sars cov 2 rbd  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike RBD Recombinant Protein
    Description:
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed
    Catalog Number:
    40592-VNAH
    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
    Buy from Supplier


    Structured Review

    Sino Biological sars cov 2 rbd
    Biodistribution of Sad23L-nCoV-S and Ad49L-nCoV-S vaccines in inoculated animals. ( A ) Serum NAb titers to Sad23L and Ad49L vectors were measured in macaques immunized by prime-boost inoculation with two vaccines at 4 week interval, or ( B ) in C57BL/6 and BALB/c mice 4 weeks post prime only or prime-boost vaccination with two vaccines or vectorial controls. ( C ) Nested-PCR amplification of Sad23L or Ad49L-hexon gene (500bp) in tissues of C57BL/6 mice 4 weeks after inoculation by prime only or prime-boost immunization with Sad23L-nCoV-S and Ad49L-nCoV-S vaccines. ( D ) Expression of S protein in splenocytes and hepatocytes of tissue frozen sections from vaccine immunized or control C57BL/6 mice by immunofluorescence staining with a human monoclonal antibody to <t>SARS-CoV-2</t> S and DAPI.
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed
    https://www.bioz.com/result/sars cov 2 rbd/product/Sino Biological
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    sars cov 2 rbd - by Bioz Stars, 2021-04
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    Images

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.09.28.311480

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

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

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

    Techniques Used: Mouse Assay, Immunofluorescence, Staining

    Characterization of Sad23L-nCoV-S and Ad49L-nCoV-S vaccines. ( A ) Recombinant adenovirus constructs Sad23L-nCoV-S and Ad49L-nCoV-S carrying the full-length S gene of SARS-CoV-2 under CMV promotor regulation within the deleted E1 region of Sad23L or Ad49L vector. ( B ) Western blot analysis for the expression of S protein from Sad23L-nCoV-S or Ad49L-nCoV-S infected HEK-293A cell lysates by rabbit polyclonal antibody to RBD and heat-inactivated COVID-19 patent’s serum IgG. Sad23L-GFP or Ad49L-GFP virus infected cells were used as mock controls. ( C ) Expression of S protein in HEK-293A cells detected by immunofluorescence staining. ( D ) Seroprevalence of neutralizing antibody to Ad5, Ad49L or Sad23L vector in 600 healthy blood donors.
    Figure Legend Snippet: Characterization of Sad23L-nCoV-S and Ad49L-nCoV-S vaccines. ( A ) Recombinant adenovirus constructs Sad23L-nCoV-S and Ad49L-nCoV-S carrying the full-length S gene of SARS-CoV-2 under CMV promotor regulation within the deleted E1 region of Sad23L or Ad49L vector. ( B ) Western blot analysis for the expression of S protein from Sad23L-nCoV-S or Ad49L-nCoV-S infected HEK-293A cell lysates by rabbit polyclonal antibody to RBD and heat-inactivated COVID-19 patent’s serum IgG. Sad23L-GFP or Ad49L-GFP virus infected cells were used as mock controls. ( C ) Expression of S protein in HEK-293A cells detected by immunofluorescence staining. ( D ) Seroprevalence of neutralizing antibody to Ad5, Ad49L or Sad23L vector in 600 healthy blood donors.

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

    2) 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: bioRxiv

    doi: 10.1101/2020.03.26.010165

    Neutralizing mechanisms of MERS VHH-55 and SARS VHH-72. A ) The MERS-CoV spike (PDB ID: 5W9H) is shown as a molecular surface, with each monomer colored either white, gray or tan. The tan and white monomers are 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 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 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 due to 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 molecular surface, with each monomer colored either white, gray or tan. The tan and white monomers are 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 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 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 due to 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

    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-SD1. 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-SD1. 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 is cross-reactive against 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 is cross-reactive against 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

    Bivalency overcomes the high off-rate constant of SARS VHH-72. A ) SARS-CoV-1 S and B ) SARS-CoV-2 S 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 ) Binding of bivalent VHHs was tested by ELISA against SARS-CoV-1 S and D ) SARS-CoV-2 RBD-SD1. 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 ) SARS-CoV-1 S and F ) SARS-CoV-2 S 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: Bivalency overcomes the high off-rate constant of SARS VHH-72. A ) SARS-CoV-1 S and B ) SARS-CoV-2 S 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 ) Binding of bivalent VHHs was tested by ELISA against SARS-CoV-1 S and D ) SARS-CoV-2 RBD-SD1. 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 ) SARS-CoV-1 S and F ) SARS-CoV-2 S 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

    3) Product Images from "Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein"

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.0c01394

    Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.
    Figure Legend Snippet: Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.

    Techniques Used: Selection

    Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.
    Figure Legend Snippet: Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.

    Techniques Used: Flow Cytometry, Binding Assay

    4) Product Images from "A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein"

    Article Title: A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20602-5

    The structure of CT-P59 Fab in complex with SARS-CoV-2 RBD. a The overall structure of the CT-P59 Fab/SARS-CoV-2 RBD complex. The RBD domain is green for the core subdomain, and orange for RBM. The heavy and light chains of CT-P59 are magenta and yellow, respectively. b Superposition of the neutralizing antibodies in complex with RBD. RBD is shown as a surface model. CT-P59 is shown as a cartoon, and the other antibodies (CR3022: PDB 6XC3, PB2-2F6: PDB 7BWJ, REGN10933: PDB 6XDG) are shown as a ribbon model. The heavy and light chains of Fab are magenta and yellow, respectively. c Assignment of the epitope residues for RBD-targeting neutralizing antibodies with a distance cutoff of 4.5 Å. RBD residues interacting with ACE2 are highlighted in red. d The detailed interactions between the RBD and CDR loops of CT-P59. The interfaces between RBD and CDR H1/2 or H3 are shown in the top and bottom panels, respectively. The RBD domain is shown as a surface model with a semi-transparent representation. The CDR loops and interacting residues on the interfaces are shown in ribbons and sticks, respectively. The residues are colored as in a . Dashed lines indicate hydrogen bonds. Water molecules are shown as red spheres.
    Figure Legend Snippet: The structure of CT-P59 Fab in complex with SARS-CoV-2 RBD. a The overall structure of the CT-P59 Fab/SARS-CoV-2 RBD complex. The RBD domain is green for the core subdomain, and orange for RBM. The heavy and light chains of CT-P59 are magenta and yellow, respectively. b Superposition of the neutralizing antibodies in complex with RBD. RBD is shown as a surface model. CT-P59 is shown as a cartoon, and the other antibodies (CR3022: PDB 6XC3, PB2-2F6: PDB 7BWJ, REGN10933: PDB 6XDG) are shown as a ribbon model. The heavy and light chains of Fab are magenta and yellow, respectively. c Assignment of the epitope residues for RBD-targeting neutralizing antibodies with a distance cutoff of 4.5 Å. RBD residues interacting with ACE2 are highlighted in red. d The detailed interactions between the RBD and CDR loops of CT-P59. The interfaces between RBD and CDR H1/2 or H3 are shown in the top and bottom panels, respectively. The RBD domain is shown as a surface model with a semi-transparent representation. The CDR loops and interacting residues on the interfaces are shown in ribbons and sticks, respectively. The residues are colored as in a . Dashed lines indicate hydrogen bonds. Water molecules are shown as red spheres.

    Techniques Used:

    Virus titration and quantitation in the lower respiratory tract of animal models. Three ferrets per group were euthanized at 3 and 7 dpi, and lungs were collected to measure viral titers ( a ) and the number of viral RNA copies ( d ). Golden Syrian hamsters ( n = 12/group) were challenged intranasally with 6.4 × 10 4 PFU/80 μL of SARS-CoV-2. Vehicle and 15, 30, 60, and 90 mg/kg of CT-P59 were intraperitoneally administered 24 h after virus inoculation. Four animals were euthanized for virus titration ( b ) and quantitation of viral RNA copies ( e ) from each group at 3 and 5 dpi. Rhesus monkeys (control n = 3, 45 mg/kg n = 2, 90 mg/kg n = 3) were infected with 10 6.4 TCID 50 /ml of SARS-CoV-2 via in a combination of intranasal (0.5 ml), intratracheal (4 ml), ocular (0.25 ml/eye), and oral (1 ml) routes. Vehicle, 45 mg/kg and 90 mg/kg of CT-P59 were administered intravenously after 24 h of virus infection. All rhesus monkeys were euthanized at 6 dpi, and lungs were collected to measure viral titers ( c ) and the number of viral RNA copies ( f ). Viral titers in the lung were determined by TCID 50 assessment in Vero cells and viral RNA copy number measurement using qRT-PCR. Viral titers and RNA copy numbers are shown as mean value + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0309, ** P = 0.0131, and *** P
    Figure Legend Snippet: Virus titration and quantitation in the lower respiratory tract of animal models. Three ferrets per group were euthanized at 3 and 7 dpi, and lungs were collected to measure viral titers ( a ) and the number of viral RNA copies ( d ). Golden Syrian hamsters ( n = 12/group) were challenged intranasally with 6.4 × 10 4 PFU/80 μL of SARS-CoV-2. Vehicle and 15, 30, 60, and 90 mg/kg of CT-P59 were intraperitoneally administered 24 h after virus inoculation. Four animals were euthanized for virus titration ( b ) and quantitation of viral RNA copies ( e ) from each group at 3 and 5 dpi. Rhesus monkeys (control n = 3, 45 mg/kg n = 2, 90 mg/kg n = 3) were infected with 10 6.4 TCID 50 /ml of SARS-CoV-2 via in a combination of intranasal (0.5 ml), intratracheal (4 ml), ocular (0.25 ml/eye), and oral (1 ml) routes. Vehicle, 45 mg/kg and 90 mg/kg of CT-P59 were administered intravenously after 24 h of virus infection. All rhesus monkeys were euthanized at 6 dpi, and lungs were collected to measure viral titers ( c ) and the number of viral RNA copies ( f ). Viral titers in the lung were determined by TCID 50 assessment in Vero cells and viral RNA copy number measurement using qRT-PCR. Viral titers and RNA copy numbers are shown as mean value + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0309, ** P = 0.0131, and *** P

    Techniques Used: Titration, Quantitation Assay, Infection, Quantitative RT-PCR

    In vivo efficacy of CT-P59 in the upper respiratory tract in an animal model. Female ferrets ( n = 6/group), and rhesus monkeys (three control; two 45 mg/kg; three 90 mg/kg) were challenged with 10 5.8 TCID 50 /ml and 10 6.4 TCID 50 of SARS-CoV-2, respectively. Control (ferrets: 30 mg/kg of human IgG isotype and rhesus monkeys: vehicle) and CT-P59 (ferrets: 3, and 30 mg/kg, rhesus monkeys: 45, and 90 mg/kg) were administered intravenously after 24 h of virus inoculation, respectively. To compare the efficacy of CT-P59, remdesivir (18 mg/kg per ferret) was administered daily via oral gavage after 24 h of virus inoculation in ferrets for 5 days. To detect viral load in the upper respiratory tract, nasal wash specimens were collected from ferrets at 2, 4, and 6 dpi, and nose and throat swab specimens from monkeys were collected daily up to 6 dpi. Virus titers (TCID 50 ) were measured in nasal wash/swab and throat swabs specimens from each group of ( a ) ferrets and, c , d rhesus monkeys. The viral RNA copy numbers were measured in nasal washes from ( b ) ferrets. Viral titers and RNA copy numbers are shown as mean values + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). The asterisks and daggers indicate significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0001 and ** P
    Figure Legend Snippet: In vivo efficacy of CT-P59 in the upper respiratory tract in an animal model. Female ferrets ( n = 6/group), and rhesus monkeys (three control; two 45 mg/kg; three 90 mg/kg) were challenged with 10 5.8 TCID 50 /ml and 10 6.4 TCID 50 of SARS-CoV-2, respectively. Control (ferrets: 30 mg/kg of human IgG isotype and rhesus monkeys: vehicle) and CT-P59 (ferrets: 3, and 30 mg/kg, rhesus monkeys: 45, and 90 mg/kg) were administered intravenously after 24 h of virus inoculation, respectively. To compare the efficacy of CT-P59, remdesivir (18 mg/kg per ferret) was administered daily via oral gavage after 24 h of virus inoculation in ferrets for 5 days. To detect viral load in the upper respiratory tract, nasal wash specimens were collected from ferrets at 2, 4, and 6 dpi, and nose and throat swab specimens from monkeys were collected daily up to 6 dpi. Virus titers (TCID 50 ) were measured in nasal wash/swab and throat swabs specimens from each group of ( a ) ferrets and, c , d rhesus monkeys. The viral RNA copy numbers were measured in nasal washes from ( b ) ferrets. Viral titers and RNA copy numbers are shown as mean values + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). The asterisks and daggers indicate significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0001 and ** P

    Techniques Used: In Vivo, Animal Model

    CT-P59 can effectively neutralize SARS-CoV-2 in vitro by blocking RBD-ACE2 binding. a Serial twofold-diluted CT-P59 were incubated with SARS-CoV-2 live viruses; wild type (blue) and D614G (red). The mixture was added to VeroE6 cells. After 2–3 days of incubation, the neutralization activity was evaluated by counting plaques. Two independent experiments were performed in duplicate. b SARS-CoV-2 RBD immobilized on biosensor was saturated with CT-P59. Then, CT-P59 flowed over the biosensor surface in the presence (red) or absence (blue) of the ACE2 receptor. As a positive control, the buffer was loaded onto SARS-CoV-2 RBD immobilized biosensor, and ACE2 flowed over the biosensor surface (black).
    Figure Legend Snippet: CT-P59 can effectively neutralize SARS-CoV-2 in vitro by blocking RBD-ACE2 binding. a Serial twofold-diluted CT-P59 were incubated with SARS-CoV-2 live viruses; wild type (blue) and D614G (red). The mixture was added to VeroE6 cells. After 2–3 days of incubation, the neutralization activity was evaluated by counting plaques. Two independent experiments were performed in duplicate. b SARS-CoV-2 RBD immobilized on biosensor was saturated with CT-P59. Then, CT-P59 flowed over the biosensor surface in the presence (red) or absence (blue) of the ACE2 receptor. As a positive control, the buffer was loaded onto SARS-CoV-2 RBD immobilized biosensor, and ACE2 flowed over the biosensor surface (black).

    Techniques Used: In Vitro, Blocking Assay, Binding Assay, Incubation, Neutralization, Activity Assay, Positive Control

    No evidence of ADE caused by CT-P59 bound to SARS-CoV-2 in permissive cells and Fc receptor (FcR)-bearing cells. a FcR-independent ADE. SARS-CoV-2 was mixed with a wide range of antibodies; CT-P59 (red circle), CR3022 (blue square), and CT-P27 (black triangle). VeroE6 was infected with a virus–antibody complex. The virus titers were determined by optical density (OD) using an anti-nucleocapsid antibody. b FcγR II-dependent ADE. In vitro ADE assay was performed as described in ( a ) except Raji cells. c FcγR I II-dependent ADE. In vitro ADE assay was carried out as described in ( a ), except U937 cells. The experiments were performed in triplicates (CR3022 and CT-P27) or in quadruplicates (CT-P59). Average and standard deviation of virus titers are depicted as dot and error bar, respectively.
    Figure Legend Snippet: No evidence of ADE caused by CT-P59 bound to SARS-CoV-2 in permissive cells and Fc receptor (FcR)-bearing cells. a FcR-independent ADE. SARS-CoV-2 was mixed with a wide range of antibodies; CT-P59 (red circle), CR3022 (blue square), and CT-P27 (black triangle). VeroE6 was infected with a virus–antibody complex. The virus titers were determined by optical density (OD) using an anti-nucleocapsid antibody. b FcγR II-dependent ADE. In vitro ADE assay was performed as described in ( a ) except Raji cells. c FcγR I II-dependent ADE. In vitro ADE assay was carried out as described in ( a ), except U937 cells. The experiments were performed in triplicates (CR3022 and CT-P27) or in quadruplicates (CT-P59). Average and standard deviation of virus titers are depicted as dot and error bar, respectively.

    Techniques Used: Infection, In Vitro, Standard Deviation

    5) 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

    6) Product Images from "A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-021-21037-2

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

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

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

    Techniques Used: Mouse Assay, Transgenic Assay, Two Tailed Test

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

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

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

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

    7) Product Images from "A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein"

    Article Title: A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20602-5

    The structure of CT-P59 Fab in complex with SARS-CoV-2 RBD. a The overall structure of the CT-P59 Fab/SARS-CoV-2 RBD complex. The RBD domain is green for the core subdomain, and orange for RBM. The heavy and light chains of CT-P59 are magenta and yellow, respectively. b Superposition of the neutralizing antibodies in complex with RBD. RBD is shown as a surface model. CT-P59 is shown as a cartoon, and the other antibodies (CR3022: PDB 6XC3, PB2-2F6: PDB 7BWJ, REGN10933: PDB 6XDG) are shown as a ribbon model. The heavy and light chains of Fab are magenta and yellow, respectively. c Assignment of the epitope residues for RBD-targeting neutralizing antibodies with a distance cutoff of 4.5 Å. RBD residues interacting with ACE2 are highlighted in red. d The detailed interactions between the RBD and CDR loops of CT-P59. The interfaces between RBD and CDR H1/2 or H3 are shown in the top and bottom panels, respectively. The RBD domain is shown as a surface model with a semi-transparent representation. The CDR loops and interacting residues on the interfaces are shown in ribbons and sticks, respectively. The residues are colored as in a . Dashed lines indicate hydrogen bonds. Water molecules are shown as red spheres.
    Figure Legend Snippet: The structure of CT-P59 Fab in complex with SARS-CoV-2 RBD. a The overall structure of the CT-P59 Fab/SARS-CoV-2 RBD complex. The RBD domain is green for the core subdomain, and orange for RBM. The heavy and light chains of CT-P59 are magenta and yellow, respectively. b Superposition of the neutralizing antibodies in complex with RBD. RBD is shown as a surface model. CT-P59 is shown as a cartoon, and the other antibodies (CR3022: PDB 6XC3, PB2-2F6: PDB 7BWJ, REGN10933: PDB 6XDG) are shown as a ribbon model. The heavy and light chains of Fab are magenta and yellow, respectively. c Assignment of the epitope residues for RBD-targeting neutralizing antibodies with a distance cutoff of 4.5 Å. RBD residues interacting with ACE2 are highlighted in red. d The detailed interactions between the RBD and CDR loops of CT-P59. The interfaces between RBD and CDR H1/2 or H3 are shown in the top and bottom panels, respectively. The RBD domain is shown as a surface model with a semi-transparent representation. The CDR loops and interacting residues on the interfaces are shown in ribbons and sticks, respectively. The residues are colored as in a . Dashed lines indicate hydrogen bonds. Water molecules are shown as red spheres.

    Techniques Used:

    Virus titration and quantitation in the lower respiratory tract of animal models. Three ferrets per group were euthanized at 3 and 7 dpi, and lungs were collected to measure viral titers ( a ) and the number of viral RNA copies ( d ). Golden Syrian hamsters ( n = 12/group) were challenged intranasally with 6.4 × 10 4 PFU/80 μL of SARS-CoV-2. Vehicle and 15, 30, 60, and 90 mg/kg of CT-P59 were intraperitoneally administered 24 h after virus inoculation. Four animals were euthanized for virus titration ( b ) and quantitation of viral RNA copies ( e ) from each group at 3 and 5 dpi. Rhesus monkeys (control n = 3, 45 mg/kg n = 2, 90 mg/kg n = 3) were infected with 10 6.4 TCID 50 /ml of SARS-CoV-2 via in a combination of intranasal (0.5 ml), intratracheal (4 ml), ocular (0.25 ml/eye), and oral (1 ml) routes. Vehicle, 45 mg/kg and 90 mg/kg of CT-P59 were administered intravenously after 24 h of virus infection. All rhesus monkeys were euthanized at 6 dpi, and lungs were collected to measure viral titers ( c ) and the number of viral RNA copies ( f ). Viral titers in the lung were determined by TCID 50 assessment in Vero cells and viral RNA copy number measurement using qRT-PCR. Viral titers and RNA copy numbers are shown as mean value + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0309, ** P = 0.0131, and *** P
    Figure Legend Snippet: Virus titration and quantitation in the lower respiratory tract of animal models. Three ferrets per group were euthanized at 3 and 7 dpi, and lungs were collected to measure viral titers ( a ) and the number of viral RNA copies ( d ). Golden Syrian hamsters ( n = 12/group) were challenged intranasally with 6.4 × 10 4 PFU/80 μL of SARS-CoV-2. Vehicle and 15, 30, 60, and 90 mg/kg of CT-P59 were intraperitoneally administered 24 h after virus inoculation. Four animals were euthanized for virus titration ( b ) and quantitation of viral RNA copies ( e ) from each group at 3 and 5 dpi. Rhesus monkeys (control n = 3, 45 mg/kg n = 2, 90 mg/kg n = 3) were infected with 10 6.4 TCID 50 /ml of SARS-CoV-2 via in a combination of intranasal (0.5 ml), intratracheal (4 ml), ocular (0.25 ml/eye), and oral (1 ml) routes. Vehicle, 45 mg/kg and 90 mg/kg of CT-P59 were administered intravenously after 24 h of virus infection. All rhesus monkeys were euthanized at 6 dpi, and lungs were collected to measure viral titers ( c ) and the number of viral RNA copies ( f ). Viral titers in the lung were determined by TCID 50 assessment in Vero cells and viral RNA copy number measurement using qRT-PCR. Viral titers and RNA copy numbers are shown as mean value + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0309, ** P = 0.0131, and *** P

    Techniques Used: Titration, Quantitation Assay, Infection, Quantitative RT-PCR

    In vivo efficacy of CT-P59 in the upper respiratory tract in an animal model. Female ferrets ( n = 6/group), and rhesus monkeys (three control; two 45 mg/kg; three 90 mg/kg) were challenged with 10 5.8 TCID 50 /ml and 10 6.4 TCID 50 of SARS-CoV-2, respectively. Control (ferrets: 30 mg/kg of human IgG isotype and rhesus monkeys: vehicle) and CT-P59 (ferrets: 3, and 30 mg/kg, rhesus monkeys: 45, and 90 mg/kg) were administered intravenously after 24 h of virus inoculation, respectively. To compare the efficacy of CT-P59, remdesivir (18 mg/kg per ferret) was administered daily via oral gavage after 24 h of virus inoculation in ferrets for 5 days. To detect viral load in the upper respiratory tract, nasal wash specimens were collected from ferrets at 2, 4, and 6 dpi, and nose and throat swab specimens from monkeys were collected daily up to 6 dpi. Virus titers (TCID 50 ) were measured in nasal wash/swab and throat swabs specimens from each group of ( a ) ferrets and, c , d rhesus monkeys. The viral RNA copy numbers were measured in nasal washes from ( b ) ferrets. Viral titers and RNA copy numbers are shown as mean values + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). The asterisks and daggers indicate significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0001 and ** P
    Figure Legend Snippet: In vivo efficacy of CT-P59 in the upper respiratory tract in an animal model. Female ferrets ( n = 6/group), and rhesus monkeys (three control; two 45 mg/kg; three 90 mg/kg) were challenged with 10 5.8 TCID 50 /ml and 10 6.4 TCID 50 of SARS-CoV-2, respectively. Control (ferrets: 30 mg/kg of human IgG isotype and rhesus monkeys: vehicle) and CT-P59 (ferrets: 3, and 30 mg/kg, rhesus monkeys: 45, and 90 mg/kg) were administered intravenously after 24 h of virus inoculation, respectively. To compare the efficacy of CT-P59, remdesivir (18 mg/kg per ferret) was administered daily via oral gavage after 24 h of virus inoculation in ferrets for 5 days. To detect viral load in the upper respiratory tract, nasal wash specimens were collected from ferrets at 2, 4, and 6 dpi, and nose and throat swab specimens from monkeys were collected daily up to 6 dpi. Virus titers (TCID 50 ) were measured in nasal wash/swab and throat swabs specimens from each group of ( a ) ferrets and, c , d rhesus monkeys. The viral RNA copy numbers were measured in nasal washes from ( b ) ferrets. Viral titers and RNA copy numbers are shown as mean values + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). The asterisks and daggers indicate significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0001 and ** P

    Techniques Used: In Vivo, Animal Model

    CT-P59 can effectively neutralize SARS-CoV-2 in vitro by blocking RBD-ACE2 binding. a Serial twofold-diluted CT-P59 were incubated with SARS-CoV-2 live viruses; wild type (blue) and D614G (red). The mixture was added to VeroE6 cells. After 2–3 days of incubation, the neutralization activity was evaluated by counting plaques. Two independent experiments were performed in duplicate. b SARS-CoV-2 RBD immobilized on biosensor was saturated with CT-P59. Then, CT-P59 flowed over the biosensor surface in the presence (red) or absence (blue) of the ACE2 receptor. As a positive control, the buffer was loaded onto SARS-CoV-2 RBD immobilized biosensor, and ACE2 flowed over the biosensor surface (black).
    Figure Legend Snippet: CT-P59 can effectively neutralize SARS-CoV-2 in vitro by blocking RBD-ACE2 binding. a Serial twofold-diluted CT-P59 were incubated with SARS-CoV-2 live viruses; wild type (blue) and D614G (red). The mixture was added to VeroE6 cells. After 2–3 days of incubation, the neutralization activity was evaluated by counting plaques. Two independent experiments were performed in duplicate. b SARS-CoV-2 RBD immobilized on biosensor was saturated with CT-P59. Then, CT-P59 flowed over the biosensor surface in the presence (red) or absence (blue) of the ACE2 receptor. As a positive control, the buffer was loaded onto SARS-CoV-2 RBD immobilized biosensor, and ACE2 flowed over the biosensor surface (black).

    Techniques Used: In Vitro, Blocking Assay, Binding Assay, Incubation, Neutralization, Activity Assay, Positive Control

    No evidence of ADE caused by CT-P59 bound to SARS-CoV-2 in permissive cells and Fc receptor (FcR)-bearing cells. a FcR-independent ADE. SARS-CoV-2 was mixed with a wide range of antibodies; CT-P59 (red circle), CR3022 (blue square), and CT-P27 (black triangle). VeroE6 was infected with a virus–antibody complex. The virus titers were determined by optical density (OD) using an anti-nucleocapsid antibody. b FcγR II-dependent ADE. In vitro ADE assay was performed as described in ( a ) except Raji cells. c FcγR I II-dependent ADE. In vitro ADE assay was carried out as described in ( a ), except U937 cells. The experiments were performed in triplicates (CR3022 and CT-P27) or in quadruplicates (CT-P59). Average and standard deviation of virus titers are depicted as dot and error bar, respectively.
    Figure Legend Snippet: No evidence of ADE caused by CT-P59 bound to SARS-CoV-2 in permissive cells and Fc receptor (FcR)-bearing cells. a FcR-independent ADE. SARS-CoV-2 was mixed with a wide range of antibodies; CT-P59 (red circle), CR3022 (blue square), and CT-P27 (black triangle). VeroE6 was infected with a virus–antibody complex. The virus titers were determined by optical density (OD) using an anti-nucleocapsid antibody. b FcγR II-dependent ADE. In vitro ADE assay was performed as described in ( a ) except Raji cells. c FcγR I II-dependent ADE. In vitro ADE assay was carried out as described in ( a ), except U937 cells. The experiments were performed in triplicates (CR3022 and CT-P27) or in quadruplicates (CT-P59). Average and standard deviation of virus titers are depicted as dot and error bar, respectively.

    Techniques Used: Infection, In Vitro, Standard Deviation

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.11.16.385849

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

    Techniques Used: Expressing, Transduction

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

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

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

    Techniques Used: Expressing, Incubation

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

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

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

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

    9) Product Images from "Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein"

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.0c01394

    Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.
    Figure Legend Snippet: Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.

    Techniques Used: Selection

    Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.
    Figure Legend Snippet: Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.

