sars cov2 s1  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike S1 His Recombinant Protein HPLC verified COVID 19 Spike S1 Research
    Description:
    A DNA sequence encoding the SARS CoV 2 2019 nCoV spike protein S1 Subunit YP 009724390 1 Val16 Arg685 was expressed with a polyhistidine tag at the C terminus
    Catalog Number:
    40591-V08H
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus spike Protein 2019-nCoV, cov spike Protein 2019-nCoV, ncov RBD Protein 2019-nCoV, ncov s1 Protein 2019-nCoV, ncov s2 Protein 2019-nCoV, ncov spike Protein 2019-nCoV, NCP-CoV RBD Protein 2019-nCoV, NCP-CoV s1 Protein 2019-nCoV, NCP-CoV s2 Protein 2019-nCoV, NCP-CoV Spike Protein 2019-nCoV, novel coronavirus RBD Protein 2019-nCoV, novel coronavirus s1 Protein 2019-nCoV, novel coronavirus s2 Protein 2019-nCoV, novel coronavirus spike Protein 2019-nCoV, RBD Protein 2019-nCoV, S1 Protein 2019-nCoV, S2 Protein 2019-nCoV, Spike RBD Protein 2019-nCoV
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological sars cov2 s1
    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with <t>SARS‐CoV2‐RBG</t> and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and <t>SARS‐CoV2‐S1</t> proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.
    A DNA sequence encoding the SARS CoV 2 2019 nCoV spike protein S1 Subunit YP 009724390 1 Val16 Arg685 was expressed with a polyhistidine tag at the C terminus
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    Images

    1) Product Images from "Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors, Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors"

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

    Journal: Small Methods

    doi: 10.1002/smtd.202001031

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

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

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

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

    Journal: The Journal of Infectious Diseases

    doi: 10.1093/infdis/jiaa479

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

    Techniques Used: Infection, Concentration Assay, Binding Assay

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.07.24.220715

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

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

    4) Product Images from "Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies"

    Article Title: Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies

    Journal: Cell

    doi: 10.1016/j.cell.2020.04.031

    SARS VHH-72 Cross-Reacts with SARS-CoV-2 (A) An SPR sensorgram measuring the binding of SARS VHH-72 to the SARS-CoV-2 RBD-SD1. Binding curves are colored black, and fit of the data to a 1:1 binding model is colored red. (B) The crystal structure of SARS VHH-72 bound to the SARS-CoV-1 RBD is shown with SARS VHH-72 as dark blue ribbons and the RBD as a pink molecular surface. Amino acids that vary between SARS-CoV-1 and SARS-CoV-2 are colored green.
    Figure Legend Snippet: SARS VHH-72 Cross-Reacts with SARS-CoV-2 (A) An SPR sensorgram measuring the binding of SARS VHH-72 to the SARS-CoV-2 RBD-SD1. Binding curves are colored black, and fit of the data to a 1:1 binding model is colored red. (B) The crystal structure of SARS VHH-72 bound to the SARS-CoV-1 RBD is shown with SARS VHH-72 as dark blue ribbons and the RBD as a pink molecular surface. Amino acids that vary between SARS-CoV-1 and SARS-CoV-2 are colored green.

    Techniques Used: SPR Assay, Binding Assay

    Neutralizing Mechanisms of MERS VHH-55 and SARS VHH-72 (A) The MERS-CoV spike (PDB ID: 5W9H ) is shown as a transparent molecular surface, with each monomer colored either white, gray, or tan. Each monomer is bound by MERS VHH-55, shown as blue ribbons. The clash between MERS VHH-55 bound to the white monomer and the neighboring tan RBD is highlighted by the red ellipse. (B) The SARS-CoV-1 spike (PDB ID: 5X58 ) is shown as a transparent molecular surface, with each protomer colored either white, gray, or pink. Every monomer is bound by a copy of SARS VHH-72, shown as dark blue ribbons. The clashes between copies of SARS VHH-72 and the two neighboring spike monomers are highlighted by the red circle. (C) The SARS-CoV-2 spike (PDB ID: 6VXX ) is shown as a transparent molecular surface, with each protomer colored either white, gray, or green. Every monomer is bound by a copy of SARS VHH-72, shown as dark blue ribbons. The clashes between copies of SARS VHH-72 and the two neighboring spike monomers are highlighted by the red circle. The SARS-CoV-2 trimer appears smaller than SARS-CoV-1 S because of the absence of flexible NTD-distal loops, which could not be built during cryo-EM analysis. (D) CoV VHHs prevent MERS-CoV RBD, SARS-CoV-1 RBD, and SARS-CoV-2 RBD-SD1 from interacting with their receptors. The results of the BLI-based receptor-blocking experiment are shown. The legend lists the immobilized RBDs and the VHHs or receptors that correspond to each curve.
    Figure Legend Snippet: Neutralizing Mechanisms of MERS VHH-55 and SARS VHH-72 (A) The MERS-CoV spike (PDB ID: 5W9H ) is shown as a transparent molecular surface, with each monomer colored either white, gray, or tan. Each monomer is bound by MERS VHH-55, shown as blue ribbons. The clash between MERS VHH-55 bound to the white monomer and the neighboring tan RBD is highlighted by the red ellipse. (B) The SARS-CoV-1 spike (PDB ID: 5X58 ) is shown as a transparent molecular surface, with each protomer colored either white, gray, or pink. Every monomer is bound by a copy of SARS VHH-72, shown as dark blue ribbons. The clashes between copies of SARS VHH-72 and the two neighboring spike monomers are highlighted by the red circle. (C) The SARS-CoV-2 spike (PDB ID: 6VXX ) is shown as a transparent molecular surface, with each protomer colored either white, gray, or green. Every monomer is bound by a copy of SARS VHH-72, shown as dark blue ribbons. The clashes between copies of SARS VHH-72 and the two neighboring spike monomers are highlighted by the red circle. The SARS-CoV-2 trimer appears smaller than SARS-CoV-1 S because of the absence of flexible NTD-distal loops, which could not be built during cryo-EM analysis. (D) CoV VHHs prevent MERS-CoV RBD, SARS-CoV-1 RBD, and SARS-CoV-2 RBD-SD1 from interacting with their receptors. The results of the BLI-based receptor-blocking experiment are shown. The legend lists the immobilized RBDs and the VHHs or receptors that correspond to each curve.

    Techniques Used: Blocking Assay

    Engineering a Functional Bivalent VHH Construct, Related to Figure 6 (A) Flow cytometry measuring the binding of the bivalent SARS VHH-72 tail-to-head fusion (VHH-72-VHH-72) to SARS-CoV-1 or SARS-CoV-2 S expressed on the cell surface. VHH-23-VHH-23, a bivalent tail-to-head fusion of an irrelevant nanobody, was included as a negative control. (B) Binding of SARS-CoV-2 RBD-SD1 to Vero E6 cells is prevented by VHH-72-VHH-72 in a dose-dependent fashion. Binding of SARS-CoV-2 RBD-SD1 to Vero E6 cells was detected by flow cytometry in the presence of the indicated bivalent VHHs (n = 2 except VHH-72-VHH-72 and VHH-23-VHH-23 at 5 μg/mL, n = 5). (C) Binding of SARS-CoV-2 RBD-SD1 to Vero E6 cells is prevented by bivalent VHH-72-Fc fusion proteins in a dose-dependent fashion. Binding of SARS-CoV-2 RBD-SD1-Fc to Vero E6 cells was detected by flow cytometry in the presence of the indicated constructs and amounts (n = 2 except no RBD, n = 4). (D) Cell surface binding of SARS VHH-72 to SARS-CoV-1 S. 293T cells were transfected with a GFP expression plasmid together with a SARS-CoV-1 S expression plasmid. Binding of the indicated protein is expressed as the median fluorescent intensity (MFI), measured to detect the His-tagged MERS VHH-55 or SARS VHH-72 or the SARS VHH-72-Fc fusions, of the GFP positive cells divided by the MFI of the GFP negative cells. (E) Cell surface binding of SARS VHH-72 to SARS-CoV-2. MFI was calculated using the same equation as Figure S6 D.
    Figure Legend Snippet: Engineering a Functional Bivalent VHH Construct, Related to Figure 6 (A) Flow cytometry measuring the binding of the bivalent SARS VHH-72 tail-to-head fusion (VHH-72-VHH-72) to SARS-CoV-1 or SARS-CoV-2 S expressed on the cell surface. VHH-23-VHH-23, a bivalent tail-to-head fusion of an irrelevant nanobody, was included as a negative control. (B) Binding of SARS-CoV-2 RBD-SD1 to Vero E6 cells is prevented by VHH-72-VHH-72 in a dose-dependent fashion. Binding of SARS-CoV-2 RBD-SD1 to Vero E6 cells was detected by flow cytometry in the presence of the indicated bivalent VHHs (n = 2 except VHH-72-VHH-72 and VHH-23-VHH-23 at 5 μg/mL, n = 5). (C) Binding of SARS-CoV-2 RBD-SD1 to Vero E6 cells is prevented by bivalent VHH-72-Fc fusion proteins in a dose-dependent fashion. Binding of SARS-CoV-2 RBD-SD1-Fc to Vero E6 cells was detected by flow cytometry in the presence of the indicated constructs and amounts (n = 2 except no RBD, n = 4). (D) Cell surface binding of SARS VHH-72 to SARS-CoV-1 S. 293T cells were transfected with a GFP expression plasmid together with a SARS-CoV-1 S expression plasmid. Binding of the indicated protein is expressed as the median fluorescent intensity (MFI), measured to detect the His-tagged MERS VHH-55 or SARS VHH-72 or the SARS VHH-72-Fc fusions, of the GFP positive cells divided by the MFI of the GFP negative cells. (E) Cell surface binding of SARS VHH-72 to SARS-CoV-2. MFI was calculated using the same equation as Figure S6 D.

