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
    The structure of CT-P59 Fab in complex with <t>SARS-CoV-2</t> 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.
    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
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 rbd - by Bioz Stars, 2021-04
    99/100 stars

    Images

    1) 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

    2) 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

    3) Product Images from "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 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

    Journal: bioRxiv

    doi: 10.1101/2020.08.19.253369

    Isolation of RBD-specific memory B cells using flow cytometry. A. The heatmap depicts the specificity of convalescent patients’ plasma against S1 and RBD from SARS-CoV-2, SARS-CoV and MERS-CoV, measured by ELISA. Serial dilutions of plasma samples were performed to test the reactivity of antibodies in plasma. The plasma of healthy donors was used as the control. Data were shown with the mean of representative experiments. B. Gating strategy for SARS-CoV-2 RBD-specific IgG + B cells in PBMCs of the convalescent patients. Living CD19+ IgD − IgG + cells were gated, and cells with positive SARS-CoV-2 RBD staining were selected for single-cell sorting. C. FACS analysis of RBD-specific memory B cells in CD19 + IgD − IgG + memory B cells from PBMCs of three batch convalescent patients. Plots show CD19 + IgD − IgG + RBD+ populations using gating strategy described in B .
    Figure Legend Snippet: Isolation of RBD-specific memory B cells using flow cytometry. A. The heatmap depicts the specificity of convalescent patients’ plasma against S1 and RBD from SARS-CoV-2, SARS-CoV and MERS-CoV, measured by ELISA. Serial dilutions of plasma samples were performed to test the reactivity of antibodies in plasma. The plasma of healthy donors was used as the control. Data were shown with the mean of representative experiments. B. Gating strategy for SARS-CoV-2 RBD-specific IgG + B cells in PBMCs of the convalescent patients. Living CD19+ IgD − IgG + cells were gated, and cells with positive SARS-CoV-2 RBD staining were selected for single-cell sorting. C. FACS analysis of RBD-specific memory B cells in CD19 + IgD − IgG + memory B cells from PBMCs of three batch convalescent patients. Plots show CD19 + IgD − IgG + RBD+ populations using gating strategy described in B .

    Techniques Used: Isolation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, FACS

    The optimization of the screening platform. A. The conventional screening of neutralizing antibodies. Antigen-specific B cells from PBMCs were sorted on day 1. The single-cell BCR genes were amplified by PCR on day 2. The antibody expression vectors were constructed in the next three days, including the PCR product sequencing, the primer synthesis, the ligation of genes and vectors, the DNA transformation and the plasmid extraction. The purified plasmids were transfected into HEK293T cells on day 5. After 48 hours, the cell supernatants were collected and analyzed with specific antigens by ELISA. Specific antibodies are used for following antibody expression and purification. Purified antibodies were screened as neutralizing candidates. B. The key parameters affecting screening efficiency. The following steps of the screening processes were carefully modified: multi-step sorting for the individual samples or the pooled samples, labeling S or S-RBD specific B cells, expressing antibodies using linear expression cassettes or plasmids, and designing preferred primers for the single-cell BCR cloning. To reduce time-consuming and workload, it is the critical step to screen neutralizing antibodies during the initial screening in the sixty days. Two methods for neutralization evaluation, competitive ELISA method in 3 hours or pseudovirus assay for 48 hours, were used side by side for confirmation of nAb neutralizing capability. C. The optimized strategy of neutralizing antibodies development. One day after PBMC thawing, specific B cell sorting was performed on day 1. A single BCR gene was cloned on day 2, using the 2 nd PCR product to construct the linear expression cassettes, which were termed as the transfection targets to be introduced directly into HEK293T cells with liposome, without constructing plasmid, to shorten the screening duration. After 48 hours, the supernatants of each transfected samples were harvested and analyzed via ELISA and pseudovirus neutralization assay for evaluating specificity and neutralization.
    Figure Legend Snippet: The optimization of the screening platform. A. The conventional screening of neutralizing antibodies. Antigen-specific B cells from PBMCs were sorted on day 1. The single-cell BCR genes were amplified by PCR on day 2. The antibody expression vectors were constructed in the next three days, including the PCR product sequencing, the primer synthesis, the ligation of genes and vectors, the DNA transformation and the plasmid extraction. The purified plasmids were transfected into HEK293T cells on day 5. After 48 hours, the cell supernatants were collected and analyzed with specific antigens by ELISA. Specific antibodies are used for following antibody expression and purification. Purified antibodies were screened as neutralizing candidates. B. The key parameters affecting screening efficiency. The following steps of the screening processes were carefully modified: multi-step sorting for the individual samples or the pooled samples, labeling S or S-RBD specific B cells, expressing antibodies using linear expression cassettes or plasmids, and designing preferred primers for the single-cell BCR cloning. To reduce time-consuming and workload, it is the critical step to screen neutralizing antibodies during the initial screening in the sixty days. Two methods for neutralization evaluation, competitive ELISA method in 3 hours or pseudovirus assay for 48 hours, were used side by side for confirmation of nAb neutralizing capability. C. The optimized strategy of neutralizing antibodies development. One day after PBMC thawing, specific B cell sorting was performed on day 1. A single BCR gene was cloned on day 2, using the 2 nd PCR product to construct the linear expression cassettes, which were termed as the transfection targets to be introduced directly into HEK293T cells with liposome, without constructing plasmid, to shorten the screening duration. After 48 hours, the supernatants of each transfected samples were harvested and analyzed via ELISA and pseudovirus neutralization assay for evaluating specificity and neutralization.

    Techniques Used: Amplification, Polymerase Chain Reaction, Expressing, Construct, Sequencing, Ligation, Transformation Assay, Plasmid Preparation, Purification, Transfection, Enzyme-linked Immunosorbent Assay, Modification, Labeling, Clone Assay, Neutralization, Competitive ELISA, FACS

    Identification of RBD specific monoclonal antibodies from convalescent COVID-19 patients. A. Screening of specific Abs against SARS-CoV-2 S1 and RBD. The heatmap reveals that the binding ability of 198 Ab supernatants produced by HEK239T cells transfected with linear Ab gene expression cassette. The mAbs rank as the screening sequence, and binding activity of mAbs against SARS-CoV-2 S1 and RBD were tested by ELISA. The brightness of blue represents the binding strength, which reflected the OD405 nm value tested by ELISA. The neutralizing activity of mAbs was discriminated according to the neutralizing value. Antibody-mediated blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus transfected into hACE2/ HEK293T cells were measured by values of relative light units (RUL). The Green columns indicate potential neutralization (neutralizing activity > 75%), while white indicate partial or not neutralization (neutralizing activity
    Figure Legend Snippet: Identification of RBD specific monoclonal antibodies from convalescent COVID-19 patients. A. Screening of specific Abs against SARS-CoV-2 S1 and RBD. The heatmap reveals that the binding ability of 198 Ab supernatants produced by HEK239T cells transfected with linear Ab gene expression cassette. The mAbs rank as the screening sequence, and binding activity of mAbs against SARS-CoV-2 S1 and RBD were tested by ELISA. The brightness of blue represents the binding strength, which reflected the OD405 nm value tested by ELISA. The neutralizing activity of mAbs was discriminated according to the neutralizing value. Antibody-mediated blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus transfected into hACE2/ HEK293T cells were measured by values of relative light units (RUL). The Green columns indicate potential neutralization (neutralizing activity > 75%), while white indicate partial or not neutralization (neutralizing activity

    Techniques Used: Binding Assay, Produced, Transfection, Expressing, Sequencing, Activity Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Luciferase, Neutralization

    The binding activity and inhibition of ACE2-RBD interaction of mAbs tested by ELISA and competitive ELISA. A. The OD 405 nm value refects a binding strength of purified mAbs to 1 μg/ml SARS-CoV-2 S1 or RBD. Plates were coated with recombinant S1 or RBD protein of SARS-CoV-2, then incubated with purified mAbs. A SARS specific mAb (CR3022) was set as the positive control. The blue dashed lines indicated the OD 405nm value of a negative sample. B. The inhibitory effect of purified mAbs against the interaction between SARS-CoV-2 RBD and hACE2 was tested via competitive ELISA analysis. Blocking efficacy was determined by comparing response units with and without prior antibody incubation. The green dashed lines indicated 50% inhibition on blocking the interaction ACE2 and RBD interaction.
    Figure Legend Snippet: The binding activity and inhibition of ACE2-RBD interaction of mAbs tested by ELISA and competitive ELISA. A. The OD 405 nm value refects a binding strength of purified mAbs to 1 μg/ml SARS-CoV-2 S1 or RBD. Plates were coated with recombinant S1 or RBD protein of SARS-CoV-2, then incubated with purified mAbs. A SARS specific mAb (CR3022) was set as the positive control. The blue dashed lines indicated the OD 405nm value of a negative sample. B. The inhibitory effect of purified mAbs against the interaction between SARS-CoV-2 RBD and hACE2 was tested via competitive ELISA analysis. Blocking efficacy was determined by comparing response units with and without prior antibody incubation. The green dashed lines indicated 50% inhibition on blocking the interaction ACE2 and RBD interaction.

    Techniques Used: Binding Assay, Activity Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Competitive ELISA, Purification, Recombinant, Incubation, Positive Control, Blocking Assay

    Schematic model depicting a rapid and efficient screening system of neutralizing Abs. Rapid neutralizing antibody screening workflows and timelines are shown, representing the multiple workflows conducted in parallel. PBMC were isolated from collected convalescent patients’ blood, and the RBD-specific memory B cells in the PBMCs were sorted as single-cell via flow-cytometric sorter (day 1). Then, the IgG heavy and light chains of monoclonal antibody genes were amplified by RT-PCR on the same day. 2 nd PCR products were cloned into linear expression cassettes on the second day. Antibodies were expressed by transient transfection with equal amounts of paired heavy and light chain linear expression cassettes in HEK293T cells and culture for two days. The cell supernatants in HEK293T cells were detected for the specificity of antibodies by ELISA in 384-well plates on the fourth day. The neutralizing activity of antibodies was detected with pseudovirus bearing SARS-CoV-2 S in 96-well plates on the sixth day. The potential neutralization antibody expression plasmids were transfected into Exi293F cells for large-scale production of Ab proteins. The cell supernatants in Exi293F cells were collected, and antibody proteins were purified by protein G. They were further measured for the binding ability and neutralizing activity via ELISA and competitive ELISA in vitro . Additionally, virus neutralization assay was performed. Created with Biorender.com.
    Figure Legend Snippet: Schematic model depicting a rapid and efficient screening system of neutralizing Abs. Rapid neutralizing antibody screening workflows and timelines are shown, representing the multiple workflows conducted in parallel. PBMC were isolated from collected convalescent patients’ blood, and the RBD-specific memory B cells in the PBMCs were sorted as single-cell via flow-cytometric sorter (day 1). Then, the IgG heavy and light chains of monoclonal antibody genes were amplified by RT-PCR on the same day. 2 nd PCR products were cloned into linear expression cassettes on the second day. Antibodies were expressed by transient transfection with equal amounts of paired heavy and light chain linear expression cassettes in HEK293T cells and culture for two days. The cell supernatants in HEK293T cells were detected for the specificity of antibodies by ELISA in 384-well plates on the fourth day. The neutralizing activity of antibodies was detected with pseudovirus bearing SARS-CoV-2 S in 96-well plates on the sixth day. The potential neutralization antibody expression plasmids were transfected into Exi293F cells for large-scale production of Ab proteins. The cell supernatants in Exi293F cells were collected, and antibody proteins were purified by protein G. They were further measured for the binding ability and neutralizing activity via ELISA and competitive ELISA in vitro . Additionally, virus neutralization assay was performed. Created with Biorender.com.

    Techniques Used: Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Clone Assay, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Activity Assay, Neutralization, Purification, Binding Assay, Competitive ELISA, In Vitro

    4) 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

    5) 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

    6) Product Images from "An engineered stable mini-protein to plug SARS-Cov-2 Spikes"

    Article Title: An engineered stable mini-protein to plug SARS-Cov-2 Spikes

    Journal: bioRxiv

    doi: 10.1101/2020.04.29.067728

    Cartoon representation of the complex between the receptor binding (RBD) domain of SARS-CoV-2 Spike protein (blue/cyan) and the human ACE2 receptor (grey/green). The receptor binding motif (RBM) is drawn in cyan. The green portion of the ACE2 domain including helices H1, H2 and H3 is drawn in green.
    Figure Legend Snippet: Cartoon representation of the complex between the receptor binding (RBD) domain of SARS-CoV-2 Spike protein (blue/cyan) and the human ACE2 receptor (grey/green). The receptor binding motif (RBM) is drawn in cyan. The green portion of the ACE2 domain including helices H1, H2 and H3 is drawn in green.

    Techniques Used: Binding Assay

    7) 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

    8) Product Images from "A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering"

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    Journal: bioRxiv

    doi: 10.1101/2021.01.19.427310

    Immune responses of T4-SARS-CoV-2 immunized mice. a. Anti-RBD IgG antibody titers in the sera from group G5 (T4-HocΔ-SocΔ-S-ecto-Ee-NP) at weeks 2 (prime), 5 (boost-1), and 8 (boost-2). For boost-2, T4-S-trimers particles were used. **P
    Figure Legend Snippet: Immune responses of T4-SARS-CoV-2 immunized mice. a. Anti-RBD IgG antibody titers in the sera from group G5 (T4-HocΔ-SocΔ-S-ecto-Ee-NP) at weeks 2 (prime), 5 (boost-1), and 8 (boost-2). For boost-2, T4-S-trimers particles were used. **P

    Techniques Used: Mouse Assay

    Immunogenicity and protective efficacy of T4-SARS-CoV-2 vaccine candidates in mice. a. Schematic diagram showing BALB/c mice immunized by the intramuscular (i.m.) route using T4-SARS-CoV-2 vaccine formulations. b. I. Formulations and mouse groups used for vaccinations. HSΔ indicates Hoc deletion and Soc deletion. Blue color (S-ecto, S-fl, and RBD) indicates the insertion of mammalian gene expression cassette into T4 genome as DNA vaccine. Red color indicates the capsid-displayed Ee, S-trimers, or E.coli -produced rRBD or sRBD protein, or the capsid-encapsidated NP protein. Naïve mice and mice immunized with the phage lacking any CoV-2 genes served as negative controls whereas mice immunized with S-trimers adjuvanted with Alhydrogel served as a positive control. II. Prime-boost immunization scheme. BALB/c mice (5 per group) were immunized on weeks 0, 3, and 6 and challenged intranasally (i.n.) with a mouse-adapted SARS-CoV-2 strain (SARS-CoV-2 MA10) 47 on week 14. c to f . The boost-2 sera (week 8 bleeding) from various groups were assessed by ELISA for antigen-specific IgG antibody titers (endpoint) against S-ecto (c), RBD (d), NP (e), and E (f). *P
    Figure Legend Snippet: Immunogenicity and protective efficacy of T4-SARS-CoV-2 vaccine candidates in mice. a. Schematic diagram showing BALB/c mice immunized by the intramuscular (i.m.) route using T4-SARS-CoV-2 vaccine formulations. b. I. Formulations and mouse groups used for vaccinations. HSΔ indicates Hoc deletion and Soc deletion. Blue color (S-ecto, S-fl, and RBD) indicates the insertion of mammalian gene expression cassette into T4 genome as DNA vaccine. Red color indicates the capsid-displayed Ee, S-trimers, or E.coli -produced rRBD or sRBD protein, or the capsid-encapsidated NP protein. Naïve mice and mice immunized with the phage lacking any CoV-2 genes served as negative controls whereas mice immunized with S-trimers adjuvanted with Alhydrogel served as a positive control. II. Prime-boost immunization scheme. BALB/c mice (5 per group) were immunized on weeks 0, 3, and 6 and challenged intranasally (i.n.) with a mouse-adapted SARS-CoV-2 strain (SARS-CoV-2 MA10) 47 on week 14. c to f . The boost-2 sera (week 8 bleeding) from various groups were assessed by ELISA for antigen-specific IgG antibody titers (endpoint) against S-ecto (c), RBD (d), NP (e), and E (f). *P

    Techniques Used: Mouse Assay, Expressing, Produced, Positive Control, Enzyme-linked Immunosorbent Assay