    Techniques Used: Flow Cytometry, Binding Assay

    10) Product Images from "Potent human neutralizing antibodies elicited by SARS-CoV-2 infection"

    Article Title: Potent human neutralizing antibodies elicited by SARS-CoV-2 infection

    Journal: bioRxiv

    doi: 10.1101/2020.03.21.990770

    Heavy chain repertoires of SARS-CoV-2 RBD-specific antibodies analyzed (A) by individual subject or (B) across the eight subjects. (A) Distribution and frequency of heavy chain variable (VH) genes usage in each subject shown along the horizontal bar. The same color scheme is used for each VH family across all study subjects. The VHs that dominate across isolated antibodies are indicated by actual frequencies in their respective color boxes. The number of RBD-binding antibodies versus total antibodies isolated are shown on the right. (B) Clustering of VH genes and their association with ELISA binding activity across the eight subjects. Unrooted phylogenetic tree depicting the genetic relationships among all VH genes of the RBD-binding antibodies. Branch lengths are drawn to scale so that sequence relatedness can be readily assessed. Sequences from the same study subject are shown in the same color at the branch tips. Colored circles represent the proportion (light orange, > 80%; light yellow, 60%-80%; light green
    Figure Legend Snippet: Heavy chain repertoires of SARS-CoV-2 RBD-specific antibodies analyzed (A) by individual subject or (B) across the eight subjects. (A) Distribution and frequency of heavy chain variable (VH) genes usage in each subject shown along the horizontal bar. The same color scheme is used for each VH family across all study subjects. The VHs that dominate across isolated antibodies are indicated by actual frequencies in their respective color boxes. The number of RBD-binding antibodies versus total antibodies isolated are shown on the right. (B) Clustering of VH genes and their association with ELISA binding activity across the eight subjects. Unrooted phylogenetic tree depicting the genetic relationships among all VH genes of the RBD-binding antibodies. Branch lengths are drawn to scale so that sequence relatedness can be readily assessed. Sequences from the same study subject are shown in the same color at the branch tips. Colored circles represent the proportion (light orange, > 80%; light yellow, 60%-80%; light green

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

    Antibody and ACE2 competition for binding to SARS-CoV-2 RBD measured by SPR. The sensorgrams show distinct binding patterns of ACE2 to SARS-CoV-2 RBD with (red curve) or without (black curve) prior incubation with each testing antibody. The competition capacity of each antibody is indicated by the level of reduction in response unit of ACE2 comparing with or without prior antibody incubation.
    Figure Legend Snippet: Antibody and ACE2 competition for binding to SARS-CoV-2 RBD measured by SPR. The sensorgrams show distinct binding patterns of ACE2 to SARS-CoV-2 RBD with (red curve) or without (black curve) prior incubation with each testing antibody. The competition capacity of each antibody is indicated by the level of reduction in response unit of ACE2 comparing with or without prior antibody incubation.

    Techniques Used: Binding Assay, SPR Assay, Incubation

    Analysis of plasma binding to cell surface expressed trimeric Spike protein. HEK 293T cells transfected with expression plasmid encoding the full length spike of SARS-CoV-2, SARS-CoV or MERS-CoV were incubated with 1:100 dilutions of plasma from the study subjects. The cells were then stained with PE labeled anti-human IgG Fc secondary antibody and analyzed by FACS. Positive control antibodies include S230 and m396 targeting the RBD of SARS-CoV Spike, and Mab-GD33 targeting the RBD of MERS-CoV Spike. VRC01 is negative control antibody targeting HIV-1 envelope glycoprotein.
    Figure Legend Snippet: Analysis of plasma binding to cell surface expressed trimeric Spike protein. HEK 293T cells transfected with expression plasmid encoding the full length spike of SARS-CoV-2, SARS-CoV or MERS-CoV were incubated with 1:100 dilutions of plasma from the study subjects. The cells were then stained with PE labeled anti-human IgG Fc secondary antibody and analyzed by FACS. Positive control antibodies include S230 and m396 targeting the RBD of SARS-CoV Spike, and Mab-GD33 targeting the RBD of MERS-CoV Spike. VRC01 is negative control antibody targeting HIV-1 envelope glycoprotein.

    Techniques Used: Binding Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Staining, Labeling, FACS, Positive Control, Negative Control

    Antibody neutralization analyzed by pseudovirus and live SARS-CoV-2. (A) Quality control of antibody through ELISA analysis prior to neutralization assay. A serial dilution of each antibody was evaluated against SARS-CoV-2 RBD coated on the ELISA plate and their binding activity was recorded at an optical density (OD) of 450nm and 630nm. (B-C) Antibody neutralization analyzed by pseudovirus (B) or live SARS-CoV-2 (C). A serial dilution of each antibody was tested against pseudovirus while two dilutions against live SARS-CoV-2. Cytopathic effects (CPE) were observed daily and recorded on Day 2 post-exposure. Selected antibodies and their concentrations tested are indicated at the upper left corner.
    Figure Legend Snippet: Antibody neutralization analyzed by pseudovirus and live SARS-CoV-2. (A) Quality control of antibody through ELISA analysis prior to neutralization assay. A serial dilution of each antibody was evaluated against SARS-CoV-2 RBD coated on the ELISA plate and their binding activity was recorded at an optical density (OD) of 450nm and 630nm. (B-C) Antibody neutralization analyzed by pseudovirus (B) or live SARS-CoV-2 (C). A serial dilution of each antibody was tested against pseudovirus while two dilutions against live SARS-CoV-2. Cytopathic effects (CPE) were observed daily and recorded on Day 2 post-exposure. Selected antibodies and their concentrations tested are indicated at the upper left corner.

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

    Analyses of plasma and B cell responses specific to SARS-CoV-2. Serial dilutions of plasma samples were analyzed for binding to the (A) RBDs or (B) trimeric Spikes of SARS-CoV-2, SARS-CoV and MERS-CoV by ELISA and (C) for neutralizing activity against pseudoviruses bearing envelope glycoprotein of SARS-CoV-2, SARS-CoV and MERS-CoV. Binding to SARS-CoV-2 NP protein was also evaluated (A). All results were derived from at least two independent experiments. (D) Gating strategy for analysis and isolation of RBD-specific memory B cells and (E) their representation among the total and memory subpopulation of B cells in the eight study subjects. Samples were named as either A, B, or C depending on collection sequence. FSC-W, forward scatter width; FSC-A, forward scatter area; and SSC-A side scatter area.
    Figure Legend Snippet: Analyses of plasma and B cell responses specific to SARS-CoV-2. Serial dilutions of plasma samples were analyzed for binding to the (A) RBDs or (B) trimeric Spikes of SARS-CoV-2, SARS-CoV and MERS-CoV by ELISA and (C) for neutralizing activity against pseudoviruses bearing envelope glycoprotein of SARS-CoV-2, SARS-CoV and MERS-CoV. Binding to SARS-CoV-2 NP protein was also evaluated (A). All results were derived from at least two independent experiments. (D) Gating strategy for analysis and isolation of RBD-specific memory B cells and (E) their representation among the total and memory subpopulation of B cells in the eight study subjects. Samples were named as either A, B, or C depending on collection sequence. FSC-W, forward scatter width; FSC-A, forward scatter area; and SSC-A side scatter area.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Derivative Assay, Isolation, Sequencing

    ELISA screening of SARS-CoV-2 RBD-specific antibodies in the supernatant of transfected cells. The study subjects and the date of sampling are indicated on the top. Samples were named as either A, B, or C depending on collection sequence. Antibodies tested for each sample are aligned in one vertical column whenever possible. For each evaluated antibody, at least two independent measurements were performed and are presented adjacently on the same row. Binding activities were assessed by OD 450 and indicated by the color scheme on the right. Negatives (no binding activity) are shown in gray for OD 450 values less than 0.1.
    Figure Legend Snippet: ELISA screening of SARS-CoV-2 RBD-specific antibodies in the supernatant of transfected cells. The study subjects and the date of sampling are indicated on the top. Samples were named as either A, B, or C depending on collection sequence. Antibodies tested for each sample are aligned in one vertical column whenever possible. For each evaluated antibody, at least two independent measurements were performed and are presented adjacently on the same row. Binding activities were assessed by OD 450 and indicated by the color scheme on the right. Negatives (no binding activity) are shown in gray for OD 450 values less than 0.1.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Transfection, Sampling, Sequencing, Binding Assay, Activity Assay

    Binding kinetics of isolated mAbs with SARS-CoV-2 RBD measured by SPR. The purified soluble SARS-CoV-2 RBD were covalently immobilized onto a CM5 sensor chip followed by injection of individual antibody at four or five different concentrations. The black lines indicate the experimentally derived curves while the red lines represent fitted curves based on the experimental data.
    Figure Legend Snippet: Binding kinetics of isolated mAbs with SARS-CoV-2 RBD measured by SPR. The purified soluble SARS-CoV-2 RBD were covalently immobilized onto a CM5 sensor chip followed by injection of individual antibody at four or five different concentrations. The black lines indicate the experimentally derived curves while the red lines represent fitted curves based on the experimental data.

    Techniques Used: Binding Assay, Isolation, SPR Assay, Purification, Chromatin Immunoprecipitation, Injection, Derivative Assay

    11) Product Images from "Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM"

    Article Title: Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM

    Journal: Science Advances

    doi: 10.1126/sciadv.abe5575

    The proposed mechanism of ACE2-induced conformational transitions of SARS-CoV-2 S trimer. Conformational transitions from the closed ground prefusion state (with packed FP, in red) to the transiently open state (step 1) with an untwisting motion (highlighted in dark gray arrow) associated with a downward movement of S1 (red arrow), from the open state to the dynamic ACE2 engaged state (step 2), and then all the way to the refolded postfusion state (step 3). The continuous swing motions of ACE2-RBD within S trimer are indicated by red arrows. The S trimer associated with ACE2 dimer (third panel) was generated by aligning the ACE2 of our S-ACE2 structure with the available full-length ACE2 dimer structure (PDB: 6M1D). The postfusion state was illustrated as a cartoon (PDB: 6XRA).
    Figure Legend Snippet: The proposed mechanism of ACE2-induced conformational transitions of SARS-CoV-2 S trimer. Conformational transitions from the closed ground prefusion state (with packed FP, in red) to the transiently open state (step 1) with an untwisting motion (highlighted in dark gray arrow) associated with a downward movement of S1 (red arrow), from the open state to the dynamic ACE2 engaged state (step 2), and then all the way to the refolded postfusion state (step 3). The continuous swing motions of ACE2-RBD within S trimer are indicated by red arrows. The S trimer associated with ACE2 dimer (third panel) was generated by aligning the ACE2 of our S-ACE2 structure with the available full-length ACE2 dimer structure (PDB: 6M1D). The postfusion state was illustrated as a cartoon (PDB: 6XRA).

    Techniques Used: Generated

    A tightly closed conformation of SARS-CoV-2 S trimer. ( A and B ) Cryo-EM map and model of SARS-CoV-2 S trimer in a tightly closed state, with three protomers shown in different color. ( C ) Close-up view of the model map fitting in the NTD and RBD regions of the S1 subunit, illustrating that most of the NTD region was well resolved. ( D ) Overlaid RBD structures of our S-closed (blue) with a cryo-EM structure of SARS-CoV-2 S in closed state (6VXX, gray), illustrating that the RBM S469-C488 loop was captured in our structure (indicated by dotted ellipsoid). ( E ) Top view of the overlaid structures as in (D) (left) and zoom-in views of specific domains, showing that there is a marked counterclockwise rotation in S1 especially in NTD, resulting in a twisted, tightly closed conformation. ( F ) Protomer interaction interface analysis by PISA. ( G ) Location of the captured FP fragment (in deep pink) within the S trimer (left) and one protomer. S1 and S2 subunits are colored steel blue and gold, respectively. ( H ) Model map fitting for the FP fragment. ( I ) Close-up view of the interactions between D614 from SD2 and FP, with the hydrogen bonds labeled in dotted lines and the L828-F855 region in FP in deep pink.
    Figure Legend Snippet: A tightly closed conformation of SARS-CoV-2 S trimer. ( A and B ) Cryo-EM map and model of SARS-CoV-2 S trimer in a tightly closed state, with three protomers shown in different color. ( C ) Close-up view of the model map fitting in the NTD and RBD regions of the S1 subunit, illustrating that most of the NTD region was well resolved. ( D ) Overlaid RBD structures of our S-closed (blue) with a cryo-EM structure of SARS-CoV-2 S in closed state (6VXX, gray), illustrating that the RBM S469-C488 loop was captured in our structure (indicated by dotted ellipsoid). ( E ) Top view of the overlaid structures as in (D) (left) and zoom-in views of specific domains, showing that there is a marked counterclockwise rotation in S1 especially in NTD, resulting in a twisted, tightly closed conformation. ( F ) Protomer interaction interface analysis by PISA. ( G ) Location of the captured FP fragment (in deep pink) within the S trimer (left) and one protomer. S1 and S2 subunits are colored steel blue and gold, respectively. ( H ) Model map fitting for the FP fragment. ( I ) Close-up view of the interactions between D614 from SD2 and FP, with the hydrogen bonds labeled in dotted lines and the L828-F855 region in FP in deep pink.

    Techniques Used: Labeling

    The architecture of the SARS-CoV-2 S-ACE2 complex. ( A and B ) Cryo-EM map and model of SARS-CoV-2 S-ACE2 complex. We named the RBD up protomer as protomer 1 (light green), and the other two RBD down ones as protomer 2 (royal blue) and protomer 3 (gold). ACE2 was colored in violet red. ( C ) Side and top views of the overlaid S-open (color) and S-closed (dark gray) structures, showing that in the open process, there is a 71.0° upward/outward rotation of RBD associated with a downward shift of SD1 in protomer 1. ( D ) Rotations of NTD and CH from the S-closed (gray) to the S-open (in color) state, with the NTD also showing a downward/outward movement (right). ( E ) Side view of the overlaid S-ACE2 (violet red) and S-open (light green) protomer 1 structures, showing that the angle between the long axis of RBD and the horizontal plane of S trimer reduces from the S-open to the S-ACE2. ( F ) Top and side views of the overlaid S-ACE2 (violet red) and S-open (color) RBD structures, showing the coordinated movements of RBDs. ( G ) Protomer interaction interface analysis of S-ACE2 by PISA. ( H ) Aromatic interactions between the core region of the up RBD-1 (green) and the RBM T470-F490 loop of the neighboring RBD-2 (blue). ( I ) Overlaid structures of S-ACE2 (gray) and S-closed (color, with the FP fragment in deep pink), indicating a downward shift of SD1 and most of the FP is missing in S-ACE2. Close-up view (right) of the potential clashes between the downward-shifted SD1 β34 and α8 helix of FP. ( J ) Population shift between the ACE2-unpresented and ACE2-presented S trimer samples.
    Figure Legend Snippet: The architecture of the SARS-CoV-2 S-ACE2 complex. ( A and B ) Cryo-EM map and model of SARS-CoV-2 S-ACE2 complex. We named the RBD up protomer as protomer 1 (light green), and the other two RBD down ones as protomer 2 (royal blue) and protomer 3 (gold). ACE2 was colored in violet red. ( C ) Side and top views of the overlaid S-open (color) and S-closed (dark gray) structures, showing that in the open process, there is a 71.0° upward/outward rotation of RBD associated with a downward shift of SD1 in protomer 1. ( D ) Rotations of NTD and CH from the S-closed (gray) to the S-open (in color) state, with the NTD also showing a downward/outward movement (right). ( E ) Side view of the overlaid S-ACE2 (violet red) and S-open (light green) protomer 1 structures, showing that the angle between the long axis of RBD and the horizontal plane of S trimer reduces from the S-open to the S-ACE2. ( F ) Top and side views of the overlaid S-ACE2 (violet red) and S-open (color) RBD structures, showing the coordinated movements of RBDs. ( G ) Protomer interaction interface analysis of S-ACE2 by PISA. ( H ) Aromatic interactions between the core region of the up RBD-1 (green) and the RBM T470-F490 loop of the neighboring RBD-2 (blue). ( I ) Overlaid structures of S-ACE2 (gray) and S-closed (color, with the FP fragment in deep pink), indicating a downward shift of SD1 and most of the FP is missing in S-ACE2. Close-up view (right) of the potential clashes between the downward-shifted SD1 β34 and α8 helix of FP. ( J ) Population shift between the ACE2-unpresented and ACE2-presented S trimer samples.

    Techniques Used:

    The T470-T478 loop and residue Y505 within RBM play important roles in the engagement of SARS-CoV-2 spike with receptor ACE2. ( A ) The overall view of ACE2 (violet red) bound protomer 1 (light green) from our S-ACE2 structure, and zoom-in view of the interaction interface between ACE2 and RBD, with the key contacting elements T470-F490 loop and Q498-Y505 within RBM highlighted in black ellipsoid and blue ellipsoid, respectively. ( B ) Superposition of our SARS-CoV-2 S-ACE2 structure with the crystal structure of SARS-CoV RBD-ACE2 (PDB: 2AJF), suggesting that the RBM T470-F490 loop has obvious conformational variations. ( C ) Binding activities of ACE2-hFc fusion protein to wild-type (wt) and mutant SARS-CoV-2 RBD proteins determined by ELISA. Different structural elements of RBD were colored in the left. Anti-RBD sera and a cross-reactive monoclonal antibody (MAb) 1A10 served as positive controls. Ctr, an irrelevant antibody. The black arrow indicates that mutations in the RBD (RBM-R3) mutant significantly reduced the binding of ACE2-hFc compared with wild-type RBD. ( D ) Binding of ACE2-hFc fusion protein to wt and single-point mutant forms of SARS-CoV-2 RBD protein measured by ELISA. RBD (Q498A), RBD (V503A), and RBD (Y505A), RBD residues Q498, V503, and Y505 were mutated to Ala, respectively. The downward arrow indicates that the mutation at Y505 completely abolished the binding of ACE2 to RBD protein. OD 450 , optical density at 450 nm.
    Figure Legend Snippet: The T470-T478 loop and residue Y505 within RBM play important roles in the engagement of SARS-CoV-2 spike with receptor ACE2. ( A ) The overall view of ACE2 (violet red) bound protomer 1 (light green) from our S-ACE2 structure, and zoom-in view of the interaction interface between ACE2 and RBD, with the key contacting elements T470-F490 loop and Q498-Y505 within RBM highlighted in black ellipsoid and blue ellipsoid, respectively. ( B ) Superposition of our SARS-CoV-2 S-ACE2 structure with the crystal structure of SARS-CoV RBD-ACE2 (PDB: 2AJF), suggesting that the RBM T470-F490 loop has obvious conformational variations. ( C ) Binding activities of ACE2-hFc fusion protein to wild-type (wt) and mutant SARS-CoV-2 RBD proteins determined by ELISA. Different structural elements of RBD were colored in the left. Anti-RBD sera and a cross-reactive monoclonal antibody (MAb) 1A10 served as positive controls. Ctr, an irrelevant antibody. The black arrow indicates that mutations in the RBD (RBM-R3) mutant significantly reduced the binding of ACE2-hFc compared with wild-type RBD. ( D ) Binding of ACE2-hFc fusion protein to wt and single-point mutant forms of SARS-CoV-2 RBD protein measured by ELISA. RBD (Q498A), RBD (V503A), and RBD (Y505A), RBD residues Q498, V503, and Y505 were mutated to Ala, respectively. The downward arrow indicates that the mutation at Y505 completely abolished the binding of ACE2 to RBD protein. OD 450 , optical density at 450 nm.

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

    Organization of the resolved N-linked glycans of SARS-CoV-2 S trimer. ( A ) Schematic representation of SARS-CoV-2 S glycoprotein. The positions of N-linked glycosylation sequons are shown as branches. A total of 18 N-linked glycans detected in our S-closed cryo-EM map are shown in red, and the remaining undetected ones in black. After ACE2 binding, the glycan density that appears weaker is indicated (*). ( B ) Surface representation of the glycosylated S trimer in the S-closed state with N-linked glycans shown in red. The location of glycan hole is indicated in black dotted ellipsoid, with the locations of S1/S2 and FP, and glycan at N657 site near the glycan hole indicated. The newly captured glycans at the N17 and N149 sites are indicated in the top view. ( C ) Surface representation of the glycosylated S-ACE2 complex with N-linked glycans in red.
    Figure Legend Snippet: Organization of the resolved N-linked glycans of SARS-CoV-2 S trimer. ( A ) Schematic representation of SARS-CoV-2 S glycoprotein. The positions of N-linked glycosylation sequons are shown as branches. A total of 18 N-linked glycans detected in our S-closed cryo-EM map are shown in red, and the remaining undetected ones in black. After ACE2 binding, the glycan density that appears weaker is indicated (*). ( B ) Surface representation of the glycosylated S trimer in the S-closed state with N-linked glycans shown in red. The location of glycan hole is indicated in black dotted ellipsoid, with the locations of S1/S2 and FP, and glycan at N657 site near the glycan hole indicated. The newly captured glycans at the N17 and N149 sites are indicated in the top view. ( C ) Surface representation of the glycosylated S-ACE2 complex with N-linked glycans in red.

    Techniques Used: Binding Assay

    12) Product Images from "Identification of Human Single-Domain Antibodies against SARS-CoV-2"

    Article Title: Identification of Human Single-Domain Antibodies against SARS-CoV-2

    Journal: Cell Host & Microbe

    doi: 10.1016/j.chom.2020.04.023

    Neutralization and Epitope Mapping of Single-Domain Antibodies (A) Antibody-mediated neutralization against luciferase-encoding pseudotyped virus with spike protein of SARS-CoV-2. Pseudotyped viruses preincubated with antibodies at indicated concentrations were used to infect Huh-7 cells, and inhibitory rates (%) of infection were calculated by luciferase activities in cell lysates. Error bars indicate mean ± SD from three independent experiments. (B) Neutralization of SARS-CoV-2 pseudotyped virus by single-domain antibody cocktails. Group D antibody n3088 or n3130 was combined with an equimolar amount of n3113. For cocktails, the concentration on the x axis indicates that of the individual single-domain antibody. Data are shown as mean ± SD. (C) Neutralization of live SARS-CoV-2 (clinical isolate nCoV-SH01) by 20 μg/mL of single-domain antibodies. (D) Neutralization activities of group D antibodies n3088 and n3130 against live SARS-CoV-2. Data are shown as mean ± SD. (E) Representative single-domain antibody from competition groups A, D, and E are listed with residues critical for binding. The critical residues are highlighted as spheres from epitope mapping experiments and shown by colors which correspond to the competition group designation as (A). The ecto-domian of SARS-CoV-2 spike glycoprotein (PBD entry 6VSB) is shown as surface with RBD colored in magenta or cyan for up or down conformation. ACE2-binding site is shown as slate spheres. (F) Comparison of the binding model of n3088 and CR3022 Fab to homotrimeric S protein with the RBD protomers adopt a single “up” or double “up” conformation. CR3022 Fab and single-domain antibody n3088 were represented as yellow and blue surface, respectively.
    Figure Legend Snippet: Neutralization and Epitope Mapping of Single-Domain Antibodies (A) Antibody-mediated neutralization against luciferase-encoding pseudotyped virus with spike protein of SARS-CoV-2. Pseudotyped viruses preincubated with antibodies at indicated concentrations were used to infect Huh-7 cells, and inhibitory rates (%) of infection were calculated by luciferase activities in cell lysates. Error bars indicate mean ± SD from three independent experiments. (B) Neutralization of SARS-CoV-2 pseudotyped virus by single-domain antibody cocktails. Group D antibody n3088 or n3130 was combined with an equimolar amount of n3113. For cocktails, the concentration on the x axis indicates that of the individual single-domain antibody. Data are shown as mean ± SD. (C) Neutralization of live SARS-CoV-2 (clinical isolate nCoV-SH01) by 20 μg/mL of single-domain antibodies. (D) Neutralization activities of group D antibodies n3088 and n3130 against live SARS-CoV-2. Data are shown as mean ± SD. (E) Representative single-domain antibody from competition groups A, D, and E are listed with residues critical for binding. The critical residues are highlighted as spheres from epitope mapping experiments and shown by colors which correspond to the competition group designation as (A). The ecto-domian of SARS-CoV-2 spike glycoprotein (PBD entry 6VSB) is shown as surface with RBD colored in magenta or cyan for up or down conformation. ACE2-binding site is shown as slate spheres. (F) Comparison of the binding model of n3088 and CR3022 Fab to homotrimeric S protein with the RBD protomers adopt a single “up” or double “up” conformation. CR3022 Fab and single-domain antibody n3088 were represented as yellow and blue surface, respectively.

    Techniques Used: Neutralization, Luciferase, Infection, Concentration Assay, Binding Assay

    Characterization of Single-Domain Antibodies Identified from Antibody Library Using SARS-CoV-2 RBD and S1 as Panning Antigens (A) Eighteen single-domain antibodies identified by panning against SARS-CoV-2 RBD and 5 antibodies by using SARS-CoV-2 S1 as panning antigens were tested in competition binding assay. Competition of these antibodies with each other, or ACE2, or the antibody CR3022 for RBD binding were measured by BLI. The antibodies are displayed in 5 groups (A, B, C, D, or E). The values are the percentage of binding that occurred during competition in comparison with non-competed binding, which was normalized to 100%, and the range of competition is indicated by the box colors. Black-filled boxes indicate strongly competing pairs (residual binding
    Figure Legend Snippet: Characterization of Single-Domain Antibodies Identified from Antibody Library Using SARS-CoV-2 RBD and S1 as Panning Antigens (A) Eighteen single-domain antibodies identified by panning against SARS-CoV-2 RBD and 5 antibodies by using SARS-CoV-2 S1 as panning antigens were tested in competition binding assay. Competition of these antibodies with each other, or ACE2, or the antibody CR3022 for RBD binding were measured by BLI. The antibodies are displayed in 5 groups (A, B, C, D, or E). The values are the percentage of binding that occurred during competition in comparison with non-competed binding, which was normalized to 100%, and the range of competition is indicated by the box colors. Black-filled boxes indicate strongly competing pairs (residual binding

    Techniques Used: Binding Assay

    13) Product Images from "A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein"

    Article Title: A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20602-5

    The structure of CT-P59 Fab in complex with SARS-CoV-2 RBD. a The overall structure of the CT-P59 Fab/SARS-CoV-2 RBD complex. The RBD domain is green for the core subdomain, and orange for RBM. The heavy and light chains of CT-P59 are magenta and yellow, respectively. b Superposition of the neutralizing antibodies in complex with RBD. RBD is shown as a surface model. CT-P59 is shown as a cartoon, and the other antibodies (CR3022: PDB 6XC3, PB2-2F6: PDB 7BWJ, REGN10933: PDB 6XDG) are shown as a ribbon model. The heavy and light chains of Fab are magenta and yellow, respectively. c Assignment of the epitope residues for RBD-targeting neutralizing antibodies with a distance cutoff of 4.5 Å. RBD residues interacting with ACE2 are highlighted in red. d The detailed interactions between the RBD and CDR loops of CT-P59. The interfaces between RBD and CDR H1/2 or H3 are shown in the top and bottom panels, respectively. The RBD domain is shown as a surface model with a semi-transparent representation. The CDR loops and interacting residues on the interfaces are shown in ribbons and sticks, respectively. The residues are colored as in a . Dashed lines indicate hydrogen bonds. Water molecules are shown as red spheres.
    Figure Legend Snippet: The structure of CT-P59 Fab in complex with SARS-CoV-2 RBD. a The overall structure of the CT-P59 Fab/SARS-CoV-2 RBD complex. The RBD domain is green for the core subdomain, and orange for RBM. The heavy and light chains of CT-P59 are magenta and yellow, respectively. b Superposition of the neutralizing antibodies in complex with RBD. RBD is shown as a surface model. CT-P59 is shown as a cartoon, and the other antibodies (CR3022: PDB 6XC3, PB2-2F6: PDB 7BWJ, REGN10933: PDB 6XDG) are shown as a ribbon model. The heavy and light chains of Fab are magenta and yellow, respectively. c Assignment of the epitope residues for RBD-targeting neutralizing antibodies with a distance cutoff of 4.5 Å. RBD residues interacting with ACE2 are highlighted in red. d The detailed interactions between the RBD and CDR loops of CT-P59. The interfaces between RBD and CDR H1/2 or H3 are shown in the top and bottom panels, respectively. The RBD domain is shown as a surface model with a semi-transparent representation. The CDR loops and interacting residues on the interfaces are shown in ribbons and sticks, respectively. The residues are colored as in a . Dashed lines indicate hydrogen bonds. Water molecules are shown as red spheres.