    Techniques Used: Functional Assay, Construct, Flow Cytometry, Binding Assay, Negative Control, Transfection, Expressing, Plasmid Preparation

    VHH-72-Fc Neutralizes SARS-CoV-2 S Pseudoviruses (A) BLI sensorgram measuring apparent binding affinity of VHH-72-Fc to immobilized SARS-CoV-2 RBD-Fc. Binding curves are colored black, buffer-only blanks are colored gray, and the fit of the data to a 1:1 binding curve is colored red. (B) Time course analysis of VHH-72-Fc expression in ExpiCHO cells. Cell culture supernatants of transiently transfected ExpiCHO cells were removed on days 3–7 after transfection (or until cell viability dropped below 75%), as indicated. Two control mAbs were included for comparison, along with the indicated amounts of purified GBP-Fc as a loading control. (C) SARS-CoV-2 S pseudotyped VSV neutralization assay. Monolayers of Vero E6 cells were infected with pseudoviruses that had been pre-incubated with the mixtures indicated by the legend. The VHH-72-Fc used in this assay was purified after expression in ExpiCHO cells (n = 4). VHH-23-Fc is an irrelevant control VHH-Fc (n = 3). NI, cells were not infected. Luciferase activity is reported in counts per second (c.p.s.) ± SEM.
    Figure Legend Snippet: VHH-72-Fc Neutralizes SARS-CoV-2 S Pseudoviruses (A) BLI sensorgram measuring apparent binding affinity of VHH-72-Fc to immobilized SARS-CoV-2 RBD-Fc. Binding curves are colored black, buffer-only blanks are colored gray, and the fit of the data to a 1:1 binding curve is colored red. (B) Time course analysis of VHH-72-Fc expression in ExpiCHO cells. Cell culture supernatants of transiently transfected ExpiCHO cells were removed on days 3–7 after transfection (or until cell viability dropped below 75%), as indicated. Two control mAbs were included for comparison, along with the indicated amounts of purified GBP-Fc as a loading control. (C) SARS-CoV-2 S pseudotyped VSV neutralization assay. Monolayers of Vero E6 cells were infected with pseudoviruses that had been pre-incubated with the mixtures indicated by the legend. The VHH-72-Fc used in this assay was purified after expression in ExpiCHO cells (n = 4). VHH-23-Fc is an irrelevant control VHH-Fc (n = 3). NI, cells were not infected. Luciferase activity is reported in counts per second (c.p.s.) ± SEM.

    Techniques Used: Binding Assay, Expressing, Cell Culture, Transfection, Purification, Neutralization, Infection, Incubation, Luciferase, Activity Assay

    SARS VHH-72 Bivalency Permits SARS-CoV-2 Pseudovirus Neutralization (A and B) SARS-CoV-1 S (A) and SARS-CoV-2 S (B) VSV pseudoviruses were used to evaluate the neutralization capacity of SARS VHH-72. MERS VHH-55 and PBS were included as negative controls. Luciferase activity is reported in counts per second (c.p.s.). NI, cells were not infected. (C and D) Binding of bivalent VHHs was tested by ELISA against SARS-CoV-1 S (C) and SARS-CoV-2 RBD-SD1 (D). VHH-72-Fc refers to SARS VHH-72 fused to a human IgG1 Fc domain by a GS(GGGGS) 2 linker. VHH-72-Fc (S) is the same Fc fusion with a GS, rather than a GS(GGGGS) 2 , linker. GBP is an irrelevant GFP-binding protein. VHH-72-VHH-72 refers to the tail-to-head construct with two SARS VHH-72 proteins connected by a (GGGGS) 3 linker. VHH-23-VHH-23 refers to the two irrelevant VHHs linked via the same (GGGGS) 3 linker. (E and F) SARS-CoV-1 S (E) and SARS-CoV-2 S (F) pseudoviruses were used to evaluate the neutralization capacity of bivalent VHH-72-Fc. GBP and PBS were included as negative controls. NI, cells were not infected.
    Figure Legend Snippet: SARS VHH-72 Bivalency Permits SARS-CoV-2 Pseudovirus Neutralization (A and B) SARS-CoV-1 S (A) and SARS-CoV-2 S (B) VSV pseudoviruses were used to evaluate the neutralization capacity of SARS VHH-72. MERS VHH-55 and PBS were included as negative controls. Luciferase activity is reported in counts per second (c.p.s.). NI, cells were not infected. (C and D) Binding of bivalent VHHs was tested by ELISA against SARS-CoV-1 S (C) and SARS-CoV-2 RBD-SD1 (D). VHH-72-Fc refers to SARS VHH-72 fused to a human IgG1 Fc domain by a GS(GGGGS) 2 linker. VHH-72-Fc (S) is the same Fc fusion with a GS, rather than a GS(GGGGS) 2 , linker. GBP is an irrelevant GFP-binding protein. VHH-72-VHH-72 refers to the tail-to-head construct with two SARS VHH-72 proteins connected by a (GGGGS) 3 linker. VHH-23-VHH-23 refers to the two irrelevant VHHs linked via the same (GGGGS) 3 linker. (E and F) SARS-CoV-1 S (E) and SARS-CoV-2 S (F) pseudoviruses were used to evaluate the neutralization capacity of bivalent VHH-72-Fc. GBP and PBS were included as negative controls. NI, cells were not infected.

    Techniques Used: Neutralization, Luciferase, Activity Assay, Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, Construct

    5) Product Images from "Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor"

    Article Title: Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor

    Journal: ACS Nano

    doi: 10.1021/acsnano.0c02823

    Detection of SARS-CoV-2 antigen protein. (A) Schematic diagram for the COVID-19 FET sensor for detection of SARS-CoV-2 spike protein. (B) Real-time response of COVID-19 FET toward SARS-CoV-2 antigen protein in PBS and (C) related dose-dependent response curve ( V DS = 0.01 V). Graphene-based FET without SARS-CoV-2 antibody is presented as negative control. (D) Selective response of COVID-19 FET sensor toward target SARS-CoV-2 antigen protein and MERS-CoV protein. (E) Real-time response of COVID-19 FET toward SARS-CoV-2 antigen protein in UTM and (F) related dose-dependent response curve.
    Figure Legend Snippet: Detection of SARS-CoV-2 antigen protein. (A) Schematic diagram for the COVID-19 FET sensor for detection of SARS-CoV-2 spike protein. (B) Real-time response of COVID-19 FET toward SARS-CoV-2 antigen protein in PBS and (C) related dose-dependent response curve ( V DS = 0.01 V). Graphene-based FET without SARS-CoV-2 antibody is presented as negative control. (D) Selective response of COVID-19 FET sensor toward target SARS-CoV-2 antigen protein and MERS-CoV protein. (E) Real-time response of COVID-19 FET toward SARS-CoV-2 antigen protein in UTM and (F) related dose-dependent response curve.