    A pipeline of SARS-CoV-2 vaccine candidates generated by sequential CRISPR engineering. a. Schematic showing a representative sequence in which the WT phage was used as a starting infection of CRISPR E. coli containing spacer 1 and donor 1. The resultant T4-mutant 1 (T4-M1) was used to infect bacteria containing spacer 2 and donor 2 to produce recombinant T4-mutant 2 (T4-M2) which has two insertion/deletion mutations, and so forth. By sequential CRISPR engineering and simple phage infections, recombinant phages with multiple desired mutations were created. Each color on phage capsid here represents a mutation. b. One example of sequential phage CRISPR engineering for creating the T4-SARS-CoV-2 nanovaccine. Numerous CoV-2 components, including CAGpromoter-S-ecto insertion, CAGpromoter-S-fl insertion, CMVpromoter-RBD insertion, Hoc deletion, Ee-Hoc insertion, Ec-Hoc insertion, Soc deletion, Soc-sRBD display, M21-Soc-sRBD display, Soc-SpyCatcher display, refolding SUMO-RBD-Spy display, S-trimer display, IPIII deletion, IPII deletion, and NP encapsidation, were permutated and combined as needed. The resultant SARS-CoV-2 vaccine candidates were characterized by PCR, DNA sequencing and/or SDS-PAGE, and some of these were then tested in a mouse study. M21 indicates a potential T cell 21 aa epitope (SYFIASFRLFARTRSMWSFNP) from SARS-CoV-2 membrane protein. c. WB showing NP protein encapsidation in the phages containing CTSam-NP insertion at IPIII deletion site.
    Figure Legend Snippet: A pipeline of SARS-CoV-2 vaccine candidates generated by sequential CRISPR engineering. a. Schematic showing a representative sequence in which the WT phage was used as a starting infection of CRISPR E. coli containing spacer 1 and donor 1. The resultant T4-mutant 1 (T4-M1) was used to infect bacteria containing spacer 2 and donor 2 to produce recombinant T4-mutant 2 (T4-M2) which has two insertion/deletion mutations, and so forth. By sequential CRISPR engineering and simple phage infections, recombinant phages with multiple desired mutations were created. Each color on phage capsid here represents a mutation. b. One example of sequential phage CRISPR engineering for creating the T4-SARS-CoV-2 nanovaccine. Numerous CoV-2 components, including CAGpromoter-S-ecto insertion, CAGpromoter-S-fl insertion, CMVpromoter-RBD insertion, Hoc deletion, Ee-Hoc insertion, Ec-Hoc insertion, Soc deletion, Soc-sRBD display, M21-Soc-sRBD display, Soc-SpyCatcher display, refolding SUMO-RBD-Spy display, S-trimer display, IPIII deletion, IPII deletion, and NP encapsidation, were permutated and combined as needed. The resultant SARS-CoV-2 vaccine candidates were characterized by PCR, DNA sequencing and/or SDS-PAGE, and some of these were then tested in a mouse study. M21 indicates a potential T cell 21 aa epitope (SYFIASFRLFARTRSMWSFNP) from SARS-CoV-2 membrane protein. c. WB showing NP protein encapsidation in the phages containing CTSam-NP insertion at IPIII deletion site.

    Techniques Used: Generated, CRISPR, Sequencing, Infection, Mutagenesis, Recombinant, Polymerase Chain Reaction, DNA Sequencing, SDS Page, Western Blot

    Serum antibody responses in various T4-SARS-CoV-2 immunized mice. a and b. Anti-S-ecto IgG1 (a) and IgG2a (b) antibody titers in the boost-2 sera (week 8 bleeding) from various groups. c and d. Anti-RBD IgG1 (c) and IgG2a (d) antibody titers in the boost-2 sera. e and f . Anti-NP IgG1 (e) and IgG2a (f) antibody titers in the boost-2 sera. g and h . Anti-E IgG1 (g) and IgG2a (h) antibody titers in the boost-2 sera. *P
    Figure Legend Snippet: Serum antibody responses in various T4-SARS-CoV-2 immunized mice. a and b. Anti-S-ecto IgG1 (a) and IgG2a (b) antibody titers in the boost-2 sera (week 8 bleeding) from various groups. c and d. Anti-RBD IgG1 (c) and IgG2a (d) antibody titers in the boost-2 sera. e and f . Anti-NP IgG1 (e) and IgG2a (f) antibody titers in the boost-2 sera. g and h . Anti-E IgG1 (g) and IgG2a (h) antibody titers in the boost-2 sera. *P

    Techniques Used: Mouse Assay

    Virus neutralization titers of rabbit sera. Infection of Vero E6 cells by live SARS-CoV-2 US-WA-1/2020 was determined in the presence of rabbit sera at a series of two-fold dilutions starting from 1:4. Culture medium only and CoV-2 virus only were used as negative and positive controls, respectively. R1442 to R1457 refer to tag numbers of rabbits. The data in control groups were presented as means ± SD of 32 wells. The data in rabbit sera groups were shown as means of duplicates.
    Figure Legend Snippet: Virus neutralization titers of rabbit sera. Infection of Vero E6 cells by live SARS-CoV-2 US-WA-1/2020 was determined in the presence of rabbit sera at a series of two-fold dilutions starting from 1:4. Culture medium only and CoV-2 virus only were used as negative and positive controls, respectively. R1442 to R1457 refer to tag numbers of rabbits. The data in control groups were presented as means ± SD of 32 wells. The data in rabbit sera groups were shown as means of duplicates.

    Techniques Used: Neutralization, Infection

    Construction and screening of various truncated SARS-CoV-2 RBDs. a. Structural models of recombinant WT RBD and various truncated RBDs bound to human ACE2. ACE2 is shown in green. The truncated RBD clones are shown in red and the WT RBD and deleted regions are shown in cyan. The Protein Data Bank (PDB) code for the SARS-CoV-2 RBD–ACE2 complex is 6M0J 34 . The truncated RBDs were generated using Chimera software. b. Solubility analysis of Soc-fused truncated RBDs after cloning and expression in E. coli under the control of the phage T7 promoter. After lysis of E. coli and centrifugation, the supernatant and pellet were analyzed by SDS-PAGE. The presence of Soc-truncated RBDs in the pellet and their absence in the supernatant demonstrated insolubility. The red arrowheads indicate the band positions of various Soc-truncated RBDs.
    Figure Legend Snippet: Construction and screening of various truncated SARS-CoV-2 RBDs. a. Structural models of recombinant WT RBD and various truncated RBDs bound to human ACE2. ACE2 is shown in green. The truncated RBD clones are shown in red and the WT RBD and deleted regions are shown in cyan. The Protein Data Bank (PDB) code for the SARS-CoV-2 RBD–ACE2 complex is 6M0J 34 . The truncated RBDs were generated using Chimera software. b. Solubility analysis of Soc-fused truncated RBDs after cloning and expression in E. coli under the control of the phage T7 promoter. After lysis of E. coli and centrifugation, the supernatant and pellet were analyzed by SDS-PAGE. The presence of Soc-truncated RBDs in the pellet and their absence in the supernatant demonstrated insolubility. The red arrowheads indicate the band positions of various Soc-truncated RBDs.

    Techniques Used: Recombinant, Clone Assay, Generated, Software, Solubility, Expressing, Lysis, Centrifugation, SDS Page

    Incorporation of various SARS-CoV-2 vaccine payloads into phage T4 nanoparticle. a. Schematic showing steps in T4 phage head morphogenesis. Mem, E. coli membrane; CTS, capsid targeting sequence. b and c. SDS-PAGE and Western Blot (WB) analysis of phage particles with IPII and IPIII deletions (IPIIΔIPIIIΔ) and NP encapsidation. Since NP has a very similar molecular size to T4 major capsid protein gp23*, an NP-specific antibody was used to detect NP. d. Structural model of viroporin-like tetrameric assembly of CoV-2 E protein 32 . The N-terminal seven residues and C-terminal ten residues are not shown due to the lack of a corresponding segment in the structural template used for homology modeling. Ee* indicates amino acids (aa) 8-12 and Ec* indicates aa 53-65. e. SDS-PAGE of Hoc deletion and Soc deletion phage (HocΔSocΔ). f. SDS-PAGE of recombinant phages displaying Ee-Hoc or Ec-Hoc fusion proteins. g. Schematic showing Soc-sRBD or Soc-SpyCatcher (SpyC) in vivo display on T4-SocΔ capsid. Soc-sRBD or Soc-SpyCatcher expression under the control of phage T7 promoter was induced by IPTG. Most of the expressed Soc-RBD was in the inclusion body (IB). Soluble Soc-sRBD (minor amount) or Soc-SpyC can be efficiently displayed on capsid. h. SDS-PAGE showing ~100 copies of Soc-sRBD displayed on T4 capsid. i. SDS-PAGE showing ~500 copies of Soc-SpyCatcher displayed on T4 capsid. j. Schematic diagram showing the solubilization and refolding of SUMO (small ubiquitin like modifiers)-RBD-Spytag inclusion body. Refolded SUMO-RBD-Spytag (rRBD) protein was efficiently displayed on T4-SpyCatcher phage via Spytag-SpyCatcher bridging. k. Display of rRBD on the T4-SpyCacher surface at increasing ratios of rRBD molecules to capsid Soc binding sites (0:1 to 2:1). RBD specific antibody was used to verify the displayed rRBD and rRBD-SpyCatcher-Soc complexes. T4* indicates T4-S-ecto-NP-Ec-SocΔ recombinant phage. Blue and red arrows indicate rRBD/complexes and Soc-SpyCatcher, respectively. l to o . Comparison of binding of T4-sRBD, and T4-rRBD phages to soluble human ACE2 receptor (l), monoclonal antibody (mAb) 1 (human IgG Clone #bcb03, Thermo Fisher) (m), mAb2 (rabbit IgG Clone #007, Sino Bio) (n), and polyclonal antibodies (pAb) (rabbit PAb, Sino Bio) (o) using BSA and T4 phage as controls. p. Comparison of binding of E. coli -produced rRBD to human ACE2 with the HEK293-produced RBD. **P
    Figure Legend Snippet: Incorporation of various SARS-CoV-2 vaccine payloads into phage T4 nanoparticle. a. Schematic showing steps in T4 phage head morphogenesis. Mem, E. coli membrane; CTS, capsid targeting sequence. b and c. SDS-PAGE and Western Blot (WB) analysis of phage particles with IPII and IPIII deletions (IPIIΔIPIIIΔ) and NP encapsidation. Since NP has a very similar molecular size to T4 major capsid protein gp23*, an NP-specific antibody was used to detect NP. d. Structural model of viroporin-like tetrameric assembly of CoV-2 E protein 32 . The N-terminal seven residues and C-terminal ten residues are not shown due to the lack of a corresponding segment in the structural template used for homology modeling. Ee* indicates amino acids (aa) 8-12 and Ec* indicates aa 53-65. e. SDS-PAGE of Hoc deletion and Soc deletion phage (HocΔSocΔ). f. SDS-PAGE of recombinant phages displaying Ee-Hoc or Ec-Hoc fusion proteins. g. Schematic showing Soc-sRBD or Soc-SpyCatcher (SpyC) in vivo display on T4-SocΔ capsid. Soc-sRBD or Soc-SpyCatcher expression under the control of phage T7 promoter was induced by IPTG. Most of the expressed Soc-RBD was in the inclusion body (IB). Soluble Soc-sRBD (minor amount) or Soc-SpyC can be efficiently displayed on capsid. h. SDS-PAGE showing ~100 copies of Soc-sRBD displayed on T4 capsid. i. SDS-PAGE showing ~500 copies of Soc-SpyCatcher displayed on T4 capsid. j. Schematic diagram showing the solubilization and refolding of SUMO (small ubiquitin like modifiers)-RBD-Spytag inclusion body. Refolded SUMO-RBD-Spytag (rRBD) protein was efficiently displayed on T4-SpyCatcher phage via Spytag-SpyCatcher bridging. k. Display of rRBD on the T4-SpyCacher surface at increasing ratios of rRBD molecules to capsid Soc binding sites (0:1 to 2:1). RBD specific antibody was used to verify the displayed rRBD and rRBD-SpyCatcher-Soc complexes. T4* indicates T4-S-ecto-NP-Ec-SocΔ recombinant phage. Blue and red arrows indicate rRBD/complexes and Soc-SpyCatcher, respectively. l to o . Comparison of binding of T4-sRBD, and T4-rRBD phages to soluble human ACE2 receptor (l), monoclonal antibody (mAb) 1 (human IgG Clone #bcb03, Thermo Fisher) (m), mAb2 (rabbit IgG Clone #007, Sino Bio) (n), and polyclonal antibodies (pAb) (rabbit PAb, Sino Bio) (o) using BSA and T4 phage as controls. p. Comparison of binding of E. coli -produced rRBD to human ACE2 with the HEK293-produced RBD. **P

    Techniques Used: Sequencing, SDS Page, Western Blot, Recombinant, In Vivo, Expressing, Binding Assay, Produced

    Design of T4-SARS-CoV-2 nanovaccine by CRISPR engineering. Engineered DNAs corresponding to various components of SARS-CoV-2 virion are incorporated into bacteriophage T4 genome. Each DNA was introduced into E. coli as a donor plasmid (a) , recombined into injected phage genome through CRISPR-targeted genome editing (b) . Different combinations of CoV-2 inserts were then generated by simple phage infections and identifying the recombinant phages in the progeny (c) . For example, recombinant phage containing CoV-2 insert #1 (dark blue) can be used to infect CRISPR E. coli containing Co-V2 insert containing donor plasmid #2 (dark red). The progeny plaques obtained will contain recombinant phage #3 with both inserts #1 and #2 (dark blue plus dark red) in the same genome. This process was repeated to rapidly construct a pipeline of multiplex T4-SARS-CoV-2 vaccine phages (d) . Selected vaccine candidates were then screened in a mouse model (e) to identify the most potent vaccine (f) . Structural model of T4-SARS-CoV-2 Nanovaccine showing an enlarged view of a single hexameric capsomer (g) . The capsomer shows six subunits of major capsid protein gp23* (green), trimers of Soc (blue), and a Hoc fiber (yellow) at the center of capsomer. The expressible spike genes are inserted into phage genome, the 12 aa E external peptide (red) is displayed at the tip of Hoc fiber, S-trimers (cyan) are attached to Soc subunits, and nucleocapsid proteins (yellow) are packaged in genome core. See Results , Materials and Methods, and Supplementary Video for additional details.
    Figure Legend Snippet: Design of T4-SARS-CoV-2 nanovaccine by CRISPR engineering. Engineered DNAs corresponding to various components of SARS-CoV-2 virion are incorporated into bacteriophage T4 genome. Each DNA was introduced into E. coli as a donor plasmid (a) , recombined into injected phage genome through CRISPR-targeted genome editing (b) . Different combinations of CoV-2 inserts were then generated by simple phage infections and identifying the recombinant phages in the progeny (c) . For example, recombinant phage containing CoV-2 insert #1 (dark blue) can be used to infect CRISPR E. coli containing Co-V2 insert containing donor plasmid #2 (dark red). The progeny plaques obtained will contain recombinant phage #3 with both inserts #1 and #2 (dark blue plus dark red) in the same genome. This process was repeated to rapidly construct a pipeline of multiplex T4-SARS-CoV-2 vaccine phages (d) . Selected vaccine candidates were then screened in a mouse model (e) to identify the most potent vaccine (f) . Structural model of T4-SARS-CoV-2 Nanovaccine showing an enlarged view of a single hexameric capsomer (g) . The capsomer shows six subunits of major capsid protein gp23* (green), trimers of Soc (blue), and a Hoc fiber (yellow) at the center of capsomer. The expressible spike genes are inserted into phage genome, the 12 aa E external peptide (red) is displayed at the tip of Hoc fiber, S-trimers (cyan) are attached to Soc subunits, and nucleocapsid proteins (yellow) are packaged in genome core. See Results , Materials and Methods, and Supplementary Video for additional details.