    Techniques Used:

    Virus titration and quantitation in the lower respiratory tract of animal models. Three ferrets per group were euthanized at 3 and 7 dpi, and lungs were collected to measure viral titers ( a ) and the number of viral RNA copies ( d ). Golden Syrian hamsters ( n = 12/group) were challenged intranasally with 6.4 × 10 4 PFU/80 μL of SARS-CoV-2. Vehicle and 15, 30, 60, and 90 mg/kg of CT-P59 were intraperitoneally administered 24 h after virus inoculation. Four animals were euthanized for virus titration ( b ) and quantitation of viral RNA copies ( e ) from each group at 3 and 5 dpi. Rhesus monkeys (control n = 3, 45 mg/kg n = 2, 90 mg/kg n = 3) were infected with 10 6.4 TCID 50 /ml of SARS-CoV-2 via in a combination of intranasal (0.5 ml), intratracheal (4 ml), ocular (0.25 ml/eye), and oral (1 ml) routes. Vehicle, 45 mg/kg and 90 mg/kg of CT-P59 were administered intravenously after 24 h of virus infection. All rhesus monkeys were euthanized at 6 dpi, and lungs were collected to measure viral titers ( c ) and the number of viral RNA copies ( f ). Viral titers in the lung were determined by TCID 50 assessment in Vero cells and viral RNA copy number measurement using qRT-PCR. Viral titers and RNA copy numbers are shown as mean value + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0309, ** P = 0.0131, and *** P
    Figure Legend Snippet: Virus titration and quantitation in the lower respiratory tract of animal models. Three ferrets per group were euthanized at 3 and 7 dpi, and lungs were collected to measure viral titers ( a ) and the number of viral RNA copies ( d ). Golden Syrian hamsters ( n = 12/group) were challenged intranasally with 6.4 × 10 4 PFU/80 μL of SARS-CoV-2. Vehicle and 15, 30, 60, and 90 mg/kg of CT-P59 were intraperitoneally administered 24 h after virus inoculation. Four animals were euthanized for virus titration ( b ) and quantitation of viral RNA copies ( e ) from each group at 3 and 5 dpi. Rhesus monkeys (control n = 3, 45 mg/kg n = 2, 90 mg/kg n = 3) were infected with 10 6.4 TCID 50 /ml of SARS-CoV-2 via in a combination of intranasal (0.5 ml), intratracheal (4 ml), ocular (0.25 ml/eye), and oral (1 ml) routes. Vehicle, 45 mg/kg and 90 mg/kg of CT-P59 were administered intravenously after 24 h of virus infection. All rhesus monkeys were euthanized at 6 dpi, and lungs were collected to measure viral titers ( c ) and the number of viral RNA copies ( f ). Viral titers in the lung were determined by TCID 50 assessment in Vero cells and viral RNA copy number measurement using qRT-PCR. Viral titers and RNA copy numbers are shown as mean value + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0309, ** P = 0.0131, and *** P

    Techniques Used: Titration, Quantitation Assay, Infection, Quantitative RT-PCR

    In vivo efficacy of CT-P59 in the upper respiratory tract in an animal model. Female ferrets ( n = 6/group), and rhesus monkeys (three control; two 45 mg/kg; three 90 mg/kg) were challenged with 10 5.8 TCID 50 /ml and 10 6.4 TCID 50 of SARS-CoV-2, respectively. Control (ferrets: 30 mg/kg of human IgG isotype and rhesus monkeys: vehicle) and CT-P59 (ferrets: 3, and 30 mg/kg, rhesus monkeys: 45, and 90 mg/kg) were administered intravenously after 24 h of virus inoculation, respectively. To compare the efficacy of CT-P59, remdesivir (18 mg/kg per ferret) was administered daily via oral gavage after 24 h of virus inoculation in ferrets for 5 days. To detect viral load in the upper respiratory tract, nasal wash specimens were collected from ferrets at 2, 4, and 6 dpi, and nose and throat swab specimens from monkeys were collected daily up to 6 dpi. Virus titers (TCID 50 ) were measured in nasal wash/swab and throat swabs specimens from each group of ( a ) ferrets and, c , d rhesus monkeys. The viral RNA copy numbers were measured in nasal washes from ( b ) ferrets. Viral titers and RNA copy numbers are shown as mean values + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). The asterisks and daggers indicate significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0001 and ** P
    Figure Legend Snippet: In vivo efficacy of CT-P59 in the upper respiratory tract in an animal model. Female ferrets ( n = 6/group), and rhesus monkeys (three control; two 45 mg/kg; three 90 mg/kg) were challenged with 10 5.8 TCID 50 /ml and 10 6.4 TCID 50 of SARS-CoV-2, respectively. Control (ferrets: 30 mg/kg of human IgG isotype and rhesus monkeys: vehicle) and CT-P59 (ferrets: 3, and 30 mg/kg, rhesus monkeys: 45, and 90 mg/kg) were administered intravenously after 24 h of virus inoculation, respectively. To compare the efficacy of CT-P59, remdesivir (18 mg/kg per ferret) was administered daily via oral gavage after 24 h of virus inoculation in ferrets for 5 days. To detect viral load in the upper respiratory tract, nasal wash specimens were collected from ferrets at 2, 4, and 6 dpi, and nose and throat swab specimens from monkeys were collected daily up to 6 dpi. Virus titers (TCID 50 ) were measured in nasal wash/swab and throat swabs specimens from each group of ( a ) ferrets and, c , d rhesus monkeys. The viral RNA copy numbers were measured in nasal washes from ( b ) ferrets. Viral titers and RNA copy numbers are shown as mean values + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). The asterisks and daggers indicate significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0001 and ** P

    Techniques Used: In Vivo, Animal Model

    CT-P59 can effectively neutralize SARS-CoV-2 in vitro by blocking RBD-ACE2 binding. a Serial twofold-diluted CT-P59 were incubated with SARS-CoV-2 live viruses; wild type (blue) and D614G (red). The mixture was added to VeroE6 cells. After 2–3 days of incubation, the neutralization activity was evaluated by counting plaques. Two independent experiments were performed in duplicate. b SARS-CoV-2 RBD immobilized on biosensor was saturated with CT-P59. Then, CT-P59 flowed over the biosensor surface in the presence (red) or absence (blue) of the ACE2 receptor. As a positive control, the buffer was loaded onto SARS-CoV-2 RBD immobilized biosensor, and ACE2 flowed over the biosensor surface (black).
    Figure Legend Snippet: CT-P59 can effectively neutralize SARS-CoV-2 in vitro by blocking RBD-ACE2 binding. a Serial twofold-diluted CT-P59 were incubated with SARS-CoV-2 live viruses; wild type (blue) and D614G (red). The mixture was added to VeroE6 cells. After 2–3 days of incubation, the neutralization activity was evaluated by counting plaques. Two independent experiments were performed in duplicate. b SARS-CoV-2 RBD immobilized on biosensor was saturated with CT-P59. Then, CT-P59 flowed over the biosensor surface in the presence (red) or absence (blue) of the ACE2 receptor. As a positive control, the buffer was loaded onto SARS-CoV-2 RBD immobilized biosensor, and ACE2 flowed over the biosensor surface (black).

    Techniques Used: In Vitro, Blocking Assay, Binding Assay, Incubation, Neutralization, Activity Assay, Positive Control

    No evidence of ADE caused by CT-P59 bound to SARS-CoV-2 in permissive cells and Fc receptor (FcR)-bearing cells. a FcR-independent ADE. SARS-CoV-2 was mixed with a wide range of antibodies; CT-P59 (red circle), CR3022 (blue square), and CT-P27 (black triangle). VeroE6 was infected with a virus–antibody complex. The virus titers were determined by optical density (OD) using an anti-nucleocapsid antibody. b FcγR II-dependent ADE. In vitro ADE assay was performed as described in ( a ) except Raji cells. c FcγR I II-dependent ADE. In vitro ADE assay was carried out as described in ( a ), except U937 cells. The experiments were performed in triplicates (CR3022 and CT-P27) or in quadruplicates (CT-P59). Average and standard deviation of virus titers are depicted as dot and error bar, respectively.
    Figure Legend Snippet: No evidence of ADE caused by CT-P59 bound to SARS-CoV-2 in permissive cells and Fc receptor (FcR)-bearing cells. a FcR-independent ADE. SARS-CoV-2 was mixed with a wide range of antibodies; CT-P59 (red circle), CR3022 (blue square), and CT-P27 (black triangle). VeroE6 was infected with a virus–antibody complex. The virus titers were determined by optical density (OD) using an anti-nucleocapsid antibody. b FcγR II-dependent ADE. In vitro ADE assay was performed as described in ( a ) except Raji cells. c FcγR I II-dependent ADE. In vitro ADE assay was carried out as described in ( a ), except U937 cells. The experiments were performed in triplicates (CR3022 and CT-P27) or in quadruplicates (CT-P59). Average and standard deviation of virus titers are depicted as dot and error bar, respectively.

    Techniques Used: Infection, In Vitro, Standard Deviation

    14) Product Images from "Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein"

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.0c01394

    Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.
    Figure Legend Snippet: Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.

    Techniques Used: Selection

    Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.
    Figure Legend Snippet: Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.

    Techniques Used: Flow Cytometry, Binding Assay

    15) Product Images from "Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein"

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.0c01394

    Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.
    Figure Legend Snippet: Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.

    Techniques Used: Selection

    Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.
    Figure Legend Snippet: Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.

    Techniques Used: Flow Cytometry, Binding Assay

    16) Product Images from "Cross-reactive coronavirus antibodies with diverse epitope specificities and extra-neutralization functions"

    Article Title: Cross-reactive coronavirus antibodies with diverse epitope specificities and extra-neutralization functions

    Journal: bioRxiv

    doi: 10.1101/2020.12.20.414748

    In vivo effects of cross-reactive antibodies ( A ) Timeline of the prophylactic antibody experiment in SARS-CoV-2 mouse adapted (MA) in vivo infection model. 200 μg antibody was given via intraperitoneal route to 12-month old female BALB/c mice 12 hours prior to virus inoculation (n= 4 or 5 per group). 1×10 3 or 1×10 4 PFU infectious dose of SARS-CoV-2 MA was administered intranasally for the low dose and high dose experiments, respectively. Weights were measured daily, and on day 4 tissue was collected for histopathology and viral load quantification. ( B ) Lung hemorrhage scores of gross pathology are shown for each low dose (1×10 3 PFU of SARS-CoV-2 MA) treatment group. An ordinary one-way ANOVA test with multiple comparisons was performed. ( C ) For the experiment treating with 1×10 4 PFU of SARS-CoV-2 MA, percent survival for each antibody group is shown. 2/5, 4/5, 3/5, and 2/5 mice survived to day 4 for antibodies 46472-4, 46472-12, CR3022 and isotype control DENV-2D22 respectively. (D) Lung hemorrhage scores of gross pathology are shown for each high dose (1×10 4 PFU of SARS-CoV-2 MA) treatment group. An ordinary one-way ANOVA test with multiple comparisons was performed.
    Figure Legend Snippet: In vivo effects of cross-reactive antibodies ( A ) Timeline of the prophylactic antibody experiment in SARS-CoV-2 mouse adapted (MA) in vivo infection model. 200 μg antibody was given via intraperitoneal route to 12-month old female BALB/c mice 12 hours prior to virus inoculation (n= 4 or 5 per group). 1×10 3 or 1×10 4 PFU infectious dose of SARS-CoV-2 MA was administered intranasally for the low dose and high dose experiments, respectively. Weights were measured daily, and on day 4 tissue was collected for histopathology and viral load quantification. ( B ) Lung hemorrhage scores of gross pathology are shown for each low dose (1×10 3 PFU of SARS-CoV-2 MA) treatment group. An ordinary one-way ANOVA test with multiple comparisons was performed. ( C ) For the experiment treating with 1×10 4 PFU of SARS-CoV-2 MA, percent survival for each antibody group is shown. 2/5, 4/5, 3/5, and 2/5 mice survived to day 4 for antibodies 46472-4, 46472-12, CR3022 and isotype control DENV-2D22 respectively. (D) Lung hemorrhage scores of gross pathology are shown for each high dose (1×10 4 PFU of SARS-CoV-2 MA) treatment group. An ordinary one-way ANOVA test with multiple comparisons was performed.

    Techniques Used: In Vivo, Infection, Mouse Assay, Histopathology

    Functional activity of cross-reactive coronavirus antibodies ( A ) Cross-reactive coronavirus antibodies were tested for antibody-dependent cellular phagocytosis activity (ADCP) against SARS-CoV-2 S, compared to positive control antibody CR3022 and negative control Palivizumab, an anti-RSV antibody. Area under the curve of the phagocytosis score is shown, calculated from data in Figure S3C . ( B ) 46472-4 and 46472-12 were tested for antibody-dependent cellular phagocytosis activity against SARS-CoV-1 S, compared to CR3022 antibody and anti-RSV antibody Palivizumab. Area under the curve of the phagocytosis score is shown, calculated from data in Figure S3D . ( C ) Cross-reactive coronavirus antibodies were tested for antibody-dependent cellular trogocytosis (ADCT) activity against SARS-CoV-2 S coated on cells, compared to positive control CR3022 and anti-RSV antibody Palivizumab. Area under the curve of the trogocytosis score is shown, calculated from data in Figure S3E . ( D ) Cross-reactive coronavirus antibodies were tested for antibody-dependent cellular trogocytosis activity against SARS-CoV-2 S displayed on transfected cells, compared to positive control CR3022 and anti-RSV antibody Palivizumab. Area under the curve of the trogocytosis score is shown, calculated from data in Figure S3F . ( E ) Cross-reactive coronavirus antibodies were tested for antibody-dependent complement deposition (ADCD) activity against SARS-CoV-2 S, compared to positive control CR3022 and anti-RSV antibody Palivizumab. Area under the curve of the C3b deposition score is shown, calculated from data in Figure S3G .
    Figure Legend Snippet: Functional activity of cross-reactive coronavirus antibodies ( A ) Cross-reactive coronavirus antibodies were tested for antibody-dependent cellular phagocytosis activity (ADCP) against SARS-CoV-2 S, compared to positive control antibody CR3022 and negative control Palivizumab, an anti-RSV antibody. Area under the curve of the phagocytosis score is shown, calculated from data in Figure S3C . ( B ) 46472-4 and 46472-12 were tested for antibody-dependent cellular phagocytosis activity against SARS-CoV-1 S, compared to CR3022 antibody and anti-RSV antibody Palivizumab. Area under the curve of the phagocytosis score is shown, calculated from data in Figure S3D . ( C ) Cross-reactive coronavirus antibodies were tested for antibody-dependent cellular trogocytosis (ADCT) activity against SARS-CoV-2 S coated on cells, compared to positive control CR3022 and anti-RSV antibody Palivizumab. Area under the curve of the trogocytosis score is shown, calculated from data in Figure S3E . ( D ) Cross-reactive coronavirus antibodies were tested for antibody-dependent cellular trogocytosis activity against SARS-CoV-2 S displayed on transfected cells, compared to positive control CR3022 and anti-RSV antibody Palivizumab. Area under the curve of the trogocytosis score is shown, calculated from data in Figure S3F . ( E ) Cross-reactive coronavirus antibodies were tested for antibody-dependent complement deposition (ADCD) activity against SARS-CoV-2 S, compared to positive control CR3022 and anti-RSV antibody Palivizumab. Area under the curve of the C3b deposition score is shown, calculated from data in Figure S3G .

    Techniques Used: Functional Assay, Activity Assay, Positive Control, Negative Control, Transfection

    Identification of coronavirus cross-reactive antibodies from SARS-CoV-1 convalescent PBMC sample using LIBRA-seq ( A ) Schematic of DNA-barcoded antigens used to probe a SARS-CoV-1 donor PBMC sample. The LIBRA-seq experiment setup consisted of eight oligo-labelled antigens in the screening library: SARS-CoV-2 S, SARS-CoV-1 S, MERS-CoV S, MERS-CoV S1, OC43-CoV S, HKU1-CoV S, and two HIV negative controls (ZM197, and CZA97). ( B ) LIBRA-seq scores for SARS-CoV-1 (x-axis) and SARS-CoV-2 (y-axis) for all IgG cells recovered from sequencing are shown as circles. The 15 lead antibody candidates are highlighted in purple. ( C ) Antibodies were tested for binding to SARS-CoV-2 S (S-2P), SARS-CoV-1 S (S-2P), OC43-CoV S (S-2P), HKU1-CoV S (S-2P), and SARS-CoV-2 S (HexaPro) by ELISA. HIV-specific antibody VRC01 is used as a negative control. Anti-SARS-CoV-1 mouse antibody 240CD was also used (BEI Resources). ELISAs were performed in technical duplicates with at least two biological duplicates. ( D ) ELISA binding data against the antigens are displayed as a heatmap of the AUC analysis calculated from the data in Figure 1C , with AUC of 0 displayed as white, and maximum AUC as purple. ELISAs were performed in technical duplicates with at least two biological duplicates.
    Figure Legend Snippet: Identification of coronavirus cross-reactive antibodies from SARS-CoV-1 convalescent PBMC sample using LIBRA-seq ( A ) Schematic of DNA-barcoded antigens used to probe a SARS-CoV-1 donor PBMC sample. The LIBRA-seq experiment setup consisted of eight oligo-labelled antigens in the screening library: SARS-CoV-2 S, SARS-CoV-1 S, MERS-CoV S, MERS-CoV S1, OC43-CoV S, HKU1-CoV S, and two HIV negative controls (ZM197, and CZA97). ( B ) LIBRA-seq scores for SARS-CoV-1 (x-axis) and SARS-CoV-2 (y-axis) for all IgG cells recovered from sequencing are shown as circles. The 15 lead antibody candidates are highlighted in purple. ( C ) Antibodies were tested for binding to SARS-CoV-2 S (S-2P), SARS-CoV-1 S (S-2P), OC43-CoV S (S-2P), HKU1-CoV S (S-2P), and SARS-CoV-2 S (HexaPro) by ELISA. HIV-specific antibody VRC01 is used as a negative control. Anti-SARS-CoV-1 mouse antibody 240CD was also used (BEI Resources). ELISAs were performed in technical duplicates with at least two biological duplicates. ( D ) ELISA binding data against the antigens are displayed as a heatmap of the AUC analysis calculated from the data in Figure 1C , with AUC of 0 displayed as white, and maximum AUC as purple. ELISAs were performed in technical duplicates with at least two biological duplicates.

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

    Epitope mapping of cross-reactive antibodies ( A ) For cross-reactive coronavirus antibodies, ELISA binding data against the antigens are displayed as a heatmap of the AUC analysis calculated from the data in Figure S2A and ( B ) for SARS-CoV-2 S1 reactive antibodies, ELISA binding data against the RBD and NTD are displayed as a heatmap of the AUC analysis calculated from the data in Figure S2B . ELISA AUC is displayed as a heat map. AUC of 0 is displayed as white and maximum AUC as purple. ELISA data are representative of at least two independent experiments. Anti-HIV antibody VRC01 and anti-VEGF antibody are shown as a negative control and anti-SARS-CoV-1 antibody 240CD is shown as positive control. ( C ) Surface plasmon resonance binding of 46472-12 Fab to SARS-CoV-2 RBD. Affinity measurements are shown to the right of the graph. ( D ) Cross-reactive antibodies were used in a competition ELISA to determine if binding of one antibody affected binding of another. Competitor antibodies were added at 10 μg/ml, and then detected antibodies were added at 0.1 μg/ml. The percent reduction in binding compared to binding without a competitor is shown. An anti-HIV antibody was also used as a negative control. ELISAs were performed in technical duplicates with at least two biological duplicates. ( E ) Antibodies were tested for autoreactivity against a variety of antigens in the Luminex AtheNA assay. Anti-HIV antibody 4E10 was used as a positive control and Ab82 was used as a negative control. ( F ) Cross-reactive coronavirus antibodies target a variety of epitopes on the SARS-CoV-2 S protein, including the RBD, NTD, and S2 domains, highlighted on the structure (PDB: 6VSB). Antibodies targeting each epitope are listed and color coded for each domain.
    Figure Legend Snippet: Epitope mapping of cross-reactive antibodies ( A ) For cross-reactive coronavirus antibodies, ELISA binding data against the antigens are displayed as a heatmap of the AUC analysis calculated from the data in Figure S2A and ( B ) for SARS-CoV-2 S1 reactive antibodies, ELISA binding data against the RBD and NTD are displayed as a heatmap of the AUC analysis calculated from the data in Figure S2B . ELISA AUC is displayed as a heat map. AUC of 0 is displayed as white and maximum AUC as purple. ELISA data are representative of at least two independent experiments. Anti-HIV antibody VRC01 and anti-VEGF antibody are shown as a negative control and anti-SARS-CoV-1 antibody 240CD is shown as positive control. ( C ) Surface plasmon resonance binding of 46472-12 Fab to SARS-CoV-2 RBD. Affinity measurements are shown to the right of the graph. ( D ) Cross-reactive antibodies were used in a competition ELISA to determine if binding of one antibody affected binding of another. Competitor antibodies were added at 10 μg/ml, and then detected antibodies were added at 0.1 μg/ml. The percent reduction in binding compared to binding without a competitor is shown. An anti-HIV antibody was also used as a negative control. ELISAs were performed in technical duplicates with at least two biological duplicates. ( E ) Antibodies were tested for autoreactivity against a variety of antigens in the Luminex AtheNA assay. Anti-HIV antibody 4E10 was used as a positive control and Ab82 was used as a negative control. ( F ) Cross-reactive coronavirus antibodies target a variety of epitopes on the SARS-CoV-2 S protein, including the RBD, NTD, and S2 domains, highlighted on the structure (PDB: 6VSB). Antibodies targeting each epitope are listed and color coded for each domain.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Negative Control, Positive Control, SPR Assay, Luminex

    17) Product Images from "Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein"

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.0c01394

    Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.
    Figure Legend Snippet: Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.

    Techniques Used: Selection

    Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.
    Figure Legend Snippet: Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.

    Techniques Used: Flow Cytometry, Binding Assay

    18) Product Images from "Sexually dimorphic placental responses to maternal SARS-CoV-2 infection"

    Article Title: Sexually dimorphic placental responses to maternal SARS-CoV-2 infection

    Journal: bioRxiv

    doi: 10.1101/2021.03.29.437516

    SARS-CoV-2 infected mothers with male fetuses have lower plasma levels of SARS-CoV-2-specific antibodies. A. Plots showing maternal Spike-, RBD-, S1-, S2-, and N-specific maternal blood IgG2 levels. Female neonates of mothers with SARS-CoV-2 are shown as white bars with an orange border while male neonates are shown as orange shaded bars with orange border. Two-way ANOVA followed by post-hoc analyses were performed to determine significance. There was a main effect of fetal/neonatal sex on maternal IgG1 levels (p = 0.003). *p
    Figure Legend Snippet: SARS-CoV-2 infected mothers with male fetuses have lower plasma levels of SARS-CoV-2-specific antibodies. A. Plots showing maternal Spike-, RBD-, S1-, S2-, and N-specific maternal blood IgG2 levels. Female neonates of mothers with SARS-CoV-2 are shown as white bars with an orange border while male neonates are shown as orange shaded bars with orange border. Two-way ANOVA followed by post-hoc analyses were performed to determine significance. There was a main effect of fetal/neonatal sex on maternal IgG1 levels (p = 0.003). *p

    Techniques Used: Infection

    SARS-CoV-2 infected mothers with male fetuses demonstrate reduced placental transfer of SARS-CoV-2 antibodies compared to those with female fetuses. A. Dot plots showing relative Spike-, RBD-, S1-, S2-, N-, and influenza (HA)-specific maternal blood (M) and cord blood (C) titers of IgG1. Female neonates of SARS-CoV-2 negative mothers are shown in light blue, female neonates of SARS-CoV-2 positive mothers are shown in light orange. Males born to SARS-CoV-2 negative mothers are shown in dark blue, and males born to SARS-CoV-2 positive mothers are shown in dark orange. Units are Mean Fluorescence Intensity or MFI. All values reflect PBS background correction. Wilcoxon matched-pairs signed rank test was performed to determine significance. * p
    Figure Legend Snippet: SARS-CoV-2 infected mothers with male fetuses demonstrate reduced placental transfer of SARS-CoV-2 antibodies compared to those with female fetuses. A. Dot plots showing relative Spike-, RBD-, S1-, S2-, N-, and influenza (HA)-specific maternal blood (M) and cord blood (C) titers of IgG1. Female neonates of SARS-CoV-2 negative mothers are shown in light blue, female neonates of SARS-CoV-2 positive mothers are shown in light orange. Males born to SARS-CoV-2 negative mothers are shown in dark blue, and males born to SARS-CoV-2 positive mothers are shown in dark orange. Units are Mean Fluorescence Intensity or MFI. All values reflect PBS background correction. Wilcoxon matched-pairs signed rank test was performed to determine significance. * p

    Techniques Used: Infection, Fluorescence

    Sexually dimorphic regulation of Fc receptor gene expression, protein expression, and colocalization. (A-C) RTqPCR analyses of male or female placental expression of FCGRT (A) , FCGR1 (B), and FCGR3A (C) in placental biopsies from SARS-CoV-2 negative (blue) or SARS-CoV-2 positive (orange) pregnancies. Expression levels shown are relative to reference gene YWHAZ . (D-F) Representative immunoblots and quantification of fetal female or fetal male expression of FcRn (A), FCγR1 (B), and FCγR3 (C) in placental biopsies from SARS-CoV-2 negative (blue) or SARS-CoV-2 positive (orange) pregnancies. Neg and Pos on western blot designates SARS-CoV-2 negative and positive pregnancies. (G) Placental tissue sections from SARS-CoV-2 + and SARS-CoV-2 – mothers were stained for FCγR3 (red), FcRn (purple), and placental alkaline phosphatase (PLAP, green), a trophoblast marker, and DAPI (blue). (H) Box-and-whisker plots showing FCγR3/FcRn co-localization in placental villi. (I) Placental tissue sections from SARS-CoV-2 + and SARS-CoV-2 – mothers were stained for FCγR1 (purple), FcRn (red), and placental alkaline phosphatase (PLAP, green), a trophoblast marker, and DAPI (blue). (J) Box-and-whisker plots showing (J) FCγR1/FcRn or (K) FCγR2/FcRn co-localization in placental villi. Two-way ANOVA followed by Bonferroni’s post-hoc analyses were performed to determine significance. * p
    Figure Legend Snippet: Sexually dimorphic regulation of Fc receptor gene expression, protein expression, and colocalization. (A-C) RTqPCR analyses of male or female placental expression of FCGRT (A) , FCGR1 (B), and FCGR3A (C) in placental biopsies from SARS-CoV-2 negative (blue) or SARS-CoV-2 positive (orange) pregnancies. Expression levels shown are relative to reference gene YWHAZ . (D-F) Representative immunoblots and quantification of fetal female or fetal male expression of FcRn (A), FCγR1 (B), and FCγR3 (C) in placental biopsies from SARS-CoV-2 negative (blue) or SARS-CoV-2 positive (orange) pregnancies. Neg and Pos on western blot designates SARS-CoV-2 negative and positive pregnancies. (G) Placental tissue sections from SARS-CoV-2 + and SARS-CoV-2 – mothers were stained for FCγR3 (red), FcRn (purple), and placental alkaline phosphatase (PLAP, green), a trophoblast marker, and DAPI (blue). (H) Box-and-whisker plots showing FCγR3/FcRn co-localization in placental villi. (I) Placental tissue sections from SARS-CoV-2 + and SARS-CoV-2 – mothers were stained for FCγR1 (purple), FcRn (red), and placental alkaline phosphatase (PLAP, green), a trophoblast marker, and DAPI (blue). (J) Box-and-whisker plots showing (J) FCγR1/FcRn or (K) FCγR2/FcRn co-localization in placental villi. Two-way ANOVA followed by Bonferroni’s post-hoc analyses were performed to determine significance. * p

    Techniques Used: Expressing, Western Blot, Staining, Marker, Whisker Assay

    Maternal and cord blood titers of HA and PTN in SARS-CoV-2 infected and non-infected mothers. Dot plots showing relative hemagglutinin (HA)- and pertussis (PTN)-specific maternal blood (M) and cord blood (C) titers of (A) IgG1, (B) IgG2, (C) IgG3, (D) FcRn, (E) FCGR2A, (F) FCGR2B, (G) FCGR3A, and (H) FCGR3B. SARS-CoV-2 - females are shown in light blue, SARS-CoV-2 + females are shown in light orange, SARS-CoV-2 – males are shown in dark blue, and SARS-CoV-2 + males are shown in dark orange. Y-axis units for all plots are PBS-corrected mean fluorescence intensity (MFI). Wilcoxon matched-pairs signed rank test was performed to determine significance. * p
    Figure Legend Snippet: Maternal and cord blood titers of HA and PTN in SARS-CoV-2 infected and non-infected mothers. Dot plots showing relative hemagglutinin (HA)- and pertussis (PTN)-specific maternal blood (M) and cord blood (C) titers of (A) IgG1, (B) IgG2, (C) IgG3, (D) FcRn, (E) FCGR2A, (F) FCGR2B, (G) FCGR3A, and (H) FCGR3B. SARS-CoV-2 - females are shown in light blue, SARS-CoV-2 + females are shown in light orange, SARS-CoV-2 – males are shown in dark blue, and SARS-CoV-2 + males are shown in dark orange. Y-axis units for all plots are PBS-corrected mean fluorescence intensity (MFI). Wilcoxon matched-pairs signed rank test was performed to determine significance. * p

