RT-qPCR for VACV B10R, HSV-1 ICP-27, and RV NSP5, respectively. (E) Viral RNA copy numbers with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 6 hr and infected with porcine rotavirus (MOI=0.01) for 6 hr. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with transmissible gastroenteritis virus (TGEV) (MOI=0.01) for 12 hr. Viral RNA copy numbers were measured by RT-qPCR. (F) TGEV titers in the cell supernatant with JIB-04 treatment. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with TGEV (MOI=0.01). Virus titers at 6 and 12 hpi were measured by plaque assays. (G) Intracellular viral RNA levels with NTZ or JIB-04 treatment in different cell types. HEK293-hACE2, HEK293-hACE2-TMPRSS2, and Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1). VSV RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 2A was performed once. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Article Snippet: Proteins were resolved in SDS-PAGE and detected as describedusing the following antibodies: GAPDH (631402, Biolegend), rotavirus VP6 (rabbit polyclonal, ABclonal technology), and SARS-CoV-2 S2 (40592-T62, Sino Biological).

    Techniques: Infection, Inhibition, Flow Cytometry, Quantitative RT-PCR

    Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Small molecule inhibitor screen. Vero E6 cells were treated with individual compounds (listed in Table S1) at 10 μM for 1 hour (hr) and infected with SARS-CoV-2-mNeonGreen (MOI=0.5). At 24 hr post infection (hpi), cells were fixed and nuclei were stained by Hoechst 33342. The intensities of mNeonGreen and Hoechst were quantified by the Typhoon biomolecular imager and Cytation plate reader, respectively. The ratio of mNeonGreen and Hoechst is plotted as percentage of inhibition. (B) Dose-response analysis of wild-type SARS-CoV-2 replication with NTZ or JIB-04 treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels were quantified at 24 hpi based on immunofluorescence. For CC 50 measurement, cells were treated with inhibitors at 0.3 μM to 1 mM for 25 hr. SI: selectivity index. (C) Dose-response analysis of intracellular viral RNA levels with compounds. Vero E6 cells were treated with NTZ (10 μM), JIB-04 (10 μM), chloroquine (10 μM), remdesivir (3 μM), or camostat (10 μM) for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). SARS-CoV-2 RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 1A was performed once with raw data included in Dataset S1. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05).

    Journal: bioRxiv

    Article Title: Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model

    doi: 10.1101/2020.09.24.312165

    Figure Lengend Snippet: Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Small molecule inhibitor screen. Vero E6 cells were treated with individual compounds (listed in Table S1) at 10 μM for 1 hour (hr) and infected with SARS-CoV-2-mNeonGreen (MOI=0.5). At 24 hr post infection (hpi), cells were fixed and nuclei were stained by Hoechst 33342. The intensities of mNeonGreen and Hoechst were quantified by the Typhoon biomolecular imager and Cytation plate reader, respectively. The ratio of mNeonGreen and Hoechst is plotted as percentage of inhibition. (B) Dose-response analysis of wild-type SARS-CoV-2 replication with NTZ or JIB-04 treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels were quantified at 24 hpi based on immunofluorescence. For CC 50 measurement, cells were treated with inhibitors at 0.3 μM to 1 mM for 25 hr. SI: selectivity index. (C) Dose-response analysis of intracellular viral RNA levels with compounds. Vero E6 cells were treated with NTZ (10 μM), JIB-04 (10 μM), chloroquine (10 μM), remdesivir (3 μM), or camostat (10 μM) for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). SARS-CoV-2 RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 1A was performed once with raw data included in Dataset S1. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05).

    Article Snippet: Proteins were resolved in SDS-PAGE and detected as describedusing the following antibodies: GAPDH (631402, Biolegend), rotavirus VP6 (rabbit polyclonal, ABclonal technology), and SARS-CoV-2 S2 (40592-T62, Sino Biological).

    Techniques: Infection, Staining, Inhibition, Immunofluorescence, Quantitative RT-PCR