    Techniques Used: CRISPR, Plasmid Preparation, Injection, Generated, Recombinant, Construct, Multiplex Assay

    Construction of T4-SARS-CoV-2 recombinant phages by CRISPR engineering. a. Schematic of T4 CRISPR engineering. b. Four nonessential regions of T4 genome are chosen for deletion and insertion of various SARS-CoV-2 genes (shown in red; SegF/Soc, FarP, IP, and Hoc). 6P, six proline substitutions in S-ecto (F817P, A892P, A899P, A942P, K986P, and V987P). Fol, T4 fibritin motif Foldon for efficient trimerization. Tag, octa-histidine and twin-strep tags. Furin cleavage site RRAR was mutated to GSAS to stabilize trimers in a prefusion state 31 . c. Efficiency of plating (EOP) of representative Cpf1-FarP7K and Cpf1-SegF spacers. d. Plate showing plaques from phage infection of bacteria containing Cpf1-FarP7K spacer only, S-ecto donor only, or Cpf1-FarP7K spacer plus S-ecto donor. e. Recombination frequency of three spike gene (RBD, S-ecto, and S-fl) insertions. f. DNA sequencing of thirty independent plaques showed that > 95% of the plaques generated in S-ecto recombination contained the correct S-ecto insert. g. Plate showing that the wild-type (WT), T4-RBD, T4-S-fl, T4-S-ecto, and T4-(S-ecto)-RBD recombinant phages had similar plaque size.
    Figure Legend Snippet: Construction of T4-SARS-CoV-2 recombinant phages by CRISPR engineering. a. Schematic of T4 CRISPR engineering. b. Four nonessential regions of T4 genome are chosen for deletion and insertion of various SARS-CoV-2 genes (shown in red; SegF/Soc, FarP, IP, and Hoc). 6P, six proline substitutions in S-ecto (F817P, A892P, A899P, A942P, K986P, and V987P). Fol, T4 fibritin motif Foldon for efficient trimerization. Tag, octa-histidine and twin-strep tags. Furin cleavage site RRAR was mutated to GSAS to stabilize trimers in a prefusion state 31 . c. Efficiency of plating (EOP) of representative Cpf1-FarP7K and Cpf1-SegF spacers. d. Plate showing plaques from phage infection of bacteria containing Cpf1-FarP7K spacer only, S-ecto donor only, or Cpf1-FarP7K spacer plus S-ecto donor. e. Recombination frequency of three spike gene (RBD, S-ecto, and S-fl) insertions. f. DNA sequencing of thirty independent plaques showed that > 95% of the plaques generated in S-ecto recombination contained the correct S-ecto insert. g. Plate showing that the wild-type (WT), T4-RBD, T4-S-fl, T4-S-ecto, and T4-(S-ecto)-RBD recombinant phages had similar plaque size.

    Techniques Used: Recombinant, CRISPR, Infection, DNA Sequencing, Generated

    CRISPR engineering of non-essential T4 genome. a. Schematic showing the 18-kb nonessential segment FarP and 11-kb nonessential segment 39-56 on T4 genome. b. Plaque size of wild-type (WT), T4- FarP 18 kb del. , T4- 39-56 11 kb del. , and T4- FarP 39-56 29 kb del. phages. Note the small size of T4- 39-56 11 kb del. and T4- FarP 39-56 29 kb del. plaques. c. Structural models of SARS-CoV-2 virus, spike trimer, and receptor binding domain (RBD). d. Schematics of S-full length (S-fl) and S-ectodomain (S-ecto) expression cassettes used for insertion into T4 genome. e. Efficiency of plating of three sets of Cpf1-FarP7K spacers and three sets of Cpf1-SegF spacers. f. Efficiency of plating of various spacers used for T4 genome engineering in this study.
    Figure Legend Snippet: CRISPR engineering of non-essential T4 genome. a. Schematic showing the 18-kb nonessential segment FarP and 11-kb nonessential segment 39-56 on T4 genome. b. Plaque size of wild-type (WT), T4- FarP 18 kb del. , T4- 39-56 11 kb del. , and T4- FarP 39-56 29 kb del. phages. Note the small size of T4- 39-56 11 kb del. and T4- FarP 39-56 29 kb del. plaques. c. Structural models of SARS-CoV-2 virus, spike trimer, and receptor binding domain (RBD). d. Schematics of S-full length (S-fl) and S-ectodomain (S-ecto) expression cassettes used for insertion into T4 genome. e. Efficiency of plating of three sets of Cpf1-FarP7K spacers and three sets of Cpf1-SegF spacers. f. Efficiency of plating of various spacers used for T4 genome engineering in this study.

    Techniques Used: CRISPR, Binding Assay, Expressing

    9) Product Images from "Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma"

    Article Title: Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma

    Journal: bioRxiv

    doi: 10.1101/2020.12.20.423708

    Genetic basis of a shared, or public, class of IGHV1-24 plasma antibodies targeting the spike NTD. a , IGHV usage of plasma anti-S-ECD or anti-RBD antibodies in all subjects (n=4). b , IGHV1-24 antibodies as a percentage of the circulating IgG plasma antibody repertoire: reactivity to SARS-CoV-2 spike S-ECD or RBD in COVID-19 subjects, or reactivity in healthy subjects to vaccine spike antigens for either respiratory syncytial virus (RSV) or trivalent influenza vaccine hemagglutinin HA1 (TIV). ** p
    Figure Legend Snippet: Genetic basis of a shared, or public, class of IGHV1-24 plasma antibodies targeting the spike NTD. a , IGHV usage of plasma anti-S-ECD or anti-RBD antibodies in all subjects (n=4). b , IGHV1-24 antibodies as a percentage of the circulating IgG plasma antibody repertoire: reactivity to SARS-CoV-2 spike S-ECD or RBD in COVID-19 subjects, or reactivity in healthy subjects to vaccine spike antigens for either respiratory syncytial virus (RSV) or trivalent influenza vaccine hemagglutinin HA1 (TIV). ** p

    Techniques Used:

    Protective spike NTD-targeting antibodies are prevalent in the plasma of convalescent COVID-19 study subjects. a , Temporal dynamics of the anti-S-ECD IgG repertoire at days 12 and 56 post-symptom onset. Titer had increased two-fold by day 56 (data not shown). b , Biolayer interferometry (BLI) sensorgrams to S-ECD ligand of anti-NTD mAbs CM17 and CM25 (subject P2), CM58 (subject P4), and anti-RBD control mAb S309 35 . c , In vitro live virus neutralization. d–f , Prophylactic protection of 12 m.o. BALB/C mice against intranasal challenge with 10 4 PFU of mouse-adapted (MA10) SARS-CoV-2. In vivo prophylactic protection to MA10 challenge; experimental conditions as in Fig 1f,g except challenge dose was 10 5 PFU. *** p
    Figure Legend Snippet: Protective spike NTD-targeting antibodies are prevalent in the plasma of convalescent COVID-19 study subjects. a , Temporal dynamics of the anti-S-ECD IgG repertoire at days 12 and 56 post-symptom onset. Titer had increased two-fold by day 56 (data not shown). b , Biolayer interferometry (BLI) sensorgrams to S-ECD ligand of anti-NTD mAbs CM17 and CM25 (subject P2), CM58 (subject P4), and anti-RBD control mAb S309 35 . c , In vitro live virus neutralization. d–f , Prophylactic protection of 12 m.o. BALB/C mice against intranasal challenge with 10 4 PFU of mouse-adapted (MA10) SARS-CoV-2. In vivo prophylactic protection to MA10 challenge; experimental conditions as in Fig 1f,g except challenge dose was 10 5 PFU. *** p

    Techniques Used: In Vitro, Neutralization, Mouse Assay, In Vivo

    Ig-seq plasma IgG lineage profiles of study subjects at early and late convalescent time points. On the left, the first time point Ig-seq profile (days 11-19) for each subject (subject P3 found in Fig.1 ) shows both the SARS-CoV-2 spike ECD (S-ECD) and RBD abundance for each plasma IgG lineage detected at > 0.5% anti-S-ECD plasma IgG (summed lineage XIC). Similarly, on the right, the second time point data for S-ECD (days 45-56) is shown for each lineage detected at > 0.5% S-ECD plasma IgG abundance (time point 2), alongside early time point S-ECD data for comparison (subject P2 found in Fig.2 ).
    Figure Legend Snippet: Ig-seq plasma IgG lineage profiles of study subjects at early and late convalescent time points. On the left, the first time point Ig-seq profile (days 11-19) for each subject (subject P3 found in Fig.1 ) shows both the SARS-CoV-2 spike ECD (S-ECD) and RBD abundance for each plasma IgG lineage detected at > 0.5% anti-S-ECD plasma IgG (summed lineage XIC). Similarly, on the right, the second time point data for S-ECD (days 45-56) is shown for each lineage detected at > 0.5% S-ECD plasma IgG abundance (time point 2), alongside early time point S-ECD data for comparison (subject P2 found in Fig.2 ).

    Techniques Used:

    Independent live virus neutralization titers of recombinant plasma IgG mAbs CM17, CM25, and CM32. In vitro live virus neutralization curves for CM17, CM25, and CM32 repeated in second independent laboratory demonstrate similar levels of inhibition (as compared to data in Fig.1e and 2c) of live SARS-CoV-2 virus infection of monolayered Vero E6 cells. The percent of infected Vero E6 cells in each sample dilution was normalized relative to the virus-only (no plasma) negative control sample.
    Figure Legend Snippet: Independent live virus neutralization titers of recombinant plasma IgG mAbs CM17, CM25, and CM32. In vitro live virus neutralization curves for CM17, CM25, and CM32 repeated in second independent laboratory demonstrate similar levels of inhibition (as compared to data in Fig.1e and 2c) of live SARS-CoV-2 virus infection of monolayered Vero E6 cells. The percent of infected Vero E6 cells in each sample dilution was normalized relative to the virus-only (no plasma) negative control sample.

    Techniques Used: Neutralization, Recombinant, In Vitro, Inhibition, Infection, Negative Control

    A single spike NTD-targeting IgG antibody in plasma can confer protection without a need for RBD-directed activity. a , Polyclonal IgG plasma antibodies were affinity purified using stabilized spike S-2P S-ECD 2 or RBD, and binding specificity was mapped using purified S subdomains; anti-RBD (green); anti-S2 (blue); anti-NTD (red). b ,The majority of the plasma anti-S-ECD response is directed to non-RBD epitopes: Binding (1:150 plasma dilution) to S-ECD alone, or in the presence of 50 µg/mL (∼1.7 µM) RBD, or S-ΔRBD deletion mutant. c , Quantitative determination of plasma anti-RBD and non-RBD antibody abundance in early convalescence. Abundance normalized to the entire anti-S-ECD plasma IgG repertoire. d , Composition, binding specificity and relative abundance of antibodies in early convalescent plasma (subject P3). e , Authentic virus neutralization of the four topmost abundant plasma IgGs (CM29, CM30, CM31, CM32) from plasma lineages Lin.1, Lin.2, Lin.3, Lin.4 in 1d that account for > 90% of the plasma anti-S-ECD repertoire. f , g Prophylactic protection of 12 m.o. BALB/C mice against lethal challenge with 10 4 PFU mouse-adapted (MA10) SARS-CoV-2 using 200µg/mouse of non-RBD mAbs CM29, CM30, and CM31. Antibody cocktail (200 µg/mouse) consisted of 2:1:1 ratio of CM29, CM30, CM31, reflecting their relative plasma abundance ( 1d ). ** p
    Figure Legend Snippet: A single spike NTD-targeting IgG antibody in plasma can confer protection without a need for RBD-directed activity. a , Polyclonal IgG plasma antibodies were affinity purified using stabilized spike S-2P S-ECD 2 or RBD, and binding specificity was mapped using purified S subdomains; anti-RBD (green); anti-S2 (blue); anti-NTD (red). b ,The majority of the plasma anti-S-ECD response is directed to non-RBD epitopes: Binding (1:150 plasma dilution) to S-ECD alone, or in the presence of 50 µg/mL (∼1.7 µM) RBD, or S-ΔRBD deletion mutant. c , Quantitative determination of plasma anti-RBD and non-RBD antibody abundance in early convalescence. Abundance normalized to the entire anti-S-ECD plasma IgG repertoire. d , Composition, binding specificity and relative abundance of antibodies in early convalescent plasma (subject P3). e , Authentic virus neutralization of the four topmost abundant plasma IgGs (CM29, CM30, CM31, CM32) from plasma lineages Lin.1, Lin.2, Lin.3, Lin.4 in 1d that account for > 90% of the plasma anti-S-ECD repertoire. f , g Prophylactic protection of 12 m.o. BALB/C mice against lethal challenge with 10 4 PFU mouse-adapted (MA10) SARS-CoV-2 using 200µg/mouse of non-RBD mAbs CM29, CM30, and CM31. Antibody cocktail (200 µg/mouse) consisted of 2:1:1 ratio of CM29, CM30, CM31, reflecting their relative plasma abundance ( 1d ). ** p

    Techniques Used: Activity Assay, Affinity Purification, Binding Assay, Purification, Mutagenesis, Neutralization, Mouse Assay

    Live virus neutralization titers of four COVID+ study subjects’ plasma at each collection time point. Serial dilutions of plasma were tested in duplicate (SD error bars) for inhibition of live SARS-CoV-2 virus infection of in vitro monolayered Vero E6 cells. The percent of infected Vero E6 cells in each sample dilution was normalized relative to the virus-only (no plasma) negative control sample.
    Figure Legend Snippet: Live virus neutralization titers of four COVID+ study subjects’ plasma at each collection time point. Serial dilutions of plasma were tested in duplicate (SD error bars) for inhibition of live SARS-CoV-2 virus infection of in vitro monolayered Vero E6 cells. The percent of infected Vero E6 cells in each sample dilution was normalized relative to the virus-only (no plasma) negative control sample.

    Techniques Used: Neutralization, Inhibition, Infection, In Vitro, Negative Control

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

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

    Journal: bioRxiv

    doi: 10.1101/2021.01.29.428890

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

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

    11) 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

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.09.08.280818

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

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

    13) Product Images from "Transmission and protection against re-infection in the ferret model with the SARS-CoV-2 USA-WA1/2020 reference isolate"

    Article Title: Transmission and protection against re-infection in the ferret model with the SARS-CoV-2 USA-WA1/2020 reference isolate

    Journal: bioRxiv

    doi: 10.1101/2020.11.20.392381

    Antibody and viral titers in SARS-CoV-2 infected mock and RBD vaccinated ferrets. Panels A displays binding antibody titers against the S-protein RBD determined by ELISA on days 0, 14, 28, 42, and 56 post-primary vaccination. Red open symbols represent RBD vaccinated ferrets. Closed black symbols represent mock vaccinated animals. Animals were given a secondary vaccination on day 28. Panel B displays neutralizing antibody titers on day 56. Panel C and D display nasal wash titers in mock and RBD vaccinated animals challenged with SARS-CoV-2, respectively. Line graphs indicate levels of vRNA determined via N2 gene qRT-PCR (left Y-axis) and bar graphs indicated infectious titers (right Y-axis) determined via TCID50 on Vero cells. Horizontal dashed line indicates limit of detection.
    Figure Legend Snippet: Antibody and viral titers in SARS-CoV-2 infected mock and RBD vaccinated ferrets. Panels A displays binding antibody titers against the S-protein RBD determined by ELISA on days 0, 14, 28, 42, and 56 post-primary vaccination. Red open symbols represent RBD vaccinated ferrets. Closed black symbols represent mock vaccinated animals. Animals were given a secondary vaccination on day 28. Panel B displays neutralizing antibody titers on day 56. Panel C and D display nasal wash titers in mock and RBD vaccinated animals challenged with SARS-CoV-2, respectively. Line graphs indicate levels of vRNA determined via N2 gene qRT-PCR (left Y-axis) and bar graphs indicated infectious titers (right Y-axis) determined via TCID50 on Vero cells. Horizontal dashed line indicates limit of detection.

    Techniques Used: Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    Direct contact and respiratory transmission of the SARS-CoV-2 USA-WA1/2020 isolate in ferrets. Panels A-C, D-F, and G-I display three separate transmission studies. Panels A-C, and D-F each represent a contact transmission study, while panels G-I display data from a respiratory transmission study. Panels A, D, G display nasal wash titers determined by qRT-PCR (left Y-axis) and TCID50 (right Y-axis) for the SARS-CoV-2 inoculated donor animals in each experiment. Line graphs indicate levels of vRNA and bar graphs indicated infectious titers. Panels B, E, and H similarly display nasal wash titers for contact animals. In a given panel, each shaded bar or symbol represents the same animal sampled over multiple time points. Paired donor and contact animals have the same shaded bar or symbol between panels. Panels C, F, and I show neutralizing antibody titers for each donor and contact animal. For all experiments, 4 pairs of ferrets (2 pairs of males and 2 pairs of females) were used, and nasal wash samples were collected every other day. Blood was collected on day 21 post-contact. In the first direct contact transmission study, panels A-C, one direct contact animal was removed due to fighting with its cage mate. Horizontal dashed line indicates limit of detection.
    Figure Legend Snippet: Direct contact and respiratory transmission of the SARS-CoV-2 USA-WA1/2020 isolate in ferrets. Panels A-C, D-F, and G-I display three separate transmission studies. Panels A-C, and D-F each represent a contact transmission study, while panels G-I display data from a respiratory transmission study. Panels A, D, G display nasal wash titers determined by qRT-PCR (left Y-axis) and TCID50 (right Y-axis) for the SARS-CoV-2 inoculated donor animals in each experiment. Line graphs indicate levels of vRNA and bar graphs indicated infectious titers. Panels B, E, and H similarly display nasal wash titers for contact animals. In a given panel, each shaded bar or symbol represents the same animal sampled over multiple time points. Paired donor and contact animals have the same shaded bar or symbol between panels. Panels C, F, and I show neutralizing antibody titers for each donor and contact animal. For all experiments, 4 pairs of ferrets (2 pairs of males and 2 pairs of females) were used, and nasal wash samples were collected every other day. Blood was collected on day 21 post-contact. In the first direct contact transmission study, panels A-C, one direct contact animal was removed due to fighting with its cage mate. Horizontal dashed line indicates limit of detection.