    Techniques Used: Infection, Fluorescence

    Male-specific upregulation of interferon stimulated genes in placentas exposed to maternal SARS-CoV-2 infection. (A) Interferon stimulated gene pathway diagram. Production of interferon stimulated genes (ISGs) of interest can occur through activation of Type I IFN (left) or Type III IFN (right). Image created using Biorender. (B-G) RTqPCR analyses of male or female placental expression of (B) IFI6, (C) CXCL10, (D) OAS1 , (E) CCL2 , (F) MX1 , and (G) IL10 in placental biopsies from SARS-CoV-2 negative (blue) or SARS-CoV-2 positive (orange) pregnancies. Expression levels shown are relative to reference gene YWHAZ. (H-I) Representative immunohistochemistry images and quantification of CD163-positive cells in placental slices from SARS-CoV-2 negative (blue) or SARS-CoV-2 positive (orange) pregnancies. Two-way ANOVA followed by Bonferroni’s post-hoc analyses were performed to determine significance. * p
    Figure Legend Snippet: Male-specific upregulation of interferon stimulated genes in placentas exposed to maternal SARS-CoV-2 infection. (A) Interferon stimulated gene pathway diagram. Production of interferon stimulated genes (ISGs) of interest can occur through activation of Type I IFN (left) or Type III IFN (right). Image created using Biorender. (B-G) RTqPCR analyses of male or female placental expression of (B) IFI6, (C) CXCL10, (D) OAS1 , (E) CCL2 , (F) MX1 , and (G) IL10 in placental biopsies from SARS-CoV-2 negative (blue) or SARS-CoV-2 positive (orange) pregnancies. Expression levels shown are relative to reference gene YWHAZ. (H-I) Representative immunohistochemistry images and quantification of CD163-positive cells in placental slices from SARS-CoV-2 negative (blue) or SARS-CoV-2 positive (orange) pregnancies. Two-way ANOVA followed by Bonferroni’s post-hoc analyses were performed to determine significance. * p

    Techniques Used: Infection, Activation Assay, Expressing, Immunohistochemistry

    No effect of maternal SARS-CoV-2 infection on expression or localization of FCγR2. (A-C) qPCR analyses of fetal male or fetal female expression of ( A ) FCGR3B, ( B ) FCGR2A, and ( C ) FCGR2B in placental biopsies from SARS-CoV-2 negative (blue) or SARS-CoV-2 positive (orange) pregnancies. Expression levels shown are relative to reference gene YWHAZ . (D) Representative immunoblots and quantification of FCγR2 in female or male placental biopsies from mothers testing negative (blue) or positive (orange) for SARS-CoV-2. (E) Placental tissue sections from SARS-CoV-2 + and SARS-CoV-2 – mothers were stained for FCγR2 (purple), FcRn (red), and placental alkaline phosphatase (PLAP, green), a trophoblast marker, and DAPI (blue). (F) Box-and-whisker plots showing FCγR2/FcRn co-localization in placental villi. Two-way ANOVA followed by post-hoc analyses were performed to determine significance. * p
    Figure Legend Snippet: No effect of maternal SARS-CoV-2 infection on expression or localization of FCγR2. (A-C) qPCR analyses of fetal male or fetal female expression of ( A ) FCGR3B, ( B ) FCGR2A, and ( C ) FCGR2B in placental biopsies from SARS-CoV-2 negative (blue) or SARS-CoV-2 positive (orange) pregnancies. Expression levels shown are relative to reference gene YWHAZ . (D) Representative immunoblots and quantification of FCγR2 in female or male placental biopsies from mothers testing negative (blue) or positive (orange) for SARS-CoV-2. (E) Placental tissue sections from SARS-CoV-2 + and SARS-CoV-2 – mothers were stained for FCγR2 (purple), FcRn (red), and placental alkaline phosphatase (PLAP, green), a trophoblast marker, and DAPI (blue). (F) Box-and-whisker plots showing FCγR2/FcRn co-localization in placental villi. Two-way ANOVA followed by post-hoc analyses were performed to determine significance. * p

    Techniques Used: Infection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Marker, Whisker Assay

    SARS-CoV-2 infected mothers with male fetuses demonstrate reduced placental transfer of SARS-CoV-2 antibodies compared to those with female fetuses. Dot plots showing relative Spike-, RBD-, S1-, S2-, and N-specific maternal blood (M) and cord blood (C) titers of (A) IgG2, (B), IgG3, (C) FcRn, (D) FCGR2A, (E) FCGR2B, (F) FCGR3A, and (G) FCGR3B. Females are shown in light orange while males are shown in dark orange with black border. Wilcoxon matched pairs signed rank test was performed to determine significance. *p
    Figure Legend Snippet: SARS-CoV-2 infected mothers with male fetuses demonstrate reduced placental transfer of SARS-CoV-2 antibodies compared to those with female fetuses. Dot plots showing relative Spike-, RBD-, S1-, S2-, and N-specific maternal blood (M) and cord blood (C) titers of (A) IgG2, (B), IgG3, (C) FcRn, (D) FCGR2A, (E) FCGR2B, (F) FCGR3A, and (G) FCGR3B. Females are shown in light orange while males are shown in dark orange with black border. Wilcoxon matched pairs signed rank test was performed to determine significance. *p

    Techniques Used: Infection

    Maternal SARS-CoV-2 infection does not impact placental expression of TNF, IL6, or CCL7 . (A-C) qPCR analyses of fetal male or fetal female expression of (A) TNF, (B) IL6, and (C) CCL7 in placental biopsies from SARS-CoV-2 negative (blue) or SARS-CoV-2 positive (orange) pregnancies. Two-way ANOVA followed by post-hoc analyses were performed to determine significance. Expression levels shown are relative to reference gene YWHAZ . * p
    Figure Legend Snippet: Maternal SARS-CoV-2 infection does not impact placental expression of TNF, IL6, or CCL7 . (A-C) qPCR analyses of fetal male or fetal female expression of (A) TNF, (B) IL6, and (C) CCL7 in placental biopsies from SARS-CoV-2 negative (blue) or SARS-CoV-2 positive (orange) pregnancies. Two-way ANOVA followed by post-hoc analyses were performed to determine significance. Expression levels shown are relative to reference gene YWHAZ . * p

    Techniques Used: Infection, Expressing, Real-time Polymerase Chain Reaction

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

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

    Journal: bioRxiv

    doi: 10.1101/2021.01.05.422952

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

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

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

    Techniques Used: Binding Assay, Concentration Assay, Titration

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

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

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

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

    20) Product Images from "Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein"

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.0c01394

    Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.
    Figure Legend Snippet: Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.

    Techniques Used: Selection

    Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.
    Figure Legend Snippet: Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.

    Techniques Used: Flow Cytometry, Binding Assay

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

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

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112643

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

    Techniques Used: Polymerase Chain Reaction

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

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

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

    Techniques Used: Titration, Concentration Assay, Standard Deviation

    Representative AIR array images of (A) 5% FBS; (B) 10% PNHS; (C) a negative single-donor sample, and (D) one convalescent serum sample. Strong responses to SARS-CoV-2 antigens are readily observed in (D), but not in (A), (B), or (C), while responses to circulating coronaviruses HKU, OC43, and 229E are observed in (B), (C), and (D). In each case, samples were diluted 1:20 in Adarza diluent, and incubated with the arrays overnight at 4 °C. See Fig. 1 for key to the array. All arrays in this figure were imaged at an exposure of 100 ms.
    Figure Legend Snippet: Representative AIR array images of (A) 5% FBS; (B) 10% PNHS; (C) a negative single-donor sample, and (D) one convalescent serum sample. Strong responses to SARS-CoV-2 antigens are readily observed in (D), but not in (A), (B), or (C), while responses to circulating coronaviruses HKU, OC43, and 229E are observed in (B), (C), and (D). In each case, samples were diluted 1:20 in Adarza diluent, and incubated with the arrays overnight at 4 °C. See Fig. 1 for key to the array. All arrays in this figure were imaged at an exposure of 100 ms.

    Techniques Used: Incubation

    22) Product Images from "Development and pre-clinical evaluation of Newcastle disease virus-vectored SARS-CoV-2 intranasal vaccine candidate"

    Article Title: Development and pre-clinical evaluation of Newcastle disease virus-vectored SARS-CoV-2 intranasal vaccine candidate

    Journal: bioRxiv

    doi: 10.1101/2021.03.07.434276

    Efficacy of live NDV vaccines against SARS-CoV-2 infection in hamsters. Golden Syrian hamsters groups vaccinated with rLS1-S1-F, rLS1-HN-RBD, the mixture rLS1-S1-F/rLS1-HN-RBD, and negative control (not immunized) were challenged 30 days after the boost with SARS-CoV-2, also a not immunized and not challenge group was included (Mock). ( A ) Viral isolate (%) was done from the lung of each hamster group (n=4) at days 2, 5, and 10 post-challenge. Two-way ANOVA and Tukey’s post hoc were performed. *: p
    Figure Legend Snippet: Efficacy of live NDV vaccines against SARS-CoV-2 infection in hamsters. Golden Syrian hamsters groups vaccinated with rLS1-S1-F, rLS1-HN-RBD, the mixture rLS1-S1-F/rLS1-HN-RBD, and negative control (not immunized) were challenged 30 days after the boost with SARS-CoV-2, also a not immunized and not challenge group was included (Mock). ( A ) Viral isolate (%) was done from the lung of each hamster group (n=4) at days 2, 5, and 10 post-challenge. Two-way ANOVA and Tukey’s post hoc were performed. *: p

    Techniques Used: Infection, Negative Control

    Expression of SARS-CoV-2 RBD and S1 proteins in infected Vero E6 cells and NDV particles. (A) Western blot detection for the HN-RBD and S1-F proteins expression. Vero E6 cells were infected with the rLS1, rLS1 rLS1-HN-RBD, and rLS1-S1-F viruses at an MOI of 1. Then after 48 hpi, the cells were lysed and analyzed by western blotting. (B) To verify the incorporation of the HN-RBD and S1-F proteins into rLS1-HN-RBD, and rLS1-S1-F viruses, the viral particles in FA from infected SPF chicken embryonated eggs with the recombinant viruses and rLS1, was concentrated by ultracentrifugation, and partially purified on 25 % sucrose cushion. Western blot analysis was carried out using partially purified viruses and lysate from infected cells, using a rabbit antibody specific to SARS-CoV-2 RBD protein and Anti Rabbit IgG conjugated to HRP. The black arrow indicates the expected protein band. (C) Vero-E6 cells infected with the rLS1, rLS1-HN-RBD, and rLS1-S1-F at an MOI of 0.5. After 48 h, the expression of RBD and S1 proteins was detected by Immunofluorescence assay using a rabbit antibody specific to SARS-CoV-2 RBD protein, and a Donkey Anti-Rabbit IgG H L-Alexa Fluor 594. Therefore, the NDV was detected using a chicken antiserum specific to the NDV, and a Goat Anti-Chicken IgY H L-Alexa Fluor® 488. Cell nuclei were stained with DAPI. A scale bar of 50 µm. Image magnification 200x. (D) Detection of S1 or RBD proteins on the viral surface of rLS1-S1-F and rLS1-HN-RBD viruses’ attachment to Vero E6 cells. The cells were incubated with purified viruses rLS1-HN-RBD or rLS1-S1-F, for 30 min. Subsequently, the cells were labeled with rabbit monoclonal antibody anti-SARS-COV-2 S1 as the primary antibody, followed by secondary antibody goat anti-rabbit IgG Alexa Fluor 488. The cells were then analyzed by a flow cytometer. The percentage of positive cells indicates the detection of S1 or RBD proteins on the viral surface of viruses bound to Vero E6 and is shown in the dot plot for rLS1-S1-F virus and sLS1-HN-RBD virus; including negative controls for each assay determined by cells incubated with phosphate-buffered saline (PBS) or rLS1 virus.
    Figure Legend Snippet: Expression of SARS-CoV-2 RBD and S1 proteins in infected Vero E6 cells and NDV particles. (A) Western blot detection for the HN-RBD and S1-F proteins expression. Vero E6 cells were infected with the rLS1, rLS1 rLS1-HN-RBD, and rLS1-S1-F viruses at an MOI of 1. Then after 48 hpi, the cells were lysed and analyzed by western blotting. (B) To verify the incorporation of the HN-RBD and S1-F proteins into rLS1-HN-RBD, and rLS1-S1-F viruses, the viral particles in FA from infected SPF chicken embryonated eggs with the recombinant viruses and rLS1, was concentrated by ultracentrifugation, and partially purified on 25 % sucrose cushion. Western blot analysis was carried out using partially purified viruses and lysate from infected cells, using a rabbit antibody specific to SARS-CoV-2 RBD protein and Anti Rabbit IgG conjugated to HRP. The black arrow indicates the expected protein band. (C) Vero-E6 cells infected with the rLS1, rLS1-HN-RBD, and rLS1-S1-F at an MOI of 0.5. After 48 h, the expression of RBD and S1 proteins was detected by Immunofluorescence assay using a rabbit antibody specific to SARS-CoV-2 RBD protein, and a Donkey Anti-Rabbit IgG H L-Alexa Fluor 594. Therefore, the NDV was detected using a chicken antiserum specific to the NDV, and a Goat Anti-Chicken IgY H L-Alexa Fluor® 488. Cell nuclei were stained with DAPI. A scale bar of 50 µm. Image magnification 200x. (D) Detection of S1 or RBD proteins on the viral surface of rLS1-S1-F and rLS1-HN-RBD viruses’ attachment to Vero E6 cells. The cells were incubated with purified viruses rLS1-HN-RBD or rLS1-S1-F, for 30 min. Subsequently, the cells were labeled with rabbit monoclonal antibody anti-SARS-COV-2 S1 as the primary antibody, followed by secondary antibody goat anti-rabbit IgG Alexa Fluor 488. The cells were then analyzed by a flow cytometer. The percentage of positive cells indicates the detection of S1 or RBD proteins on the viral surface of viruses bound to Vero E6 and is shown in the dot plot for rLS1-S1-F virus and sLS1-HN-RBD virus; including negative controls for each assay determined by cells incubated with phosphate-buffered saline (PBS) or rLS1 virus.

    Techniques Used: Expressing, Infection, Western Blot, Recombinant, Purification, Immunofluorescence, Staining, Incubation, Labeling, Flow Cytometry

    The intranasal vaccine elicits specific antibodies against RBD protein and neutralizing antibodies against SARS-CoV-2 in hamsters. ( A ) Immunization regimen. To evaluate the immunogenicity of the NDV vaccines, five-week-old female and males golden Syrian hamsters were used in this study. The hamsters were randomly divided into five groups. The Hamsters were vaccinated intranasal route with live NDV vaccine following a prime-boost-regimen with a two-week interval. Group 1 received rLS1-HN-RBD ( n =12), Group 2 received the rLS1-S1-F ( n =12), Group 3 received the mixture of rLS1-HN-RBD/rLS1-S1-F ( n =12), Group 4 did not receive vaccine ( n =12) since group served controls, and Group 5 receive no vaccine and was not challenged, hence serving as healthy control ( n =12). One boost immunization with the same concentration of each vaccine was applied in all vaccinated groups at the second week. ( B ) ELISA assay to measure SARS-CoV-2 RBD-specific serum IgG antibody, and ( C ) S1 subunit-specific serum IgG antibody. Sera from hamsters at pre-boost and 15 days after boost were evaluated. SARS-CoV-2 RBD purified recombinant protein was used for ELISA. The cutoff was set at 0.06. ( D ). Immunized hamsters were bled pre-boost and 15 days after boost. All sera were isolated by low-speed centrifugation. Serum samples were processed to evaluate the neutralizing antibody titers against SARS-CoV-2 RBD protein using the surrogate virus neutralization test (sVNT). The positive cut-off and negative cut-off for SARS-CoV-2 neutralizing antibody detection were interpreted as the inhibition rate. The cut-off interpretation of results: result positive ≥20% (neutralizing antibody detected), result negative
    Figure Legend Snippet: The intranasal vaccine elicits specific antibodies against RBD protein and neutralizing antibodies against SARS-CoV-2 in hamsters. ( A ) Immunization regimen. To evaluate the immunogenicity of the NDV vaccines, five-week-old female and males golden Syrian hamsters were used in this study. The hamsters were randomly divided into five groups. The Hamsters were vaccinated intranasal route with live NDV vaccine following a prime-boost-regimen with a two-week interval. Group 1 received rLS1-HN-RBD ( n =12), Group 2 received the rLS1-S1-F ( n =12), Group 3 received the mixture of rLS1-HN-RBD/rLS1-S1-F ( n =12), Group 4 did not receive vaccine ( n =12) since group served controls, and Group 5 receive no vaccine and was not challenged, hence serving as healthy control ( n =12). One boost immunization with the same concentration of each vaccine was applied in all vaccinated groups at the second week. ( B ) ELISA assay to measure SARS-CoV-2 RBD-specific serum IgG antibody, and ( C ) S1 subunit-specific serum IgG antibody. Sera from hamsters at pre-boost and 15 days after boost were evaluated. SARS-CoV-2 RBD purified recombinant protein was used for ELISA. The cutoff was set at 0.06. ( D ). Immunized hamsters were bled pre-boost and 15 days after boost. All sera were isolated by low-speed centrifugation. Serum samples were processed to evaluate the neutralizing antibody titers against SARS-CoV-2 RBD protein using the surrogate virus neutralization test (sVNT). The positive cut-off and negative cut-off for SARS-CoV-2 neutralizing antibody detection were interpreted as the inhibition rate. The cut-off interpretation of results: result positive ≥20% (neutralizing antibody detected), result negative

    Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay, Purification, Recombinant, Isolation, Centrifugation, Neutralization, Inhibition

    S tability of the lyophilized NDV vaccine. The expression of S1-F and HN-RBD proteins in Vero E6 cells infected with the lyophilized NDV vaccine was confirmed at day 1, 30, and 50 days post-lyophilized by Western blot assay using a rabbit antibody specific to SARS-CoV-2 RBD protein and Anti Rabbit IgG conjugated to HRP. The black arrow indicates the expected protein band.
    Figure Legend Snippet: S tability of the lyophilized NDV vaccine. The expression of S1-F and HN-RBD proteins in Vero E6 cells infected with the lyophilized NDV vaccine was confirmed at day 1, 30, and 50 days post-lyophilized by Western blot assay using a rabbit antibody specific to SARS-CoV-2 RBD protein and Anti Rabbit IgG conjugated to HRP. The black arrow indicates the expected protein band.

    Techniques Used: Expressing, Infection, Western Blot

    The strategy used for the generation of the recombinant NDVs expressing SARS-CoV-2 RBD and S1. (A) The schematic representation of the strategy of construction recombinant NDVs. Two cassettes transcriptional were designed for expressing RBD and S1: 1) HN-RBD was fused with the complete transmembrane domain (TM) and the cytoplasmic tail (CT) of the gene haemagglutinin– neuraminidase (HN), 2) S1-F was fused with the TM/CT of the gene fusion (F) from of full-length pFLC-LS1. (B) The full-length antigenome of NDV strain LaSota was used as a clone (pFLC-LS1) was used as back clone, the pFLC-LS1-HN-RBD and pFLC-LS1-S1-F were generated from cassettes that expressing RBD and S1 inserted into genome NDV under control of transcriptional gene end (GE) and gene start (GS) signals. The names, position, and direction of the primers used are shown with arrows (blacks) indicating size products. (C) The insertion of the expression cassette into the non-coding region between the P/M genes of NDV genome was verified by RT-PCR using the junction primers NDV-3LS1-2020-F1 and NDV-3LS1-2020-R1 as shown in (B).
    Figure Legend Snippet: The strategy used for the generation of the recombinant NDVs expressing SARS-CoV-2 RBD and S1. (A) The schematic representation of the strategy of construction recombinant NDVs. Two cassettes transcriptional were designed for expressing RBD and S1: 1) HN-RBD was fused with the complete transmembrane domain (TM) and the cytoplasmic tail (CT) of the gene haemagglutinin– neuraminidase (HN), 2) S1-F was fused with the TM/CT of the gene fusion (F) from of full-length pFLC-LS1. (B) The full-length antigenome of NDV strain LaSota was used as a clone (pFLC-LS1) was used as back clone, the pFLC-LS1-HN-RBD and pFLC-LS1-S1-F were generated from cassettes that expressing RBD and S1 inserted into genome NDV under control of transcriptional gene end (GE) and gene start (GS) signals. The names, position, and direction of the primers used are shown with arrows (blacks) indicating size products. (C) The insertion of the expression cassette into the non-coding region between the P/M genes of NDV genome was verified by RT-PCR using the junction primers NDV-3LS1-2020-F1 and NDV-3LS1-2020-R1 as shown in (B).

    Techniques Used: Recombinant, Expressing, Generated, Reverse Transcription Polymerase Chain Reaction

    Body weight and mobility analysis of SARS-CoV-2 challenged golden Syrian hamsters. ( A ) Changes in body weight (percent weight change compared to day 0) of hamsters inoculated with SARS-CoV-2 and Mock group, at days 2, 5, and 10 post-challenged. Mobility assessment results shown ( B ) average velocity, ( C ) average acceleration, and ( D ) average displacement. Mean ± s.d. are shown. Asterisks indicate that results were statistically significant compared to the control group (P
    Figure Legend Snippet: Body weight and mobility analysis of SARS-CoV-2 challenged golden Syrian hamsters. ( A ) Changes in body weight (percent weight change compared to day 0) of hamsters inoculated with SARS-CoV-2 and Mock group, at days 2, 5, and 10 post-challenged. Mobility assessment results shown ( B ) average velocity, ( C ) average acceleration, and ( D ) average displacement. Mean ± s.d. are shown. Asterisks indicate that results were statistically significant compared to the control group (P

    Techniques Used:

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.09.28.311480

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

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

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

    Techniques Used: Mouse Assay, Immunofluorescence, Staining

    Characterization of Sad23L-nCoV-S and Ad49L-nCoV-S vaccines. ( A ) Recombinant adenovirus constructs Sad23L-nCoV-S and Ad49L-nCoV-S carrying the full-length S gene of SARS-CoV-2 under CMV promotor regulation within the deleted E1 region of Sad23L or Ad49L vector. ( B ) Western blot analysis for the expression of S protein from Sad23L-nCoV-S or Ad49L-nCoV-S infected HEK-293A cell lysates by rabbit polyclonal antibody to RBD and heat-inactivated COVID-19 patent’s serum IgG. Sad23L-GFP or Ad49L-GFP virus infected cells were used as mock controls. ( C ) Expression of S protein in HEK-293A cells detected by immunofluorescence staining. ( D ) Seroprevalence of neutralizing antibody to Ad5, Ad49L or Sad23L vector in 600 healthy blood donors.
    Figure Legend Snippet: Characterization of Sad23L-nCoV-S and Ad49L-nCoV-S vaccines. ( A ) Recombinant adenovirus constructs Sad23L-nCoV-S and Ad49L-nCoV-S carrying the full-length S gene of SARS-CoV-2 under CMV promotor regulation within the deleted E1 region of Sad23L or Ad49L vector. ( B ) Western blot analysis for the expression of S protein from Sad23L-nCoV-S or Ad49L-nCoV-S infected HEK-293A cell lysates by rabbit polyclonal antibody to RBD and heat-inactivated COVID-19 patent’s serum IgG. Sad23L-GFP or Ad49L-GFP virus infected cells were used as mock controls. ( C ) Expression of S protein in HEK-293A cells detected by immunofluorescence staining. ( D ) Seroprevalence of neutralizing antibody to Ad5, Ad49L or Sad23L vector in 600 healthy blood donors.

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

    24) Product Images from "Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM"

    Article Title: Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM

    Journal: bioRxiv

    doi: 10.1101/2020.06.30.177097

    ACE2 binding induced conformational dynamics of the SARS-CoV-2 S-ACE2 complex determined by multi-body refinement. (A) The contributions of all eigenvectors to the motion in the S-ACE2 complex, with eigenvectors 1-3 dominant the contributions. (B) Top view of the map showing the three swing motions of the first 3 eigenvectors, with S trimer following the color schema as in Fig. 2 , and the two extreme locations of ACE2 illustrated in deep pink and light blue densities. The swing angular range and direction are indicated in dark red arrow. (C) Histograms of the amplitudes along the first 3 eigenvectors. (D) Atomic models of S-ACE2 and S-closed, colored according to the B factor distribution (ranging from 100Å 2 [blue] to 130Å 2 [red]).
    Figure Legend Snippet: ACE2 binding induced conformational dynamics of the SARS-CoV-2 S-ACE2 complex determined by multi-body refinement. (A) The contributions of all eigenvectors to the motion in the S-ACE2 complex, with eigenvectors 1-3 dominant the contributions. (B) Top view of the map showing the three swing motions of the first 3 eigenvectors, with S trimer following the color schema as in Fig. 2 , and the two extreme locations of ACE2 illustrated in deep pink and light blue densities. The swing angular range and direction are indicated in dark red arrow. (C) Histograms of the amplitudes along the first 3 eigenvectors. (D) Atomic models of S-ACE2 and S-closed, colored according to the B factor distribution (ranging from 100Å 2 [blue] to 130Å 2 [red]).

    Techniques Used: Binding Assay

    Organization of the resolved N-Linked glycans of SARS-CoV-2 S trimer. (A) Schematic representation of SARS-CoV-2 S glycoprotein. The positions of N-linked glycosylation sequons are shown as branches. 18 N-linked glycans detected in our cryo-EM map of the S-closed state are shown in red, the remaining undetected ones in black. After ACE2 binding, the glycan density that appears weaker is indicated (*). (B) Surface representation of the glycosylated SARS-CoV-2 S trimer in the S-closed state with N-linked glycans shown in red. The location of glycan hole is indicated in black doted ellipsoid, with the locations of S1/S2 and FP, and glycan at N657 site near the glycan hole indicated. The newly captured glycans at N17 and N149 sites are indicated in the top view. (C) Surface representation of the glycosylated S-ACE2 complex with N-linked glycans in red. After ACE2 binding, the glycan density that appears weaker is indicated.
    Figure Legend Snippet: Organization of the resolved N-Linked glycans of SARS-CoV-2 S trimer. (A) Schematic representation of SARS-CoV-2 S glycoprotein. The positions of N-linked glycosylation sequons are shown as branches. 18 N-linked glycans detected in our cryo-EM map of the S-closed state are shown in red, the remaining undetected ones in black. After ACE2 binding, the glycan density that appears weaker is indicated (*). (B) Surface representation of the glycosylated SARS-CoV-2 S trimer in the S-closed state with N-linked glycans shown in red. The location of glycan hole is indicated in black doted ellipsoid, with the locations of S1/S2 and FP, and glycan at N657 site near the glycan hole indicated. The newly captured glycans at N17 and N149 sites are indicated in the top view. (C) Surface representation of the glycosylated S-ACE2 complex with N-linked glycans in red. After ACE2 binding, the glycan density that appears weaker is indicated.

    Techniques Used: Binding Assay

    Cryo-EM data processing procedure for SARS-CoV-2 S trimer in the presence of ACE2.
    Figure Legend Snippet: Cryo-EM data processing procedure for SARS-CoV-2 S trimer in the presence of ACE2.

    Techniques Used:

    A complete architecture of the SARS-CoV-2 S-ACE2 complex. (A-B) Cryo-EM map and pseudo atomic model of SARS-CoV-2 S-ACE2 complex. We named the RBD up protomer as protomer 1 (light green), and the other two RBD down ones as protomer 2 (royal blue) and protomer 3 (gold). ACE2 was colored in violet red. (C) Side and top views of the overlaid S-open (color) and S-closed (dark grey) structures, showing in the open process there is a 71.0° upwards/outwards rotation of RBD associated with a downwards shift of SD1 in protomer 1. (D) Rotations of NTD and CH from S-closed (grey) to S-open (in color) state, with the NTD also showing a downwards/outwards movement. (E) Side view of the overlaid S-ACE2 (violet red) and S-open (light green) protomer 1 structures, showing the angle between the long axis of RBD and the horizontal plane of S trimer reduces from the S-open to the S-ACE2 state. (F) Top and side views of the overlaid S-ACE2 (violet red) and S-open (color) RBD structures, showing the coordinated movements of RBDs. (G) Protomer interaction interface analysis of S-ACE2 by PISA. (H) Aromatic interactions between the core region of the up RBD-1 (green) and the RBM T470-F490 loop of the neighboring RBD-2 (blue). (I) Overlaid structures of S-ACE2 (grey) and S-closed (color, with the FP fragment in deep pink), indicating a downwards shift of SD1 and most of the FP is missing in S-ACE2 state. Close up view (right panel) of the potential clashes between the downwards shifted SD1 β34 and α8 helix of FP. (J) Population shift between the ACE2-unpresented and ACE2-presented S trimer samples.
    Figure Legend Snippet: A complete architecture of the SARS-CoV-2 S-ACE2 complex. (A-B) Cryo-EM map and pseudo atomic model of SARS-CoV-2 S-ACE2 complex. We named the RBD up protomer as protomer 1 (light green), and the other two RBD down ones as protomer 2 (royal blue) and protomer 3 (gold). ACE2 was colored in violet red. (C) Side and top views of the overlaid S-open (color) and S-closed (dark grey) structures, showing in the open process there is a 71.0° upwards/outwards rotation of RBD associated with a downwards shift of SD1 in protomer 1. (D) Rotations of NTD and CH from S-closed (grey) to S-open (in color) state, with the NTD also showing a downwards/outwards movement. (E) Side view of the overlaid S-ACE2 (violet red) and S-open (light green) protomer 1 structures, showing the angle between the long axis of RBD and the horizontal plane of S trimer reduces from the S-open to the S-ACE2 state. (F) Top and side views of the overlaid S-ACE2 (violet red) and S-open (color) RBD structures, showing the coordinated movements of RBDs. (G) Protomer interaction interface analysis of S-ACE2 by PISA. (H) Aromatic interactions between the core region of the up RBD-1 (green) and the RBM T470-F490 loop of the neighboring RBD-2 (blue). (I) Overlaid structures of S-ACE2 (grey) and S-closed (color, with the FP fragment in deep pink), indicating a downwards shift of SD1 and most of the FP is missing in S-ACE2 state. Close up view (right panel) of the potential clashes between the downwards shifted SD1 β34 and α8 helix of FP. (J) Population shift between the ACE2-unpresented and ACE2-presented S trimer samples.