    Inhibition or knockdown of specific KDM histone demethylases inhibits virus replication (A) Expression of IFN and IFN-stimulated genes with NTZ or JIB-04 treatment. HEK293 cells were treated with NTZ (10 μM), JIB-04 (3 μM), or transfected with low-molecular-weight poly(I:C) (100 ng/ml) for 24 hr. mRNA levels of IFNL3 and CXCL10 were measured by RT-qPCR. (B) Autophagy formation with compound treatment. HEK293 cells were transfected with EGFP-LC3 plasmid for 24 hr and treated with rapamycin (100 nM), NTZ (10 μM), or JIB-04 (3 μM) for another 18 hr. GFP positive punctate structures indicate autophagy activation. Scale bar, 20 μm. (C) Intracellular viral RNA levels with JIB-04 and camostat treatment. Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (D) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. S RNA levels were measured by RT-qPCR. (E) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. FL: full-length. S2: cleaved S2 fragment. (F) siRNA-mediated knockdown of JIB-04 target histone demethylases. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr. mRNA levels of indicated histone demethylases were measured by RT-qPCR. (G) Western blot analysis of RV antigen VP6 levels in cells with histone demethylase siRNA knockdown. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01) for 12 hr. (H) Pathway enrichment analysis of gene expression regulated by JIB-04 treatment. Downregulated genes in Fig. 3F with p values

    Journal: bioRxiv

    Article Title: Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model

    doi: 10.1101/2020.09.24.312165

    Figure Lengend Snippet: Inhibition or knockdown of specific KDM histone demethylases inhibits virus replication (A) Expression of IFN and IFN-stimulated genes with NTZ or JIB-04 treatment. HEK293 cells were treated with NTZ (10 μM), JIB-04 (3 μM), or transfected with low-molecular-weight poly(I:C) (100 ng/ml) for 24 hr. mRNA levels of IFNL3 and CXCL10 were measured by RT-qPCR. (B) Autophagy formation with compound treatment. HEK293 cells were transfected with EGFP-LC3 plasmid for 24 hr and treated with rapamycin (100 nM), NTZ (10 μM), or JIB-04 (3 μM) for another 18 hr. GFP positive punctate structures indicate autophagy activation. Scale bar, 20 μm. (C) Intracellular viral RNA levels with JIB-04 and camostat treatment. Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (D) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. S RNA levels were measured by RT-qPCR. (E) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. FL: full-length. S2: cleaved S2 fragment. (F) siRNA-mediated knockdown of JIB-04 target histone demethylases. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr. mRNA levels of indicated histone demethylases were measured by RT-qPCR. (G) Western blot analysis of RV antigen VP6 levels in cells with histone demethylase siRNA knockdown. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01) for 12 hr. (H) Pathway enrichment analysis of gene expression regulated by JIB-04 treatment. Downregulated genes in Fig. 3F with p values

    Article Snippet: Proteins were resolved in SDS-PAGE and detected as describedusing the following antibodies: GAPDH (631402, Biolegend), rotavirus VP6 (rabbit polyclonal, ABclonal technology), and SARS-CoV-2 S2 (40592-T62, Sino Biological).

    Techniques: Inhibition, Expressing, Transfection, Molecular Weight, Quantitative RT-PCR, Plasmid Preparation, Activation Assay, Infection, Western Blot

    Principal findings and conceptual model. ( A ) ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293 T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. ( B ) SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral entry ( B ) assay are listed. ( C ) Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.

    Journal: eLife

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

    doi: 10.7554/eLife.61552

    Figure Lengend Snippet: Principal findings and conceptual model. ( A ) ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293 T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. ( B ) SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral entry ( B ) assay are listed. ( C ) Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.

    Article Snippet: Rabbit anti-SARS-CoV-2 RBD (40592-T62) and anti-S2 (40590-T62) pAbs were from Sino Biologicals (Beijing, China).

    Techniques: Binding Assay, Expressing, Generated

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

    Journal: eLife

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

    doi: 10.7554/eLife.61552

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

    Article Snippet: Rabbit anti-SARS-CoV-2 RBD (40592-T62) and anti-S2 (40590-T62) pAbs were from Sino Biologicals (Beijing, China).

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

    Glycan coverage of Spike-ACE2 co-complex. SARS-CoV-2 Spike protein trimer (pink) bound to ACE2 (green). ( A ) Without glycans. ( B ) With N-glycans (red) identified using LC-MS on Spike and ACE2. ( C ) Molecular dynamics simulation analyzed the range of movement of each glycan. The space sampled by glycans is represented by a gray cloud. Glycans cover the Spike-ACE2 interface. They also surround the putative proteolysis site of furin (‘S1-S2’, yellow) and S2’ (blue).

    Journal: eLife

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

    doi: 10.7554/eLife.61552

    Figure Lengend Snippet: Glycan coverage of Spike-ACE2 co-complex. SARS-CoV-2 Spike protein trimer (pink) bound to ACE2 (green). ( A ) Without glycans. ( B ) With N-glycans (red) identified using LC-MS on Spike and ACE2. ( C ) Molecular dynamics simulation analyzed the range of movement of each glycan. The space sampled by glycans is represented by a gray cloud. Glycans cover the Spike-ACE2 interface. They also surround the putative proteolysis site of furin (‘S1-S2’, yellow) and S2’ (blue).