    Techniques Used: Transmission Assay, Quantitative RT-PCR

    Viral and antibody titers in ferrets re-challenged with SARS-CoV-2 on day 28 and 56 post-primary infection. Panel A and B display nasal wash titers in ferrets re-challenged with SARS-CoV-2 on days 28 and 56 post-primary infection, respectively. Line graphs indicate levels of vRNA determined via N2 gene qRT-PCR (left Y-axis) and bar graphs indicated infectious titers (right Y-axis) determined via TCID50 on Vero cells. Panel C displays neutralizing antibody titers prior to primary infection (day 0), at the time of re-challenge (day 28 or 56) and 14 days post re-challenge (days 42 and 70). Horizontal dashed line indicates limit of detection.
    Figure Legend Snippet: Viral and antibody titers in ferrets re-challenged with SARS-CoV-2 on day 28 and 56 post-primary infection. Panel A and B display nasal wash titers in ferrets re-challenged with SARS-CoV-2 on days 28 and 56 post-primary infection, respectively. Line graphs indicate levels of vRNA determined via N2 gene qRT-PCR (left Y-axis) and bar graphs indicated infectious titers (right Y-axis) determined via TCID50 on Vero cells. Panel C displays neutralizing antibody titers prior to primary infection (day 0), at the time of re-challenge (day 28 or 56) and 14 days post re-challenge (days 42 and 70). Horizontal dashed line indicates limit of detection.

    Techniques Used: Infection, Quantitative RT-PCR

    14) Product Images from "Immunization with the receptor–binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement"

    Article Title: Immunization with the receptor–binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement

    Journal: bioRxiv

    doi: 10.1101/2020.05.21.107565

    Treatment with the anti-RBD-Fc sera #1 inhibited SARS-CoV-2 infection-triggered CPE. VeroE6 cells were inoculated with mixtures of authentic SARS-CoV-2 and serially diluted anti-RBD-Fc sera #1. The cells were checked daily for CPE. Data presented are images taken at 48 hours post-infection. The test concentrations of the anti-RBD-Fc sera #1 are indicated.
    Figure Legend Snippet: Treatment with the anti-RBD-Fc sera #1 inhibited SARS-CoV-2 infection-triggered CPE. VeroE6 cells were inoculated with mixtures of authentic SARS-CoV-2 and serially diluted anti-RBD-Fc sera #1. The cells were checked daily for CPE. Data presented are images taken at 48 hours post-infection. The test concentrations of the anti-RBD-Fc sera #1 are indicated.

    Techniques Used: Infection

    Neutralization potency and breadth of the anti-RBD sera. ( A ) Anti-RBD sera neutralized SARS2-PV infection in vitro. The day-40 pooled sera were serially diluted and tested for neutralization of retrovirus pseudotyped with SARS-CoV-2 S protein. Data (means±SD) from three independent experiments are shown. ( B ) Anti-RBD sera cross-neutralized SARS-PV infection in vitro. The day-40 pooled sera were serially diluted and tested for neutralization of retrovirus pseudotyped with SARS-CoV S protein. Data (means±SD) from three independent experiments are shown. ( C ) Neutralization efficiency of the anti-RBD sera against authentic SARS-CoV-2 infection. Serially diluted anti-RBD sera were mixed with 200 PFU of live SARS-CoV-2 and then incubated for 1 hr at 37°C. The antisera/virus mixtures were added to pre-seeded VeroE6 cells, followed by incubation for three days. The cells were then analyzed for viral RNA copy number by qPCR analysis. Data are expressed as percentage of the viral RNA copy number of the treatment groups in relation to that of the virus-only control. Means ± SD of triplicate wells are shown. Significant differences between treatment groups and the virus-only control were calculated using student’s two-tail t test and shown as: ***, P
    Figure Legend Snippet: Neutralization potency and breadth of the anti-RBD sera. ( A ) Anti-RBD sera neutralized SARS2-PV infection in vitro. The day-40 pooled sera were serially diluted and tested for neutralization of retrovirus pseudotyped with SARS-CoV-2 S protein. Data (means±SD) from three independent experiments are shown. ( B ) Anti-RBD sera cross-neutralized SARS-PV infection in vitro. The day-40 pooled sera were serially diluted and tested for neutralization of retrovirus pseudotyped with SARS-CoV S protein. Data (means±SD) from three independent experiments are shown. ( C ) Neutralization efficiency of the anti-RBD sera against authentic SARS-CoV-2 infection. Serially diluted anti-RBD sera were mixed with 200 PFU of live SARS-CoV-2 and then incubated for 1 hr at 37°C. The antisera/virus mixtures were added to pre-seeded VeroE6 cells, followed by incubation for three days. The cells were then analyzed for viral RNA copy number by qPCR analysis. Data are expressed as percentage of the viral RNA copy number of the treatment groups in relation to that of the virus-only control. Means ± SD of triplicate wells are shown. Significant differences between treatment groups and the virus-only control were calculated using student’s two-tail t test and shown as: ***, P

    Techniques Used: Neutralization, Infection, In Vitro, Incubation, Real-time Polymerase Chain Reaction

    Assessment of the anti-RBD sera for potential ADE. ( A-C ) ADE assays with SARS2-PV as the inoculum. Serial dilutions of the anti-RBD or the control sera were incubated with SARS2-S pseudotyped retrovirus for 1 hour at 37 °C. The mixtures were added to ( A ) A20, ( B ) THP-1, or ( C ) K562 cell suspensions, followed by incubation at 37°C for three days. Infected cells were subjected to flow cytometry analysis. Data are expressed as percentage of the GFP-expressing cells in relation to the total cells counted. Means ± SD of triplicate wells are shown. ( D ) ADE assay with live SARS-CoV-2 virus as the inoculum. Serial dilutions of the anti-RBD or the control sera were mixed with the live SARS-CoV-2 virus and incubated for 1 hour at 37 °C. The mixtures were added to K562 cell suspensions, followed by incubation at 37 °C for three days. Infected cell cultures were subjected to RNA extraction and qPCR analysis. Data are expressed as percentage of the viral RNA copy number of the treatment groups in relation to that of the virus-only control. Means ± SD of triplicate wells are shown. Significant differences between the virus only (without antisera treatment) group and each of the antisera treatment groups were indicated: n.s., P > 0.05.
    Figure Legend Snippet: Assessment of the anti-RBD sera for potential ADE. ( A-C ) ADE assays with SARS2-PV as the inoculum. Serial dilutions of the anti-RBD or the control sera were incubated with SARS2-S pseudotyped retrovirus for 1 hour at 37 °C. The mixtures were added to ( A ) A20, ( B ) THP-1, or ( C ) K562 cell suspensions, followed by incubation at 37°C for three days. Infected cells were subjected to flow cytometry analysis. Data are expressed as percentage of the GFP-expressing cells in relation to the total cells counted. Means ± SD of triplicate wells are shown. ( D ) ADE assay with live SARS-CoV-2 virus as the inoculum. Serial dilutions of the anti-RBD or the control sera were mixed with the live SARS-CoV-2 virus and incubated for 1 hour at 37 °C. The mixtures were added to K562 cell suspensions, followed by incubation at 37 °C for three days. Infected cell cultures were subjected to RNA extraction and qPCR analysis. Data are expressed as percentage of the viral RNA copy number of the treatment groups in relation to that of the virus-only control. Means ± SD of triplicate wells are shown. Significant differences between the virus only (without antisera treatment) group and each of the antisera treatment groups were indicated: n.s., P > 0.05.

    Techniques Used: Incubation, Infection, Flow Cytometry, Expressing, RNA Extraction, Real-time Polymerase Chain Reaction

    Treatment with the anti-RBD sera inhibited SARS-CoV-2 infection-triggered CPE. VeroE6 cells were inoculated with mixtures of the authentic SARS-CoV-2 virus and serially diluted anti-RBD sera. The cells were checked daily for CPE. Data presented are images taken at 48 hours post-infection. The test concentrations of the anti-RBD sera are indicated.
    Figure Legend Snippet: Treatment with the anti-RBD sera inhibited SARS-CoV-2 infection-triggered CPE. VeroE6 cells were inoculated with mixtures of the authentic SARS-CoV-2 virus and serially diluted anti-RBD sera. The cells were checked daily for CPE. Data presented are images taken at 48 hours post-infection. The test concentrations of the anti-RBD sera are indicated.

    Techniques Used: Infection

    Immunization with recombinant RBD-Fc fusion protein potently elicited SARS-CoV-2 neutralizing antibodies in mice. ( A ) RBD-binding activities of the sera from the three RBD-Fc-immunized mice and the control (naïve) mouse. The sera were serially diluted and then analyzed by ELISA with recombinant SARS2-RBD protein as the coating antigen. Data shown are means and SD of triplicate wells. ( B ) Inhibitory effect of the anti-RBD-Fc sera on the RBD/ACE2 interaction. The anti-RBD-Fc sera #1 and the control sera were serially diluted and then subjected to ACE2 competition ELISA. Data shown are means and SD of triplicate wells. ( C ) Neutralization potency of the antisera against SARS-CoV-2 pseudovirus infection. The antisera were serially diluted and then evaluated for neutralization of SARS-CoV-2 spike-pseudotyped retrovirus. Results from three independent experiments are shown. ( D ) Neutralization potency of the antisera against authentic SARS-CoV-2 infection. Serially diluted antisera were subjected to neutralization assay with SARS-CoV-2 strain nCoV-SH01 as the challenge virus. Data shown are means and SD of triplicate wells. Significant differences were calculated using student’s two-tail t test and shown as: ***, P
    Figure Legend Snippet: Immunization with recombinant RBD-Fc fusion protein potently elicited SARS-CoV-2 neutralizing antibodies in mice. ( A ) RBD-binding activities of the sera from the three RBD-Fc-immunized mice and the control (naïve) mouse. The sera were serially diluted and then analyzed by ELISA with recombinant SARS2-RBD protein as the coating antigen. Data shown are means and SD of triplicate wells. ( B ) Inhibitory effect of the anti-RBD-Fc sera on the RBD/ACE2 interaction. The anti-RBD-Fc sera #1 and the control sera were serially diluted and then subjected to ACE2 competition ELISA. Data shown are means and SD of triplicate wells. ( C ) Neutralization potency of the antisera against SARS-CoV-2 pseudovirus infection. The antisera were serially diluted and then evaluated for neutralization of SARS-CoV-2 spike-pseudotyped retrovirus. Results from three independent experiments are shown. ( D ) Neutralization potency of the antisera against authentic SARS-CoV-2 infection. Serially diluted antisera were subjected to neutralization assay with SARS-CoV-2 strain nCoV-SH01 as the challenge virus. Data shown are means and SD of triplicate wells. Significant differences were calculated using student’s two-tail t test and shown as: ***, P

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

    15) Product Images from "Presence of antibodies against SARS-CoV-2 spike protein in bovine whey IgG enriched fraction"

    Article Title: Presence of antibodies against SARS-CoV-2 spike protein in bovine whey IgG enriched fraction

    Journal: International Dairy Journal

    doi: 10.1016/j.idairyj.2021.105002

    Bovine IgG enriched fraction containing IgG against SARS-CoV-2 assessed by direct enzyme-linked immunosorbent assays (ELISA) using a partial-length of recombinant SARS-CoV-2 S (aa 177–512, 288–512, 348–578, 387–516 and 408–664), full-recombinant SARS-CoV-2 N (aa 1-419) and partial-length of recombinant SARS-CoV-2 N (aa 1–120, 111–220, 1–220 and 210–419) ( Fig.1 ) as coating antigens. Two different lots of bovine IgG enriched fraction prepared in 2019 and 2018 were used (2a and 2b, respectively): Image 1 , 0.003 μg mL -1 ; Image 2 , 0.03 μg mL -1 ; Image 3 , 0.3 μg mL -1 ; Image 4 , 3 μg mL -1 ; Image 5 , 30 μg mL -1 . A picture of a representative ELISA result is shown in 2c.
    Figure Legend Snippet: Bovine IgG enriched fraction containing IgG against SARS-CoV-2 assessed by direct enzyme-linked immunosorbent assays (ELISA) using a partial-length of recombinant SARS-CoV-2 S (aa 177–512, 288–512, 348–578, 387–516 and 408–664), full-recombinant SARS-CoV-2 N (aa 1-419) and partial-length of recombinant SARS-CoV-2 N (aa 1–120, 111–220, 1–220 and 210–419) ( Fig.1 ) as coating antigens. Two different lots of bovine IgG enriched fraction prepared in 2019 and 2018 were used (2a and 2b, respectively): Image 1 , 0.003 μg mL -1 ; Image 2 , 0.03 μg mL -1 ; Image 3 , 0.3 μg mL -1 ; Image 4 , 3 μg mL -1 ; Image 5 , 30 μg mL -1 . A picture of a representative ELISA result is shown in 2c.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Recombinant

    Competitive inhibition ELISA of the bovine IgG enriched fraction (IgG 0.3 μg mL -1 ), incubated with one of three peptides of S protein of SARS-CoV-2 ( Image 7 , aa 382–401; Image 8 , aa 427–446; Image 9 , aa 502–520) at concentrations 0.001, 0.01, 0,1, 1, and 10 μg mL -1 , the remaining free IgG against the S protein of SARS-CoV-2 was assayed by direct ELISA, using plates coated with the peptide corresponding to aa 288–512 of S protein of SARS-CoV-2. Two lots of bovine IgG enriched fraction prepared in 2019 ( Image 10 ) and 2018 ( Image 11 ) were tested.
    Figure Legend Snippet: Competitive inhibition ELISA of the bovine IgG enriched fraction (IgG 0.3 μg mL -1 ), incubated with one of three peptides of S protein of SARS-CoV-2 ( Image 7 , aa 382–401; Image 8 , aa 427–446; Image 9 , aa 502–520) at concentrations 0.001, 0.01, 0,1, 1, and 10 μg mL -1 , the remaining free IgG against the S protein of SARS-CoV-2 was assayed by direct ELISA, using plates coated with the peptide corresponding to aa 288–512 of S protein of SARS-CoV-2. Two lots of bovine IgG enriched fraction prepared in 2019 ( Image 10 ) and 2018 ( Image 11 ) were tested.

    Techniques Used: Inhibition, Enzyme-linked Immunosorbent Assay, Incubation, Direct ELISA

    Determination of epitopes by direct ELISA using nine peptides of SARS-CoV-2 S protein, corresponding to aa 382–401, 397–416, 427–446, 442–461, 457–476, 472–491, 487–506 and 502–520, with plates coated with a recombinant protein covering the RBD of SARS-CoV-2 S protein. Two lots of bovine IgG enriched fraction prepared in 2019 and 2018 (3a and 3b, respectively), were tested: Image 6 , 0.3 μg mL -1 ; Image 2 , 3 μg mL -1 ; Image 3 , 30 μg mL -1 .
    Figure Legend Snippet: Determination of epitopes by direct ELISA using nine peptides of SARS-CoV-2 S protein, corresponding to aa 382–401, 397–416, 427–446, 442–461, 457–476, 472–491, 487–506 and 502–520, with plates coated with a recombinant protein covering the RBD of SARS-CoV-2 S protein. Two lots of bovine IgG enriched fraction prepared in 2019 and 2018 (3a and 3b, respectively), were tested: Image 6 , 0.3 μg mL -1 ; Image 2 , 3 μg mL -1 ; Image 3 , 30 μg mL -1 .

    Techniques Used: Direct ELISA, Recombinant

    Overall topology of (a) SARS-CoV-2 spike protein (S) and five regions of recombinant SARS-CoV-2 S and (b) SARS-CoV-2 nucleocapsid protein (N) and regions of recombinant SARS-CoV-2 N. NTD, N-terminal domain; CTD, C-terminal domain; RBD, receptor binding domain; RDM, receptor binding motif; SD1, subdomain 1; SD2, subdomain 2; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane region; IC, intracellular domain.
    Figure Legend Snippet: Overall topology of (a) SARS-CoV-2 spike protein (S) and five regions of recombinant SARS-CoV-2 S and (b) SARS-CoV-2 nucleocapsid protein (N) and regions of recombinant SARS-CoV-2 N. NTD, N-terminal domain; CTD, C-terminal domain; RBD, receptor binding domain; RDM, receptor binding motif; SD1, subdomain 1; SD2, subdomain 2; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane region; IC, intracellular domain.