    Techniques Used: Negative Control

    Detection of cultured SARS-CoV-2 virus. (A) Schematic diagram for the COVID-19 FET sensor for detection of SARS-CoV-2 cultured virus. (B) Real-time response of COVID-19 FET toward SARS-CoV-2 cultured virus and (C) related dose-dependent response curve.
    Figure Legend Snippet: Detection of cultured SARS-CoV-2 virus. (A) Schematic diagram for the COVID-19 FET sensor for detection of SARS-CoV-2 cultured virus. (B) Real-time response of COVID-19 FET toward SARS-CoV-2 cultured virus and (C) related dose-dependent response curve.

    Techniques Used: Cell Culture

    Electrical characterization of pristine, PBASE-modified, and SARS-CoV-2 spike antibody-immobilized graphene. (A) Schematic diagram of the aqueous-solution-gated FET (COVID-19 FET sensor) configuration using the antibody-conjugated graphene. (B) I DS – V DS output curves of the antibody-conjugated FET with various gating voltages from 0 to −1.5 V in steps of −0.3 V. I DS negatively increased as V GS negatively increased. (C) Current–voltage ( I–V ) characteristics of the graphene-based device of each functionalization process for the antibody modification. (D) Measurement of transfer curves of the COVID-19 FET sensor in steps of the antibody conjugation ( V DS = 0.01 V).
    Figure Legend Snippet: Electrical characterization of pristine, PBASE-modified, and SARS-CoV-2 spike antibody-immobilized graphene. (A) Schematic diagram of the aqueous-solution-gated FET (COVID-19 FET sensor) configuration using the antibody-conjugated graphene. (B) I DS – V DS output curves of the antibody-conjugated FET with various gating voltages from 0 to −1.5 V in steps of −0.3 V. I DS negatively increased as V GS negatively increased. (C) Current–voltage ( I–V ) characteristics of the graphene-based device of each functionalization process for the antibody modification. (D) Measurement of transfer curves of the COVID-19 FET sensor in steps of the antibody conjugation ( V DS = 0.01 V).

    Techniques Used: Modification, Conjugation Assay

    Detection of SARS-CoV-2 virus from clinical samples. (A) Schematic diagram for the COVID-19 FET sensor for detection of SARS-CoV-2 virus from COVID-19 patients. (B,C) Comparison of response signal between normal samples and patient ones. (D) Real-time response of COVID-19 FET toward SARS-CoV-2 clinical sample and (C) related dose-dependent response curve.
    Figure Legend Snippet: Detection of SARS-CoV-2 virus from clinical samples. (A) Schematic diagram for the COVID-19 FET sensor for detection of SARS-CoV-2 virus from COVID-19 patients. (B,C) Comparison of response signal between normal samples and patient ones. (D) Real-time response of COVID-19 FET toward SARS-CoV-2 clinical sample and (C) related dose-dependent response curve.

    Techniques Used:

    6) Product Images from "CoVaccine HT™ Adjuvant Potentiates Robust Immune Responses to Recombinant SARS-CoV-2 Spike S1 Immunization"

    Article Title: CoVaccine HT™ Adjuvant Potentiates Robust Immune Responses to Recombinant SARS-CoV-2 Spike S1 Immunization

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.599587

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

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

    7) Product Images from "Ocular conjunctival inoculation of SARS-CoV-2 can cause mild COVID-19 in Rhesus macaques"

    Article Title: Ocular conjunctival inoculation of SARS-CoV-2 can cause mild COVID-19 in Rhesus macaques

    Journal: bioRxiv

    doi: 10.1101/2020.03.13.990036

    Graphic outline of experimental design and sample collection. Five male rhesus macaques ( Macaca Mulatta ) between the ages of 3 and 5 years were inoculated with 10 6 TCID 50 /ml SARS-CoV-2. Two rhesus macaques via ocular conjunctival route named CJ-1 and CJ-2, one was inoculated via intratracheal route named IT-1, two were inoculated via intragastric route in sequence named IG-1 and IG-2, respectively. The macaques were observed daily for clinical signs (body weight temperature were tested as shown). On 0, 1, 3, 5, and 7 dpi, the conjunctival, nasal, throat and anal swabs were collected. CJ-1, IT-1, and IG-1 were euthanized and necropsied on 7 dpi. Tissues were collected to analysis the virus distributions. All sera were collected on 0, 7, 14 and 21 dpi for serologic detection to exam the SARS-CoV-2 specific IgG antibodies.
    Figure Legend Snippet: Graphic outline of experimental design and sample collection. Five male rhesus macaques ( Macaca Mulatta ) between the ages of 3 and 5 years were inoculated with 10 6 TCID 50 /ml SARS-CoV-2. Two rhesus macaques via ocular conjunctival route named CJ-1 and CJ-2, one was inoculated via intratracheal route named IT-1, two were inoculated via intragastric route in sequence named IG-1 and IG-2, respectively. The macaques were observed daily for clinical signs (body weight temperature were tested as shown). On 0, 1, 3, 5, and 7 dpi, the conjunctival, nasal, throat and anal swabs were collected. CJ-1, IT-1, and IG-1 were euthanized and necropsied on 7 dpi. Tissues were collected to analysis the virus distributions. All sera were collected on 0, 7, 14 and 21 dpi for serologic detection to exam the SARS-CoV-2 specific IgG antibodies.

    Techniques Used: Sequencing

    Clinical features, viral distributions and antibody detection in the sera from the rhesus macaques inoculated with SARS-CoV-2 via three routes. Clinical signs including body weight (A) and temperature (B) were observed. The viral load of the conjunctival, nasal, throat, and anal swabs specimens (C) from the five inoculated macaques on 0, 1, 3, 5, and 7 dpi. The comparison of viral distributionsin the majority of organs and tissues (D) from CJ-1 and IT-1 on 7 dpi. The darker the blue color, the higher the viral load. The specific IgG antibody against SARS-CoV-2 in the sera of the inoculated-macaques were tested by ELISA on 0, 7, 14, and 21 dpi (E). According to unpaired Welch’s t -test, the specific IgG antibody in the sera of conjunctival inoculated macaque exhibited a significant increase compared with prior to inoculation (**p=0.0027) and 21 dpi (**p=0.0039) . CJ-1 and CJ-2 were the two macaques that inoculated via conjunctival route, IT-1 was the macaque that inoculated via intratracheal route. IG-1 and IG-2 were the two macaques that inoculated via intragastric route. ND, not detected. NC, negative control (unpaired Welch’s t -test, ** p
    Figure Legend Snippet: Clinical features, viral distributions and antibody detection in the sera from the rhesus macaques inoculated with SARS-CoV-2 via three routes. Clinical signs including body weight (A) and temperature (B) were observed. The viral load of the conjunctival, nasal, throat, and anal swabs specimens (C) from the five inoculated macaques on 0, 1, 3, 5, and 7 dpi. The comparison of viral distributionsin the majority of organs and tissues (D) from CJ-1 and IT-1 on 7 dpi. The darker the blue color, the higher the viral load. The specific IgG antibody against SARS-CoV-2 in the sera of the inoculated-macaques were tested by ELISA on 0, 7, 14, and 21 dpi (E). According to unpaired Welch’s t -test, the specific IgG antibody in the sera of conjunctival inoculated macaque exhibited a significant increase compared with prior to inoculation (**p=0.0027) and 21 dpi (**p=0.0039) . CJ-1 and CJ-2 were the two macaques that inoculated via conjunctival route, IT-1 was the macaque that inoculated via intratracheal route. IG-1 and IG-2 were the two macaques that inoculated via intragastric route. ND, not detected. NC, negative control (unpaired Welch’s t -test, ** p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Negative Control