    Article Snippet: Rabbit anti-SARS-CoV-2 RBD (40592-T62) and anti-S2 (40590-T62) pAbs were from Sino Biologicals (Beijing, China).

    Techniques: Liquid Chromatography with Mass Spectroscopy

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

    Journal: Cell

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

    doi: 10.1016/j.cell.2020.09.033

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

    Article Snippet: The SARS-CoV-2 Spike Protein Binds Heparin through the RBD To test experimentally if the SARS-CoV-2 S protein interacts with heparin/HS, recombinant ectodomain and RBD proteins were prepared and characterized.

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

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

    Journal: Cell

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

    doi: 10.1016/j.cell.2020.09.033

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

    Article Snippet: The SARS-CoV-2 Spike Protein Binds Heparin through the RBD To test experimentally if the SARS-CoV-2 S protein interacts with heparin/HS, recombinant ectodomain and RBD proteins were prepared and characterized.

    Techniques: Binding Assay, Flow Cytometry

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

    Journal: Cell

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

    doi: 10.1016/j.cell.2020.09.033

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

    Article Snippet: The SARS-CoV-2 Spike Protein Binds Heparin through the RBD To test experimentally if the SARS-CoV-2 S protein interacts with heparin/HS, recombinant ectodomain and RBD proteins were prepared and characterized.

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

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

    Journal: Cell

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

    doi: 10.1016/j.cell.2020.09.033

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

    Article Snippet: The SARS-CoV-2 Spike Protein Binds Heparin through the RBD To test experimentally if the SARS-CoV-2 S protein interacts with heparin/HS, recombinant ectodomain and RBD proteins were prepared and characterized.

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

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

    Journal: Cell

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

    doi: 10.1016/j.cell.2020.09.033

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

    Article Snippet: The SARS-CoV-2 Spike Protein Binds Heparin through the RBD To test experimentally if the SARS-CoV-2 S protein interacts with heparin/HS, recombinant ectodomain and RBD proteins were prepared and characterized.

    Techniques: Binding Assay, Sequencing

    Gross pathological examination of the lungs of SARS-CoV-2 infected hamsters. Foci (arrowheads) of pulmonary consolidation in untreated SARS-CoV-2 infected animals (A) and animals treated with control MAb (D) or low dose plasma (F). Protection against pulmonary lesions in hamsters treated with MAb 47D11 (C) and high dose plasma (E), similar to mock infected animals (B). Images are from representative animals of each treatment group.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

    doi: 10.1101/2020.08.24.264630

    Figure Lengend Snippet: Gross pathological examination of the lungs of SARS-CoV-2 infected hamsters. Foci (arrowheads) of pulmonary consolidation in untreated SARS-CoV-2 infected animals (A) and animals treated with control MAb (D) or low dose plasma (F). Protection against pulmonary lesions in hamsters treated with MAb 47D11 (C) and high dose plasma (E), similar to mock infected animals (B). Images are from representative animals of each treatment group.

    Article Snippet: Quantitative assessment of virus antigen expression in the lung was performed according to the same method, but using lung sections stained by immunohistochemistry for SARS-CoV-2 antigen.

    Techniques: Infection

    Effect of preventive treatment with MAb or high dose convalescent plasma on severity of pneumonia and level of virus antigen expression in lung parenchyma of hamsters after challenge with SARS-CoV-2. Comparison of extent of histopathological changes (HE) and virus antigen expression (IHC) at four days after SARS-CoV-2 inoculation at low magnification (two left columns) and high magnification (two right columns) in hamsters treated 24 hours before virus inoculation with neutralizing antibodies (second, third and fourth rows) compared to no treatment before SARS-CoV-2 inoculation (first row) and sham inoculation (fifth row).

    Journal: bioRxiv

    Article Title: SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

    doi: 10.1101/2020.08.24.264630

    Figure Lengend Snippet: Effect of preventive treatment with MAb or high dose convalescent plasma on severity of pneumonia and level of virus antigen expression in lung parenchyma of hamsters after challenge with SARS-CoV-2. Comparison of extent of histopathological changes (HE) and virus antigen expression (IHC) at four days after SARS-CoV-2 inoculation at low magnification (two left columns) and high magnification (two right columns) in hamsters treated 24 hours before virus inoculation with neutralizing antibodies (second, third and fourth rows) compared to no treatment before SARS-CoV-2 inoculation (first row) and sham inoculation (fifth row).