    Techniques Used: Recombinant, Binding Assay

    16) 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

    17) Product Images from "Testing-on-a-probe biosensors reveal association of early SARS-CoV-2 total antibodies and surrogate neutralizing antibodies with mortality in COVID-19 patients"

    Article Title: Testing-on-a-probe biosensors reveal association of early SARS-CoV-2 total antibodies and surrogate neutralizing antibodies with mortality in COVID-19 patients

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2021.113008

    Correlation of the TOP-SNAb assay with two well-established SARS-CoV-2 NAb assays . The correlations between the PsV and PRNT, between TOP-SNAb and PRNT, and between TOP-SNAb and PsV are shown in Fig. 2 A, B, C, respectively. The readout of TOP-SNAb is the percentage of RBD-ACE2 binding, which inversely correlates with the SNAb binding inhibition (neutralizing activity). The titers of PRNT and PsV were reported as PRNT 50 and IC50, respectively. The results were presented as Log10 scale. Correlations between two assays were assessed by Spearman correlation coefficient.
    Figure Legend Snippet: Correlation of the TOP-SNAb assay with two well-established SARS-CoV-2 NAb assays . The correlations between the PsV and PRNT, between TOP-SNAb and PRNT, and between TOP-SNAb and PsV are shown in Fig. 2 A, B, C, respectively. The readout of TOP-SNAb is the percentage of RBD-ACE2 binding, which inversely correlates with the SNAb binding inhibition (neutralizing activity). The titers of PRNT and PsV were reported as PRNT 50 and IC50, respectively. The results were presented as Log10 scale. Correlations between two assays were assessed by Spearman correlation coefficient.

    Techniques Used: Plaque Reduction Neutralization Test, Binding Assay, Inhibition, Activity Assay

    Survival probability among SARS-CoV-2 infected patients with positive and negative (A) TOP-TAb and (B) TOP-SNAb at initial hospital ED presentation . Data were analyzed using Cox proportional hazards regression adjusting for age and cancer comorbidity.
    Figure Legend Snippet: Survival probability among SARS-CoV-2 infected patients with positive and negative (A) TOP-TAb and (B) TOP-SNAb at initial hospital ED presentation . Data were analyzed using Cox proportional hazards regression adjusting for age and cancer comorbidity.

    Techniques Used: Infection

    TOP-TAb and TOP-SNAb at initial hospital ED presentation stratified by in-hospital mortality and viral load . (A) At the initial hospital ED presentation, TAb and SNAb positivity rates were higher in patients who survived than who died. (B) TAb level and (C) SNAb binding inhibition were higher in patients who survived than who died. Association of (D) TAb and (E) SNAb with SARS-Cov-2 ORF1a/b target C T values at the time of hospital ED presentation. Data were expressed as Log10 scale with box and whisker (10–90 percentile) plots.
    Figure Legend Snippet: TOP-TAb and TOP-SNAb at initial hospital ED presentation stratified by in-hospital mortality and viral load . (A) At the initial hospital ED presentation, TAb and SNAb positivity rates were higher in patients who survived than who died. (B) TAb level and (C) SNAb binding inhibition were higher in patients who survived than who died. Association of (D) TAb and (E) SNAb with SARS-Cov-2 ORF1a/b target C T values at the time of hospital ED presentation. Data were expressed as Log10 scale with box and whisker (10–90 percentile) plots.

    Techniques Used: Binding Assay, Inhibition, Whisker Assay

    Detection rates of SARS-CoV-2 TOP-TAb, TOP-SNAb, and Roche Tab . Detection rates were evaluated based on (A) days after initial ED visit (DAED) and (B) days after onset of symptoms (DAOS).
    Figure Legend Snippet: Detection rates of SARS-CoV-2 TOP-TAb, TOP-SNAb, and Roche Tab . Detection rates were evaluated based on (A) days after initial ED visit (DAED) and (B) days after onset of symptoms (DAOS).

    Techniques Used:

    Principles of fully automated, testing-on-a-probe (TOP) Total Antibody (TAb) and Surrogate Neutralizing Antibody (SNAb) Assays . The assay cartridge consists of an RBD pre-coated probe and preloaded reagent microwells. (A) TAb assay: The instrument sequentially transfers and incubates RBD pre-coated biosensor probe in a well with diluted sample to capture SARS-CoV-2 specific antibodies, a wash well, a biotinylated RBD well, a wash well, a Cy5-Streptavidin-polysacharide (Cy5-SA-PS) well, and a wash well. At the end, the probe is transferred to a well where the fluorescence bound on the tip of the biosensor is measured. (B) SNAb assay: the instrument sequentially transfers and incubates RBD pre-coated biosensor probe in a well containing a mixture of patient sample and biotinylated ACE2, a wash well, a Cy5-SA-PS well and a wash well. At the end, the biosensor probe is transferred to a read well where the fluorescence bound on the biosensor tip is measured. (C and D) Assay sensitivity enhancement by conjugation of Cy5-SA to a high molecular weight PS. Samples of SARS-CoV-2 negative human serum spiked with monoclonal SARS-CoV-2 IgG (C) or IgM (D) at different concentrations were measured on the TOP-TAb assay with Cy5-SA or Cy5-SA-PS as the signaling element. The Cy5-SA-PS showed enhanced signal sensitive by up to 20-fold and reduced background noise by 3-fold compared to Cy5-SA.
    Figure Legend Snippet: Principles of fully automated, testing-on-a-probe (TOP) Total Antibody (TAb) and Surrogate Neutralizing Antibody (SNAb) Assays . The assay cartridge consists of an RBD pre-coated probe and preloaded reagent microwells. (A) TAb assay: The instrument sequentially transfers and incubates RBD pre-coated biosensor probe in a well with diluted sample to capture SARS-CoV-2 specific antibodies, a wash well, a biotinylated RBD well, a wash well, a Cy5-Streptavidin-polysacharide (Cy5-SA-PS) well, and a wash well. At the end, the probe is transferred to a well where the fluorescence bound on the tip of the biosensor is measured. (B) SNAb assay: the instrument sequentially transfers and incubates RBD pre-coated biosensor probe in a well containing a mixture of patient sample and biotinylated ACE2, a wash well, a Cy5-SA-PS well and a wash well. At the end, the biosensor probe is transferred to a read well where the fluorescence bound on the biosensor tip is measured. (C and D) Assay sensitivity enhancement by conjugation of Cy5-SA to a high molecular weight PS. Samples of SARS-CoV-2 negative human serum spiked with monoclonal SARS-CoV-2 IgG (C) or IgM (D) at different concentrations were measured on the TOP-TAb assay with Cy5-SA or Cy5-SA-PS as the signaling element. The Cy5-SA-PS showed enhanced signal sensitive by up to 20-fold and reduced background noise by 3-fold compared to Cy5-SA.

    Techniques Used: Fluorescence, Conjugation Assay, Molecular Weight

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

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

    Journal: Cell

    doi: 10.1016/j.cell.2020.09.033

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

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

    ACE2 and Cellular Heparan Sulfate Are Both Necessary for Binding of SARS-CoV-2 Spike Ectodomain (A) Western blot shows overexpression of ACE2 in the A375 and A375 B4GALT7 −/− cells. A representative blot is shown. (B) Binding of SARS-CoV-2 spike protein to cells with and without ACE2 overexpression. Note that binding is reduced in the cells deficient in HS. (C) Gene targeting of ACE2 in A549 using CRISPR-Cas9. The bars show spike binding to two independent ACE2 CRISPR-Cas9 knockout clones with and without HSase treatment. Note that binding depends on ACE2 expression and that residual binding depends in part on HS. All analyses were done by flow cytometry. The graphs show representative experiments performed in triplicate technical replicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S5 .
    Figure Legend Snippet: ACE2 and Cellular Heparan Sulfate Are Both Necessary for Binding of SARS-CoV-2 Spike Ectodomain (A) Western blot shows overexpression of ACE2 in the A375 and A375 B4GALT7 −/− cells. A representative blot is shown. (B) Binding of SARS-CoV-2 spike protein to cells with and without ACE2 overexpression. Note that binding is reduced in the cells deficient in HS. (C) Gene targeting of ACE2 in A549 using CRISPR-Cas9. The bars show spike binding to two independent ACE2 CRISPR-Cas9 knockout clones with and without HSase treatment. Note that binding depends on ACE2 expression and that residual binding depends in part on HS. All analyses were done by flow cytometry. The graphs show representative experiments performed in triplicate technical replicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S5 .

    Techniques Used: Binding Assay, Western Blot, Over Expression, CRISPR, Knock-Out, Clone Assay, Expressing, Flow Cytometry

    SARS-CoV-2 Spike Binds Heparin through the RBD (A) Recombinant trimeric SARS-CoV-2 spike and RBD proteins were bound to heparin-Sepharose and eluted with a gradient of sodium chloride. (B) RBD protein from SARS-CoV-1 and SARS-CoV-2 binding to immobilized heparin-BSA. (C) Binding of spike protein or ACE2 to heparin-BSA. Insert shows SARS-CoV-2 spike protein binding to heparin-BSA in the presence of ACE2. (D) Binding of spike protein in the active RBD open (Mut7) and inactive RBD closed (Mut2) conformation to heparin-BSA. (E) Binding of ACE2 to spike protein in active RBD open (Mut7) and inactive RBD closed (Mut 2) conformation. (F) ACE2 binding to spike protein immobilized on heparin-BSA. The broken line represents background binding. Statistical analysis was by one-way ANOVA. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). (G and H) Negative stain-electron microscopy analysis of binding of heparin and ACE2 to spike protein. ACE2 binding to spike protein increases in the presence of heparin. 3D class averages of SARS-CoV-2 spike bound to zero, one, two, or three ACE2 (white, orange, blue, or gray) when complexed with and without a heparin dp20. The incubation was done for 15 min (G) or 60 min (H). The percentage of particles belonging to each class is shown in pie charts. See also Figure S3 .
    Figure Legend Snippet: SARS-CoV-2 Spike Binds Heparin through the RBD (A) Recombinant trimeric SARS-CoV-2 spike and RBD proteins were bound to heparin-Sepharose and eluted with a gradient of sodium chloride. (B) RBD protein from SARS-CoV-1 and SARS-CoV-2 binding to immobilized heparin-BSA. (C) Binding of spike protein or ACE2 to heparin-BSA. Insert shows SARS-CoV-2 spike protein binding to heparin-BSA in the presence of ACE2. (D) Binding of spike protein in the active RBD open (Mut7) and inactive RBD closed (Mut2) conformation to heparin-BSA. (E) Binding of ACE2 to spike protein in active RBD open (Mut7) and inactive RBD closed (Mut 2) conformation. (F) ACE2 binding to spike protein immobilized on heparin-BSA. The broken line represents background binding. Statistical analysis was by one-way ANOVA. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). (G and H) Negative stain-electron microscopy analysis of binding of heparin and ACE2 to spike protein. ACE2 binding to spike protein increases in the presence of heparin. 3D class averages of SARS-CoV-2 spike bound to zero, one, two, or three ACE2 (white, orange, blue, or gray) when complexed with and without a heparin dp20. The incubation was done for 15 min (G) or 60 min (H). The percentage of particles belonging to each class is shown in pie charts. See also Figure S3 .

    Techniques Used: Recombinant, Binding Assay, Protein Binding, Staining, Electron Microscopy, Incubation

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

    Techniques Used: Binding Assay, Flow Cytometry

    Infection of Hep3B by MERS Pseudovirus, Related to Figure 6 Infection of Hep3B cells with SARS-CoV-2 and MERS-CoV S protein pseudotyped viruses carrying luciferase with and without treatment with heparin lyases.
    Figure Legend Snippet: Infection of Hep3B by MERS Pseudovirus, Related to Figure 6 Infection of Hep3B cells with SARS-CoV-2 and MERS-CoV S protein pseudotyped viruses carrying luciferase with and without treatment with heparin lyases.

    Techniques Used: Infection, Luciferase

    Manipulation of Cellular Heparan Sulfate Decreases Infection of Authentic SARS-CoV-2 Virus (A,) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) Vero cells stained with antibodies against SARS-CoV-2 nucleocapsid and spike protein. (B) SARS-CoV-2 infection of Vero cells performed in the absence and presence of HSase, or with incubation with different concentrations of unfractionated heparin (UFH). The extent of infection was analyzed by flow cytometry as in (A). The graph shows a composite of five separate experiments, each performed in triplicate. The MOI was 0.5, but the extent of infection varied. The MOI in the experiment shown in maroon and blue was 0.2. The mean data from the individual experiments are colorized to allow for separate visualization (C) Same data as in (B), but with the experimental data normalized to the mock infection for each respective experiment. (D) SARS-CoV-2 infection of Hep3B mutants altered in HS biosynthesis enzymes. Cells were infected for 1 h and incubated 48 h, allowing for new virus to form. The resulting viral titers in the culture supernatants were determined by plaque assays on Vero E6 cells. Average values with standard error mean are shown, along with the individual data points. The experiment was initially optimized and then performed in triplicate. (E) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) human bronchial epithelial cells at an air-liquid interface stained with antibodies against SARS-CoV-2 nucleocapsid. (F) SARS-CoV-2 infection of human bronchial epithelial cells at an air-liquid interface was performed in the absence and presence of HSase, or with incubation UFH. The extent of infection was analyzed by flow cytometry. The graph shows a composite of three separate experiments, each performed in triplicate. The mean data from the individual experiments are colorized to allow for separate visualization. (G) Same data as in (F), but with each experimental dataset normalized to the uninfected control. Statistical analysis by one-way ANOVA (B, C, and G) or unpaired t test (D); ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S7 .
    Figure Legend Snippet: Manipulation of Cellular Heparan Sulfate Decreases Infection of Authentic SARS-CoV-2 Virus (A,) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) Vero cells stained with antibodies against SARS-CoV-2 nucleocapsid and spike protein. (B) SARS-CoV-2 infection of Vero cells performed in the absence and presence of HSase, or with incubation with different concentrations of unfractionated heparin (UFH). The extent of infection was analyzed by flow cytometry as in (A). The graph shows a composite of five separate experiments, each performed in triplicate. The MOI was 0.5, but the extent of infection varied. The MOI in the experiment shown in maroon and blue was 0.2. The mean data from the individual experiments are colorized to allow for separate visualization (C) Same data as in (B), but with the experimental data normalized to the mock infection for each respective experiment. (D) SARS-CoV-2 infection of Hep3B mutants altered in HS biosynthesis enzymes. Cells were infected for 1 h and incubated 48 h, allowing for new virus to form. The resulting viral titers in the culture supernatants were determined by plaque assays on Vero E6 cells. Average values with standard error mean are shown, along with the individual data points. The experiment was initially optimized and then performed in triplicate. (E) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) human bronchial epithelial cells at an air-liquid interface stained with antibodies against SARS-CoV-2 nucleocapsid. (F) SARS-CoV-2 infection of human bronchial epithelial cells at an air-liquid interface was performed in the absence and presence of HSase, or with incubation UFH. The extent of infection was analyzed by flow cytometry. The graph shows a composite of three separate experiments, each performed in triplicate. The mean data from the individual experiments are colorized to allow for separate visualization. (G) Same data as in (F), but with each experimental dataset normalized to the uninfected control. Statistical analysis by one-way ANOVA (B, C, and G) or unpaired t test (D); ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S7 .

    Techniques Used: Infection, Flow Cytometry, Staining, Incubation

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

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

    Analysis of Recombinant Spike Proteins and Receptor-Binding Domain, Related to Star Methods, Protein Production (A) SDS-PAGE gel of recombinant SARS-CoV-2 spike ectodomain protein produced in ExpiCho cells and commercial recombinant SARS-CoV-2 RBD. (B) Transmission electron micrographs of recombinant SARS-CoV-2 spike ectodomain protein. (C) Size exclusion chromatography of recombinant SARS-CoV-2 spike ectodomain protein on a Superose 6 column. (D) SDS-PAGE gel of recombinant SARS-CoV-2 RBD produced in ExpiHEK cells. (E) Size exclusion chromatography of recombinant SARS-CoV-2 RBD on a Superdex200 column.
    Figure Legend Snippet: Analysis of Recombinant Spike Proteins and Receptor-Binding Domain, Related to Star Methods, Protein Production (A) SDS-PAGE gel of recombinant SARS-CoV-2 spike ectodomain protein produced in ExpiCho cells and commercial recombinant SARS-CoV-2 RBD. (B) Transmission electron micrographs of recombinant SARS-CoV-2 spike ectodomain protein. (C) Size exclusion chromatography of recombinant SARS-CoV-2 spike ectodomain protein on a Superose 6 column. (D) SDS-PAGE gel of recombinant SARS-CoV-2 RBD produced in ExpiHEK cells. (E) Size exclusion chromatography of recombinant SARS-CoV-2 RBD on a Superdex200 column.