    Compare the lesions in lungs from CJ-1 and IT-1 by radiographic alterations, histopathological and Immunohistochemical observation. The anterior-posterior and right lateral chest radiographs (A) from rhesus macaque imaged prior to SARS-CoV-2 inoculation (day 0) and 7 dpi. Areas of interstitial infiltration, indicative of pneumonia, are highlighted (red circle); obscures costophrenic angle (red arrows); patchy lesions (blue circle). Positional indicators are included (R=right). The histopathological and immunohistochemical observations in the lungs (B). Both the two macaques exhibited interstitial pneumonia with thickened alveolar septa, filtration of inflammatory cells mainly including lymphocytes and macrophages, some amounts of exudation (red arrows) in the alveolar cavities on 7dpi. Conjunctival route caused relatively mild pneumonia. The sequential sections were stained by HE and IHC, respectively. The viral antigens were observed primarily in the alveolar epithelia (black arrows) and the detached-degenerative cellular debris (green arrows). The H E stained-sections under 400 magnification were the fractionated gain (black frame) of these sections under 100 magnification. The IHC section showed the same field with the black frame section under 400 magnification. Black scale bar = 100 μm, red scale bar = 50 μm.
    Figure Legend Snippet: Compare the lesions in lungs from CJ-1 and IT-1 by radiographic alterations, histopathological and Immunohistochemical observation. The anterior-posterior and right lateral chest radiographs (A) from rhesus macaque imaged prior to SARS-CoV-2 inoculation (day 0) and 7 dpi. Areas of interstitial infiltration, indicative of pneumonia, are highlighted (red circle); obscures costophrenic angle (red arrows); patchy lesions (blue circle). Positional indicators are included (R=right). The histopathological and immunohistochemical observations in the lungs (B). Both the two macaques exhibited interstitial pneumonia with thickened alveolar septa, filtration of inflammatory cells mainly including lymphocytes and macrophages, some amounts of exudation (red arrows) in the alveolar cavities on 7dpi. Conjunctival route caused relatively mild pneumonia. The sequential sections were stained by HE and IHC, respectively. The viral antigens were observed primarily in the alveolar epithelia (black arrows) and the detached-degenerative cellular debris (green arrows). The H E stained-sections under 400 magnification were the fractionated gain (black frame) of these sections under 100 magnification. The IHC section showed the same field with the black frame section under 400 magnification. Black scale bar = 100 μm, red scale bar = 50 μm.

    Techniques Used: Immunohistochemistry, Filtration, Staining

    8) Product Images from "Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies"

    Article Title: Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies

    Journal: bioRxiv

    doi: 10.1101/2020.12.13.422550

    The predicted linear B-cell epitopes in the Spike protein of SARS-CoV-2. a , The number of linear B-cell epitopes shared among the distinct methods and literature mining. The pink, green and light blue represent epitopes with antigenicity scores > 0.9, 0.4 and 0.9, and
    Figure Legend Snippet: The predicted linear B-cell epitopes in the Spike protein of SARS-CoV-2. a , The number of linear B-cell epitopes shared among the distinct methods and literature mining. The pink, green and light blue represent epitopes with antigenicity scores > 0.9, 0.4 and 0.9, and

    Techniques Used:

    Measurements of the selected Linear B cell epitope binding to antibody and neutralization efficiency of selected epitopes against SARS-CoV-2. a-d, The binding affinity assessed by ELISA between linear B-cell epitopes and serum antibodies from immunized horse with S1-based vaccines (a), immunized mouse with RBD-based vaccines (b), immunized monkey with RBD-based vaccines (c), and a patient recovering from COVID-19 (d). e, The binding affinity assessed by ELISA between the linear B-cell epitopes and serum antibodies from immunized mice with corresponding epitopes of ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. f, Neutralization assay against SARS-CoV-2 pseudovirus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of EC 50 . g, Neutralization assay against SARS-CoV-2 live virus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of NT 50 .
    Figure Legend Snippet: Measurements of the selected Linear B cell epitope binding to antibody and neutralization efficiency of selected epitopes against SARS-CoV-2. a-d, The binding affinity assessed by ELISA between linear B-cell epitopes and serum antibodies from immunized horse with S1-based vaccines (a), immunized mouse with RBD-based vaccines (b), immunized monkey with RBD-based vaccines (c), and a patient recovering from COVID-19 (d). e, The binding affinity assessed by ELISA between the linear B-cell epitopes and serum antibodies from immunized mice with corresponding epitopes of ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. f, Neutralization assay against SARS-CoV-2 pseudovirus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of EC 50 . g, Neutralization assay against SARS-CoV-2 live virus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of NT 50 .

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

    The characteristics of the 18 selected linear B cell epitopes. a , The sequences of 18 selected linear B-cell epitopes. The bold is the mutated site in less than ten of 118,694 virus strains; The red is the predicted discontinuous residues. The bars on the right side are the Wilcoxon test p value for the comparisons of IgG or IgA antibody enrichment scores associated with each linear B-cell epitope between COVID-19 patients and negative controls. b , The digesting enzymes profile of the epitope sequence. Red indicated not digest, blue indicated digest. c-d , The localization of the 18 selected epitopes mapped on SARS-CoV-2 S (PDB: 6VSB) protein (c) and ACE-RBD complex (d). e-f , The localizations of B cell discontinuous epitopes on SARS-CoV-2 S (PDB: 6VSB) protein (e) and ACE-RBD complex (f). The spike protein is grey, the RBD region is wheat color, the selected epitopes are green, the mutation sites are red, the human ACE domain is blue, and the discontinuous B-cell epitopes are purple.
    Figure Legend Snippet: The characteristics of the 18 selected linear B cell epitopes. a , The sequences of 18 selected linear B-cell epitopes. The bold is the mutated site in less than ten of 118,694 virus strains; The red is the predicted discontinuous residues. The bars on the right side are the Wilcoxon test p value for the comparisons of IgG or IgA antibody enrichment scores associated with each linear B-cell epitope between COVID-19 patients and negative controls. b , The digesting enzymes profile of the epitope sequence. Red indicated not digest, blue indicated digest. c-d , The localization of the 18 selected epitopes mapped on SARS-CoV-2 S (PDB: 6VSB) protein (c) and ACE-RBD complex (d). e-f , The localizations of B cell discontinuous epitopes on SARS-CoV-2 S (PDB: 6VSB) protein (e) and ACE-RBD complex (f). The spike protein is grey, the RBD region is wheat color, the selected epitopes are green, the mutation sites are red, the human ACE domain is blue, and the discontinuous B-cell epitopes are purple.

    Techniques Used: Sequencing, Mutagenesis

    9) Product Images from "Age‐related rhesus macaque models of COVID‐19, et al. Age‐related rhesus macaque models of COVID‐19"

    Article Title: Age‐related rhesus macaque models of COVID‐19, et al. Age‐related rhesus macaque models of COVID‐19

    Journal: Animal Models and Experimental Medicine

    doi: 10.1002/ame2.12108

    Viral load of the SARS‐CoV‐2‐infected rhesus macaque model. A. Average viral loads of swabs from the younger group (YG, n = 3, red line) monkeys. B. Average viral load of swabs from the elder group (EG, n = 2, blue line) monkeys. Viral loads of nasal, throat, and anal swab specimens collected from the inoculated macaques on 0, 3, 5, 7, 9, 11, and 14 dpi. C. Viral loads in varies lobe of lung tissue from YG and EG monkeys at day 7 post‐inoculation. RNA was extracted and viral load was determined by qRT‐PCR. All data are presented as mean ± SEM
    Figure Legend Snippet: Viral load of the SARS‐CoV‐2‐infected rhesus macaque model. A. Average viral loads of swabs from the younger group (YG, n = 3, red line) monkeys. B. Average viral load of swabs from the elder group (EG, n = 2, blue line) monkeys. Viral loads of nasal, throat, and anal swab specimens collected from the inoculated macaques on 0, 3, 5, 7, 9, 11, and 14 dpi. C. Viral loads in varies lobe of lung tissue from YG and EG monkeys at day 7 post‐inoculation. RNA was extracted and viral load was determined by qRT‐PCR. All data are presented as mean ± SEM

    Techniques Used: Infection, Quantitative RT-PCR

    Hematological analysis in rhesus macaques inoculated with SARS‐CoV‐2. A. The counts of white blood cells (WBC) were analysed. B. The percentage and counts of monocytes were determined. C. The percentage and counts of lymphocytes were detected. D. The percentage and counts of CD3 + CD8 + T cells, CD3 + CD4 + T cells were shown. YG (red line) and EG (blue line) were indicated in the upper right corner of each panel. All data are presented as mean ± SEM
    Figure Legend Snippet: Hematological analysis in rhesus macaques inoculated with SARS‐CoV‐2. A. The counts of white blood cells (WBC) were analysed. B. The percentage and counts of monocytes were determined. C. The percentage and counts of lymphocytes were detected. D. The percentage and counts of CD3 + CD8 + T cells, CD3 + CD4 + T cells were shown. YG (red line) and EG (blue line) were indicated in the upper right corner of each panel. All data are presented as mean ± SEM