    Techniques Used:

    Cryo-EM analysis of the SARS-CoV-2 S trimer in the tightly closed state. (A) Representative cryo-EM image of the SARS-CoV-2 S trimer. (B) Reference-free 2D class averages of the S trimer. (C) Euler angular distribution of 3D reconstructions before and after adding tilt data, with the dotted circle indicating the sparsely distributed tilted top views in the non-tilt data. (D-E) Local resolution evaluation (D) and resolution assessment of our S-closed cryo-EM map by Fourier shell correlation (FSC) at 0.143 criterion (E). (F) Close up view of the model-map fitting in S2 subunit. (G) Compared with the recent structure of SARS-CoV-2 S in closed state (gray, 6VXX), our S-closed structure (blue) showed a slight inward tilt leading the peripheral edge of NTD exhibiting a 12.4 Å inward movement (for the Cα of T124). (H) Top view of the overlaid structures between our SARS-CoV-2 S-closed structure and the SARS-CoV S-closed structure (PDB: 5XLR) and zoom in views of the overlaid structures in NTD, RBD, CH domains. (I) N-linked glycans resolved in our S-closed cryo-EM map, with the densities corresponding to glycans colored in red.
    Figure Legend Snippet: Cryo-EM analysis of the SARS-CoV-2 S trimer in the tightly closed state. (A) Representative cryo-EM image of the SARS-CoV-2 S trimer. (B) Reference-free 2D class averages of the S trimer. (C) Euler angular distribution of 3D reconstructions before and after adding tilt data, with the dotted circle indicating the sparsely distributed tilted top views in the non-tilt data. (D-E) Local resolution evaluation (D) and resolution assessment of our S-closed cryo-EM map by Fourier shell correlation (FSC) at 0.143 criterion (E). (F) Close up view of the model-map fitting in S2 subunit. (G) Compared with the recent structure of SARS-CoV-2 S in closed state (gray, 6VXX), our S-closed structure (blue) showed a slight inward tilt leading the peripheral edge of NTD exhibiting a 12.4 Å inward movement (for the Cα of T124). (H) Top view of the overlaid structures between our SARS-CoV-2 S-closed structure and the SARS-CoV S-closed structure (PDB: 5XLR) and zoom in views of the overlaid structures in NTD, RBD, CH domains. (I) N-linked glycans resolved in our S-closed cryo-EM map, with the densities corresponding to glycans colored in red.

    Techniques Used:

    Amino acid sequence alignment of SARS-CoV-2 S to SARS-CoV S. The secondary structure elements were defined based on an ESPript ( Robert and Gouet, 2014 ) algorithm and are labeled based on our SARS-CoV-2 S-closed structure. The RBD domain is labeled in green frames, and the subdomains of RBM are also labeled.
    Figure Legend Snippet: Amino acid sequence alignment of SARS-CoV-2 S to SARS-CoV S. The secondary structure elements were defined based on an ESPript ( Robert and Gouet, 2014 ) algorithm and are labeled based on our SARS-CoV-2 S-closed structure. The RBD domain is labeled in green frames, and the subdomains of RBM are also labeled.

    Techniques Used: Sequencing, Labeling

    The T470-T478 loop and residue Y505 within RBM play vital roles in the engagement of SARS-CoV-2 spike with receptor ACE2. (A) The overall view of ACE2 (violet red) bound protomer 1 (light green) from our S-ACE2 structure, and zoom in view of the interaction interface between ACE2 and RBD, with the key contacting elements T470-F490 loop and Q498-Y505 within RBM highlighted in black ellipsoid and blue ellipsoid, respectively. (B) Superposition of our SARS-CoV-2 S-ACE2 structure with the crystal structure of SARS-CoV RBD-ACE2 (PDB: 2AJF), suggesting the RBM T470-F490 loop has obvious conformational variations. (C) Binding activities of ACE2-hFc fusion protein to wild-type (wt) and mutant SARS-CoV-2 RBD proteins determined by ELISA. Different structural elements of RBD were colored in the left panel. Anti-RBD sera and a cross-reactive MAb 1A10 served as positive controls. Ctr, an irrelevant antibody. The black arrow indicates that mutations in the RBD (RBM-R3) mutant significantly reduced the binding of ACE2-hFc compared to wild-type RBD. (D) Binding of ACE2-hFc fusion protein to wt and single-point mutant forms of SARS-CoV-2 RBD protein measured by ELISA. RBD (Q498A), RBD (V503A), and RBD (Y505A), RBD residues Q498, V503, and Y505 were mutated to Ala, respectively. The downward arrow indicates that the mutation at Y505 completely abolished the binding of ACE2 to RBD protein.
    Figure Legend Snippet: The T470-T478 loop and residue Y505 within RBM play vital roles in the engagement of SARS-CoV-2 spike with receptor ACE2. (A) The overall view of ACE2 (violet red) bound protomer 1 (light green) from our S-ACE2 structure, and zoom in view of the interaction interface between ACE2 and RBD, with the key contacting elements T470-F490 loop and Q498-Y505 within RBM highlighted in black ellipsoid and blue ellipsoid, respectively. (B) Superposition of our SARS-CoV-2 S-ACE2 structure with the crystal structure of SARS-CoV RBD-ACE2 (PDB: 2AJF), suggesting the RBM T470-F490 loop has obvious conformational variations. (C) Binding activities of ACE2-hFc fusion protein to wild-type (wt) and mutant SARS-CoV-2 RBD proteins determined by ELISA. Different structural elements of RBD were colored in the left panel. Anti-RBD sera and a cross-reactive MAb 1A10 served as positive controls. Ctr, an irrelevant antibody. The black arrow indicates that mutations in the RBD (RBM-R3) mutant significantly reduced the binding of ACE2-hFc compared to wild-type RBD. (D) Binding of ACE2-hFc fusion protein to wt and single-point mutant forms of SARS-CoV-2 RBD protein measured by ELISA. RBD (Q498A), RBD (V503A), and RBD (Y505A), RBD residues Q498, V503, and Y505 were mutated to Ala, respectively. The downward arrow indicates that the mutation at Y505 completely abolished the binding of ACE2 to RBD protein.

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

    Cryo-EM analysis on the SARS-CoV-2 S-ACE2 complex. (A) Representative cryo-EM image of the SARS-CoV-2 S trimer in the presence of ACE2. (B) Reference-free 2D class averages of the sample. (C-D) Local resolution evaluation of the S-ACE2 map (C) and S-open map (D). (E) Resolution assessment of the cryo-EM reconstructions by Fourier shell correlation (FSC) at 0.143 criterion. (F) Unliganded S-open map obtained from this dataset. (G) Cryo-EM map of S-ACE2 complex without cross linker (left, colored), and its overlay with S-ACE2 map with cross linker (pink, low pass filtered to similar resolution, right panel), suggesting they are in similar conformation. (H) ACE2 binding induced motions of S-ACE2 without cross linker. Left, contributions of all eigenvectors to motions of S-ACE2; right three panels, top view of the map showing the three swing motions along the first 3 eigenvectors.
    Figure Legend Snippet: Cryo-EM analysis on the SARS-CoV-2 S-ACE2 complex. (A) Representative cryo-EM image of the SARS-CoV-2 S trimer in the presence of ACE2. (B) Reference-free 2D class averages of the sample. (C-D) Local resolution evaluation of the S-ACE2 map (C) and S-open map (D). (E) Resolution assessment of the cryo-EM reconstructions by Fourier shell correlation (FSC) at 0.143 criterion. (F) Unliganded S-open map obtained from this dataset. (G) Cryo-EM map of S-ACE2 complex without cross linker (left, colored), and its overlay with S-ACE2 map with cross linker (pink, low pass filtered to similar resolution, right panel), suggesting they are in similar conformation. (H) ACE2 binding induced motions of S-ACE2 without cross linker. Left, contributions of all eigenvectors to motions of S-ACE2; right three panels, top view of the map showing the three swing motions along the first 3 eigenvectors.

    Techniques Used: Binding Assay

    The proposed mechanism of ACE2 induced conformational transitions of SARS-CoV-2 S trimer. Conformational transitions from the ground prefusion closed state (with packed FP, in red) to the transiently open state (Step 1) with an untwisting motion of the S trimer (highlighted in dark grey arrow) associated with a downwards movement of S1 (red arrow), from the open state to the dynamic ACE2 engaged state (Step 2), and from the ACE2 engaged state all the way to the refolded postfusion state (Step 3). The continuous swing motions of the associated ACE2-RBD within the S trimer are indicated by red arrows. The SARS-CoV-2 S trimer associated with ACE2 dimer (in the third panel) was generated by aligning the ACE2 of our SARS-CoV-2 S-ACE2 complex structure with the available full length ACE2 dimer structure (PDB: 6M1D). The postfusion state was illustrated as a carton.
    Figure Legend Snippet: The proposed mechanism of ACE2 induced conformational transitions of SARS-CoV-2 S trimer. Conformational transitions from the ground prefusion closed state (with packed FP, in red) to the transiently open state (Step 1) with an untwisting motion of the S trimer (highlighted in dark grey arrow) associated with a downwards movement of S1 (red arrow), from the open state to the dynamic ACE2 engaged state (Step 2), and from the ACE2 engaged state all the way to the refolded postfusion state (Step 3). The continuous swing motions of the associated ACE2-RBD within the S trimer are indicated by red arrows. The SARS-CoV-2 S trimer associated with ACE2 dimer (in the third panel) was generated by aligning the ACE2 of our SARS-CoV-2 S-ACE2 complex structure with the available full length ACE2 dimer structure (PDB: 6M1D). The postfusion state was illustrated as a carton.

    Techniques Used: Generated

    Purification of SARS-CoV-2 S ectodomain, human ACE2 PD domain, and SARS-CoV-2 S-ACE2 complex. (A) Schematic diagram of SARS-CoV-2 S organization in this study. S1/S2 protease cleavage site (S1/S2), N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), and central helix (CH) are labeled. (B-C) SDS-PAGE analysis of the purified S protein (B) and ACE2 (C). (D) Size-exclusion chromatogram and SDS-PAGE analysis of the formed SARS-CoV-2 S-ACE2 complex.
    Figure Legend Snippet: Purification of SARS-CoV-2 S ectodomain, human ACE2 PD domain, and SARS-CoV-2 S-ACE2 complex. (A) Schematic diagram of SARS-CoV-2 S organization in this study. S1/S2 protease cleavage site (S1/S2), N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), and central helix (CH) are labeled. (B-C) SDS-PAGE analysis of the purified S protein (B) and ACE2 (C). (D) Size-exclusion chromatogram and SDS-PAGE analysis of the formed SARS-CoV-2 S-ACE2 complex.

    Techniques Used: Purification, Binding Assay, Labeling, SDS Page

    An uncharacterized tightly closed conformation of SARS-CoV-2 S trimer. (A-B) Cryo-EM map and pseudo atomic model of SARS-CoV-2 S trimer in a tightly closed state, with three protomers shown in different color. (C) Close up view of the model-map fitting in the NTD and RBD regions of S1 subunit, illustrating most of the NTD region was well resolved. (D) Overlaid structures of our S-closed structure (blue) with the recent structure of SARS-CoV-2 S in closed state (gray, 6VXX), illustrating the RBM S469-C488 loop was newly captured in our structure (highlighted in dotted ellipsoid). (E) Top view of the overlaid structures as in (D) (left panel) and zoom-in views of specific domains, showing there is a dramatic anti-clockwise rotation in S1 especially in NTD, and a slight clockwise rotation in the central CH, resulting in a twisted tightly closed conformation. (F) Protomer interaction interface analysis by PISA. (G)The location of the newly resolved FP fragment (in deep pink) within the S trimer (left) and one protomer. S1 and S2 subunit is colored steel blue and gold, respectively. (H) Model-map fitting for the newly resolved FP fragment. (I) Close up view of the interactions between D614 from SD2 and FP, with the hydrogen bonds labeled in dotted lines and the L828-F855 region in FP in deep pink.
    Figure Legend Snippet: An uncharacterized tightly closed conformation of SARS-CoV-2 S trimer. (A-B) Cryo-EM map and pseudo atomic model of SARS-CoV-2 S trimer in a tightly closed state, with three protomers shown in different color. (C) Close up view of the model-map fitting in the NTD and RBD regions of S1 subunit, illustrating most of the NTD region was well resolved. (D) Overlaid structures of our S-closed structure (blue) with the recent structure of SARS-CoV-2 S in closed state (gray, 6VXX), illustrating the RBM S469-C488 loop was newly captured in our structure (highlighted in dotted ellipsoid). (E) Top view of the overlaid structures as in (D) (left panel) and zoom-in views of specific domains, showing there is a dramatic anti-clockwise rotation in S1 especially in NTD, and a slight clockwise rotation in the central CH, resulting in a twisted tightly closed conformation. (F) Protomer interaction interface analysis by PISA. (G)The location of the newly resolved FP fragment (in deep pink) within the S trimer (left) and one protomer. S1 and S2 subunit is colored steel blue and gold, respectively. (H) Model-map fitting for the newly resolved FP fragment. (I) Close up view of the interactions between D614 from SD2 and FP, with the hydrogen bonds labeled in dotted lines and the L828-F855 region in FP in deep pink.

    Techniques Used: Labeling

    Cryo-EM data processing procedure for SARS-CoV-2 S trimer.
    Figure Legend Snippet: Cryo-EM data processing procedure for SARS-CoV-2 S trimer.

    Techniques Used:

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

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

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20465-w

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

    Techniques Used: Labeling, Binding Assay

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

    Techniques Used: Binding Assay, Generated

    Binding properties, receptor-binding inhibitory activity, and neutralization activity of the MAbs. a Reactivities of anti-SARS-CoV-2 MAbs to the SARS-CoV-2 RBD measured by ELISA. Data are mean ± SEM of triplicate wells. Zika virus (ZIKV)-specific MAb 5F8 served as IgG1 isotype control (IgG-ctr) and was used as a control in all subsequent experiments. b Isotypes, binding affinities, and neutralization activity of the MAbs. Binding affinities of the MAbs to immobilized SARS-CoV-2 RBD and S trimer were determined by bio-layer interferometry (BLI). c Competition between the MAbs and ACE2 for binding to SARS-CoV-2 RBD was measured by ELISA. Biotinylated ACE2-hFc fusion protein was tested for the ability to bind to immobilized RBD in presence of the MAbs, and the signal was detected using HRP-conjugated streptavidin. Data are mean ± SEM of triplicate wells. d The MAbs neutralized SARS-CoV-2 pseudovirus infection in vitro. The purified MAbs were fourfold serially diluted and evaluated for neutralization of murine leukemia virus (MLV) pseudotyped with SARS-CoV-2 spike protein. Luciferase activity was measured 2 days after infection. Results shown are representative of two independent experiments. Data are expressed as mean ± SEM of five replicate wells. e The MAbs neutralized authentic SARS-CoV-2 infection in vitro. Serially diluted purified MAbs were subjected to live SARS-CoV-2 virus neutralization assay. After 48 h culture, viral RNA in cells were detected by RT-qPCR. Data are mean ± SEM of triplicate wells.
    Figure Legend Snippet: Binding properties, receptor-binding inhibitory activity, and neutralization activity of the MAbs. a Reactivities of anti-SARS-CoV-2 MAbs to the SARS-CoV-2 RBD measured by ELISA. Data are mean ± SEM of triplicate wells. Zika virus (ZIKV)-specific MAb 5F8 served as IgG1 isotype control (IgG-ctr) and was used as a control in all subsequent experiments. b Isotypes, binding affinities, and neutralization activity of the MAbs. Binding affinities of the MAbs to immobilized SARS-CoV-2 RBD and S trimer were determined by bio-layer interferometry (BLI). c Competition between the MAbs and ACE2 for binding to SARS-CoV-2 RBD was measured by ELISA. Biotinylated ACE2-hFc fusion protein was tested for the ability to bind to immobilized RBD in presence of the MAbs, and the signal was detected using HRP-conjugated streptavidin. Data are mean ± SEM of triplicate wells. d The MAbs neutralized SARS-CoV-2 pseudovirus infection in vitro. The purified MAbs were fourfold serially diluted and evaluated for neutralization of murine leukemia virus (MLV) pseudotyped with SARS-CoV-2 spike protein. Luciferase activity was measured 2 days after infection. Results shown are representative of two independent experiments. Data are expressed as mean ± SEM of five replicate wells. e The MAbs neutralized authentic SARS-CoV-2 infection in vitro. Serially diluted purified MAbs were subjected to live SARS-CoV-2 virus neutralization assay. After 48 h culture, viral RNA in cells were detected by RT-qPCR. Data are mean ± SEM of triplicate wells.

    Techniques Used: Binding Assay, Activity Assay, Neutralization, Enzyme-linked Immunosorbent Assay, Infection, In Vitro, Purification, Luciferase, Quantitative RT-PCR

    Antibody competition, epitope mapping, and generation of antibody cocktail. a , b Antibody binding competition assay. Antibody competition for binding to SARS-CoV-2 RBD was measured by BLI. Immobilized RBD was first saturated with the first antibody MAb 3C1 ( a ) or MAb 2H2 ( b ), and then a second MAb (MAb names were shown after the plus sign) or dissociation buffer (control) was added and allowed to react with the RBD. c Diagrams of chimeric RBD mutants (cRBD). cRBD (core), the N-terminal residues R319 to N437 of core region in the SARS-CoV-2 RBD were mutated into the corresponding part of SARS-CoV. cRBD (RBM-R2) and cRBD (RBM-R3), residues L452 to K462, and residues T470 to T478 of RBM region in the SARS-CoV-2 RBD were separately substituted by the corresponding residues of SARS-CoV. The positions of the mutated amino acids are shown in the wild-type RBD crystal structure (PDB: 6M0J; right panel). d Reactivities of the MAbs to wild-type (wt) and mutant SARS-CoV-2 RBD proteins measured by ELISA. RBD-mFc immune sera (anti-RBD) served as positive control. The downward arrow indicates that substitutions in RBD mutants significantly reduced the binding of the MAbs compared to wild-type RBD. The reactivity level of wild-type SARS-CoV-2 RBD and anti-RBD sera was set to 100%, and the red dashed line represents 50% reduction relative to wild type. Data are mean ± SEM of triplicate wells. Each symbol represents one well. e Grouping of the MAbs. Group 1, MAb16-3C1; group 2, the other MAbs. Antibody epitopes were shown in brackets. f Neutralization activity of the murine 2H2/3C1 cocktail. 2H2 alone, 3C1 alone, and the 2H2/3C1 (1:1) cocktail were serially diluted and evaluated for neutralization of SARS-CoV-2 pseudovirus. g Neutralization activity of the chimeric MAb cocktail against SARS-CoV-2 pseudovirus. c2H2 alone, c3C1 alone, and the c2H2/c3C1 (1:1) cocktail were serially diluted and assessed for neutralization of SARS-CoV-2 pseudovirus. For f and g luciferase activity was measured 2 days after infection. Data are expressed as mean ± SEM of five replicate wells. h Neutralization activity of the chimeric MAb cocktail against authentic SARS-CoV-2. Serially diluted purified MAbs were subjected to live SARS-CoV-2 virus neutralization assay. After 48 h culture, viral RNA in cells were detected by RT-qPCR. Data are mean ± SEM of triplicate wells. For f – h , for MAb cocktails the concentration on the x -axis is that of the 2H2 or c2H2 antibody.
    Figure Legend Snippet: Antibody competition, epitope mapping, and generation of antibody cocktail. a , b Antibody binding competition assay. Antibody competition for binding to SARS-CoV-2 RBD was measured by BLI. Immobilized RBD was first saturated with the first antibody MAb 3C1 ( a ) or MAb 2H2 ( b ), and then a second MAb (MAb names were shown after the plus sign) or dissociation buffer (control) was added and allowed to react with the RBD. c Diagrams of chimeric RBD mutants (cRBD). cRBD (core), the N-terminal residues R319 to N437 of core region in the SARS-CoV-2 RBD were mutated into the corresponding part of SARS-CoV. cRBD (RBM-R2) and cRBD (RBM-R3), residues L452 to K462, and residues T470 to T478 of RBM region in the SARS-CoV-2 RBD were separately substituted by the corresponding residues of SARS-CoV. The positions of the mutated amino acids are shown in the wild-type RBD crystal structure (PDB: 6M0J; right panel). d Reactivities of the MAbs to wild-type (wt) and mutant SARS-CoV-2 RBD proteins measured by ELISA. RBD-mFc immune sera (anti-RBD) served as positive control. The downward arrow indicates that substitutions in RBD mutants significantly reduced the binding of the MAbs compared to wild-type RBD. The reactivity level of wild-type SARS-CoV-2 RBD and anti-RBD sera was set to 100%, and the red dashed line represents 50% reduction relative to wild type. Data are mean ± SEM of triplicate wells. Each symbol represents one well. e Grouping of the MAbs. Group 1, MAb16-3C1; group 2, the other MAbs. Antibody epitopes were shown in brackets. f Neutralization activity of the murine 2H2/3C1 cocktail. 2H2 alone, 3C1 alone, and the 2H2/3C1 (1:1) cocktail were serially diluted and evaluated for neutralization of SARS-CoV-2 pseudovirus. g Neutralization activity of the chimeric MAb cocktail against SARS-CoV-2 pseudovirus. c2H2 alone, c3C1 alone, and the c2H2/c3C1 (1:1) cocktail were serially diluted and assessed for neutralization of SARS-CoV-2 pseudovirus. For f and g luciferase activity was measured 2 days after infection. Data are expressed as mean ± SEM of five replicate wells. h Neutralization activity of the chimeric MAb cocktail against authentic SARS-CoV-2. Serially diluted purified MAbs were subjected to live SARS-CoV-2 virus neutralization assay. After 48 h culture, viral RNA in cells were detected by RT-qPCR. Data are mean ± SEM of triplicate wells. For f – h , for MAb cocktails the concentration on the x -axis is that of the 2H2 or c2H2 antibody.

    Techniques Used: Binding Assay, Competitive Binding Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Positive Control, Neutralization, Activity Assay, Luciferase, Infection, Purification, Quantitative RT-PCR, Concentration Assay

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

    Techniques Used: Labeling, Binding Assay

    Protective efficacy of MAb 2H2 and the chimeric antibody cocktail against authentic SARS-CoV-2 infection in mice. a , b In vivo prophylactic efficacy ( a ) and therapeutic efficacy ( b ) of MAb 2H2, c2H2, and/or the c2H2/c3C1 cocktail against SARS-CoV-2 infection. Upper left panel: study outline. Upper right panel: qRT-PCR analysis of viral RNA copies present in lung tissues after 3 days of infection. Lower panel: H E staining of lung tissue sections at 3 d.p.i. For a , qPCR results are shown as fold increase relative to wide-type Balb/c group (without Ad5-hACE2 treatment). For b , qPCR results are expressed as viral RNA levels in different antibody treatment groups relative to that in the PBS control group. For top right panels in a and b , each symbol represents one mouse. Error bars represent SEM. Statistical significance was determined by a two-tailed Student’s t test and indicated as follows: ns not significant; * p
    Figure Legend Snippet: Protective efficacy of MAb 2H2 and the chimeric antibody cocktail against authentic SARS-CoV-2 infection in mice. a , b In vivo prophylactic efficacy ( a ) and therapeutic efficacy ( b ) of MAb 2H2, c2H2, and/or the c2H2/c3C1 cocktail against SARS-CoV-2 infection. Upper left panel: study outline. Upper right panel: qRT-PCR analysis of viral RNA copies present in lung tissues after 3 days of infection. Lower panel: H E staining of lung tissue sections at 3 d.p.i. For a , qPCR results are shown as fold increase relative to wide-type Balb/c group (without Ad5-hACE2 treatment). For b , qPCR results are expressed as viral RNA levels in different antibody treatment groups relative to that in the PBS control group. For top right panels in a and b , each symbol represents one mouse. Error bars represent SEM. Statistical significance was determined by a two-tailed Student’s t test and indicated as follows: ns not significant; * p

    Techniques Used: Infection, Mouse Assay, In Vivo, Quantitative RT-PCR, Staining, Real-time Polymerase Chain Reaction, Two Tailed Test

    26) Product Images from "Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein"

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.0c01394

    Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.
    Figure Legend Snippet: Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.

    Techniques Used: Selection

    Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.
    Figure Legend Snippet: Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.

    Techniques Used: Flow Cytometry, Binding Assay

    27) Product Images from "Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein"

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.0c01394

    Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.
    Figure Legend Snippet: Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.

    Techniques Used: Selection

    Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.
    Figure Legend Snippet: Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.

    Techniques Used: Flow Cytometry, Binding Assay

    28) Product Images from "Methylene Blue Inhibits In Vitro the SARS-CoV-2 Spike – ACE2 Protein-Protein Interaction – A Mechanism That Can Contribute to Its Antiviral Activity Against COVID-19"

    Article Title: Methylene Blue Inhibits In Vitro the SARS-CoV-2 Spike – ACE2 Protein-Protein Interaction – A Mechanism That Can Contribute to Its Antiviral Activity Against COVID-19

    Journal: bioRxiv

    doi: 10.1101/2020.08.29.273441

    Concentration-dependent inhibition of SARS-CoV-2 RBD binding to ACE2 by selected compounds. Concentration-response curves obtained in ELISA-type assay with Fc-conjugated ACE2 coated on the plate (1 μg/mL) and His-tagged RBD (0.5 μg/mL) added and amount bound in the presence of increasing concentrations of test compounds detected. As before, erythrosine B (ErB) and sunset yellow FCF (SY(FD C#6)) were included as positive and negative controls, respectively. Data (mean ± SD for two experiments in duplicates) were normalized and fitted with standard inhibition curves; obtained IC 50 values are shown at right.
    Figure Legend Snippet: Concentration-dependent inhibition of SARS-CoV-2 RBD binding to ACE2 by selected compounds. Concentration-response curves obtained in ELISA-type assay with Fc-conjugated ACE2 coated on the plate (1 μg/mL) and His-tagged RBD (0.5 μg/mL) added and amount bound in the presence of increasing concentrations of test compounds detected. As before, erythrosine B (ErB) and sunset yellow FCF (SY(FD C#6)) were included as positive and negative controls, respectively. Data (mean ± SD for two experiments in duplicates) were normalized and fitted with standard inhibition curves; obtained IC 50 values are shown at right.

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

    Concentration-response curves for binding of SARS-CoV-2 spike protein S1 and RBD to ACE2 in our ELISA-based assay format. Data obtained with Fc-conjugated ACE2 coated on the plate and His-tagged S1 or RBD added in increasing amounts as shown with the amount bound detected using an anti-His–HRP conjugate (mean ± SD for two experiments in duplicates).
    Figure Legend Snippet: Concentration-response curves for binding of SARS-CoV-2 spike protein S1 and RBD to ACE2 in our ELISA-based assay format. Data obtained with Fc-conjugated ACE2 coated on the plate and His-tagged S1 or RBD added in increasing amounts as shown with the amount bound detected using an anti-His–HRP conjugate (mean ± SD for two experiments in duplicates).

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

    Inhibitory effect of selected compounds on SARS-CoV-2 RBD binding to hACE2 in our screening assay. Percent inhibition values obtained at 5 μM concentration shown normalized to control (100%). Erythrosine B, a known promiscuous SMI of PPIs ( Ganesan et al., 2011 ) and sunset yellow FCF (FD C yellow no. 6), a food colorant likely to be inactive, were included as positive and negative controls, respectively. Chemical structures are shown for comparison purposes.
    Figure Legend Snippet: Inhibitory effect of selected compounds on SARS-CoV-2 RBD binding to hACE2 in our screening assay. Percent inhibition values obtained at 5 μM concentration shown normalized to control (100%). Erythrosine B, a known promiscuous SMI of PPIs ( Ganesan et al., 2011 ) and sunset yellow FCF (FD C yellow no. 6), a food colorant likely to be inactive, were included as positive and negative controls, respectively. Chemical structures are shown for comparison purposes.