    Article Snippet: Quantitative assessment of virus antigen expression in the lung was performed according to the same method, but using lung sections stained by immunohistochemistry for SARS-CoV-2 antigen.

    Techniques: Expressing, Immunohistochemistry

    Effect of prophylactic neutralizing antibody treatment on weight loss and virus replication following SARS-CoV-2 infection in hamsters. A. Body weights of hamsters treated with antibodies were measured at indicated days after inoculation with SARS-CoV-2. SARS-CoV-2 viral RNA (B, C, E and G) or infectious virus (D, F and H) was detected in throat (B), nasal washes (C and D), lung (E and F) and nasal turbinates (G and H). The mean % of starting weight, the mean copy number or the mean infectious titer is shown, error bars represent the standard error of mean. n = 4. * = P

    Journal: bioRxiv

    Article Title: SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

    doi: 10.1101/2020.08.24.264630

    Figure Lengend Snippet: Effect of prophylactic neutralizing antibody treatment on weight loss and virus replication following SARS-CoV-2 infection in hamsters. A. Body weights of hamsters treated with antibodies were measured at indicated days after inoculation with SARS-CoV-2. SARS-CoV-2 viral RNA (B, C, E and G) or infectious virus (D, F and H) was detected in throat (B), nasal washes (C and D), lung (E and F) and nasal turbinates (G and H). The mean % of starting weight, the mean copy number or the mean infectious titer is shown, error bars represent the standard error of mean. n = 4. * = P

    Article Snippet: Quantitative assessment of virus antigen expression in the lung was performed according to the same method, but using lung sections stained by immunohistochemistry for SARS-CoV-2 antigen.

    Techniques: Infection

    Quantitative assessment of histopathological changes and virus antigen expression. Percentage of inflamed lung tissue (A) and percentage of lung tissue expressing SARS-CoV-2 antigen (B) estimated by microscopic examination in different groups of hamsters at four days after SARS-CoV-2 inoculation. Individual (symbols) and mean (horizontal lines) percentages are shown. Error bars represent the standard error of mean. n = 4. * = P

    Journal: bioRxiv

    Article Title: SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

    doi: 10.1101/2020.08.24.264630

    Figure Lengend Snippet: Quantitative assessment of histopathological changes and virus antigen expression. Percentage of inflamed lung tissue (A) and percentage of lung tissue expressing SARS-CoV-2 antigen (B) estimated by microscopic examination in different groups of hamsters at four days after SARS-CoV-2 inoculation. Individual (symbols) and mean (horizontal lines) percentages are shown. Error bars represent the standard error of mean. n = 4. * = P

    Article Snippet: Quantitative assessment of virus antigen expression in the lung was performed according to the same method, but using lung sections stained by immunohistochemistry for SARS-CoV-2 antigen.

    Techniques: Expressing

    Histopathological changes and virus antigen expression in nasal turbinates of hamsters after challenge with SARS-CoV-2. In the nasal turbinate of a sham-inoculated hamster (left column), the nasal cavity is empty and the histology of the olfactory mucosa is normal (A). In a serial section, there is no SARS-CoV-2 antigen expression (C). In the nasal turbinate of a non-treated SARS-CoV-2-inoculated hamster (B and D), the nasal cavity is filled with edema fluid mixed with inflammatory cells and debris and the olfactory mucosa is infiltrated by neutrophils (B). A serial section of this tissue shows SARS-CoV-2 antigen expression in many olfactory mucosal cells, as well as in cells in the lumen (C).

    Journal: bioRxiv

    Article Title: SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

    doi: 10.1101/2020.08.24.264630

    Figure Lengend Snippet: Histopathological changes and virus antigen expression in nasal turbinates of hamsters after challenge with SARS-CoV-2. In the nasal turbinate of a sham-inoculated hamster (left column), the nasal cavity is empty and the histology of the olfactory mucosa is normal (A). In a serial section, there is no SARS-CoV-2 antigen expression (C). In the nasal turbinate of a non-treated SARS-CoV-2-inoculated hamster (B and D), the nasal cavity is filled with edema fluid mixed with inflammatory cells and debris and the olfactory mucosa is infiltrated by neutrophils (B). A serial section of this tissue shows SARS-CoV-2 antigen expression in many olfactory mucosal cells, as well as in cells in the lumen (C).

    Article Snippet: Quantitative assessment of virus antigen expression in the lung was performed according to the same method, but using lung sections stained by immunohistochemistry for SARS-CoV-2 antigen.

    Techniques: Expressing