    Techniques Used:

    The comparison of lesions in the lung between younger group (YG) and elder group (EG) by radiographic alterations, histopathological and immunohistochemical (IHC) observation of the SARS‐CoV‐2‐inoculated‐rhesus macaque. A. Anterior‐posterior thoracic X‐rays from of rhesus macaque imaged prior to SARS‐CoV‐2 inoculation (day 0) and on 7 dpi of YG and 5 dpi of EG. Areas of interstitial infiltration, indicative of pneumonia, are highlighted (red circle). Positional indicators are included (R = right). B. Histopathological changes in lungs from YG and EG. Lung tissue was collected and stained with hematoxylin and eosin. Black scale bar = 40 µm. IHC staining demonstrated that SARS‐CoV‐2 antigens were mainly in the epithelial cells and macrophages. SARS‐CoV‐2 antigens were indicated by red arrows. Red scale bar = 50 µm
    Figure Legend Snippet: The comparison of lesions in the lung between younger group (YG) and elder group (EG) by radiographic alterations, histopathological and immunohistochemical (IHC) observation of the SARS‐CoV‐2‐inoculated‐rhesus macaque. A. Anterior‐posterior thoracic X‐rays from of rhesus macaque imaged prior to SARS‐CoV‐2 inoculation (day 0) and on 7 dpi of YG and 5 dpi of EG. Areas of interstitial infiltration, indicative of pneumonia, are highlighted (red circle). Positional indicators are included (R = right). B. Histopathological changes in lungs from YG and EG. Lung tissue was collected and stained with hematoxylin and eosin. Black scale bar = 40 µm. IHC staining demonstrated that SARS‐CoV‐2 antigens were mainly in the epithelial cells and macrophages. SARS‐CoV‐2 antigens were indicated by red arrows. Red scale bar = 50 µm

    Techniques Used: Immunohistochemistry, Staining

    10) Product Images from "Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies"

    Article Title: Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies

    Journal: bioRxiv

    doi: 10.1101/2020.12.13.422550

    The predicted linear B-cell epitopes in the Spike protein of SARS-CoV-2. a , The number of linear B-cell epitopes shared among the distinct methods and literature mining. The pink, green and light blue represent epitopes with antigenicity scores > 0.9, 0.4 and 0.9, and
    Figure Legend Snippet: The predicted linear B-cell epitopes in the Spike protein of SARS-CoV-2. a , The number of linear B-cell epitopes shared among the distinct methods and literature mining. The pink, green and light blue represent epitopes with antigenicity scores > 0.9, 0.4 and 0.9, and

    Techniques Used:

    Measurements of the selected Linear B cell epitope binding to antibody and neutralization efficiency of selected epitopes against SARS-CoV-2. a-d, The binding affinity assessed by ELISA between linear B-cell epitopes and serum antibodies from immunized horse with S1-based vaccines (a), immunized mouse with RBD-based vaccines (b), immunized monkey with RBD-based vaccines (c), and a patient recovering from COVID-19 (d). e, The binding affinity assessed by ELISA between the linear B-cell epitopes and serum antibodies from immunized mice with corresponding epitopes of ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. f, Neutralization assay against SARS-CoV-2 pseudovirus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of EC 50 . g, Neutralization assay against SARS-CoV-2 live virus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of NT 50 .
    Figure Legend Snippet: Measurements of the selected Linear B cell epitope binding to antibody and neutralization efficiency of selected epitopes against SARS-CoV-2. a-d, The binding affinity assessed by ELISA between linear B-cell epitopes and serum antibodies from immunized horse with S1-based vaccines (a), immunized mouse with RBD-based vaccines (b), immunized monkey with RBD-based vaccines (c), and a patient recovering from COVID-19 (d). e, The binding affinity assessed by ELISA between the linear B-cell epitopes and serum antibodies from immunized mice with corresponding epitopes of ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. f, Neutralization assay against SARS-CoV-2 pseudovirus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of EC 50 . g, Neutralization assay against SARS-CoV-2 live virus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of NT 50 .

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

    The characteristics of the 18 selected linear B cell epitopes. a , The sequences of 18 selected linear B-cell epitopes. The bold is the mutated site in less than ten of 118,694 virus strains; The red is the predicted discontinuous residues. The bars on the right side are the Wilcoxon test p value for the comparisons of IgG or IgA antibody enrichment scores associated with each linear B-cell epitope between COVID-19 patients and negative controls. b , The digesting enzymes profile of the epitope sequence. Red indicated not digest, blue indicated digest. c-d , The localization of the 18 selected epitopes mapped on SARS-CoV-2 S (PDB: 6VSB) protein (c) and ACE-RBD complex (d). e-f , The localizations of B cell discontinuous epitopes on SARS-CoV-2 S (PDB: 6VSB) protein (e) and ACE-RBD complex (f). The spike protein is grey, the RBD region is wheat color, the selected epitopes are green, the mutation sites are red, the human ACE domain is blue, and the discontinuous B-cell epitopes are purple.
    Figure Legend Snippet: The characteristics of the 18 selected linear B cell epitopes. a , The sequences of 18 selected linear B-cell epitopes. The bold is the mutated site in less than ten of 118,694 virus strains; The red is the predicted discontinuous residues. The bars on the right side are the Wilcoxon test p value for the comparisons of IgG or IgA antibody enrichment scores associated with each linear B-cell epitope between COVID-19 patients and negative controls. b , The digesting enzymes profile of the epitope sequence. Red indicated not digest, blue indicated digest. c-d , The localization of the 18 selected epitopes mapped on SARS-CoV-2 S (PDB: 6VSB) protein (c) and ACE-RBD complex (d). e-f , The localizations of B cell discontinuous epitopes on SARS-CoV-2 S (PDB: 6VSB) protein (e) and ACE-RBD complex (f). The spike protein is grey, the RBD region is wheat color, the selected epitopes are green, the mutation sites are red, the human ACE domain is blue, and the discontinuous B-cell epitopes are purple.

    Techniques Used: Sequencing, Mutagenesis

    11) Product Images from "Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies"

    Article Title: Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies

    Journal: Cell

    doi: 10.1016/j.cell.2020.04.031

    SARS VHH-72 Cross-Reacts with SARS-CoV-2 (A) An SPR sensorgram measuring the binding of SARS VHH-72 to the SARS-CoV-2 RBD-SD1. Binding curves are colored black, and fit of the data to a 1:1 binding model is colored red. (B) The crystal structure of SARS VHH-72 bound to the SARS-CoV-1 RBD is shown with SARS VHH-72 as dark blue ribbons and the RBD as a pink molecular surface. Amino acids that vary between SARS-CoV-1 and SARS-CoV-2 are colored green.
    Figure Legend Snippet: SARS VHH-72 Cross-Reacts with SARS-CoV-2 (A) An SPR sensorgram measuring the binding of SARS VHH-72 to the SARS-CoV-2 RBD-SD1. Binding curves are colored black, and fit of the data to a 1:1 binding model is colored red. (B) The crystal structure of SARS VHH-72 bound to the SARS-CoV-1 RBD is shown with SARS VHH-72 as dark blue ribbons and the RBD as a pink molecular surface. Amino acids that vary between SARS-CoV-1 and SARS-CoV-2 are colored green.