    Techniques Used: Binding Assay, Screening Assay, Inhibition, Concentration Assay

    29) Product Images from "A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein"

    Article Title: A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20602-5

    The structure of CT-P59 Fab in complex with SARS-CoV-2 RBD. a The overall structure of the CT-P59 Fab/SARS-CoV-2 RBD complex. The RBD domain is green for the core subdomain, and orange for RBM. The heavy and light chains of CT-P59 are magenta and yellow, respectively. b Superposition of the neutralizing antibodies in complex with RBD. RBD is shown as a surface model. CT-P59 is shown as a cartoon, and the other antibodies (CR3022: PDB 6XC3, PB2-2F6: PDB 7BWJ, REGN10933: PDB 6XDG) are shown as a ribbon model. The heavy and light chains of Fab are magenta and yellow, respectively. c Assignment of the epitope residues for RBD-targeting neutralizing antibodies with a distance cutoff of 4.5 Å. RBD residues interacting with ACE2 are highlighted in red. d The detailed interactions between the RBD and CDR loops of CT-P59. The interfaces between RBD and CDR H1/2 or H3 are shown in the top and bottom panels, respectively. The RBD domain is shown as a surface model with a semi-transparent representation. The CDR loops and interacting residues on the interfaces are shown in ribbons and sticks, respectively. The residues are colored as in a . Dashed lines indicate hydrogen bonds. Water molecules are shown as red spheres.
    Figure Legend Snippet: The structure of CT-P59 Fab in complex with SARS-CoV-2 RBD. a The overall structure of the CT-P59 Fab/SARS-CoV-2 RBD complex. The RBD domain is green for the core subdomain, and orange for RBM. The heavy and light chains of CT-P59 are magenta and yellow, respectively. b Superposition of the neutralizing antibodies in complex with RBD. RBD is shown as a surface model. CT-P59 is shown as a cartoon, and the other antibodies (CR3022: PDB 6XC3, PB2-2F6: PDB 7BWJ, REGN10933: PDB 6XDG) are shown as a ribbon model. The heavy and light chains of Fab are magenta and yellow, respectively. c Assignment of the epitope residues for RBD-targeting neutralizing antibodies with a distance cutoff of 4.5 Å. RBD residues interacting with ACE2 are highlighted in red. d The detailed interactions between the RBD and CDR loops of CT-P59. The interfaces between RBD and CDR H1/2 or H3 are shown in the top and bottom panels, respectively. The RBD domain is shown as a surface model with a semi-transparent representation. The CDR loops and interacting residues on the interfaces are shown in ribbons and sticks, respectively. The residues are colored as in a . Dashed lines indicate hydrogen bonds. Water molecules are shown as red spheres.

    Techniques Used:

    Virus titration and quantitation in the lower respiratory tract of animal models. Three ferrets per group were euthanized at 3 and 7 dpi, and lungs were collected to measure viral titers ( a ) and the number of viral RNA copies ( d ). Golden Syrian hamsters ( n = 12/group) were challenged intranasally with 6.4 × 10 4 PFU/80 μL of SARS-CoV-2. Vehicle and 15, 30, 60, and 90 mg/kg of CT-P59 were intraperitoneally administered 24 h after virus inoculation. Four animals were euthanized for virus titration ( b ) and quantitation of viral RNA copies ( e ) from each group at 3 and 5 dpi. Rhesus monkeys (control n = 3, 45 mg/kg n = 2, 90 mg/kg n = 3) were infected with 10 6.4 TCID 50 /ml of SARS-CoV-2 via in a combination of intranasal (0.5 ml), intratracheal (4 ml), ocular (0.25 ml/eye), and oral (1 ml) routes. Vehicle, 45 mg/kg and 90 mg/kg of CT-P59 were administered intravenously after 24 h of virus infection. All rhesus monkeys were euthanized at 6 dpi, and lungs were collected to measure viral titers ( c ) and the number of viral RNA copies ( f ). Viral titers in the lung were determined by TCID 50 assessment in Vero cells and viral RNA copy number measurement using qRT-PCR. Viral titers and RNA copy numbers are shown as mean value + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0309, ** P = 0.0131, and *** P
    Figure Legend Snippet: Virus titration and quantitation in the lower respiratory tract of animal models. Three ferrets per group were euthanized at 3 and 7 dpi, and lungs were collected to measure viral titers ( a ) and the number of viral RNA copies ( d ). Golden Syrian hamsters ( n = 12/group) were challenged intranasally with 6.4 × 10 4 PFU/80 μL of SARS-CoV-2. Vehicle and 15, 30, 60, and 90 mg/kg of CT-P59 were intraperitoneally administered 24 h after virus inoculation. Four animals were euthanized for virus titration ( b ) and quantitation of viral RNA copies ( e ) from each group at 3 and 5 dpi. Rhesus monkeys (control n = 3, 45 mg/kg n = 2, 90 mg/kg n = 3) were infected with 10 6.4 TCID 50 /ml of SARS-CoV-2 via in a combination of intranasal (0.5 ml), intratracheal (4 ml), ocular (0.25 ml/eye), and oral (1 ml) routes. Vehicle, 45 mg/kg and 90 mg/kg of CT-P59 were administered intravenously after 24 h of virus infection. All rhesus monkeys were euthanized at 6 dpi, and lungs were collected to measure viral titers ( c ) and the number of viral RNA copies ( f ). Viral titers in the lung were determined by TCID 50 assessment in Vero cells and viral RNA copy number measurement using qRT-PCR. Viral titers and RNA copy numbers are shown as mean value + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0309, ** P = 0.0131, and *** P

    Techniques Used: Titration, Quantitation Assay, Infection, Quantitative RT-PCR

    In vivo efficacy of CT-P59 in the upper respiratory tract in an animal model. Female ferrets ( n = 6/group), and rhesus monkeys (three control; two 45 mg/kg; three 90 mg/kg) were challenged with 10 5.8 TCID 50 /ml and 10 6.4 TCID 50 of SARS-CoV-2, respectively. Control (ferrets: 30 mg/kg of human IgG isotype and rhesus monkeys: vehicle) and CT-P59 (ferrets: 3, and 30 mg/kg, rhesus monkeys: 45, and 90 mg/kg) were administered intravenously after 24 h of virus inoculation, respectively. To compare the efficacy of CT-P59, remdesivir (18 mg/kg per ferret) was administered daily via oral gavage after 24 h of virus inoculation in ferrets for 5 days. To detect viral load in the upper respiratory tract, nasal wash specimens were collected from ferrets at 2, 4, and 6 dpi, and nose and throat swab specimens from monkeys were collected daily up to 6 dpi. Virus titers (TCID 50 ) were measured in nasal wash/swab and throat swabs specimens from each group of ( a ) ferrets and, c , d rhesus monkeys. The viral RNA copy numbers were measured in nasal washes from ( b ) ferrets. Viral titers and RNA copy numbers are shown as mean values + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). The asterisks and daggers indicate significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0001 and ** P
    Figure Legend Snippet: In vivo efficacy of CT-P59 in the upper respiratory tract in an animal model. Female ferrets ( n = 6/group), and rhesus monkeys (three control; two 45 mg/kg; three 90 mg/kg) were challenged with 10 5.8 TCID 50 /ml and 10 6.4 TCID 50 of SARS-CoV-2, respectively. Control (ferrets: 30 mg/kg of human IgG isotype and rhesus monkeys: vehicle) and CT-P59 (ferrets: 3, and 30 mg/kg, rhesus monkeys: 45, and 90 mg/kg) were administered intravenously after 24 h of virus inoculation, respectively. To compare the efficacy of CT-P59, remdesivir (18 mg/kg per ferret) was administered daily via oral gavage after 24 h of virus inoculation in ferrets for 5 days. To detect viral load in the upper respiratory tract, nasal wash specimens were collected from ferrets at 2, 4, and 6 dpi, and nose and throat swab specimens from monkeys were collected daily up to 6 dpi. Virus titers (TCID 50 ) were measured in nasal wash/swab and throat swabs specimens from each group of ( a ) ferrets and, c , d rhesus monkeys. The viral RNA copy numbers were measured in nasal washes from ( b ) ferrets. Viral titers and RNA copy numbers are shown as mean values + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). The asterisks and daggers indicate significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0001 and ** P

    Techniques Used: In Vivo, Animal Model

    CT-P59 can effectively neutralize SARS-CoV-2 in vitro by blocking RBD-ACE2 binding. a Serial twofold-diluted CT-P59 were incubated with SARS-CoV-2 live viruses; wild type (blue) and D614G (red). The mixture was added to VeroE6 cells. After 2–3 days of incubation, the neutralization activity was evaluated by counting plaques. Two independent experiments were performed in duplicate. b SARS-CoV-2 RBD immobilized on biosensor was saturated with CT-P59. Then, CT-P59 flowed over the biosensor surface in the presence (red) or absence (blue) of the ACE2 receptor. As a positive control, the buffer was loaded onto SARS-CoV-2 RBD immobilized biosensor, and ACE2 flowed over the biosensor surface (black).
    Figure Legend Snippet: CT-P59 can effectively neutralize SARS-CoV-2 in vitro by blocking RBD-ACE2 binding. a Serial twofold-diluted CT-P59 were incubated with SARS-CoV-2 live viruses; wild type (blue) and D614G (red). The mixture was added to VeroE6 cells. After 2–3 days of incubation, the neutralization activity was evaluated by counting plaques. Two independent experiments were performed in duplicate. b SARS-CoV-2 RBD immobilized on biosensor was saturated with CT-P59. Then, CT-P59 flowed over the biosensor surface in the presence (red) or absence (blue) of the ACE2 receptor. As a positive control, the buffer was loaded onto SARS-CoV-2 RBD immobilized biosensor, and ACE2 flowed over the biosensor surface (black).

    Techniques Used: In Vitro, Blocking Assay, Binding Assay, Incubation, Neutralization, Activity Assay, Positive Control

    No evidence of ADE caused by CT-P59 bound to SARS-CoV-2 in permissive cells and Fc receptor (FcR)-bearing cells. a FcR-independent ADE. SARS-CoV-2 was mixed with a wide range of antibodies; CT-P59 (red circle), CR3022 (blue square), and CT-P27 (black triangle). VeroE6 was infected with a virus–antibody complex. The virus titers were determined by optical density (OD) using an anti-nucleocapsid antibody. b FcγR II-dependent ADE. In vitro ADE assay was performed as described in ( a ) except Raji cells. c FcγR I II-dependent ADE. In vitro ADE assay was carried out as described in ( a ), except U937 cells. The experiments were performed in triplicates (CR3022 and CT-P27) or in quadruplicates (CT-P59). Average and standard deviation of virus titers are depicted as dot and error bar, respectively.
    Figure Legend Snippet: No evidence of ADE caused by CT-P59 bound to SARS-CoV-2 in permissive cells and Fc receptor (FcR)-bearing cells. a FcR-independent ADE. SARS-CoV-2 was mixed with a wide range of antibodies; CT-P59 (red circle), CR3022 (blue square), and CT-P27 (black triangle). VeroE6 was infected with a virus–antibody complex. The virus titers were determined by optical density (OD) using an anti-nucleocapsid antibody. b FcγR II-dependent ADE. In vitro ADE assay was performed as described in ( a ) except Raji cells. c FcγR I II-dependent ADE. In vitro ADE assay was carried out as described in ( a ), except U937 cells. The experiments were performed in triplicates (CR3022 and CT-P27) or in quadruplicates (CT-P59). Average and standard deviation of virus titers are depicted as dot and error bar, respectively.

    Techniques Used: Infection, In Vitro, Standard Deviation

    30) Product Images from "Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals"

    Article Title: Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals

    Journal: Science Translational Medicine

    doi: 10.1126/scitranslmed.abd6990

    Characteristics of the isolated nAbs, stereotypic IGH clonotypes, and RBD binding–predicted clones. ( A ) Serially diluted IgG2/4 was mixed with an equal volume of SARS-CoV-2 containing 100 TCID 50 , and the IgG2/4-virus mixture was added to Vero cells with eight repeats and incubated for 5 days. Cells infected with 100 TCID 50 of SARS-CoV-2, isotype IgG2/4 control, or without the virus were applied as positive, negative, and uninfected controls, respectively. CPE in each well was observed 5 days after infection. ( B ) Characteristics of nAbs found in patients A and E. ( C ) IGH clonotypes that are highly homologous to E-3B1 and reactive against recombinant SARS-CoV-2 S and RBD proteins. The right column shows the results of the phage ELISA. All experiments were performed in quadruplicate, and the data are presented as the means ± SD. ( D ) List of diverse Ig light chain clonotypes that can be paired with the IGH clonotypes from (B) to achieve reactivity. ( E ) J and ( F ) VJ gene usage in the IGH repertoire of patients (top) and the binding-predicted IGH clones (bottom). For the VJ gene usage heatmap, the frequency values for the IGH repertoire of all 17 patients were averaged and are displayed (top) along with those of the predicted RBD-binding IGH clones (bottom). N/A, not applicable.
    Figure Legend Snippet: Characteristics of the isolated nAbs, stereotypic IGH clonotypes, and RBD binding–predicted clones. ( A ) Serially diluted IgG2/4 was mixed with an equal volume of SARS-CoV-2 containing 100 TCID 50 , and the IgG2/4-virus mixture was added to Vero cells with eight repeats and incubated for 5 days. Cells infected with 100 TCID 50 of SARS-CoV-2, isotype IgG2/4 control, or without the virus were applied as positive, negative, and uninfected controls, respectively. CPE in each well was observed 5 days after infection. ( B ) Characteristics of nAbs found in patients A and E. ( C ) IGH clonotypes that are highly homologous to E-3B1 and reactive against recombinant SARS-CoV-2 S and RBD proteins. The right column shows the results of the phage ELISA. All experiments were performed in quadruplicate, and the data are presented as the means ± SD. ( D ) List of diverse Ig light chain clonotypes that can be paired with the IGH clonotypes from (B) to achieve reactivity. ( E ) J and ( F ) VJ gene usage in the IGH repertoire of patients (top) and the binding-predicted IGH clones (bottom). For the VJ gene usage heatmap, the frequency values for the IGH repertoire of all 17 patients were averaged and are displayed (top) along with those of the predicted RBD-binding IGH clones (bottom). N/A, not applicable.

    Techniques Used: Isolation, Binding Assay, Clone Assay, Incubation, Infection, Recombinant, Enzyme-linked Immunosorbent Assay

    Titrations of serum IgG by ELISAs specific to SARS-CoV-2. Plasma samples from 17 patients with SARS-CoV-2 were diluted (1:100) and added to plates coated with recombinant SARS-CoV-2 ( A ) N, ( B ) S, ( C ) S1, ( D ) S2, which was fused to a polyhistidine (HIS) tag, or ( E ) RBD protein, which was fused to a human hCκ domain. The amount of bound IgG was determined using anti-human IgG (Fc-specific) antibody, and ABTS (2,2’-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) was used as the substrate. All experiments were performed in duplicate, and the data are presented as the means ± SD.
    Figure Legend Snippet: Titrations of serum IgG by ELISAs specific to SARS-CoV-2. Plasma samples from 17 patients with SARS-CoV-2 were diluted (1:100) and added to plates coated with recombinant SARS-CoV-2 ( A ) N, ( B ) S, ( C ) S1, ( D ) S2, which was fused to a polyhistidine (HIS) tag, or ( E ) RBD protein, which was fused to a human hCκ domain. The amount of bound IgG was determined using anti-human IgG (Fc-specific) antibody, and ABTS (2,2’-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) was used as the substrate. All experiments were performed in duplicate, and the data are presented as the means ± SD.

    Techniques Used: Recombinant

    Reactivity of nAbs against recombinant SARS-CoV-2 spike mutants. Recombinant wild-type or mutant (V341I, F342L, N354D, V367F, R408I, A435S, G476S, V483A, and D614G) SARS-CoV-2 S, S1, or RBD protein–coated microtiter plates were incubated with varying concentrations of ( A ) E-3B1-hFc, ( B ) A-1H4-hFc, ( C ) A-2F1-hFc, ( D ) A-2H4-hFc, ( E ) E-3G9-hFc, and ( F ) irrelevant scFv-hFc. HRP-conjugated anti-human IgG antibody was used as the probe, and ABTS was used as the substrate. All experiments were performed in triplicate, and data are presented as the means ± SD.
    Figure Legend Snippet: Reactivity of nAbs against recombinant SARS-CoV-2 spike mutants. Recombinant wild-type or mutant (V341I, F342L, N354D, V367F, R408I, A435S, G476S, V483A, and D614G) SARS-CoV-2 S, S1, or RBD protein–coated microtiter plates were incubated with varying concentrations of ( A ) E-3B1-hFc, ( B ) A-1H4-hFc, ( C ) A-2F1-hFc, ( D ) A-2H4-hFc, ( E ) E-3G9-hFc, and ( F ) irrelevant scFv-hFc. HRP-conjugated anti-human IgG antibody was used as the probe, and ABTS was used as the substrate. All experiments were performed in triplicate, and data are presented as the means ± SD.

    Techniques Used: Recombinant, Mutagenesis, Incubation

    31) Product Images from "Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries"

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    Journal: bioRxiv

    doi: 10.1101/2020.07.27.224089

    RU169 output clone diversity Using the SARS-CoV-2 RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.
    Figure Legend Snippet: RU169 output clone diversity Using the SARS-CoV-2 RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.

    Techniques Used: FACS, Selection, Produced, Clone Assay

    BLI kinetics of selected scFv clones from the RU169 RBD screen. scFv were cloned into an AviTag™ biotinylation vector, as described in the Materials and Methods, expressed and purified by Ni-NTA resin. scFv were loaded onto a streptavidin BLI sensor and the association/dissociation kinetics of binding to soluble SARS-CoV-2 S1 trimer (100 nM) were measured using BLI. The K D of the scFvs for the S1 target ranged from 1 nM to 400 nM.
    Figure Legend Snippet: BLI kinetics of selected scFv clones from the RU169 RBD screen. scFv were cloned into an AviTag™ biotinylation vector, as described in the Materials and Methods, expressed and purified by Ni-NTA resin. scFv were loaded onto a streptavidin BLI sensor and the association/dissociation kinetics of binding to soluble SARS-CoV-2 S1 trimer (100 nM) were measured using BLI. The K D of the scFvs for the S1 target ranged from 1 nM to 400 nM.

    Techniques Used: Clone Assay, Plasmid Preparation, Purification, Binding Assay

    Anti-RBD clones in IgG1 format form long-lived complexes with SARS-CoV-2 S1 trimer and potently inhibit the interaction with ACE2 in vitro . A. Dissociation kinetics of IgG1 anti-RBD clones from SARS-CoV-2 S1 trimer. Biotinylated SARS-CoV-2 S1 trimer was bound to a streptavidin BLI sensor. IgG1 anti-RBD clones were bound (100 nM) and the dissociation followed for 4 hours in PBS at 25°C. B. ACE2-S1 Dynabead assay with molar equivalents of mAb clones to S1 trimer.
    Figure Legend Snippet: Anti-RBD clones in IgG1 format form long-lived complexes with SARS-CoV-2 S1 trimer and potently inhibit the interaction with ACE2 in vitro . A. Dissociation kinetics of IgG1 anti-RBD clones from SARS-CoV-2 S1 trimer. Biotinylated SARS-CoV-2 S1 trimer was bound to a streptavidin BLI sensor. IgG1 anti-RBD clones were bound (100 nM) and the dissociation followed for 4 hours in PBS at 25°C. B. ACE2-S1 Dynabead assay with molar equivalents of mAb clones to S1 trimer.

    Techniques Used: Clone Assay, In Vitro

    FACS strategy of screen RU167 for scFv inhibiting the SARS-CoV-2 RBD/ACE2 interaction The FACS-based screening strategy for screen RU167 to isolate antibodies that bound SARS-CoV-2 RBD and specifically inhibited co-binding of RBD to the human ACE2 protein. The viral RBD and the ACE2 protein were labeled with different fluorophores (A). Binding to cells expressing scFv clones that bound RBD and blocking the ACE2-binding site (B) would be observed and gated positively for in the FACS plot for events which were RBD-dye HIGH and ACE2-dye LOW (C).
    Figure Legend Snippet: FACS strategy of screen RU167 for scFv inhibiting the SARS-CoV-2 RBD/ACE2 interaction The FACS-based screening strategy for screen RU167 to isolate antibodies that bound SARS-CoV-2 RBD and specifically inhibited co-binding of RBD to the human ACE2 protein. The viral RBD and the ACE2 protein were labeled with different fluorophores (A). Binding to cells expressing scFv clones that bound RBD and blocking the ACE2-binding site (B) would be observed and gated positively for in the FACS plot for events which were RBD-dye HIGH and ACE2-dye LOW (C).

    Techniques Used: FACS, Binding Assay, Labeling, Expressing, Clone Assay, Blocking Assay

    BLI kinetics of anti-RBD diabodies AviTag™ biotinylated SARS-CoV-2 S1 trimer was loaded onto a BLI sensor and the association/dissociation kinetics of binding to anti-RBD diabodies (100 nM) were measured using BLI. The K D s of the dbs to the S1 target ranged from 84 pM to 1 nM.
    Figure Legend Snippet: BLI kinetics of anti-RBD diabodies AviTag™ biotinylated SARS-CoV-2 S1 trimer was loaded onto a BLI sensor and the association/dissociation kinetics of binding to anti-RBD diabodies (100 nM) were measured using BLI. The K D s of the dbs to the S1 target ranged from 84 pM to 1 nM.

    Techniques Used: Binding Assay

    Cytometry plots of ACE2-S1 Dynabead assay of anti-RBD diabodies The degree of inhibition of the ACE2 and SARS-CoV-2 S1 trimer interaction by stoichiometric amounts of anti-RBD diabodies was determined using a Dynabead assay as described in the Materials and Methods. The degree of bead fluorescence was indicative of the amount of dye-labeled S1 trimer that was bound to ACE2. Inhibition of the interaction by anti-RBD diabodies resulted in a reduction in fluorescence. The first panel is the SSC/FSC indicating the P1 gating of beads. The second panel is the biotin-blocked control (no ACE2/S1 interaction) and the third panel is the no anti-RBD control (maximum ACE2/S1 interaction. Each subsequent row represents a db clone at 1:1, 5:1 and 10:1 stoichiometric ratios to the soluble SARS-CoV-2 S1 trimer. The data are summarized graphically in Figure 3 .
    Figure Legend Snippet: Cytometry plots of ACE2-S1 Dynabead assay of anti-RBD diabodies The degree of inhibition of the ACE2 and SARS-CoV-2 S1 trimer interaction by stoichiometric amounts of anti-RBD diabodies was determined using a Dynabead assay as described in the Materials and Methods. The degree of bead fluorescence was indicative of the amount of dye-labeled S1 trimer that was bound to ACE2. Inhibition of the interaction by anti-RBD diabodies resulted in a reduction in fluorescence. The first panel is the SSC/FSC indicating the P1 gating of beads. The second panel is the biotin-blocked control (no ACE2/S1 interaction) and the third panel is the no anti-RBD control (maximum ACE2/S1 interaction. Each subsequent row represents a db clone at 1:1, 5:1 and 10:1 stoichiometric ratios to the soluble SARS-CoV-2 S1 trimer. The data are summarized graphically in Figure 3 .

    Techniques Used: Cytometry, Inhibition, Fluorescence, Labeling

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.06.15.153064

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

    Techniques Used: Polymerase Chain Reaction

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

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

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

    Techniques Used: Enzyme-linked Immunosorbent Assay

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

    Techniques Used: Incubation

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

    Techniques Used: Titration, Concentration Assay, Standard Deviation

    33) Product Images from "Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM"

    Article Title: Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM

    Journal: bioRxiv

    doi: 10.1101/2020.06.30.177097

    ACE2 binding induced conformational dynamics of the SARS-CoV-2 S-ACE2 complex determined by multi-body refinement. (A) The contributions of all eigenvectors to the motion in the S-ACE2 complex, with eigenvectors 1-3 dominant the contributions. (B) Top view of the map showing the three swing motions of the first 3 eigenvectors, with S trimer following the color schema as in Fig. 2 , and the two extreme locations of ACE2 illustrated in deep pink and light blue densities. The swing angular range and direction are indicated in dark red arrow. (C) Histograms of the amplitudes along the first 3 eigenvectors. (D) Atomic models of S-ACE2 and S-closed, colored according to the B factor distribution (ranging from 100Å 2 [blue] to 130Å 2 [red]).
    Figure Legend Snippet: ACE2 binding induced conformational dynamics of the SARS-CoV-2 S-ACE2 complex determined by multi-body refinement. (A) The contributions of all eigenvectors to the motion in the S-ACE2 complex, with eigenvectors 1-3 dominant the contributions. (B) Top view of the map showing the three swing motions of the first 3 eigenvectors, with S trimer following the color schema as in Fig. 2 , and the two extreme locations of ACE2 illustrated in deep pink and light blue densities. The swing angular range and direction are indicated in dark red arrow. (C) Histograms of the amplitudes along the first 3 eigenvectors. (D) Atomic models of S-ACE2 and S-closed, colored according to the B factor distribution (ranging from 100Å 2 [blue] to 130Å 2 [red]).

    Techniques Used: Binding Assay

    Organization of the resolved N-Linked glycans of SARS-CoV-2 S trimer. (A) Schematic representation of SARS-CoV-2 S glycoprotein. The positions of N-linked glycosylation sequons are shown as branches. 18 N-linked glycans detected in our cryo-EM map of the S-closed state are shown in red, the remaining undetected ones in black. After ACE2 binding, the glycan density that appears weaker is indicated (*). (B) Surface representation of the glycosylated SARS-CoV-2 S trimer in the S-closed state with N-linked glycans shown in red. The location of glycan hole is indicated in black doted ellipsoid, with the locations of S1/S2 and FP, and glycan at N657 site near the glycan hole indicated. The newly captured glycans at N17 and N149 sites are indicated in the top view. (C) Surface representation of the glycosylated S-ACE2 complex with N-linked glycans in red. After ACE2 binding, the glycan density that appears weaker is indicated.
    Figure Legend Snippet: Organization of the resolved N-Linked glycans of SARS-CoV-2 S trimer. (A) Schematic representation of SARS-CoV-2 S glycoprotein. The positions of N-linked glycosylation sequons are shown as branches. 18 N-linked glycans detected in our cryo-EM map of the S-closed state are shown in red, the remaining undetected ones in black. After ACE2 binding, the glycan density that appears weaker is indicated (*). (B) Surface representation of the glycosylated SARS-CoV-2 S trimer in the S-closed state with N-linked glycans shown in red. The location of glycan hole is indicated in black doted ellipsoid, with the locations of S1/S2 and FP, and glycan at N657 site near the glycan hole indicated. The newly captured glycans at N17 and N149 sites are indicated in the top view. (C) Surface representation of the glycosylated S-ACE2 complex with N-linked glycans in red. After ACE2 binding, the glycan density that appears weaker is indicated.

    Techniques Used: Binding Assay

    Cryo-EM data processing procedure for SARS-CoV-2 S trimer in the presence of ACE2.
    Figure Legend Snippet: Cryo-EM data processing procedure for SARS-CoV-2 S trimer in the presence of ACE2.

    Techniques Used:

    A complete architecture of the SARS-CoV-2 S-ACE2 complex. (A-B) Cryo-EM map and pseudo atomic model of SARS-CoV-2 S-ACE2 complex. We named the RBD up protomer as protomer 1 (light green), and the other two RBD down ones as protomer 2 (royal blue) and protomer 3 (gold). ACE2 was colored in violet red. (C) Side and top views of the overlaid S-open (color) and S-closed (dark grey) structures, showing in the open process there is a 71.0° upwards/outwards rotation of RBD associated with a downwards shift of SD1 in protomer 1. (D) Rotations of NTD and CH from S-closed (grey) to S-open (in color) state, with the NTD also showing a downwards/outwards movement. (E) Side view of the overlaid S-ACE2 (violet red) and S-open (light green) protomer 1 structures, showing the angle between the long axis of RBD and the horizontal plane of S trimer reduces from the S-open to the S-ACE2 state. (F) Top and side views of the overlaid S-ACE2 (violet red) and S-open (color) RBD structures, showing the coordinated movements of RBDs. (G) Protomer interaction interface analysis of S-ACE2 by PISA. (H) Aromatic interactions between the core region of the up RBD-1 (green) and the RBM T470-F490 loop of the neighboring RBD-2 (blue). (I) Overlaid structures of S-ACE2 (grey) and S-closed (color, with the FP fragment in deep pink), indicating a downwards shift of SD1 and most of the FP is missing in S-ACE2 state. Close up view (right panel) of the potential clashes between the downwards shifted SD1 β34 and α8 helix of FP. (J) Population shift between the ACE2-unpresented and ACE2-presented S trimer samples.
    Figure Legend Snippet: A complete architecture of the SARS-CoV-2 S-ACE2 complex. (A-B) Cryo-EM map and pseudo atomic model of SARS-CoV-2 S-ACE2 complex. We named the RBD up protomer as protomer 1 (light green), and the other two RBD down ones as protomer 2 (royal blue) and protomer 3 (gold). ACE2 was colored in violet red. (C) Side and top views of the overlaid S-open (color) and S-closed (dark grey) structures, showing in the open process there is a 71.0° upwards/outwards rotation of RBD associated with a downwards shift of SD1 in protomer 1. (D) Rotations of NTD and CH from S-closed (grey) to S-open (in color) state, with the NTD also showing a downwards/outwards movement. (E) Side view of the overlaid S-ACE2 (violet red) and S-open (light green) protomer 1 structures, showing the angle between the long axis of RBD and the horizontal plane of S trimer reduces from the S-open to the S-ACE2 state. (F) Top and side views of the overlaid S-ACE2 (violet red) and S-open (color) RBD structures, showing the coordinated movements of RBDs. (G) Protomer interaction interface analysis of S-ACE2 by PISA. (H) Aromatic interactions between the core region of the up RBD-1 (green) and the RBM T470-F490 loop of the neighboring RBD-2 (blue). (I) Overlaid structures of S-ACE2 (grey) and S-closed (color, with the FP fragment in deep pink), indicating a downwards shift of SD1 and most of the FP is missing in S-ACE2 state. Close up view (right panel) of the potential clashes between the downwards shifted SD1 β34 and α8 helix of FP. (J) Population shift between the ACE2-unpresented and ACE2-presented S trimer samples.