    Techniques Used: Recombinant, Binding Assay, SDS Page, Produced, Transmission Assay, Size-exclusion Chromatography

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

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

    Location of the Putative Heparin/HS Binding Site in the Spike Protein RBD from SARS-CoV-2, Related to Figure 1 PDB files 6VSB and 6M0J were used to model the spike protein. The residues colored pink on the three RBDs (444+509+346+354+356+357+355+466+ 347+348+349+353+450+448+451+352) make up a potential binding site for heparin and heparan sulfate.
    Figure Legend Snippet: Location of the Putative Heparin/HS Binding Site in the Spike Protein RBD from SARS-CoV-2, Related to Figure 1 PDB files 6VSB and 6M0J were used to model the spike protein. The residues colored pink on the three RBDs (444+509+346+354+356+357+355+466+ 347+348+349+353+450+448+451+352) make up a potential binding site for heparin and heparan sulfate.

    Techniques Used: Binding Assay

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

    Techniques Used: Binding Assay, Sequencing

    Binding of Spike Protein to Heparin and ACE2 and Electron Micrographs of the Spike-ACE2 Complexes, Related to Figure 2 (A) SARS-CoV-2 spike binding to immobilized heparin or BSA. (B) ACE2 binding to immobilized spike protein. (C) Transmission electron micrographs of stabilized spike protein treated with ACE2 and with or without dp20 for 15 min or 1 h. (D) 2D classes averages for each condition.
    Figure Legend Snippet: Binding of Spike Protein to Heparin and ACE2 and Electron Micrographs of the Spike-ACE2 Complexes, Related to Figure 2 (A) SARS-CoV-2 spike binding to immobilized heparin or BSA. (B) ACE2 binding to immobilized spike protein. (C) Transmission electron micrographs of stabilized spike protein treated with ACE2 and with or without dp20 for 15 min or 1 h. (D) 2D classes averages for each condition.

    Techniques Used: Binding Assay, Transmission Assay

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.07.07.191007

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

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

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

    Techniques Used: Infection, Expressing

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

    Techniques Used: Infection

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

    Techniques Used: Generated, Infection

    20) 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

    21) Product Images from "Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry"

    Article Title: Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

    Journal: bioRxiv

    doi: 10.1101/2021.02.28.433291

    Native top-down MS of intact S-RBD expressed from HEK 293 cell line using TIMS-MS. (A-B) Ion mobility heat map (A) and native MS 1 analysis (B) of the S-RBD. (C) Magnified ion mobility heat map for the S-RBD region from (A). The diffuse ion mobility spectrum suggests a diverse spread of multiple protein ions with various collisional cross sections crowded in the native spectrum. (D) Magnified MS 1 from (B) with charge states of the native S-RBD annotated. The inset reveals that the large cluster of various proteoforms of the S-RBD inhibits its isotopic resolution.
    Figure Legend Snippet: Native top-down MS of intact S-RBD expressed from HEK 293 cell line using TIMS-MS. (A-B) Ion mobility heat map (A) and native MS 1 analysis (B) of the S-RBD. (C) Magnified ion mobility heat map for the S-RBD region from (A). The diffuse ion mobility spectrum suggests a diverse spread of multiple protein ions with various collisional cross sections crowded in the native spectrum. (D) Magnified MS 1 from (B) with charge states of the native S-RBD annotated. The inset reveals that the large cluster of various proteoforms of the S-RBD inhibits its isotopic resolution.

    Techniques Used:

    22) 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

    23) Product Images from "Stereotypic Neutralizing VH Clonotypes Against SARS-CoV-2 RBD in COVID-19 Patients and the Healthy Population"

    Article Title: Stereotypic Neutralizing VH Clonotypes Against SARS-CoV-2 RBD in COVID-19 Patients and the Healthy Population

    Journal: bioRxiv

    doi: 10.1101/2020.06.26.174557

    Inhibition of recombinant SARS-CoV-2 S glycoprotein binding to ACE2-expressing cells, by flow cytometry. The recombinant scFv-hFc fusion proteins (200 nM or 600 nM) were mixed and incubated with recombinant SARS-CoV-2 S glycoprotein (200 nM) fused with a HIS tag at the C-terminus. After incubation with Vero E6 (ACE2 + ) cells, the relative amount of bound, recombinant SARS-CoV-2 S glycoprotein was measured using a FITC-conjugated anti-HIS antibody. For each sample, 10,000 cells were monitored.
    Figure Legend Snippet: Inhibition of recombinant SARS-CoV-2 S glycoprotein binding to ACE2-expressing cells, by flow cytometry. The recombinant scFv-hFc fusion proteins (200 nM or 600 nM) were mixed and incubated with recombinant SARS-CoV-2 S glycoprotein (200 nM) fused with a HIS tag at the C-terminus. After incubation with Vero E6 (ACE2 + ) cells, the relative amount of bound, recombinant SARS-CoV-2 S glycoprotein was measured using a FITC-conjugated anti-HIS antibody. For each sample, 10,000 cells were monitored.

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

    Characteristics of nAbs, derived from patients A and E, stereotypic IGH clonotypes that are highly homologous to E-3B1, and the predicted RBD-binding clones that were enriched through biopanning. Stereotypic nAb V H clonotypes against the SARS-CoV-2 RBD, encoded by IGHV3-53/3-66 and IGHJ6, were found in six of seven patients. a, Characteristics of nAbs discovered in patients A and E. b, 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 mean ± SD. c, List of diverse IGL clonotypes that can be paired with the IGH clonotypes from b to achieve reactivity. d, Measurement of viral RNA in the culture supernatant of Vero cells after SARS-CoV-2 infection e, J and f, VJ gene usage in the IGH repertoire of patients (upper) and the binding-predicted IGH clones (bottom). For the VJ gene usage heatmap, the frequency values for the IGH repertoire of all seven patients were averaged and are displayed (upper) along with those of the predicted RBD-binding IGH clones (bottom). N/A: not applicable
    Figure Legend Snippet: Characteristics of nAbs, derived from patients A and E, stereotypic IGH clonotypes that are highly homologous to E-3B1, and the predicted RBD-binding clones that were enriched through biopanning. Stereotypic nAb V H clonotypes against the SARS-CoV-2 RBD, encoded by IGHV3-53/3-66 and IGHJ6, were found in six of seven patients. a, Characteristics of nAbs discovered in patients A and E. b, 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 mean ± SD. c, List of diverse IGL clonotypes that can be paired with the IGH clonotypes from b to achieve reactivity. d, Measurement of viral RNA in the culture supernatant of Vero cells after SARS-CoV-2 infection e, J and f, VJ gene usage in the IGH repertoire of patients (upper) and the binding-predicted IGH clones (bottom). For the VJ gene usage heatmap, the frequency values for the IGH repertoire of all seven patients were averaged and are displayed (upper) along with those of the predicted RBD-binding IGH clones (bottom). N/A: not applicable

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

    24) Product Images from "Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins"

    Article Title: Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins

    Journal: bioRxiv

    doi: 10.1101/2020.03.28.013276

    N-glycosites characterization of SARS-CoV-2 S proteins. (A and B) N-glycosites of the recombinant SARS-CoV-2 S protein or subunits expressed in insect cells (A) and human cells (B). PSS: putative signal sequence; RBD: receptor-binding domain; S1/S2: S1/S2 protease cleavage site; Oval: potential N-glycosite; Yellow oval: ambiguously assigned N-glycosite; Red oval: unambiguously assigned N-glycosite; Blue arrow: unambiguously assigned N-glycosite using trypsin digestion; Green arrow: unambiguously assigned N-glycosite using Glu-C digestion; Yellow arrow: unambiguously assigned N-glycosite using the combination of trypsin and Glu-C digestion. The unambiguously glycosite was determined by at least twice identification within each digestion list in Table S1 and Table S2. (C) N-glycosites were demonstrated in the three-dimensional structure of the SARS-CoV-2 S protein trimers (PDB code: 6VSB). RBDs, yellow; N-glycosites, blue.
    Figure Legend Snippet: N-glycosites characterization of SARS-CoV-2 S proteins. (A and B) N-glycosites of the recombinant SARS-CoV-2 S protein or subunits expressed in insect cells (A) and human cells (B). PSS: putative signal sequence; RBD: receptor-binding domain; S1/S2: S1/S2 protease cleavage site; Oval: potential N-glycosite; Yellow oval: ambiguously assigned N-glycosite; Red oval: unambiguously assigned N-glycosite; Blue arrow: unambiguously assigned N-glycosite using trypsin digestion; Green arrow: unambiguously assigned N-glycosite using Glu-C digestion; Yellow arrow: unambiguously assigned N-glycosite using the combination of trypsin and Glu-C digestion. The unambiguously glycosite was determined by at least twice identification within each digestion list in Table S1 and Table S2. (C) N-glycosites were demonstrated in the three-dimensional structure of the SARS-CoV-2 S protein trimers (PDB code: 6VSB). RBDs, yellow; N-glycosites, blue.

    Techniques Used: Recombinant, Sequencing, Binding Assay

    Site-specific N-glycosylation of recombinant SARS-CoV-2 S proteins. (A and B) The number of intact N-glycopeptides and N-glycans in recombinant SARS-CoV-2 S proteins expressed in insect cells (A) or human cells (B). (C and D) The numbers of the N-glycosites containing one representative N-glycan and its deduced structure from the recombinant SARS-CoV-2 S protein or subunit expressed in insect cells (C) and human cells (D). (E and F) Different types and numbers of N-glycan compositions on each N-glycosite of the recombinant SARS-CoV-2 S protein or subunit expressed in insect cells (E) or human cells (F).
    Figure Legend Snippet: Site-specific N-glycosylation of recombinant SARS-CoV-2 S proteins. (A and B) The number of intact N-glycopeptides and N-glycans in recombinant SARS-CoV-2 S proteins expressed in insect cells (A) or human cells (B). (C and D) The numbers of the N-glycosites containing one representative N-glycan and its deduced structure from the recombinant SARS-CoV-2 S protein or subunit expressed in insect cells (C) and human cells (D). (E and F) Different types and numbers of N-glycan compositions on each N-glycosite of the recombinant SARS-CoV-2 S protein or subunit expressed in insect cells (E) or human cells (F).

    Techniques Used: Recombinant

    Workflow for site-specific N-glycosylation characterization of recombinant SARS-CoV-2 S proteins using two complementary proteases for digestion and an integrated N-glycoproteomic analysis.
    Figure Legend Snippet: Workflow for site-specific N-glycosylation characterization of recombinant SARS-CoV-2 S proteins using two complementary proteases for digestion and an integrated N-glycoproteomic analysis.

    Techniques Used: Recombinant

    25) 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

    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 "An engineered stable mini-protein to plug SARS-Cov-2 Spikes"

    Article Title: An engineered stable mini-protein to plug SARS-Cov-2 Spikes

    Journal: bioRxiv

    doi: 10.1101/2020.04.29.067728

    Cartoon representation of the complex between the receptor binding (RBD) domain of SARS-CoV-2 Spike protein (blue/cyan) and the human ACE2 receptor (grey/green). The receptor binding motif (RBM) is drawn in cyan. The green portion of the ACE2 domain including helices H1, H2 and H3 is drawn in green.
    Figure Legend Snippet: Cartoon representation of the complex between the receptor binding (RBD) domain of SARS-CoV-2 Spike protein (blue/cyan) and the human ACE2 receptor (grey/green). The receptor binding motif (RBM) is drawn in cyan. The green portion of the ACE2 domain including helices H1, H2 and H3 is drawn in green.

    Techniques Used: Binding Assay

    29) Product Images from "A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering"

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    Journal: bioRxiv

    doi: 10.1101/2021.01.19.427310

    Immune responses of T4-SARS-CoV-2 immunized mice. a. Anti-RBD IgG antibody titers in the sera from group G5 (T4-HocΔ-SocΔ-S-ecto-Ee-NP) at weeks 2 (prime), 5 (boost-1), and 8 (boost-2). For boost-2, T4-S-trimers particles were used. **P
    Figure Legend Snippet: Immune responses of T4-SARS-CoV-2 immunized mice. a. Anti-RBD IgG antibody titers in the sera from group G5 (T4-HocΔ-SocΔ-S-ecto-Ee-NP) at weeks 2 (prime), 5 (boost-1), and 8 (boost-2). For boost-2, T4-S-trimers particles were used. **P

    Techniques Used: Mouse Assay

    Immunogenicity and protective efficacy of T4-SARS-CoV-2 vaccine candidates in mice. a. Schematic diagram showing BALB/c mice immunized by the intramuscular (i.m.) route using T4-SARS-CoV-2 vaccine formulations. b. I. Formulations and mouse groups used for vaccinations. HSΔ indicates Hoc deletion and Soc deletion. Blue color (S-ecto, S-fl, and RBD) indicates the insertion of mammalian gene expression cassette into T4 genome as DNA vaccine. Red color indicates the capsid-displayed Ee, S-trimers, or E.coli -produced rRBD or sRBD protein, or the capsid-encapsidated NP protein. Naïve mice and mice immunized with the phage lacking any CoV-2 genes served as negative controls whereas mice immunized with S-trimers adjuvanted with Alhydrogel served as a positive control. II. Prime-boost immunization scheme. BALB/c mice (5 per group) were immunized on weeks 0, 3, and 6 and challenged intranasally (i.n.) with a mouse-adapted SARS-CoV-2 strain (SARS-CoV-2 MA10) 47 on week 14. c to f . The boost-2 sera (week 8 bleeding) from various groups were assessed by ELISA for antigen-specific IgG antibody titers (endpoint) against S-ecto (c), RBD (d), NP (e), and E (f). *P
    Figure Legend Snippet: Immunogenicity and protective efficacy of T4-SARS-CoV-2 vaccine candidates in mice. a. Schematic diagram showing BALB/c mice immunized by the intramuscular (i.m.) route using T4-SARS-CoV-2 vaccine formulations. b. I. Formulations and mouse groups used for vaccinations. HSΔ indicates Hoc deletion and Soc deletion. Blue color (S-ecto, S-fl, and RBD) indicates the insertion of mammalian gene expression cassette into T4 genome as DNA vaccine. Red color indicates the capsid-displayed Ee, S-trimers, or E.coli -produced rRBD or sRBD protein, or the capsid-encapsidated NP protein. Naïve mice and mice immunized with the phage lacking any CoV-2 genes served as negative controls whereas mice immunized with S-trimers adjuvanted with Alhydrogel served as a positive control. II. Prime-boost immunization scheme. BALB/c mice (5 per group) were immunized on weeks 0, 3, and 6 and challenged intranasally (i.n.) with a mouse-adapted SARS-CoV-2 strain (SARS-CoV-2 MA10) 47 on week 14. c to f . The boost-2 sera (week 8 bleeding) from various groups were assessed by ELISA for antigen-specific IgG antibody titers (endpoint) against S-ecto (c), RBD (d), NP (e), and E (f). *P

    Techniques Used: Mouse Assay, Expressing, Produced, Positive Control, Enzyme-linked Immunosorbent Assay

    A pipeline of SARS-CoV-2 vaccine candidates generated by sequential CRISPR engineering. a. Schematic showing a representative sequence in which the WT phage was used as a starting infection of CRISPR E. coli containing spacer 1 and donor 1. The resultant T4-mutant 1 (T4-M1) was used to infect bacteria containing spacer 2 and donor 2 to produce recombinant T4-mutant 2 (T4-M2) which has two insertion/deletion mutations, and so forth. By sequential CRISPR engineering and simple phage infections, recombinant phages with multiple desired mutations were created. Each color on phage capsid here represents a mutation. b. One example of sequential phage CRISPR engineering for creating the T4-SARS-CoV-2 nanovaccine. Numerous CoV-2 components, including CAGpromoter-S-ecto insertion, CAGpromoter-S-fl insertion, CMVpromoter-RBD insertion, Hoc deletion, Ee-Hoc insertion, Ec-Hoc insertion, Soc deletion, Soc-sRBD display, M21-Soc-sRBD display, Soc-SpyCatcher display, refolding SUMO-RBD-Spy display, S-trimer display, IPIII deletion, IPII deletion, and NP encapsidation, were permutated and combined as needed. The resultant SARS-CoV-2 vaccine candidates were characterized by PCR, DNA sequencing and/or SDS-PAGE, and some of these were then tested in a mouse study. M21 indicates a potential T cell 21 aa epitope (SYFIASFRLFARTRSMWSFNP) from SARS-CoV-2 membrane protein. c. WB showing NP protein encapsidation in the phages containing CTSam-NP insertion at IPIII deletion site.
    Figure Legend Snippet: A pipeline of SARS-CoV-2 vaccine candidates generated by sequential CRISPR engineering. a. Schematic showing a representative sequence in which the WT phage was used as a starting infection of CRISPR E. coli containing spacer 1 and donor 1. The resultant T4-mutant 1 (T4-M1) was used to infect bacteria containing spacer 2 and donor 2 to produce recombinant T4-mutant 2 (T4-M2) which has two insertion/deletion mutations, and so forth. By sequential CRISPR engineering and simple phage infections, recombinant phages with multiple desired mutations were created. Each color on phage capsid here represents a mutation. b. One example of sequential phage CRISPR engineering for creating the T4-SARS-CoV-2 nanovaccine. Numerous CoV-2 components, including CAGpromoter-S-ecto insertion, CAGpromoter-S-fl insertion, CMVpromoter-RBD insertion, Hoc deletion, Ee-Hoc insertion, Ec-Hoc insertion, Soc deletion, Soc-sRBD display, M21-Soc-sRBD display, Soc-SpyCatcher display, refolding SUMO-RBD-Spy display, S-trimer display, IPIII deletion, IPII deletion, and NP encapsidation, were permutated and combined as needed. The resultant SARS-CoV-2 vaccine candidates were characterized by PCR, DNA sequencing and/or SDS-PAGE, and some of these were then tested in a mouse study. M21 indicates a potential T cell 21 aa epitope (SYFIASFRLFARTRSMWSFNP) from SARS-CoV-2 membrane protein. c. WB showing NP protein encapsidation in the phages containing CTSam-NP insertion at IPIII deletion site.