    Techniques Used: SPR Assay, Binding Assay

    Neutralizing Mechanisms of MERS VHH-55 and SARS VHH-72 (A) The MERS-CoV spike (PDB ID: 5W9H ) is shown as a transparent molecular surface, with each monomer colored either white, gray, or tan. Each monomer is bound by MERS VHH-55, shown as blue ribbons. The clash between MERS VHH-55 bound to the white monomer and the neighboring tan RBD is highlighted by the red ellipse. (B) The SARS-CoV-1 spike (PDB ID: 5X58 ) is shown as a transparent molecular surface, with each protomer colored either white, gray, or pink. Every monomer is bound by a copy of SARS VHH-72, shown as dark blue ribbons. The clashes between copies of SARS VHH-72 and the two neighboring spike monomers are highlighted by the red circle. (C) The SARS-CoV-2 spike (PDB ID: 6VXX ) is shown as a transparent molecular surface, with each protomer colored either white, gray, or green. Every monomer is bound by a copy of SARS VHH-72, shown as dark blue ribbons. The clashes between copies of SARS VHH-72 and the two neighboring spike monomers are highlighted by the red circle. The SARS-CoV-2 trimer appears smaller than SARS-CoV-1 S because of the absence of flexible NTD-distal loops, which could not be built during cryo-EM analysis. (D) CoV VHHs prevent MERS-CoV RBD, SARS-CoV-1 RBD, and SARS-CoV-2 RBD-SD1 from interacting with their receptors. The results of the BLI-based receptor-blocking experiment are shown. The legend lists the immobilized RBDs and the VHHs or receptors that correspond to each curve.
    Figure Legend Snippet: Neutralizing Mechanisms of MERS VHH-55 and SARS VHH-72 (A) The MERS-CoV spike (PDB ID: 5W9H ) is shown as a transparent molecular surface, with each monomer colored either white, gray, or tan. Each monomer is bound by MERS VHH-55, shown as blue ribbons. The clash between MERS VHH-55 bound to the white monomer and the neighboring tan RBD is highlighted by the red ellipse. (B) The SARS-CoV-1 spike (PDB ID: 5X58 ) is shown as a transparent molecular surface, with each protomer colored either white, gray, or pink. Every monomer is bound by a copy of SARS VHH-72, shown as dark blue ribbons. The clashes between copies of SARS VHH-72 and the two neighboring spike monomers are highlighted by the red circle. (C) The SARS-CoV-2 spike (PDB ID: 6VXX ) is shown as a transparent molecular surface, with each protomer colored either white, gray, or green. Every monomer is bound by a copy of SARS VHH-72, shown as dark blue ribbons. The clashes between copies of SARS VHH-72 and the two neighboring spike monomers are highlighted by the red circle. The SARS-CoV-2 trimer appears smaller than SARS-CoV-1 S because of the absence of flexible NTD-distal loops, which could not be built during cryo-EM analysis. (D) CoV VHHs prevent MERS-CoV RBD, SARS-CoV-1 RBD, and SARS-CoV-2 RBD-SD1 from interacting with their receptors. The results of the BLI-based receptor-blocking experiment are shown. The legend lists the immobilized RBDs and the VHHs or receptors that correspond to each curve.

    Techniques Used: Blocking Assay

    Engineering a Functional Bivalent VHH Construct, Related to Figure 6 (A) Flow cytometry measuring the binding of the bivalent SARS VHH-72 tail-to-head fusion (VHH-72-VHH-72) to SARS-CoV-1 or SARS-CoV-2 S expressed on the cell surface. VHH-23-VHH-23, a bivalent tail-to-head fusion of an irrelevant nanobody, was included as a negative control. (B) Binding of SARS-CoV-2 RBD-SD1 to Vero E6 cells is prevented by VHH-72-VHH-72 in a dose-dependent fashion. Binding of SARS-CoV-2 RBD-SD1 to Vero E6 cells was detected by flow cytometry in the presence of the indicated bivalent VHHs (n = 2 except VHH-72-VHH-72 and VHH-23-VHH-23 at 5 μg/mL, n = 5). (C) Binding of SARS-CoV-2 RBD-SD1 to Vero E6 cells is prevented by bivalent VHH-72-Fc fusion proteins in a dose-dependent fashion. Binding of SARS-CoV-2 RBD-SD1-Fc to Vero E6 cells was detected by flow cytometry in the presence of the indicated constructs and amounts (n = 2 except no RBD, n = 4). (D) Cell surface binding of SARS VHH-72 to SARS-CoV-1 S. 293T cells were transfected with a GFP expression plasmid together with a SARS-CoV-1 S expression plasmid. Binding of the indicated protein is expressed as the median fluorescent intensity (MFI), measured to detect the His-tagged MERS VHH-55 or SARS VHH-72 or the SARS VHH-72-Fc fusions, of the GFP positive cells divided by the MFI of the GFP negative cells. (E) Cell surface binding of SARS VHH-72 to SARS-CoV-2. MFI was calculated using the same equation as Figure S6 D.
    Figure Legend Snippet: Engineering a Functional Bivalent VHH Construct, Related to Figure 6 (A) Flow cytometry measuring the binding of the bivalent SARS VHH-72 tail-to-head fusion (VHH-72-VHH-72) to SARS-CoV-1 or SARS-CoV-2 S expressed on the cell surface. VHH-23-VHH-23, a bivalent tail-to-head fusion of an irrelevant nanobody, was included as a negative control. (B) Binding of SARS-CoV-2 RBD-SD1 to Vero E6 cells is prevented by VHH-72-VHH-72 in a dose-dependent fashion. Binding of SARS-CoV-2 RBD-SD1 to Vero E6 cells was detected by flow cytometry in the presence of the indicated bivalent VHHs (n = 2 except VHH-72-VHH-72 and VHH-23-VHH-23 at 5 μg/mL, n = 5). (C) Binding of SARS-CoV-2 RBD-SD1 to Vero E6 cells is prevented by bivalent VHH-72-Fc fusion proteins in a dose-dependent fashion. Binding of SARS-CoV-2 RBD-SD1-Fc to Vero E6 cells was detected by flow cytometry in the presence of the indicated constructs and amounts (n = 2 except no RBD, n = 4). (D) Cell surface binding of SARS VHH-72 to SARS-CoV-1 S. 293T cells were transfected with a GFP expression plasmid together with a SARS-CoV-1 S expression plasmid. Binding of the indicated protein is expressed as the median fluorescent intensity (MFI), measured to detect the His-tagged MERS VHH-55 or SARS VHH-72 or the SARS VHH-72-Fc fusions, of the GFP positive cells divided by the MFI of the GFP negative cells. (E) Cell surface binding of SARS VHH-72 to SARS-CoV-2. MFI was calculated using the same equation as Figure S6 D.

    Techniques Used: Functional Assay, Construct, Flow Cytometry, Binding Assay, Negative Control, Transfection, Expressing, Plasmid Preparation

    VHH-72-Fc Neutralizes SARS-CoV-2 S Pseudoviruses (A) BLI sensorgram measuring apparent binding affinity of VHH-72-Fc to immobilized SARS-CoV-2 RBD-Fc. Binding curves are colored black, buffer-only blanks are colored gray, and the fit of the data to a 1:1 binding curve is colored red. (B) Time course analysis of VHH-72-Fc expression in ExpiCHO cells. Cell culture supernatants of transiently transfected ExpiCHO cells were removed on days 3–7 after transfection (or until cell viability dropped below 75%), as indicated. Two control mAbs were included for comparison, along with the indicated amounts of purified GBP-Fc as a loading control. (C) SARS-CoV-2 S pseudotyped VSV neutralization assay. Monolayers of Vero E6 cells were infected with pseudoviruses that had been pre-incubated with the mixtures indicated by the legend. The VHH-72-Fc used in this assay was purified after expression in ExpiCHO cells (n = 4). VHH-23-Fc is an irrelevant control VHH-Fc (n = 3). NI, cells were not infected. Luciferase activity is reported in counts per second (c.p.s.) ± SEM.
    Figure Legend Snippet: VHH-72-Fc Neutralizes SARS-CoV-2 S Pseudoviruses (A) BLI sensorgram measuring apparent binding affinity of VHH-72-Fc to immobilized SARS-CoV-2 RBD-Fc. Binding curves are colored black, buffer-only blanks are colored gray, and the fit of the data to a 1:1 binding curve is colored red. (B) Time course analysis of VHH-72-Fc expression in ExpiCHO cells. Cell culture supernatants of transiently transfected ExpiCHO cells were removed on days 3–7 after transfection (or until cell viability dropped below 75%), as indicated. Two control mAbs were included for comparison, along with the indicated amounts of purified GBP-Fc as a loading control. (C) SARS-CoV-2 S pseudotyped VSV neutralization assay. Monolayers of Vero E6 cells were infected with pseudoviruses that had been pre-incubated with the mixtures indicated by the legend. The VHH-72-Fc used in this assay was purified after expression in ExpiCHO cells (n = 4). VHH-23-Fc is an irrelevant control VHH-Fc (n = 3). NI, cells were not infected. Luciferase activity is reported in counts per second (c.p.s.) ± SEM.