    Techniques Used:

    Cryo-EM analysis of the SARS-CoV-2 S trimer in the tightly closed state. (A) Representative cryo-EM image of the SARS-CoV-2 S trimer. (B) Reference-free 2D class averages of the S trimer. (C) Euler angular distribution of 3D reconstructions before and after adding tilt data, with the dotted circle indicating the sparsely distributed tilted top views in the non-tilt data. (D-E) Local resolution evaluation (D) and resolution assessment of our S-closed cryo-EM map by Fourier shell correlation (FSC) at 0.143 criterion (E). (F) Close up view of the model-map fitting in S2 subunit. (G) Compared with the recent structure of SARS-CoV-2 S in closed state (gray, 6VXX), our S-closed structure (blue) showed a slight inward tilt leading the peripheral edge of NTD exhibiting a 12.4 Å inward movement (for the Cα of T124). (H) Top view of the overlaid structures between our SARS-CoV-2 S-closed structure and the SARS-CoV S-closed structure (PDB: 5XLR) and zoom in views of the overlaid structures in NTD, RBD, CH domains. (I) N-linked glycans resolved in our S-closed cryo-EM map, with the densities corresponding to glycans colored in red.
    Figure Legend Snippet: Cryo-EM analysis of the SARS-CoV-2 S trimer in the tightly closed state. (A) Representative cryo-EM image of the SARS-CoV-2 S trimer. (B) Reference-free 2D class averages of the S trimer. (C) Euler angular distribution of 3D reconstructions before and after adding tilt data, with the dotted circle indicating the sparsely distributed tilted top views in the non-tilt data. (D-E) Local resolution evaluation (D) and resolution assessment of our S-closed cryo-EM map by Fourier shell correlation (FSC) at 0.143 criterion (E). (F) Close up view of the model-map fitting in S2 subunit. (G) Compared with the recent structure of SARS-CoV-2 S in closed state (gray, 6VXX), our S-closed structure (blue) showed a slight inward tilt leading the peripheral edge of NTD exhibiting a 12.4 Å inward movement (for the Cα of T124). (H) Top view of the overlaid structures between our SARS-CoV-2 S-closed structure and the SARS-CoV S-closed structure (PDB: 5XLR) and zoom in views of the overlaid structures in NTD, RBD, CH domains. (I) N-linked glycans resolved in our S-closed cryo-EM map, with the densities corresponding to glycans colored in red.

    Techniques Used:

    Amino acid sequence alignment of SARS-CoV-2 S to SARS-CoV S. The secondary structure elements were defined based on an ESPript ( Robert and Gouet, 2014 ) algorithm and are labeled based on our SARS-CoV-2 S-closed structure. The RBD domain is labeled in green frames, and the subdomains of RBM are also labeled.
    Figure Legend Snippet: Amino acid sequence alignment of SARS-CoV-2 S to SARS-CoV S. The secondary structure elements were defined based on an ESPript ( Robert and Gouet, 2014 ) algorithm and are labeled based on our SARS-CoV-2 S-closed structure. The RBD domain is labeled in green frames, and the subdomains of RBM are also labeled.

    Techniques Used: Sequencing, Labeling

    The T470-T478 loop and residue Y505 within RBM play vital roles in the engagement of SARS-CoV-2 spike with receptor ACE2. (A) The overall view of ACE2 (violet red) bound protomer 1 (light green) from our S-ACE2 structure, and zoom in view of the interaction interface between ACE2 and RBD, with the key contacting elements T470-F490 loop and Q498-Y505 within RBM highlighted in black ellipsoid and blue ellipsoid, respectively. (B) Superposition of our SARS-CoV-2 S-ACE2 structure with the crystal structure of SARS-CoV RBD-ACE2 (PDB: 2AJF), suggesting the RBM T470-F490 loop has obvious conformational variations. (C) Binding activities of ACE2-hFc fusion protein to wild-type (wt) and mutant SARS-CoV-2 RBD proteins determined by ELISA. Different structural elements of RBD were colored in the left panel. Anti-RBD sera and a cross-reactive MAb 1A10 served as positive controls. Ctr, an irrelevant antibody. The black arrow indicates that mutations in the RBD (RBM-R3) mutant significantly reduced the binding of ACE2-hFc compared to wild-type RBD. (D) Binding of ACE2-hFc fusion protein to wt and single-point mutant forms of SARS-CoV-2 RBD protein measured by ELISA. RBD (Q498A), RBD (V503A), and RBD (Y505A), RBD residues Q498, V503, and Y505 were mutated to Ala, respectively. The downward arrow indicates that the mutation at Y505 completely abolished the binding of ACE2 to RBD protein.
    Figure Legend Snippet: The T470-T478 loop and residue Y505 within RBM play vital roles in the engagement of SARS-CoV-2 spike with receptor ACE2. (A) The overall view of ACE2 (violet red) bound protomer 1 (light green) from our S-ACE2 structure, and zoom in view of the interaction interface between ACE2 and RBD, with the key contacting elements T470-F490 loop and Q498-Y505 within RBM highlighted in black ellipsoid and blue ellipsoid, respectively. (B) Superposition of our SARS-CoV-2 S-ACE2 structure with the crystal structure of SARS-CoV RBD-ACE2 (PDB: 2AJF), suggesting the RBM T470-F490 loop has obvious conformational variations. (C) Binding activities of ACE2-hFc fusion protein to wild-type (wt) and mutant SARS-CoV-2 RBD proteins determined by ELISA. Different structural elements of RBD were colored in the left panel. Anti-RBD sera and a cross-reactive MAb 1A10 served as positive controls. Ctr, an irrelevant antibody. The black arrow indicates that mutations in the RBD (RBM-R3) mutant significantly reduced the binding of ACE2-hFc compared to wild-type RBD. (D) Binding of ACE2-hFc fusion protein to wt and single-point mutant forms of SARS-CoV-2 RBD protein measured by ELISA. RBD (Q498A), RBD (V503A), and RBD (Y505A), RBD residues Q498, V503, and Y505 were mutated to Ala, respectively. The downward arrow indicates that the mutation at Y505 completely abolished the binding of ACE2 to RBD protein.

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

    Cryo-EM analysis on the SARS-CoV-2 S-ACE2 complex. (A) Representative cryo-EM image of the SARS-CoV-2 S trimer in the presence of ACE2. (B) Reference-free 2D class averages of the sample. (C-D) Local resolution evaluation of the S-ACE2 map (C) and S-open map (D). (E) Resolution assessment of the cryo-EM reconstructions by Fourier shell correlation (FSC) at 0.143 criterion. (F) Unliganded S-open map obtained from this dataset. (G) Cryo-EM map of S-ACE2 complex without cross linker (left, colored), and its overlay with S-ACE2 map with cross linker (pink, low pass filtered to similar resolution, right panel), suggesting they are in similar conformation. (H) ACE2 binding induced motions of S-ACE2 without cross linker. Left, contributions of all eigenvectors to motions of S-ACE2; right three panels, top view of the map showing the three swing motions along the first 3 eigenvectors.
    Figure Legend Snippet: Cryo-EM analysis on the SARS-CoV-2 S-ACE2 complex. (A) Representative cryo-EM image of the SARS-CoV-2 S trimer in the presence of ACE2. (B) Reference-free 2D class averages of the sample. (C-D) Local resolution evaluation of the S-ACE2 map (C) and S-open map (D). (E) Resolution assessment of the cryo-EM reconstructions by Fourier shell correlation (FSC) at 0.143 criterion. (F) Unliganded S-open map obtained from this dataset. (G) Cryo-EM map of S-ACE2 complex without cross linker (left, colored), and its overlay with S-ACE2 map with cross linker (pink, low pass filtered to similar resolution, right panel), suggesting they are in similar conformation. (H) ACE2 binding induced motions of S-ACE2 without cross linker. Left, contributions of all eigenvectors to motions of S-ACE2; right three panels, top view of the map showing the three swing motions along the first 3 eigenvectors.

    Techniques Used: Binding Assay

    The proposed mechanism of ACE2 induced conformational transitions of SARS-CoV-2 S trimer. Conformational transitions from the ground prefusion closed state (with packed FP, in red) to the transiently open state (Step 1) with an untwisting motion of the S trimer (highlighted in dark grey arrow) associated with a downwards movement of S1 (red arrow), from the open state to the dynamic ACE2 engaged state (Step 2), and from the ACE2 engaged state all the way to the refolded postfusion state (Step 3). The continuous swing motions of the associated ACE2-RBD within the S trimer are indicated by red arrows. The SARS-CoV-2 S trimer associated with ACE2 dimer (in the third panel) was generated by aligning the ACE2 of our SARS-CoV-2 S-ACE2 complex structure with the available full length ACE2 dimer structure (PDB: 6M1D). The postfusion state was illustrated as a carton.
    Figure Legend Snippet: The proposed mechanism of ACE2 induced conformational transitions of SARS-CoV-2 S trimer. Conformational transitions from the ground prefusion closed state (with packed FP, in red) to the transiently open state (Step 1) with an untwisting motion of the S trimer (highlighted in dark grey arrow) associated with a downwards movement of S1 (red arrow), from the open state to the dynamic ACE2 engaged state (Step 2), and from the ACE2 engaged state all the way to the refolded postfusion state (Step 3). The continuous swing motions of the associated ACE2-RBD within the S trimer are indicated by red arrows. The SARS-CoV-2 S trimer associated with ACE2 dimer (in the third panel) was generated by aligning the ACE2 of our SARS-CoV-2 S-ACE2 complex structure with the available full length ACE2 dimer structure (PDB: 6M1D). The postfusion state was illustrated as a carton.

    Techniques Used: Generated

    Purification of SARS-CoV-2 S ectodomain, human ACE2 PD domain, and SARS-CoV-2 S-ACE2 complex. (A) Schematic diagram of SARS-CoV-2 S organization in this study. S1/S2 protease cleavage site (S1/S2), N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), and central helix (CH) are labeled. (B-C) SDS-PAGE analysis of the purified S protein (B) and ACE2 (C). (D) Size-exclusion chromatogram and SDS-PAGE analysis of the formed SARS-CoV-2 S-ACE2 complex.
    Figure Legend Snippet: Purification of SARS-CoV-2 S ectodomain, human ACE2 PD domain, and SARS-CoV-2 S-ACE2 complex. (A) Schematic diagram of SARS-CoV-2 S organization in this study. S1/S2 protease cleavage site (S1/S2), N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), and central helix (CH) are labeled. (B-C) SDS-PAGE analysis of the purified S protein (B) and ACE2 (C). (D) Size-exclusion chromatogram and SDS-PAGE analysis of the formed SARS-CoV-2 S-ACE2 complex.

    Techniques Used: Purification, Binding Assay, Labeling, SDS Page

    An uncharacterized tightly closed conformation of SARS-CoV-2 S trimer. (A-B) Cryo-EM map and pseudo atomic model of SARS-CoV-2 S trimer in a tightly closed state, with three protomers shown in different color. (C) Close up view of the model-map fitting in the NTD and RBD regions of S1 subunit, illustrating most of the NTD region was well resolved. (D) Overlaid structures of our S-closed structure (blue) with the recent structure of SARS-CoV-2 S in closed state (gray, 6VXX), illustrating the RBM S469-C488 loop was newly captured in our structure (highlighted in dotted ellipsoid). (E) Top view of the overlaid structures as in (D) (left panel) and zoom-in views of specific domains, showing there is a dramatic anti-clockwise rotation in S1 especially in NTD, and a slight clockwise rotation in the central CH, resulting in a twisted tightly closed conformation. (F) Protomer interaction interface analysis by PISA. (G)The location of the newly resolved FP fragment (in deep pink) within the S trimer (left) and one protomer. S1 and S2 subunit is colored steel blue and gold, respectively. (H) Model-map fitting for the newly resolved FP fragment. (I) Close up view of the interactions between D614 from SD2 and FP, with the hydrogen bonds labeled in dotted lines and the L828-F855 region in FP in deep pink.
    Figure Legend Snippet: An uncharacterized tightly closed conformation of SARS-CoV-2 S trimer. (A-B) Cryo-EM map and pseudo atomic model of SARS-CoV-2 S trimer in a tightly closed state, with three protomers shown in different color. (C) Close up view of the model-map fitting in the NTD and RBD regions of S1 subunit, illustrating most of the NTD region was well resolved. (D) Overlaid structures of our S-closed structure (blue) with the recent structure of SARS-CoV-2 S in closed state (gray, 6VXX), illustrating the RBM S469-C488 loop was newly captured in our structure (highlighted in dotted ellipsoid). (E) Top view of the overlaid structures as in (D) (left panel) and zoom-in views of specific domains, showing there is a dramatic anti-clockwise rotation in S1 especially in NTD, and a slight clockwise rotation in the central CH, resulting in a twisted tightly closed conformation. (F) Protomer interaction interface analysis by PISA. (G)The location of the newly resolved FP fragment (in deep pink) within the S trimer (left) and one protomer. S1 and S2 subunit is colored steel blue and gold, respectively. (H) Model-map fitting for the newly resolved FP fragment. (I) Close up view of the interactions between D614 from SD2 and FP, with the hydrogen bonds labeled in dotted lines and the L828-F855 region in FP in deep pink.

    Techniques Used: Labeling

    Cryo-EM data processing procedure for SARS-CoV-2 S trimer.
    Figure Legend Snippet: Cryo-EM data processing procedure for SARS-CoV-2 S trimer.

    Techniques Used:

    34) Product Images from "Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein"

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.0c01394

    Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.
    Figure Legend Snippet: Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.

    Techniques Used: Selection

    Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.
    Figure Legend Snippet: Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.

    Techniques Used: Flow Cytometry, Binding Assay

    35) Product Images from "A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein"

    Article Title: A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20602-5

    The structure of CT-P59 Fab in complex with SARS-CoV-2 RBD. a The overall structure of the CT-P59 Fab/SARS-CoV-2 RBD complex. The RBD domain is green for the core subdomain, and orange for RBM. The heavy and light chains of CT-P59 are magenta and yellow, respectively. b Superposition of the neutralizing antibodies in complex with RBD. RBD is shown as a surface model. CT-P59 is shown as a cartoon, and the other antibodies (CR3022: PDB 6XC3, PB2-2F6: PDB 7BWJ, REGN10933: PDB 6XDG) are shown as a ribbon model. The heavy and light chains of Fab are magenta and yellow, respectively. c Assignment of the epitope residues for RBD-targeting neutralizing antibodies with a distance cutoff of 4.5 Å. RBD residues interacting with ACE2 are highlighted in red. d The detailed interactions between the RBD and CDR loops of CT-P59. The interfaces between RBD and CDR H1/2 or H3 are shown in the top and bottom panels, respectively. The RBD domain is shown as a surface model with a semi-transparent representation. The CDR loops and interacting residues on the interfaces are shown in ribbons and sticks, respectively. The residues are colored as in a . Dashed lines indicate hydrogen bonds. Water molecules are shown as red spheres.
    Figure Legend Snippet: The structure of CT-P59 Fab in complex with SARS-CoV-2 RBD. a The overall structure of the CT-P59 Fab/SARS-CoV-2 RBD complex. The RBD domain is green for the core subdomain, and orange for RBM. The heavy and light chains of CT-P59 are magenta and yellow, respectively. b Superposition of the neutralizing antibodies in complex with RBD. RBD is shown as a surface model. CT-P59 is shown as a cartoon, and the other antibodies (CR3022: PDB 6XC3, PB2-2F6: PDB 7BWJ, REGN10933: PDB 6XDG) are shown as a ribbon model. The heavy and light chains of Fab are magenta and yellow, respectively. c Assignment of the epitope residues for RBD-targeting neutralizing antibodies with a distance cutoff of 4.5 Å. RBD residues interacting with ACE2 are highlighted in red. d The detailed interactions between the RBD and CDR loops of CT-P59. The interfaces between RBD and CDR H1/2 or H3 are shown in the top and bottom panels, respectively. The RBD domain is shown as a surface model with a semi-transparent representation. The CDR loops and interacting residues on the interfaces are shown in ribbons and sticks, respectively. The residues are colored as in a . Dashed lines indicate hydrogen bonds. Water molecules are shown as red spheres.

    Techniques Used:

    Virus titration and quantitation in the lower respiratory tract of animal models. Three ferrets per group were euthanized at 3 and 7 dpi, and lungs were collected to measure viral titers ( a ) and the number of viral RNA copies ( d ). Golden Syrian hamsters ( n = 12/group) were challenged intranasally with 6.4 × 10 4 PFU/80 μL of SARS-CoV-2. Vehicle and 15, 30, 60, and 90 mg/kg of CT-P59 were intraperitoneally administered 24 h after virus inoculation. Four animals were euthanized for virus titration ( b ) and quantitation of viral RNA copies ( e ) from each group at 3 and 5 dpi. Rhesus monkeys (control n = 3, 45 mg/kg n = 2, 90 mg/kg n = 3) were infected with 10 6.4 TCID 50 /ml of SARS-CoV-2 via in a combination of intranasal (0.5 ml), intratracheal (4 ml), ocular (0.25 ml/eye), and oral (1 ml) routes. Vehicle, 45 mg/kg and 90 mg/kg of CT-P59 were administered intravenously after 24 h of virus infection. All rhesus monkeys were euthanized at 6 dpi, and lungs were collected to measure viral titers ( c ) and the number of viral RNA copies ( f ). Viral titers in the lung were determined by TCID 50 assessment in Vero cells and viral RNA copy number measurement using qRT-PCR. Viral titers and RNA copy numbers are shown as mean value + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0309, ** P = 0.0131, and *** P
    Figure Legend Snippet: Virus titration and quantitation in the lower respiratory tract of animal models. Three ferrets per group were euthanized at 3 and 7 dpi, and lungs were collected to measure viral titers ( a ) and the number of viral RNA copies ( d ). Golden Syrian hamsters ( n = 12/group) were challenged intranasally with 6.4 × 10 4 PFU/80 μL of SARS-CoV-2. Vehicle and 15, 30, 60, and 90 mg/kg of CT-P59 were intraperitoneally administered 24 h after virus inoculation. Four animals were euthanized for virus titration ( b ) and quantitation of viral RNA copies ( e ) from each group at 3 and 5 dpi. Rhesus monkeys (control n = 3, 45 mg/kg n = 2, 90 mg/kg n = 3) were infected with 10 6.4 TCID 50 /ml of SARS-CoV-2 via in a combination of intranasal (0.5 ml), intratracheal (4 ml), ocular (0.25 ml/eye), and oral (1 ml) routes. Vehicle, 45 mg/kg and 90 mg/kg of CT-P59 were administered intravenously after 24 h of virus infection. All rhesus monkeys were euthanized at 6 dpi, and lungs were collected to measure viral titers ( c ) and the number of viral RNA copies ( f ). Viral titers in the lung were determined by TCID 50 assessment in Vero cells and viral RNA copy number measurement using qRT-PCR. Viral titers and RNA copy numbers are shown as mean value + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0309, ** P = 0.0131, and *** P

    Techniques Used: Titration, Quantitation Assay, Infection, Quantitative RT-PCR

    In vivo efficacy of CT-P59 in the upper respiratory tract in an animal model. Female ferrets ( n = 6/group), and rhesus monkeys (three control; two 45 mg/kg; three 90 mg/kg) were challenged with 10 5.8 TCID 50 /ml and 10 6.4 TCID 50 of SARS-CoV-2, respectively. Control (ferrets: 30 mg/kg of human IgG isotype and rhesus monkeys: vehicle) and CT-P59 (ferrets: 3, and 30 mg/kg, rhesus monkeys: 45, and 90 mg/kg) were administered intravenously after 24 h of virus inoculation, respectively. To compare the efficacy of CT-P59, remdesivir (18 mg/kg per ferret) was administered daily via oral gavage after 24 h of virus inoculation in ferrets for 5 days. To detect viral load in the upper respiratory tract, nasal wash specimens were collected from ferrets at 2, 4, and 6 dpi, and nose and throat swab specimens from monkeys were collected daily up to 6 dpi. Virus titers (TCID 50 ) were measured in nasal wash/swab and throat swabs specimens from each group of ( a ) ferrets and, c , d rhesus monkeys. The viral RNA copy numbers were measured in nasal washes from ( b ) ferrets. Viral titers and RNA copy numbers are shown as mean values + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). The asterisks and daggers indicate significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0001 and ** P
    Figure Legend Snippet: In vivo efficacy of CT-P59 in the upper respiratory tract in an animal model. Female ferrets ( n = 6/group), and rhesus monkeys (three control; two 45 mg/kg; three 90 mg/kg) were challenged with 10 5.8 TCID 50 /ml and 10 6.4 TCID 50 of SARS-CoV-2, respectively. Control (ferrets: 30 mg/kg of human IgG isotype and rhesus monkeys: vehicle) and CT-P59 (ferrets: 3, and 30 mg/kg, rhesus monkeys: 45, and 90 mg/kg) were administered intravenously after 24 h of virus inoculation, respectively. To compare the efficacy of CT-P59, remdesivir (18 mg/kg per ferret) was administered daily via oral gavage after 24 h of virus inoculation in ferrets for 5 days. To detect viral load in the upper respiratory tract, nasal wash specimens were collected from ferrets at 2, 4, and 6 dpi, and nose and throat swab specimens from monkeys were collected daily up to 6 dpi. Virus titers (TCID 50 ) were measured in nasal wash/swab and throat swabs specimens from each group of ( a ) ferrets and, c , d rhesus monkeys. The viral RNA copy numbers were measured in nasal washes from ( b ) ferrets. Viral titers and RNA copy numbers are shown as mean values + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). The asterisks and daggers indicate significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0001 and ** P

    Techniques Used: In Vivo, Animal Model

    CT-P59 can effectively neutralize SARS-CoV-2 in vitro by blocking RBD-ACE2 binding. a Serial twofold-diluted CT-P59 were incubated with SARS-CoV-2 live viruses; wild type (blue) and D614G (red). The mixture was added to VeroE6 cells. After 2–3 days of incubation, the neutralization activity was evaluated by counting plaques. Two independent experiments were performed in duplicate. b SARS-CoV-2 RBD immobilized on biosensor was saturated with CT-P59. Then, CT-P59 flowed over the biosensor surface in the presence (red) or absence (blue) of the ACE2 receptor. As a positive control, the buffer was loaded onto SARS-CoV-2 RBD immobilized biosensor, and ACE2 flowed over the biosensor surface (black).
    Figure Legend Snippet: CT-P59 can effectively neutralize SARS-CoV-2 in vitro by blocking RBD-ACE2 binding. a Serial twofold-diluted CT-P59 were incubated with SARS-CoV-2 live viruses; wild type (blue) and D614G (red). The mixture was added to VeroE6 cells. After 2–3 days of incubation, the neutralization activity was evaluated by counting plaques. Two independent experiments were performed in duplicate. b SARS-CoV-2 RBD immobilized on biosensor was saturated with CT-P59. Then, CT-P59 flowed over the biosensor surface in the presence (red) or absence (blue) of the ACE2 receptor. As a positive control, the buffer was loaded onto SARS-CoV-2 RBD immobilized biosensor, and ACE2 flowed over the biosensor surface (black).

    Techniques Used: In Vitro, Blocking Assay, Binding Assay, Incubation, Neutralization, Activity Assay, Positive Control

    No evidence of ADE caused by CT-P59 bound to SARS-CoV-2 in permissive cells and Fc receptor (FcR)-bearing cells. a FcR-independent ADE. SARS-CoV-2 was mixed with a wide range of antibodies; CT-P59 (red circle), CR3022 (blue square), and CT-P27 (black triangle). VeroE6 was infected with a virus–antibody complex. The virus titers were determined by optical density (OD) using an anti-nucleocapsid antibody. b FcγR II-dependent ADE. In vitro ADE assay was performed as described in ( a ) except Raji cells. c FcγR I II-dependent ADE. In vitro ADE assay was carried out as described in ( a ), except U937 cells. The experiments were performed in triplicates (CR3022 and CT-P27) or in quadruplicates (CT-P59). Average and standard deviation of virus titers are depicted as dot and error bar, respectively.
    Figure Legend Snippet: No evidence of ADE caused by CT-P59 bound to SARS-CoV-2 in permissive cells and Fc receptor (FcR)-bearing cells. a FcR-independent ADE. SARS-CoV-2 was mixed with a wide range of antibodies; CT-P59 (red circle), CR3022 (blue square), and CT-P27 (black triangle). VeroE6 was infected with a virus–antibody complex. The virus titers were determined by optical density (OD) using an anti-nucleocapsid antibody. b FcγR II-dependent ADE. In vitro ADE assay was performed as described in ( a ) except Raji cells. c FcγR I II-dependent ADE. In vitro ADE assay was carried out as described in ( a ), except U937 cells. The experiments were performed in triplicates (CR3022 and CT-P27) or in quadruplicates (CT-P59). Average and standard deviation of virus titers are depicted as dot and error bar, respectively.

    Techniques Used: Infection, In Vitro, Standard Deviation

    36) 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

    37) Product Images from "A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein"

    Article Title: A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20602-5

    The structure of CT-P59 Fab in complex with SARS-CoV-2 RBD. a The overall structure of the CT-P59 Fab/SARS-CoV-2 RBD complex. The RBD domain is green for the core subdomain, and orange for RBM. The heavy and light chains of CT-P59 are magenta and yellow, respectively. b Superposition of the neutralizing antibodies in complex with RBD. RBD is shown as a surface model. CT-P59 is shown as a cartoon, and the other antibodies (CR3022: PDB 6XC3, PB2-2F6: PDB 7BWJ, REGN10933: PDB 6XDG) are shown as a ribbon model. The heavy and light chains of Fab are magenta and yellow, respectively. c Assignment of the epitope residues for RBD-targeting neutralizing antibodies with a distance cutoff of 4.5 Å. RBD residues interacting with ACE2 are highlighted in red. d The detailed interactions between the RBD and CDR loops of CT-P59. The interfaces between RBD and CDR H1/2 or H3 are shown in the top and bottom panels, respectively. The RBD domain is shown as a surface model with a semi-transparent representation. The CDR loops and interacting residues on the interfaces are shown in ribbons and sticks, respectively. The residues are colored as in a . Dashed lines indicate hydrogen bonds. Water molecules are shown as red spheres.
    Figure Legend Snippet: The structure of CT-P59 Fab in complex with SARS-CoV-2 RBD. a The overall structure of the CT-P59 Fab/SARS-CoV-2 RBD complex. The RBD domain is green for the core subdomain, and orange for RBM. The heavy and light chains of CT-P59 are magenta and yellow, respectively. b Superposition of the neutralizing antibodies in complex with RBD. RBD is shown as a surface model. CT-P59 is shown as a cartoon, and the other antibodies (CR3022: PDB 6XC3, PB2-2F6: PDB 7BWJ, REGN10933: PDB 6XDG) are shown as a ribbon model. The heavy and light chains of Fab are magenta and yellow, respectively. c Assignment of the epitope residues for RBD-targeting neutralizing antibodies with a distance cutoff of 4.5 Å. RBD residues interacting with ACE2 are highlighted in red. d The detailed interactions between the RBD and CDR loops of CT-P59. The interfaces between RBD and CDR H1/2 or H3 are shown in the top and bottom panels, respectively. The RBD domain is shown as a surface model with a semi-transparent representation. The CDR loops and interacting residues on the interfaces are shown in ribbons and sticks, respectively. The residues are colored as in a . Dashed lines indicate hydrogen bonds. Water molecules are shown as red spheres.