    Techniques Used: Generated, CRISPR, Sequencing, Infection, Mutagenesis, Recombinant, Polymerase Chain Reaction, DNA Sequencing, SDS Page, Western Blot

    Serum antibody responses in various T4-SARS-CoV-2 immunized mice. a and b. Anti-S-ecto IgG1 (a) and IgG2a (b) antibody titers in the boost-2 sera (week 8 bleeding) from various groups. c and d. Anti-RBD IgG1 (c) and IgG2a (d) antibody titers in the boost-2 sera. e and f . Anti-NP IgG1 (e) and IgG2a (f) antibody titers in the boost-2 sera. g and h . Anti-E IgG1 (g) and IgG2a (h) antibody titers in the boost-2 sera. *P
    Figure Legend Snippet: Serum antibody responses in various T4-SARS-CoV-2 immunized mice. a and b. Anti-S-ecto IgG1 (a) and IgG2a (b) antibody titers in the boost-2 sera (week 8 bleeding) from various groups. c and d. Anti-RBD IgG1 (c) and IgG2a (d) antibody titers in the boost-2 sera. e and f . Anti-NP IgG1 (e) and IgG2a (f) antibody titers in the boost-2 sera. g and h . Anti-E IgG1 (g) and IgG2a (h) antibody titers in the boost-2 sera. *P

    Techniques Used: Mouse Assay

    Virus neutralization titers of rabbit sera. Infection of Vero E6 cells by live SARS-CoV-2 US-WA-1/2020 was determined in the presence of rabbit sera at a series of two-fold dilutions starting from 1:4. Culture medium only and CoV-2 virus only were used as negative and positive controls, respectively. R1442 to R1457 refer to tag numbers of rabbits. The data in control groups were presented as means ± SD of 32 wells. The data in rabbit sera groups were shown as means of duplicates.
    Figure Legend Snippet: Virus neutralization titers of rabbit sera. Infection of Vero E6 cells by live SARS-CoV-2 US-WA-1/2020 was determined in the presence of rabbit sera at a series of two-fold dilutions starting from 1:4. Culture medium only and CoV-2 virus only were used as negative and positive controls, respectively. R1442 to R1457 refer to tag numbers of rabbits. The data in control groups were presented as means ± SD of 32 wells. The data in rabbit sera groups were shown as means of duplicates.

    Techniques Used: Neutralization, Infection

    Construction and screening of various truncated SARS-CoV-2 RBDs. a. Structural models of recombinant WT RBD and various truncated RBDs bound to human ACE2. ACE2 is shown in green. The truncated RBD clones are shown in red and the WT RBD and deleted regions are shown in cyan. The Protein Data Bank (PDB) code for the SARS-CoV-2 RBD–ACE2 complex is 6M0J 34 . The truncated RBDs were generated using Chimera software. b. Solubility analysis of Soc-fused truncated RBDs after cloning and expression in E. coli under the control of the phage T7 promoter. After lysis of E. coli and centrifugation, the supernatant and pellet were analyzed by SDS-PAGE. The presence of Soc-truncated RBDs in the pellet and their absence in the supernatant demonstrated insolubility. The red arrowheads indicate the band positions of various Soc-truncated RBDs.
    Figure Legend Snippet: Construction and screening of various truncated SARS-CoV-2 RBDs. a. Structural models of recombinant WT RBD and various truncated RBDs bound to human ACE2. ACE2 is shown in green. The truncated RBD clones are shown in red and the WT RBD and deleted regions are shown in cyan. The Protein Data Bank (PDB) code for the SARS-CoV-2 RBD–ACE2 complex is 6M0J 34 . The truncated RBDs were generated using Chimera software. b. Solubility analysis of Soc-fused truncated RBDs after cloning and expression in E. coli under the control of the phage T7 promoter. After lysis of E. coli and centrifugation, the supernatant and pellet were analyzed by SDS-PAGE. The presence of Soc-truncated RBDs in the pellet and their absence in the supernatant demonstrated insolubility. The red arrowheads indicate the band positions of various Soc-truncated RBDs.

    Techniques Used: Recombinant, Clone Assay, Generated, Software, Solubility, Expressing, Lysis, Centrifugation, SDS Page

    Incorporation of various SARS-CoV-2 vaccine payloads into phage T4 nanoparticle. a. Schematic showing steps in T4 phage head morphogenesis. Mem, E. coli membrane; CTS, capsid targeting sequence. b and c. SDS-PAGE and Western Blot (WB) analysis of phage particles with IPII and IPIII deletions (IPIIΔIPIIIΔ) and NP encapsidation. Since NP has a very similar molecular size to T4 major capsid protein gp23*, an NP-specific antibody was used to detect NP. d. Structural model of viroporin-like tetrameric assembly of CoV-2 E protein 32 . The N-terminal seven residues and C-terminal ten residues are not shown due to the lack of a corresponding segment in the structural template used for homology modeling. Ee* indicates amino acids (aa) 8-12 and Ec* indicates aa 53-65. e. SDS-PAGE of Hoc deletion and Soc deletion phage (HocΔSocΔ). f. SDS-PAGE of recombinant phages displaying Ee-Hoc or Ec-Hoc fusion proteins. g. Schematic showing Soc-sRBD or Soc-SpyCatcher (SpyC) in vivo display on T4-SocΔ capsid. Soc-sRBD or Soc-SpyCatcher expression under the control of phage T7 promoter was induced by IPTG. Most of the expressed Soc-RBD was in the inclusion body (IB). Soluble Soc-sRBD (minor amount) or Soc-SpyC can be efficiently displayed on capsid. h. SDS-PAGE showing ~100 copies of Soc-sRBD displayed on T4 capsid. i. SDS-PAGE showing ~500 copies of Soc-SpyCatcher displayed on T4 capsid. j. Schematic diagram showing the solubilization and refolding of SUMO (small ubiquitin like modifiers)-RBD-Spytag inclusion body. Refolded SUMO-RBD-Spytag (rRBD) protein was efficiently displayed on T4-SpyCatcher phage via Spytag-SpyCatcher bridging. k. Display of rRBD on the T4-SpyCacher surface at increasing ratios of rRBD molecules to capsid Soc binding sites (0:1 to 2:1). RBD specific antibody was used to verify the displayed rRBD and rRBD-SpyCatcher-Soc complexes. T4* indicates T4-S-ecto-NP-Ec-SocΔ recombinant phage. Blue and red arrows indicate rRBD/complexes and Soc-SpyCatcher, respectively. l to o . Comparison of binding of T4-sRBD, and T4-rRBD phages to soluble human ACE2 receptor (l), monoclonal antibody (mAb) 1 (human IgG Clone #bcb03, Thermo Fisher) (m), mAb2 (rabbit IgG Clone #007, Sino Bio) (n), and polyclonal antibodies (pAb) (rabbit PAb, Sino Bio) (o) using BSA and T4 phage as controls. p. Comparison of binding of E. coli -produced rRBD to human ACE2 with the HEK293-produced RBD. **P
    Figure Legend Snippet: Incorporation of various SARS-CoV-2 vaccine payloads into phage T4 nanoparticle. a. Schematic showing steps in T4 phage head morphogenesis. Mem, E. coli membrane; CTS, capsid targeting sequence. b and c. SDS-PAGE and Western Blot (WB) analysis of phage particles with IPII and IPIII deletions (IPIIΔIPIIIΔ) and NP encapsidation. Since NP has a very similar molecular size to T4 major capsid protein gp23*, an NP-specific antibody was used to detect NP. d. Structural model of viroporin-like tetrameric assembly of CoV-2 E protein 32 . The N-terminal seven residues and C-terminal ten residues are not shown due to the lack of a corresponding segment in the structural template used for homology modeling. Ee* indicates amino acids (aa) 8-12 and Ec* indicates aa 53-65. e. SDS-PAGE of Hoc deletion and Soc deletion phage (HocΔSocΔ). f. SDS-PAGE of recombinant phages displaying Ee-Hoc or Ec-Hoc fusion proteins. g. Schematic showing Soc-sRBD or Soc-SpyCatcher (SpyC) in vivo display on T4-SocΔ capsid. Soc-sRBD or Soc-SpyCatcher expression under the control of phage T7 promoter was induced by IPTG. Most of the expressed Soc-RBD was in the inclusion body (IB). Soluble Soc-sRBD (minor amount) or Soc-SpyC can be efficiently displayed on capsid. h. SDS-PAGE showing ~100 copies of Soc-sRBD displayed on T4 capsid. i. SDS-PAGE showing ~500 copies of Soc-SpyCatcher displayed on T4 capsid. j. Schematic diagram showing the solubilization and refolding of SUMO (small ubiquitin like modifiers)-RBD-Spytag inclusion body. Refolded SUMO-RBD-Spytag (rRBD) protein was efficiently displayed on T4-SpyCatcher phage via Spytag-SpyCatcher bridging. k. Display of rRBD on the T4-SpyCacher surface at increasing ratios of rRBD molecules to capsid Soc binding sites (0:1 to 2:1). RBD specific antibody was used to verify the displayed rRBD and rRBD-SpyCatcher-Soc complexes. T4* indicates T4-S-ecto-NP-Ec-SocΔ recombinant phage. Blue and red arrows indicate rRBD/complexes and Soc-SpyCatcher, respectively. l to o . Comparison of binding of T4-sRBD, and T4-rRBD phages to soluble human ACE2 receptor (l), monoclonal antibody (mAb) 1 (human IgG Clone #bcb03, Thermo Fisher) (m), mAb2 (rabbit IgG Clone #007, Sino Bio) (n), and polyclonal antibodies (pAb) (rabbit PAb, Sino Bio) (o) using BSA and T4 phage as controls. p. Comparison of binding of E. coli -produced rRBD to human ACE2 with the HEK293-produced RBD. **P

    Techniques Used: Sequencing, SDS Page, Western Blot, Recombinant, In Vivo, Expressing, Binding Assay, Produced

    Design of T4-SARS-CoV-2 nanovaccine by CRISPR engineering. Engineered DNAs corresponding to various components of SARS-CoV-2 virion are incorporated into bacteriophage T4 genome. Each DNA was introduced into E. coli as a donor plasmid (a) , recombined into injected phage genome through CRISPR-targeted genome editing (b) . Different combinations of CoV-2 inserts were then generated by simple phage infections and identifying the recombinant phages in the progeny (c) . For example, recombinant phage containing CoV-2 insert #1 (dark blue) can be used to infect CRISPR E. coli containing Co-V2 insert containing donor plasmid #2 (dark red). The progeny plaques obtained will contain recombinant phage #3 with both inserts #1 and #2 (dark blue plus dark red) in the same genome. This process was repeated to rapidly construct a pipeline of multiplex T4-SARS-CoV-2 vaccine phages (d) . Selected vaccine candidates were then screened in a mouse model (e) to identify the most potent vaccine (f) . Structural model of T4-SARS-CoV-2 Nanovaccine showing an enlarged view of a single hexameric capsomer (g) . The capsomer shows six subunits of major capsid protein gp23* (green), trimers of Soc (blue), and a Hoc fiber (yellow) at the center of capsomer. The expressible spike genes are inserted into phage genome, the 12 aa E external peptide (red) is displayed at the tip of Hoc fiber, S-trimers (cyan) are attached to Soc subunits, and nucleocapsid proteins (yellow) are packaged in genome core. See Results , Materials and Methods, and Supplementary Video for additional details.
    Figure Legend Snippet: Design of T4-SARS-CoV-2 nanovaccine by CRISPR engineering. Engineered DNAs corresponding to various components of SARS-CoV-2 virion are incorporated into bacteriophage T4 genome. Each DNA was introduced into E. coli as a donor plasmid (a) , recombined into injected phage genome through CRISPR-targeted genome editing (b) . Different combinations of CoV-2 inserts were then generated by simple phage infections and identifying the recombinant phages in the progeny (c) . For example, recombinant phage containing CoV-2 insert #1 (dark blue) can be used to infect CRISPR E. coli containing Co-V2 insert containing donor plasmid #2 (dark red). The progeny plaques obtained will contain recombinant phage #3 with both inserts #1 and #2 (dark blue plus dark red) in the same genome. This process was repeated to rapidly construct a pipeline of multiplex T4-SARS-CoV-2 vaccine phages (d) . Selected vaccine candidates were then screened in a mouse model (e) to identify the most potent vaccine (f) . Structural model of T4-SARS-CoV-2 Nanovaccine showing an enlarged view of a single hexameric capsomer (g) . The capsomer shows six subunits of major capsid protein gp23* (green), trimers of Soc (blue), and a Hoc fiber (yellow) at the center of capsomer. The expressible spike genes are inserted into phage genome, the 12 aa E external peptide (red) is displayed at the tip of Hoc fiber, S-trimers (cyan) are attached to Soc subunits, and nucleocapsid proteins (yellow) are packaged in genome core. See Results , Materials and Methods, and Supplementary Video for additional details.

    Techniques Used: CRISPR, Plasmid Preparation, Injection, Generated, Recombinant, Construct, Multiplex Assay

    Construction of T4-SARS-CoV-2 recombinant phages by CRISPR engineering. a. Schematic of T4 CRISPR engineering. b. Four nonessential regions of T4 genome are chosen for deletion and insertion of various SARS-CoV-2 genes (shown in red; SegF/Soc, FarP, IP, and Hoc). 6P, six proline substitutions in S-ecto (F817P, A892P, A899P, A942P, K986P, and V987P). Fol, T4 fibritin motif Foldon for efficient trimerization. Tag, octa-histidine and twin-strep tags. Furin cleavage site RRAR was mutated to GSAS to stabilize trimers in a prefusion state 31 . c. Efficiency of plating (EOP) of representative Cpf1-FarP7K and Cpf1-SegF spacers. d. Plate showing plaques from phage infection of bacteria containing Cpf1-FarP7K spacer only, S-ecto donor only, or Cpf1-FarP7K spacer plus S-ecto donor. e. Recombination frequency of three spike gene (RBD, S-ecto, and S-fl) insertions. f. DNA sequencing of thirty independent plaques showed that > 95% of the plaques generated in S-ecto recombination contained the correct S-ecto insert. g. Plate showing that the wild-type (WT), T4-RBD, T4-S-fl, T4-S-ecto, and T4-(S-ecto)-RBD recombinant phages had similar plaque size.
    Figure Legend Snippet: Construction of T4-SARS-CoV-2 recombinant phages by CRISPR engineering. a. Schematic of T4 CRISPR engineering. b. Four nonessential regions of T4 genome are chosen for deletion and insertion of various SARS-CoV-2 genes (shown in red; SegF/Soc, FarP, IP, and Hoc). 6P, six proline substitutions in S-ecto (F817P, A892P, A899P, A942P, K986P, and V987P). Fol, T4 fibritin motif Foldon for efficient trimerization. Tag, octa-histidine and twin-strep tags. Furin cleavage site RRAR was mutated to GSAS to stabilize trimers in a prefusion state 31 . c. Efficiency of plating (EOP) of representative Cpf1-FarP7K and Cpf1-SegF spacers. d. Plate showing plaques from phage infection of bacteria containing Cpf1-FarP7K spacer only, S-ecto donor only, or Cpf1-FarP7K spacer plus S-ecto donor. e. Recombination frequency of three spike gene (RBD, S-ecto, and S-fl) insertions. f. DNA sequencing of thirty independent plaques showed that > 95% of the plaques generated in S-ecto recombination contained the correct S-ecto insert. g. Plate showing that the wild-type (WT), T4-RBD, T4-S-fl, T4-S-ecto, and T4-(S-ecto)-RBD recombinant phages had similar plaque size.