    Techniques Used: Binding Assay, Expressing, Cell Culture, Transfection, Purification, Neutralization, Infection, Incubation, Luciferase, Activity Assay

    SARS VHH-72 Binds to a Broadly Conserved Epitope on the SARS-CoV-1 RBD, Related to Figure 3 (A) The crystal structure of SARS VHH-72 bound to the SARS-CoV-1 RBD is shown, with colors corresponding to those of Figure S4 A. (B) The crystal structure of SARS VHH-72 bound to the SARS-CoV-1 RBD is shown with SARS VHH-72 as dark blue ribbons and the RBD as a pink molecular surface. Amino acids that vary between SARS-CoV-1 and WIV1-CoV are colored teal. (C) SPR sensorgram measuring the binding of SARS VHH-72 to the WIV1-CoV RBD. Binding curves are colored black and the fit of the data to a 1:1 binding model is colored red.
    Figure Legend Snippet: SARS VHH-72 Binds to a Broadly Conserved Epitope on the SARS-CoV-1 RBD, Related to Figure 3 (A) The crystal structure of SARS VHH-72 bound to the SARS-CoV-1 RBD is shown, with colors corresponding to those of Figure S4 A. (B) The crystal structure of SARS VHH-72 bound to the SARS-CoV-1 RBD is shown with SARS VHH-72 as dark blue ribbons and the RBD as a pink molecular surface. Amino acids that vary between SARS-CoV-1 and WIV1-CoV are colored teal. (C) SPR sensorgram measuring the binding of SARS VHH-72 to the WIV1-CoV RBD. Binding curves are colored black and the fit of the data to a 1:1 binding model is colored red.

    Techniques Used: SPR Assay, Binding Assay

    The Crystal Structure of SARS VHH-72 Bound to the SARS-CoV-1 RBD (A) SARS VHH-72 is shown as dark blue ribbons and the SARS-CoV-1 RBD is shown as a pink-colored molecular surface. The ACE2 binding interface on the SARS-CoV-1 RBD is colored red. (B) The structure of ACE2 bound to the SARS-CoV-1 RBD (PDB ID: 2AJF ) is aligned to the crystal structure of SARS VHH-72 bound to the SARS-CoV-1 RBD. ACE2 is shown as a red, transparent molecular surface. (C) A simulated N -linked glycan containing an energy-minimized trimannosyl core (derived from PDB ID: 1HD4 ) is modeled as red sticks, coming from Asn322 in ACE2. ACE2 is shown as a red molecular surface, the SARS-CoV-1 RBD is shown as pink ribbons, and SARS VHH-72 is shown as a dark blue, transparent molecular surface. (D) A zoomed-in view of the panel from (A) is shown, with the SARS-CoV-1 RBD now displayed as pink-colored ribbons. Residues that form interactions are shown as sticks, with nitrogen atoms colored dark blue and oxygen atoms colored red. Hydrogen bonds and salt bridges between SARS VHH-72 and the SARS-CoV-1 RBD are shown as black dots. (E) The same view from (D) has been turned by 60° to show additional contacts. Residues that form interactions are shown as sticks, with nitrogen atoms colored dark blue and oxygen atoms colored red. Interactions between SARS VHH-72 and the SARS-CoV-1 RBD are shown as black dots.
    Figure Legend Snippet: The Crystal Structure of SARS VHH-72 Bound to the SARS-CoV-1 RBD (A) SARS VHH-72 is shown as dark blue ribbons and the SARS-CoV-1 RBD is shown as a pink-colored molecular surface. The ACE2 binding interface on the SARS-CoV-1 RBD is colored red. (B) The structure of ACE2 bound to the SARS-CoV-1 RBD (PDB ID: 2AJF ) is aligned to the crystal structure of SARS VHH-72 bound to the SARS-CoV-1 RBD. ACE2 is shown as a red, transparent molecular surface. (C) A simulated N -linked glycan containing an energy-minimized trimannosyl core (derived from PDB ID: 1HD4 ) is modeled as red sticks, coming from Asn322 in ACE2. ACE2 is shown as a red molecular surface, the SARS-CoV-1 RBD is shown as pink ribbons, and SARS VHH-72 is shown as a dark blue, transparent molecular surface. (D) A zoomed-in view of the panel from (A) is shown, with the SARS-CoV-1 RBD now displayed as pink-colored ribbons. Residues that form interactions are shown as sticks, with nitrogen atoms colored dark blue and oxygen atoms colored red. Hydrogen bonds and salt bridges between SARS VHH-72 and the SARS-CoV-1 RBD are shown as black dots. (E) The same view from (D) has been turned by 60° to show additional contacts. Residues that form interactions are shown as sticks, with nitrogen atoms colored dark blue and oxygen atoms colored red. Interactions between SARS VHH-72 and the SARS-CoV-1 RBD are shown as black dots.

    Techniques Used: Binding Assay, Derivative Assay

    Comparison of the CoV VHH Epitopes with Known RBD-Directed Antibodies, Related to Figures 2 and 3 (A) The structure of MERS VHH-55 bound to the MERS-CoV RBD is shown with MERS VHH-55 as blue ribbons and the MERS-CoV RBD as a white molecular surface. Epitopes from previously reported crystal structures of the MERS-CoV RBD bound by RBD-directed antibodies are shown as colored patches on the MERS-CoV RBD surface. The LCA60 epitope is shown in yellow, the MERS S4 epitope is shown in green, the overlapping C2/MCA1/m336 epitopes are shown in red and the overlapping JC57-14/D12/4C2/MERS-27 epitopes are shown in purple. (B) The structure of SARS VHH-72 bound to the SARS-CoV-1 RBD is shown with SARS VHH-72 as dark blue ribbons and the SARS-CoV-1 RBD as a white molecular surface. Epitopes from previously reported crystal structures of the SARS-CoV-1 RBD bound by RBD-directed antibodies are shown as colored patches on the SARS-CoV-1 RBD surface. The 80R epitope is shown in blue, the S230 epitope is shown in yellow, the CR3022 epitope is shown in purple and the overlapping m396/F26G19 epitopes are shown in red. (C) The SARS-CoV-1 RBD is shown as a white molecular surface, ACE2 is shown as a transparent red molecular surface, SARS VHH-72 is shown as dark blue ribbons and CR3022 Fab is shown as purple ribbons.
    Figure Legend Snippet: Comparison of the CoV VHH Epitopes with Known RBD-Directed Antibodies, Related to Figures 2 and 3 (A) The structure of MERS VHH-55 bound to the MERS-CoV RBD is shown with MERS VHH-55 as blue ribbons and the MERS-CoV RBD as a white molecular surface. Epitopes from previously reported crystal structures of the MERS-CoV RBD bound by RBD-directed antibodies are shown as colored patches on the MERS-CoV RBD surface. The LCA60 epitope is shown in yellow, the MERS S4 epitope is shown in green, the overlapping C2/MCA1/m336 epitopes are shown in red and the overlapping JC57-14/D12/4C2/MERS-27 epitopes are shown in purple. (B) The structure of SARS VHH-72 bound to the SARS-CoV-1 RBD is shown with SARS VHH-72 as dark blue ribbons and the SARS-CoV-1 RBD as a white molecular surface. Epitopes from previously reported crystal structures of the SARS-CoV-1 RBD bound by RBD-directed antibodies are shown as colored patches on the SARS-CoV-1 RBD surface. The 80R epitope is shown in blue, the S230 epitope is shown in yellow, the CR3022 epitope is shown in purple and the overlapping m396/F26G19 epitopes are shown in red. (C) The SARS-CoV-1 RBD is shown as a white molecular surface, ACE2 is shown as a transparent red molecular surface, SARS VHH-72 is shown as dark blue ribbons and CR3022 Fab is shown as purple ribbons.