    Techniques Used:

    Virus titration and quantitation in the lower respiratory tract of animal models. Three ferrets per group were euthanized at 3 and 7 dpi, and lungs were collected to measure viral titers ( a ) and the number of viral RNA copies ( d ). Golden Syrian hamsters ( n = 12/group) were challenged intranasally with 6.4 × 10 4 PFU/80 μL of SARS-CoV-2. Vehicle and 15, 30, 60, and 90 mg/kg of CT-P59 were intraperitoneally administered 24 h after virus inoculation. Four animals were euthanized for virus titration ( b ) and quantitation of viral RNA copies ( e ) from each group at 3 and 5 dpi. Rhesus monkeys (control n = 3, 45 mg/kg n = 2, 90 mg/kg n = 3) were infected with 10 6.4 TCID 50 /ml of SARS-CoV-2 via in a combination of intranasal (0.5 ml), intratracheal (4 ml), ocular (0.25 ml/eye), and oral (1 ml) routes. Vehicle, 45 mg/kg and 90 mg/kg of CT-P59 were administered intravenously after 24 h of virus infection. All rhesus monkeys were euthanized at 6 dpi, and lungs were collected to measure viral titers ( c ) and the number of viral RNA copies ( f ). Viral titers in the lung were determined by TCID 50 assessment in Vero cells and viral RNA copy number measurement using qRT-PCR. Viral titers and RNA copy numbers are shown as mean value + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0309, ** P = 0.0131, and *** P
    Figure Legend Snippet: Virus titration and quantitation in the lower respiratory tract of animal models. Three ferrets per group were euthanized at 3 and 7 dpi, and lungs were collected to measure viral titers ( a ) and the number of viral RNA copies ( d ). Golden Syrian hamsters ( n = 12/group) were challenged intranasally with 6.4 × 10 4 PFU/80 μL of SARS-CoV-2. Vehicle and 15, 30, 60, and 90 mg/kg of CT-P59 were intraperitoneally administered 24 h after virus inoculation. Four animals were euthanized for virus titration ( b ) and quantitation of viral RNA copies ( e ) from each group at 3 and 5 dpi. Rhesus monkeys (control n = 3, 45 mg/kg n = 2, 90 mg/kg n = 3) were infected with 10 6.4 TCID 50 /ml of SARS-CoV-2 via in a combination of intranasal (0.5 ml), intratracheal (4 ml), ocular (0.25 ml/eye), and oral (1 ml) routes. Vehicle, 45 mg/kg and 90 mg/kg of CT-P59 were administered intravenously after 24 h of virus infection. All rhesus monkeys were euthanized at 6 dpi, and lungs were collected to measure viral titers ( c ) and the number of viral RNA copies ( f ). Viral titers in the lung were determined by TCID 50 assessment in Vero cells and viral RNA copy number measurement using qRT-PCR. Viral titers and RNA copy numbers are shown as mean value + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0309, ** P = 0.0131, and *** P

    Techniques Used: Titration, Quantitation Assay, Infection, Quantitative RT-PCR

    In vivo efficacy of CT-P59 in the upper respiratory tract in an animal model. Female ferrets ( n = 6/group), and rhesus monkeys (three control; two 45 mg/kg; three 90 mg/kg) were challenged with 10 5.8 TCID 50 /ml and 10 6.4 TCID 50 of SARS-CoV-2, respectively. Control (ferrets: 30 mg/kg of human IgG isotype and rhesus monkeys: vehicle) and CT-P59 (ferrets: 3, and 30 mg/kg, rhesus monkeys: 45, and 90 mg/kg) were administered intravenously after 24 h of virus inoculation, respectively. To compare the efficacy of CT-P59, remdesivir (18 mg/kg per ferret) was administered daily via oral gavage after 24 h of virus inoculation in ferrets for 5 days. To detect viral load in the upper respiratory tract, nasal wash specimens were collected from ferrets at 2, 4, and 6 dpi, and nose and throat swab specimens from monkeys were collected daily up to 6 dpi. Virus titers (TCID 50 ) were measured in nasal wash/swab and throat swabs specimens from each group of ( a ) ferrets and, c , d rhesus monkeys. The viral RNA copy numbers were measured in nasal washes from ( b ) ferrets. Viral titers and RNA copy numbers are shown as mean values + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). The asterisks and daggers indicate significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0001 and ** P
    Figure Legend Snippet: In vivo efficacy of CT-P59 in the upper respiratory tract in an animal model. Female ferrets ( n = 6/group), and rhesus monkeys (three control; two 45 mg/kg; three 90 mg/kg) were challenged with 10 5.8 TCID 50 /ml and 10 6.4 TCID 50 of SARS-CoV-2, respectively. Control (ferrets: 30 mg/kg of human IgG isotype and rhesus monkeys: vehicle) and CT-P59 (ferrets: 3, and 30 mg/kg, rhesus monkeys: 45, and 90 mg/kg) were administered intravenously after 24 h of virus inoculation, respectively. To compare the efficacy of CT-P59, remdesivir (18 mg/kg per ferret) was administered daily via oral gavage after 24 h of virus inoculation in ferrets for 5 days. To detect viral load in the upper respiratory tract, nasal wash specimens were collected from ferrets at 2, 4, and 6 dpi, and nose and throat swab specimens from monkeys were collected daily up to 6 dpi. Virus titers (TCID 50 ) were measured in nasal wash/swab and throat swabs specimens from each group of ( a ) ferrets and, c , d rhesus monkeys. The viral RNA copy numbers were measured in nasal washes from ( b ) ferrets. Viral titers and RNA copy numbers are shown as mean values + /− SEM and titers below the limit of detection are shown as 0.8 log 10 TCID 50 /ml or 0.3 log 10 viral RNA copies/ml (dashed lines). The asterisks and daggers indicate significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test. * P = 0.0001 and ** P

    Techniques Used: In Vivo, Animal Model

    CT-P59 can effectively neutralize SARS-CoV-2 in vitro by blocking RBD-ACE2 binding. a Serial twofold-diluted CT-P59 were incubated with SARS-CoV-2 live viruses; wild type (blue) and D614G (red). The mixture was added to VeroE6 cells. After 2–3 days of incubation, the neutralization activity was evaluated by counting plaques. Two independent experiments were performed in duplicate. b SARS-CoV-2 RBD immobilized on biosensor was saturated with CT-P59. Then, CT-P59 flowed over the biosensor surface in the presence (red) or absence (blue) of the ACE2 receptor. As a positive control, the buffer was loaded onto SARS-CoV-2 RBD immobilized biosensor, and ACE2 flowed over the biosensor surface (black).
    Figure Legend Snippet: CT-P59 can effectively neutralize SARS-CoV-2 in vitro by blocking RBD-ACE2 binding. a Serial twofold-diluted CT-P59 were incubated with SARS-CoV-2 live viruses; wild type (blue) and D614G (red). The mixture was added to VeroE6 cells. After 2–3 days of incubation, the neutralization activity was evaluated by counting plaques. Two independent experiments were performed in duplicate. b SARS-CoV-2 RBD immobilized on biosensor was saturated with CT-P59. Then, CT-P59 flowed over the biosensor surface in the presence (red) or absence (blue) of the ACE2 receptor. As a positive control, the buffer was loaded onto SARS-CoV-2 RBD immobilized biosensor, and ACE2 flowed over the biosensor surface (black).

    Techniques Used: In Vitro, Blocking Assay, Binding Assay, Incubation, Neutralization, Activity Assay, Positive Control

    No evidence of ADE caused by CT-P59 bound to SARS-CoV-2 in permissive cells and Fc receptor (FcR)-bearing cells. a FcR-independent ADE. SARS-CoV-2 was mixed with a wide range of antibodies; CT-P59 (red circle), CR3022 (blue square), and CT-P27 (black triangle). VeroE6 was infected with a virus–antibody complex. The virus titers were determined by optical density (OD) using an anti-nucleocapsid antibody. b FcγR II-dependent ADE. In vitro ADE assay was performed as described in ( a ) except Raji cells. c FcγR I II-dependent ADE. In vitro ADE assay was carried out as described in ( a ), except U937 cells. The experiments were performed in triplicates (CR3022 and CT-P27) or in quadruplicates (CT-P59). Average and standard deviation of virus titers are depicted as dot and error bar, respectively.
    Figure Legend Snippet: No evidence of ADE caused by CT-P59 bound to SARS-CoV-2 in permissive cells and Fc receptor (FcR)-bearing cells. a FcR-independent ADE. SARS-CoV-2 was mixed with a wide range of antibodies; CT-P59 (red circle), CR3022 (blue square), and CT-P27 (black triangle). VeroE6 was infected with a virus–antibody complex. The virus titers were determined by optical density (OD) using an anti-nucleocapsid antibody. b FcγR II-dependent ADE. In vitro ADE assay was performed as described in ( a ) except Raji cells. c FcγR I II-dependent ADE. In vitro ADE assay was carried out as described in ( a ), except U937 cells. The experiments were performed in triplicates (CR3022 and CT-P27) or in quadruplicates (CT-P59). Average and standard deviation of virus titers are depicted as dot and error bar, respectively.

    Techniques Used: Infection, In Vitro, Standard Deviation

    Related Articles

    Binding Assay:

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein
    Article Snippet: Flow cytometry results indicated that all the mutants showed weaker binding performance against RBD-Ni-beads than original aptamers ( C,G). .. This result indicated that MDS helps us to understand binding patterns at the molecular level of the two aptamers targeting RBD and suggests two aptamers may have partially identical binding sites, like ACE2 on SARS-CoV-2 RBD. .. Further study of the binding surface/sites between RBD and aptamers can help improve the relationship and enhance competitiveness with ACE2.

    Article Title: A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein
    Article Snippet: Following incubation and washing, SARS-CoV-2 RBD-bound phages were eluted and used to infect fresh ER2738 cells. .. After several rounds of biopanning, scFv phages binding to SARS-CoV-2 RBD were identified by phage ELISA for further selection. .. Preparation of scFv-Fc, full-length IgG, and S proteins Each scFv identified by phage ELISA was cloned into the Fc fusion vector and transiently expressed in Chinese hamster ovary (CHO) cells.

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

    Enzyme-linked Immunosorbent Assay:

    Article Title: A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein
    Article Snippet: Following incubation and washing, SARS-CoV-2 RBD-bound phages were eluted and used to infect fresh ER2738 cells. .. After several rounds of biopanning, scFv phages binding to SARS-CoV-2 RBD were identified by phage ELISA for further selection. .. Preparation of scFv-Fc, full-length IgG, and S proteins Each scFv identified by phage ELISA was cloned into the Fc fusion vector and transiently expressed in Chinese hamster ovary (CHO) cells.

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

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering
    Article Snippet: .. ELISA determination of IgG and IgG subtype antibodiesELISA plates (Evergreen Scientific) were coated with 100 μl per well of 1 μg/ml of SARS-CoV-2 S-ecto protein (Sino Biological), SARS-CoV-2 RBD-untagged protein (Sino Biological), SARS-CoV-2 NP protein (Sino Biological), or SARS-CoV-2 E protein (1-75 aa) (Thermo Fisher) in coating buffer [0.05 M sodium carbonate–sodium bicarbonate (pH 9.6)]. .. After overnight incubation at 4°C, the plates were washed twice with PBS buffer and blocked for 2 hr at 37°C with 200 μl per well PBS–5% BSA buffer.

    Selection:

    Article Title: A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein
    Article Snippet: Following incubation and washing, SARS-CoV-2 RBD-bound phages were eluted and used to infect fresh ER2738 cells. .. After several rounds of biopanning, scFv phages binding to SARS-CoV-2 RBD were identified by phage ELISA for further selection. .. Preparation of scFv-Fc, full-length IgG, and S proteins Each scFv identified by phage ELISA was cloned into the Fc fusion vector and transiently expressed in Chinese hamster ovary (CHO) cells.

    Recombinant:

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

    Article Title: An engineered stable mini-protein to plug SARS-Cov-2 Spikes
    Article Snippet: Microscale thermophoresis (MST) binding studies The thermophoretic measurements were performed using Monolith NT.115 device with red detection channel (NanoTemper Technologies, Munich, Germany). .. Recombinant Spikeplug was produced in our laboratory, while SARS-CoV-2 Spike RBD was purchased from Sino Biological Inc. For MST recording, the SARS-CoV-2 Spike RBD was labelled with the fluorescent dye NT647 using the protocol suggested from the NanoTemper. .. Thermophoretic experiments were conducted using Monolith NT.115 (NanoTemper Technologies, Munich, Germany).

    Article Title: Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections
    Article Snippet: All the infection experiments were performed in the biosafety level-3 (BSL-3) laboratory of Fudan University. .. Recombinant proteins and antibodiesFor mouse immunization, recombinant SARS-CoV-2 RBD (residues R319 to F541) fused with a C-terminal mouse IgG1 Fc tag (RBD-mFc) was purchased from Sino Biological Inc (Beijing, China). .. For antibody screening and characterization, several recombinant proteins were produced in our laboratory.

    Derivative Assay:

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

    other:

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein
    Article Snippet: Molecular Docking and Molecular Dynamic Simulations To better understand the interaction patterns between SARS-CoV-2 RBD and the two aptamers, we performed molecular docking and molecular dynamics simulations (MDS).

    Magnetic Beads:

    Article Title: A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein
    Article Snippet: .. Briefly, SARS-CoV-2 RBD (Sino biological) was coated on magnetic beads (Invitrogen) and incubated with the phage library. .. Following incubation and washing, SARS-CoV-2 RBD-bound phages were eluted and used to infect fresh ER2738 cells.

    Incubation:

    Article Title: A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein
    Article Snippet: .. Briefly, SARS-CoV-2 RBD (Sino biological) was coated on magnetic beads (Invitrogen) and incubated with the phage library. .. Following incubation and washing, SARS-CoV-2 RBD-bound phages were eluted and used to infect fresh ER2738 cells.

    Produced:

    Article Title: An engineered stable mini-protein to plug SARS-Cov-2 Spikes
    Article Snippet: Microscale thermophoresis (MST) binding studies The thermophoretic measurements were performed using Monolith NT.115 device with red detection channel (NanoTemper Technologies, Munich, Germany). .. Recombinant Spikeplug was produced in our laboratory, while SARS-CoV-2 Spike RBD was purchased from Sino Biological Inc. For MST recording, the SARS-CoV-2 Spike RBD was labelled with the fluorescent dye NT647 using the protocol suggested from the NanoTemper. .. Thermophoretic experiments were conducted using Monolith NT.115 (NanoTemper Technologies, Munich, Germany).

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    Sino Biological sars cov 2 rbd
    RU169 output clone diversity Using the <t>SARS-CoV-2</t> RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.
    Sars Cov 2 Rbd, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological sars cov 2 2019 ncov spike rbd antibody rabbit pab
    ELISA ( x -axis) vs. LFRET ( y -axis) results by disease severity. ( a ) Anti-NP IgA ELISA vs. anti-NP LFRET (N = 81, R = 0.25). ( b ) anti-NP IgG ELISA vs. anti-NP LFRET (N = 129, R = 0.62). ( c ) anti-NP IgM ELISA vs. anti-NP LFRET (N = 81, R = 0.13). ( d ) anti-SP IgA ELISA vs. anti-SP LFRET (N = 129, R = 0.53). ( e ) anti-SP IgG ELISA vs. anti-SP LFRET (N = 129, R = 0.62). ( f ) anti-SP IgM ELISA vs. anti-SP LFRET (N = 81, R = 0.56). Color of the dot indicates <t>SARS-CoV-2</t> PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal and vertical black lines indicate LFRET and ELISA cutoffs. On the x -axis, ELISA absorbance on a logarithmic scale and on the y -axis, LFRET signal on a logarithmic scale. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. R = Pearson’s correlation coefficient.
    Sars Cov 2 2019 Ncov Spike Rbd Antibody Rabbit Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sars cov 2 2019 ncov spike rbd antibody rabbit pab/product/Sino Biological
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    Image Search Results


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

    Journal: bioRxiv

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

    doi: 10.1101/2020.06.08.140871

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

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

    Techniques: Infection, Purification, Standard Deviation

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.06.08.140871

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

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

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

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.06.08.140871

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

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

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

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.06.08.140871

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

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

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

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.06.08.140871

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

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

    Techniques: Neutralization, Incubation, Infection, Immunostaining, Luciferase

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.06.08.140871

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

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

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

    RU169 output clone diversity Using the SARS-CoV-2 RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: RU169 output clone diversity Using the SARS-CoV-2 RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: FACS, Selection, Produced, Clone Assay

    BLI kinetics of selected scFv clones from the RU169 RBD screen. scFv were cloned into an AviTag™ biotinylation vector, as described in the Materials and Methods, expressed and purified by Ni-NTA resin. scFv were loaded onto a streptavidin BLI sensor and the association/dissociation kinetics of binding to soluble SARS-CoV-2 S1 trimer (100 nM) were measured using BLI. The K D of the scFvs for the S1 target ranged from 1 nM to 400 nM.

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: BLI kinetics of selected scFv clones from the RU169 RBD screen. scFv were cloned into an AviTag™ biotinylation vector, as described in the Materials and Methods, expressed and purified by Ni-NTA resin. scFv were loaded onto a streptavidin BLI sensor and the association/dissociation kinetics of binding to soluble SARS-CoV-2 S1 trimer (100 nM) were measured using BLI. The K D of the scFvs for the S1 target ranged from 1 nM to 400 nM.

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: Clone Assay, Plasmid Preparation, Purification, Binding Assay

    Anti-RBD clones in IgG1 format form long-lived complexes with SARS-CoV-2 S1 trimer and potently inhibit the interaction with ACE2 in vitro . A. Dissociation kinetics of IgG1 anti-RBD clones from SARS-CoV-2 S1 trimer. Biotinylated SARS-CoV-2 S1 trimer was bound to a streptavidin BLI sensor. IgG1 anti-RBD clones were bound (100 nM) and the dissociation followed for 4 hours in PBS at 25°C. B. ACE2-S1 Dynabead assay with molar equivalents of mAb clones to S1 trimer.

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: Anti-RBD clones in IgG1 format form long-lived complexes with SARS-CoV-2 S1 trimer and potently inhibit the interaction with ACE2 in vitro . A. Dissociation kinetics of IgG1 anti-RBD clones from SARS-CoV-2 S1 trimer. Biotinylated SARS-CoV-2 S1 trimer was bound to a streptavidin BLI sensor. IgG1 anti-RBD clones were bound (100 nM) and the dissociation followed for 4 hours in PBS at 25°C. B. ACE2-S1 Dynabead assay with molar equivalents of mAb clones to S1 trimer.

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: Clone Assay, In Vitro

    FACS strategy of screen RU167 for scFv inhibiting the SARS-CoV-2 RBD/ACE2 interaction The FACS-based screening strategy for screen RU167 to isolate antibodies that bound SARS-CoV-2 RBD and specifically inhibited co-binding of RBD to the human ACE2 protein. The viral RBD and the ACE2 protein were labeled with different fluorophores (A). Binding to cells expressing scFv clones that bound RBD and blocking the ACE2-binding site (B) would be observed and gated positively for in the FACS plot for events which were RBD-dye HIGH and ACE2-dye LOW (C).

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: FACS strategy of screen RU167 for scFv inhibiting the SARS-CoV-2 RBD/ACE2 interaction The FACS-based screening strategy for screen RU167 to isolate antibodies that bound SARS-CoV-2 RBD and specifically inhibited co-binding of RBD to the human ACE2 protein. The viral RBD and the ACE2 protein were labeled with different fluorophores (A). Binding to cells expressing scFv clones that bound RBD and blocking the ACE2-binding site (B) would be observed and gated positively for in the FACS plot for events which were RBD-dye HIGH and ACE2-dye LOW (C).

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: FACS, Binding Assay, Labeling, Expressing, Clone Assay, Blocking Assay

    BLI kinetics of anti-RBD diabodies AviTag™ biotinylated SARS-CoV-2 S1 trimer was loaded onto a BLI sensor and the association/dissociation kinetics of binding to anti-RBD diabodies (100 nM) were measured using BLI. The K D s of the dbs to the S1 target ranged from 84 pM to 1 nM.

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: BLI kinetics of anti-RBD diabodies AviTag™ biotinylated SARS-CoV-2 S1 trimer was loaded onto a BLI sensor and the association/dissociation kinetics of binding to anti-RBD diabodies (100 nM) were measured using BLI. The K D s of the dbs to the S1 target ranged from 84 pM to 1 nM.

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: Binding Assay

    Cytometry plots of ACE2-S1 Dynabead assay of anti-RBD diabodies The degree of inhibition of the ACE2 and SARS-CoV-2 S1 trimer interaction by stoichiometric amounts of anti-RBD diabodies was determined using a Dynabead assay as described in the Materials and Methods. The degree of bead fluorescence was indicative of the amount of dye-labeled S1 trimer that was bound to ACE2. Inhibition of the interaction by anti-RBD diabodies resulted in a reduction in fluorescence. The first panel is the SSC/FSC indicating the P1 gating of beads. The second panel is the biotin-blocked control (no ACE2/S1 interaction) and the third panel is the no anti-RBD control (maximum ACE2/S1 interaction. Each subsequent row represents a db clone at 1:1, 5:1 and 10:1 stoichiometric ratios to the soluble SARS-CoV-2 S1 trimer. The data are summarized graphically in Figure 3 .

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: Cytometry plots of ACE2-S1 Dynabead assay of anti-RBD diabodies The degree of inhibition of the ACE2 and SARS-CoV-2 S1 trimer interaction by stoichiometric amounts of anti-RBD diabodies was determined using a Dynabead assay as described in the Materials and Methods. The degree of bead fluorescence was indicative of the amount of dye-labeled S1 trimer that was bound to ACE2. Inhibition of the interaction by anti-RBD diabodies resulted in a reduction in fluorescence. The first panel is the SSC/FSC indicating the P1 gating of beads. The second panel is the biotin-blocked control (no ACE2/S1 interaction) and the third panel is the no anti-RBD control (maximum ACE2/S1 interaction. Each subsequent row represents a db clone at 1:1, 5:1 and 10:1 stoichiometric ratios to the soluble SARS-CoV-2 S1 trimer. The data are summarized graphically in Figure 3 .

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: Cytometry, Inhibition, Fluorescence, Labeling

    ELISA ( x -axis) vs. LFRET ( y -axis) results by disease severity. ( a ) Anti-NP IgA ELISA vs. anti-NP LFRET (N = 81, R = 0.25). ( b ) anti-NP IgG ELISA vs. anti-NP LFRET (N = 129, R = 0.62). ( c ) anti-NP IgM ELISA vs. anti-NP LFRET (N = 81, R = 0.13). ( d ) anti-SP IgA ELISA vs. anti-SP LFRET (N = 129, R = 0.53). ( e ) anti-SP IgG ELISA vs. anti-SP LFRET (N = 129, R = 0.62). ( f ) anti-SP IgM ELISA vs. anti-SP LFRET (N = 81, R = 0.56). Color of the dot indicates SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal and vertical black lines indicate LFRET and ELISA cutoffs. On the x -axis, ELISA absorbance on a logarithmic scale and on the y -axis, LFRET signal on a logarithmic scale. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. R = Pearson’s correlation coefficient.

    Journal: Viruses

    Article Title: A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

    doi: 10.3390/v13020143

    Figure Lengend Snippet: ELISA ( x -axis) vs. LFRET ( y -axis) results by disease severity. ( a ) Anti-NP IgA ELISA vs. anti-NP LFRET (N = 81, R = 0.25). ( b ) anti-NP IgG ELISA vs. anti-NP LFRET (N = 129, R = 0.62). ( c ) anti-NP IgM ELISA vs. anti-NP LFRET (N = 81, R = 0.13). ( d ) anti-SP IgA ELISA vs. anti-SP LFRET (N = 129, R = 0.53). ( e ) anti-SP IgG ELISA vs. anti-SP LFRET (N = 129, R = 0.62). ( f ) anti-SP IgM ELISA vs. anti-SP LFRET (N = 81, R = 0.56). Color of the dot indicates SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal and vertical black lines indicate LFRET and ELISA cutoffs. On the x -axis, ELISA absorbance on a logarithmic scale and on the y -axis, LFRET signal on a logarithmic scale. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. R = Pearson’s correlation coefficient.

    Article Snippet: At 48 h, the medium was analyzed for the presence of SARS-CoV-2 SP by dot blotting; briefly via drying 2.5 µL of the supernatant onto a nitrocellulose membrane, which then was blocked (3% skim milk in Tris-buffered saline with 0.05% Tween-20), washed, probed with rabbit anti-RBD (40592-T62, Sino Biological, Beijing, China), washed, probed with anti-rabbit IRDye800 (LI-COR Biosciences, Lincoln, NE, USA), washed, and read using Odyssey Infrared Imaging System (LI-COR Biosciences).

    Techniques: Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Förster Resonance Energy Transfer

    Microneutralization vs. LFRET and ELISA. Microneutralization titers are on the x -axis and LFRET signal or ELISA absorbance on the y -axis. Logarithmic scale is used on both axes. ( a ) Microneutralization titer vs. anti-SP LFRET signal (N = 107, ρ = 0.87). ( b – d ) Microneutralization titer vs. anti-SP IgG, IgA and IgM ELISA (N = 107, 107 and 67, ρ = 0.68, 0.86 and 0.81). ( e ) Microneutralization titer vs. anti-NP LFRET signal (N = 107, ρ = 0.83). ( f – h ) Microneutralization titer vs. anti-NP IgG, IgA and IgM ELISA (N = 107, 67 and 67, ρ = 0.81, 0.69 and 0.61). Color of the dots indicate SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal black lines indicate LFRET/ELISA cutoffs. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. ρ = Spearman’s rank correlation coefficient.

    Journal: Viruses

    Article Title: A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

    doi: 10.3390/v13020143

    Figure Lengend Snippet: Microneutralization vs. LFRET and ELISA. Microneutralization titers are on the x -axis and LFRET signal or ELISA absorbance on the y -axis. Logarithmic scale is used on both axes. ( a ) Microneutralization titer vs. anti-SP LFRET signal (N = 107, ρ = 0.87). ( b – d ) Microneutralization titer vs. anti-SP IgG, IgA and IgM ELISA (N = 107, 107 and 67, ρ = 0.68, 0.86 and 0.81). ( e ) Microneutralization titer vs. anti-NP LFRET signal (N = 107, ρ = 0.83). ( f – h ) Microneutralization titer vs. anti-NP IgG, IgA and IgM ELISA (N = 107, 67 and 67, ρ = 0.81, 0.69 and 0.61). Color of the dots indicate SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal black lines indicate LFRET/ELISA cutoffs. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. ρ = Spearman’s rank correlation coefficient.

    Article Snippet: At 48 h, the medium was analyzed for the presence of SARS-CoV-2 SP by dot blotting; briefly via drying 2.5 µL of the supernatant onto a nitrocellulose membrane, which then was blocked (3% skim milk in Tris-buffered saline with 0.05% Tween-20), washed, probed with rabbit anti-RBD (40592-T62, Sino Biological, Beijing, China), washed, probed with anti-rabbit IRDye800 (LI-COR Biosciences, Lincoln, NE, USA), washed, and read using Odyssey Infrared Imaging System (LI-COR Biosciences).

    Techniques: Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Förster Resonance Energy Transfer

    Simplified protocol for SARS-CoV-2 NP and SP LFRET assay. Eu-NP/-SP = Europium-labeled nucleoprotein/spike glycoprotein. AF-L = Alexa Fluor™ 647 -labeled protein L. TR-FRET = time-resolved Förster resonance energy transfer. RT = room temperature. TBS+BSA (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.2% BSA) was used for all dilutions. On-plate dilutions were 5 nM Eu-NP/500 nM AF-L/serum 1/25 for anti-NP and 5 nM Eu-SP/250 nM AF-L/serum 1/100 for anti-SP LFRET. For further details see the prior publication [ 5 ].

    Journal: Viruses

    Article Title: A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

    doi: 10.3390/v13020143

    Figure Lengend Snippet: Simplified protocol for SARS-CoV-2 NP and SP LFRET assay. Eu-NP/-SP = Europium-labeled nucleoprotein/spike glycoprotein. AF-L = Alexa Fluor™ 647 -labeled protein L. TR-FRET = time-resolved Förster resonance energy transfer. RT = room temperature. TBS+BSA (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.2% BSA) was used for all dilutions. On-plate dilutions were 5 nM Eu-NP/500 nM AF-L/serum 1/25 for anti-NP and 5 nM Eu-SP/250 nM AF-L/serum 1/100 for anti-SP LFRET. For further details see the prior publication [ 5 ].

    Article Snippet: At 48 h, the medium was analyzed for the presence of SARS-CoV-2 SP by dot blotting; briefly via drying 2.5 µL of the supernatant onto a nitrocellulose membrane, which then was blocked (3% skim milk in Tris-buffered saline with 0.05% Tween-20), washed, probed with rabbit anti-RBD (40592-T62, Sino Biological, Beijing, China), washed, probed with anti-rabbit IRDye800 (LI-COR Biosciences, Lincoln, NE, USA), washed, and read using Odyssey Infrared Imaging System (LI-COR Biosciences).

    Techniques: Labeling, Förster Resonance Energy Transfer