    Techniques Used:

    SARS VHH-72 Bivalency Permits SARS-CoV-2 Pseudovirus Neutralization (A and B) SARS-CoV-1 S (A) and SARS-CoV-2 S (B) VSV pseudoviruses were used to evaluate the neutralization capacity of SARS VHH-72. MERS VHH-55 and PBS were included as negative controls. Luciferase activity is reported in counts per second (c.p.s.). NI, cells were not infected. (C and D) Binding of bivalent VHHs was tested by ELISA against SARS-CoV-1 S (C) and SARS-CoV-2 RBD-SD1 (D). VHH-72-Fc refers to SARS VHH-72 fused to a human IgG1 Fc domain by a GS(GGGGS) 2 linker. VHH-72-Fc (S) is the same Fc fusion with a GS, rather than a GS(GGGGS) 2 , linker. GBP is an irrelevant GFP-binding protein. VHH-72-VHH-72 refers to the tail-to-head construct with two SARS VHH-72 proteins connected by a (GGGGS) 3 linker. VHH-23-VHH-23 refers to the two irrelevant VHHs linked via the same (GGGGS) 3 linker. (E and F) SARS-CoV-1 S (E) and SARS-CoV-2 S (F) pseudoviruses were used to evaluate the neutralization capacity of bivalent VHH-72-Fc. GBP and PBS were included as negative controls. NI, cells were not infected.
    Figure Legend Snippet: SARS VHH-72 Bivalency Permits SARS-CoV-2 Pseudovirus Neutralization (A and B) SARS-CoV-1 S (A) and SARS-CoV-2 S (B) VSV pseudoviruses were used to evaluate the neutralization capacity of SARS VHH-72. MERS VHH-55 and PBS were included as negative controls. Luciferase activity is reported in counts per second (c.p.s.). NI, cells were not infected. (C and D) Binding of bivalent VHHs was tested by ELISA against SARS-CoV-1 S (C) and SARS-CoV-2 RBD-SD1 (D). VHH-72-Fc refers to SARS VHH-72 fused to a human IgG1 Fc domain by a GS(GGGGS) 2 linker. VHH-72-Fc (S) is the same Fc fusion with a GS, rather than a GS(GGGGS) 2 , linker. GBP is an irrelevant GFP-binding protein. VHH-72-VHH-72 refers to the tail-to-head construct with two SARS VHH-72 proteins connected by a (GGGGS) 3 linker. VHH-23-VHH-23 refers to the two irrelevant VHHs linked via the same (GGGGS) 3 linker. (E and F) SARS-CoV-1 S (E) and SARS-CoV-2 S (F) pseudoviruses were used to evaluate the neutralization capacity of bivalent VHH-72-Fc. GBP and PBS were included as negative controls. NI, cells were not infected.

    Techniques Used: Neutralization, Luciferase, Activity Assay, Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, Construct

    Epitope Determination and Biophysical Characterization of MERS VHH-55 and SARS VHH-72 (A) Reactivity of MERS-CoV and SARS-CoV RBD-directed VHHs against the MERS-CoV and SARS-CoV-1 RBD, respectively. A VHH against an irrelevant antigen (F-VHH) was included as a control. Datapoints represent the mean of three replicates and error bars represent the standard errors of the mean. (B) SPR sensorgrams showing binding between the MERS-CoV RBD and MERS VHH-55 (left) and SARS-CoV-1 RBD and SARS VHH-72 (right). Binding curves are colored black, and fit of the data to a 1:1 binding model is colored red.
    Figure Legend Snippet: Epitope Determination and Biophysical Characterization of MERS VHH-55 and SARS VHH-72 (A) Reactivity of MERS-CoV and SARS-CoV RBD-directed VHHs against the MERS-CoV and SARS-CoV-1 RBD, respectively. A VHH against an irrelevant antigen (F-VHH) was included as a control. Datapoints represent the mean of three replicates and error bars represent the standard errors of the mean. (B) SPR sensorgrams showing binding between the MERS-CoV RBD and MERS VHH-55 (left) and SARS-CoV-1 RBD and SARS VHH-72 (right). Binding curves are colored black, and fit of the data to a 1:1 binding model is colored red.

    Techniques Used: SPR Assay, Binding Assay

    Lack of Binding of MERS-CoV and SARS-CoV-Directed VHHs to Non-RBD Epitopes, Related to Figure 1 ELISA data showing binding of the MERS-CoV specific VHHs to the MERS-CoV S1 protein and absence of binding of the MERS-CoV and SARS-CoV specific VHHs against the MERS-CoV NTD and SARS-CoV-1 NTD, respectively. A VHH against an irrelevant antigen (F-VHH) was included as a control.
    Figure Legend Snippet: Lack of Binding of MERS-CoV and SARS-CoV-Directed VHHs to Non-RBD Epitopes, Related to Figure 1 ELISA data showing binding of the MERS-CoV specific VHHs to the MERS-CoV S1 protein and absence of binding of the MERS-CoV and SARS-CoV specific VHHs against the MERS-CoV NTD and SARS-CoV-1 NTD, respectively. A VHH against an irrelevant antigen (F-VHH) was included as a control.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

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    Sino Biological sars cov2 s1
    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with <t>SARS‐CoV2‐RBG</t> and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and <t>SARS‐CoV2‐S1</t> proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.
    Sars Cov2 S1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological sars cov 2 s protein
    Cryo-EM structures of the <t>SARS-CoV-2</t> S trimer in complex with the 3C1 Fab. a , b S-3C1-F3b cryo-EM map ( a ) and pseudo atomic model ( b ). All the three RBDs are up and each of them binds with a 3C1 Fab. The heavy chain of the 3C1 Fab in medium blue and light chain in violet red. c , d S-3C1-F3a cryo-EM map ( c ) and pseudo atomic model ( d ). There are two up RBDs and one down RBD, with each bound with a 3C1 Fab. e Structural alignment of the three up RBDs of S-3C1-F3b (in color) and the only up RBD from S-open (gray), suggesting 3C1 induced outward tilt of the RBDs within the S trimer. f , g Conformational comparation between S-3C1-F1 and S-open ( f ), as well as between S-3C1-F3a and S-3C1-F2 ( g ). h RBD/3C1 interaction interface (take RBD-3/3C1 of S-3C1-F3b as an example), with major involved structural elements labeled. i ACE2 (coral, PDB: 6M0J) would clash with the heavy chain of 3C1 Fab (blue). They share overlapping epitopes on the RBM (dotted black circle); additionally, the framework of 3C1-VH would clash with ACE2 (dotted black frame), which could be enhanced by the presence of an N-linked glycan at site N322 of ACE2. j 3C1 showed two distinct orientations to bind RBD within S trimer, i.e., adopting orientation 1 to associate with up RBD while orientation 2 with down RBD. k Contact footprint variations of 3C1 on up RBD (left) compared with that on down RBD (right), with unique epitopes indicated by dotted black frame. l – m Potential simultaneous binding of RBD by 2H2 and 3C1 cocktail. In 3C1 orientation 1, 3C1 and 2H2 could have minor clash (indicated by black frame, l ); while in origination 2, there is no clash between 3C1 and 2H2 Fabs ( m ).
    Sars Cov 2 S Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.

    Journal: Small Methods

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

    doi: 10.1002/smtd.202001031

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

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

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

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

    Journal: The Journal of Infectious Diseases

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

    doi: 10.1093/infdis/jiaa479

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

    Article Snippet: For the multiplex bead-based immune assay the following antigens obtained from Sino Biological were used: SARS-CoV-2 monomeric spike S1 (40591-V08H), RBD (40592-V08B), and nucleoprotein (N) (40588-V08B).

    Techniques: Infection, Concentration Assay, Binding Assay

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-20465-w

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

    Article Snippet: To prepare SARS-CoV-2 S protein, mammalian codon-optimized gene coding S ectodomain (residues 1–1208) with proline substitutions at residues 986 and 987, a “GSAS” substitution at the furin cleavage site (residues 682–685) was cloned into vector pcDNA3.1+.

    Techniques: Labeling, Binding Assay

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-20465-w

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

    Article Snippet: To prepare SARS-CoV-2 S protein, mammalian codon-optimized gene coding S ectodomain (residues 1–1208) with proline substitutions at residues 986 and 987, a “GSAS” substitution at the furin cleavage site (residues 682–685) was cloned into vector pcDNA3.1+.

    Techniques: Binding Assay, Generated

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-20465-w

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

    Article Snippet: To prepare SARS-CoV-2 S protein, mammalian codon-optimized gene coding S ectodomain (residues 1–1208) with proline substitutions at residues 986 and 987, a “GSAS” substitution at the furin cleavage site (residues 682–685) was cloned into vector pcDNA3.1+.

    Techniques: Labeling, Binding Assay

    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