    Techniques Used: Recombinant, CRISPR, Infection, DNA Sequencing, Generated

    CRISPR engineering of non-essential T4 genome. a. Schematic showing the 18-kb nonessential segment FarP and 11-kb nonessential segment 39-56 on T4 genome. b. Plaque size of wild-type (WT), T4- FarP 18 kb del. , T4- 39-56 11 kb del. , and T4- FarP 39-56 29 kb del. phages. Note the small size of T4- 39-56 11 kb del. and T4- FarP 39-56 29 kb del. plaques. c. Structural models of SARS-CoV-2 virus, spike trimer, and receptor binding domain (RBD). d. Schematics of S-full length (S-fl) and S-ectodomain (S-ecto) expression cassettes used for insertion into T4 genome. e. Efficiency of plating of three sets of Cpf1-FarP7K spacers and three sets of Cpf1-SegF spacers. f. Efficiency of plating of various spacers used for T4 genome engineering in this study.
    Figure Legend Snippet: CRISPR engineering of non-essential T4 genome. a. Schematic showing the 18-kb nonessential segment FarP and 11-kb nonessential segment 39-56 on T4 genome. b. Plaque size of wild-type (WT), T4- FarP 18 kb del. , T4- 39-56 11 kb del. , and T4- FarP 39-56 29 kb del. phages. Note the small size of T4- 39-56 11 kb del. and T4- FarP 39-56 29 kb del. plaques. c. Structural models of SARS-CoV-2 virus, spike trimer, and receptor binding domain (RBD). d. Schematics of S-full length (S-fl) and S-ectodomain (S-ecto) expression cassettes used for insertion into T4 genome. e. Efficiency of plating of three sets of Cpf1-FarP7K spacers and three sets of Cpf1-SegF spacers. f. Efficiency of plating of various spacers used for T4 genome engineering in this study.

    Techniques Used: CRISPR, Binding Assay, Expressing

    30) 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

    31) Product Images from "SARS-CoV-2 induces robust germinal center CD4 T follicular helper cell responses in rhesus macaques"

    Article Title: SARS-CoV-2 induces robust germinal center CD4 T follicular helper cell responses in rhesus macaques

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20642-x

    IgG1 subclass and neutralizing antibodies induced following SARS-CoV-2 infection. A Fold increase in antibody responses in animals was determined by dividing post-infection concentrations by those measured on day 0 in each animal. Data shown are n = 8 animals for all time points. Horizontal line indicates median B Fold increase in IgG1, IgG2, IgG3, and IgG4 antibodies against S1, S2, and N show dominance of IgG1 subclass antibodies. Data shown are for n = 6 animals not given CP. C Correlations between day 10 levels of S1-specific IgG and IgM, N-specific IgA and IgG, and pseudovirus neutralizing antibody titers and anti-receptor binding domain (RBD) IgG antibodies measured by ELISA. Unique symbols identify animals in each of the experimental groups (two-tailed Pearson test p values shown; correlation for anti-RBD IgG and T h 1 T fh cells shows one-tailed Spearman test p value).
    Figure Legend Snippet: IgG1 subclass and neutralizing antibodies induced following SARS-CoV-2 infection. A Fold increase in antibody responses in animals was determined by dividing post-infection concentrations by those measured on day 0 in each animal. Data shown are n = 8 animals for all time points. Horizontal line indicates median B Fold increase in IgG1, IgG2, IgG3, and IgG4 antibodies against S1, S2, and N show dominance of IgG1 subclass antibodies. Data shown are for n = 6 animals not given CP. C Correlations between day 10 levels of S1-specific IgG and IgM, N-specific IgA and IgG, and pseudovirus neutralizing antibody titers and anti-receptor binding domain (RBD) IgG antibodies measured by ELISA. Unique symbols identify animals in each of the experimental groups (two-tailed Pearson test p values shown; correlation for anti-RBD IgG and T h 1 T fh cells shows one-tailed Spearman test p value).

    Techniques Used: Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, One-tailed Test

    Humoral responses to SARS-CoV-2 are dominated by IgG antibodies. Concentrations of A IgM, B IgG, and C IgA antibodies (Ab) specific for S1, S2, and N proteins measured by BAMA or ELISA in serum. The dashed line represents the median pre-infection (day 0) concentration for all animals. Unique symbols identify animals in each of the experimental groups. (** p = 0.007, * p = 0.015 at indicated time points relative to d0 using a Wilcoxon matched-pairs signed-rank two-tailed t test).
    Figure Legend Snippet: Humoral responses to SARS-CoV-2 are dominated by IgG antibodies. Concentrations of A IgM, B IgG, and C IgA antibodies (Ab) specific for S1, S2, and N proteins measured by BAMA or ELISA in serum. The dashed line represents the median pre-infection (day 0) concentration for all animals. Unique symbols identify animals in each of the experimental groups. (** p = 0.007, * p = 0.015 at indicated time points relative to d0 using a Wilcoxon matched-pairs signed-rank two-tailed t test).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Infection, Concentration Assay, Two Tailed Test

    CD4 T fh cells targeting spike (S) and nucleocapsid (N) in blood following SARS-CoV-2 infection. A Representative gating strategy to identify SARS-CoV-2-specific CD4 T cells following stimulation with spike (S) and nucleocapsid (N) peptide pools in PBMCs. B AIM + CXCR5 − and CXCR5 + CD4 subsets in PBMCs at Day 7.
    Figure Legend Snippet: CD4 T fh cells targeting spike (S) and nucleocapsid (N) in blood following SARS-CoV-2 infection. A Representative gating strategy to identify SARS-CoV-2-specific CD4 T cells following stimulation with spike (S) and nucleocapsid (N) peptide pools in PBMCs. B AIM + CXCR5 − and CXCR5 + CD4 subsets in PBMCs at Day 7.

    Techniques Used: Infection

    SARS-CoV-2 infection induces germinal center responses targeting spike (S) and nucleocapsid (N) in mediastinal lymph nodes. A Median fluorescence intensity (MFI) of CXCR5, CCR7, CD69 and B SLAM, ICOS, CD28 within CXCR3 - (orange) and CXCR3 + (magenta) GC T fh cells in spleen following SARS-CoV-2 infection. Naive CD4 T cells in spleen shown for comparison (grey) (SLAM; **** p
    Figure Legend Snippet: SARS-CoV-2 infection induces germinal center responses targeting spike (S) and nucleocapsid (N) in mediastinal lymph nodes. A Median fluorescence intensity (MFI) of CXCR5, CCR7, CD69 and B SLAM, ICOS, CD28 within CXCR3 - (orange) and CXCR3 + (magenta) GC T fh cells in spleen following SARS-CoV-2 infection. Naive CD4 T cells in spleen shown for comparison (grey) (SLAM; **** p

    Techniques Used: Infection, Fluorescence

    Induction of T h 1 CD4 effectors in the lungs during SARS-CoV-2 infection. A Gating strategy for identifying CD95 + CD69 + CD4 and CD8 cells expressing granzyme B, PD-1, α4β7, CCR6, and CXCR3. Fluorochromes used were CD45-A488, CD3-A700, CD20/Dead-APC-Cy7, CD8-BUV 805, CD4-BV650, CD95-BUV737, CD69-BV711, Granzyme B- BV421, PD-1-Pe Cy7, a4b7-PE, CD25-APC, CCR6-PECF594, CXCR3-BV786. B Percentage of CD4 and CD8 T cells expressing granzyme B, PD-1, CXCR3, and CCR6 in lung and blood (* p = 0.02 using a two-tailed Mann–Whitney U test). C Correlation plot of vRNA from nasal washes and either granzyme B (GzmB) or PD-1 in CD8 T cells (one-tailed Pearson test p values shown).
    Figure Legend Snippet: Induction of T h 1 CD4 effectors in the lungs during SARS-CoV-2 infection. A Gating strategy for identifying CD95 + CD69 + CD4 and CD8 cells expressing granzyme B, PD-1, α4β7, CCR6, and CXCR3. Fluorochromes used were CD45-A488, CD3-A700, CD20/Dead-APC-Cy7, CD8-BUV 805, CD4-BV650, CD95-BUV737, CD69-BV711, Granzyme B- BV421, PD-1-Pe Cy7, a4b7-PE, CD25-APC, CCR6-PECF594, CXCR3-BV786. B Percentage of CD4 and CD8 T cells expressing granzyme B, PD-1, CXCR3, and CCR6 in lung and blood (* p = 0.02 using a two-tailed Mann–Whitney U test). C Correlation plot of vRNA from nasal washes and either granzyme B (GzmB) or PD-1 in CD8 T cells (one-tailed Pearson test p values shown).

    Techniques Used: Infection, Expressing, Two Tailed Test, MANN-WHITNEY, One-tailed Test

    SARS-CoV-2 infection induces germinal center responses in mediastinal lymph nodes. A Representative multi-color immunofluorescence image of CD3, PD-1, CD20, Bcl-6 with DAPI staining in mediastinal lymph nodes. Two connecting sections were stained with CD3/PD-1 and CD20/ Bcl-6/CD3 to visualize germinal center (GC) T fh cells and GC B cells, respectively. Images in (a–d) showing GC B cells and images in (f–i) showing GC T fh cells are enlarged from white boxes in (e) and collected using a ×20 objective. Merged image in (d) shows CD20+Bcl-6+ GC B cells and image in (i) shows CD3 + PD-1+ GC T fh cells. Scale bar in (e) is 100 µm and the rest are 25 µm. CD3 stain in pink is pseudo color (original red) to distinguish from Bcl-6. B Representative gating strategy to identify follicular dendritic cells (FDC), germinal center B cells (GC B), and germinal center T fh cells (GC T fh ) in the mediastinal lymph nodes (Med) Fluorochromes used were CD45-A488, CD3-A700, CD20-BV421, Dead-BV510, CD8-BUV 805, CD4-BV650, CD95-BUV737, CXCR5-PE, PD-1-Pe-Cy7, Bcl-6-APC-Cy7, CD140b-APC, CD21-PECF594, CXCR3-BV786. C Median fluorescence intensity of Bcl-6, CD21, CD140b, and CXCR3. D Frequency of GC T fh cells, GC B cells, FDCs significantly higher in mediastinal lymph node (Med, data shown from n = 8 independent animals (GC T fh ; ** p = 0.007, * p = 0.01) relative to cervical lymph nodes (CLN, data shown from n = 8 independent animals) and mesenteric lymph nodes (Mes, data shown from n = 7 independent animals) using a two-tailed Wilcoxon matched-pairs signed-rank test, GC B cells; * p = 0.04 using a two-tailed Wilcoxon matched-pairs signed-rank test, FDCs; p = 0.039 using a two-tailed Wilcoxon matched-pairs signed-rank test. Horizontal line indicates median. E Majority of GC T fh cells in mediastinal lymph nodes express CXCR3 (GC T fh and T fh ; ** p = 0.007, * p = 0.01, and mTfh * p = 0.01 relative to CLN and Mes using a two-tailed Wilcoxon matched-pairs signed-rank test). Data shown are from n = 8 independent animals for Med, CLN, and n = 7 independent animals for Mes. Horizontal line indicates median.
    Figure Legend Snippet: SARS-CoV-2 infection induces germinal center responses in mediastinal lymph nodes. A Representative multi-color immunofluorescence image of CD3, PD-1, CD20, Bcl-6 with DAPI staining in mediastinal lymph nodes. Two connecting sections were stained with CD3/PD-1 and CD20/ Bcl-6/CD3 to visualize germinal center (GC) T fh cells and GC B cells, respectively. Images in (a–d) showing GC B cells and images in (f–i) showing GC T fh cells are enlarged from white boxes in (e) and collected using a ×20 objective. Merged image in (d) shows CD20+Bcl-6+ GC B cells and image in (i) shows CD3 + PD-1+ GC T fh cells. Scale bar in (e) is 100 µm and the rest are 25 µm. CD3 stain in pink is pseudo color (original red) to distinguish from Bcl-6. B Representative gating strategy to identify follicular dendritic cells (FDC), germinal center B cells (GC B), and germinal center T fh cells (GC T fh ) in the mediastinal lymph nodes (Med) Fluorochromes used were CD45-A488, CD3-A700, CD20-BV421, Dead-BV510, CD8-BUV 805, CD4-BV650, CD95-BUV737, CXCR5-PE, PD-1-Pe-Cy7, Bcl-6-APC-Cy7, CD140b-APC, CD21-PECF594, CXCR3-BV786. C Median fluorescence intensity of Bcl-6, CD21, CD140b, and CXCR3. D Frequency of GC T fh cells, GC B cells, FDCs significantly higher in mediastinal lymph node (Med, data shown from n = 8 independent animals (GC T fh ; ** p = 0.007, * p = 0.01) relative to cervical lymph nodes (CLN, data shown from n = 8 independent animals) and mesenteric lymph nodes (Mes, data shown from n = 7 independent animals) using a two-tailed Wilcoxon matched-pairs signed-rank test, GC B cells; * p = 0.04 using a two-tailed Wilcoxon matched-pairs signed-rank test, FDCs; p = 0.039 using a two-tailed Wilcoxon matched-pairs signed-rank test. Horizontal line indicates median. E Majority of GC T fh cells in mediastinal lymph nodes express CXCR3 (GC T fh and T fh ; ** p = 0.007, * p = 0.01, and mTfh * p = 0.01 relative to CLN and Mes using a two-tailed Wilcoxon matched-pairs signed-rank test). Data shown are from n = 8 independent animals for Med, CLN, and n = 7 independent animals for Mes. Horizontal line indicates median.

    Techniques Used: Infection, Immunofluorescence, Staining, Fluorescence, Two Tailed Test

    SARS-CoV-2 infection leads to rapid and transient shifts in innate immune responses in peripheral blood. A Representative gating strategy for innate immune subsets in whole blood after gating on singlets. Fluorochromes used were CD3/CD20- APC-Cy7, CD14-A700, CD8- BUV 805, CD66-APC, HLA-DR-BV786, CD16-BV605, CD123-BV421, CD11c-Pe-Cy7. B Kinetics of innate immune responses (pro-inflammatory monocytes; * p = 0.01 at d2 and d4 relative to d0 using a one-tailed paired t test in infected animals, ** p = 0.006 and 0.002 at d2 and d4 relative to d0 in infused animals, pDCs; ** p = 0.005 at d2 relative to d0 using a one-tailed paired t test in infected animals, * p = 0.01 at d2 relative to d0 using a one-tailed paired t test in infused animals, mDCs; * p = 0.02 at d2 relative to d0 using a one-tailed paired t test in infected animals). C Serum chemokines monocyte chemoattractant protein (MCP)-1, interferon gamma induced protein (IP)-10, and interferon induced T-cell alpha chemoattractant (I-TAC) (MCP-1; ** p = 0.001 for infected and ** p = 0.005 for infused at d2 relative to d0 using a one-tailed paired t test, IP-10; * p = 0.03 for infected and *** p = 0.0008 for infused at d2 relative to d0 using a one-tailed paired t test, ITAC; *** p = 0.0005 for infected and *** p = 0.0007 for infused at d2 relative to d0 using a one-tailed paired t test). D Correlation of innate immune cells against chemokines, and interleukin (IL)-10 vs IL-6 (two-tailed Pearson test p values shown, 95% confidence bands of the best fit line are shown).
    Figure Legend Snippet: SARS-CoV-2 infection leads to rapid and transient shifts in innate immune responses in peripheral blood. A Representative gating strategy for innate immune subsets in whole blood after gating on singlets. Fluorochromes used were CD3/CD20- APC-Cy7, CD14-A700, CD8- BUV 805, CD66-APC, HLA-DR-BV786, CD16-BV605, CD123-BV421, CD11c-Pe-Cy7. B Kinetics of innate immune responses (pro-inflammatory monocytes; * p = 0.01 at d2 and d4 relative to d0 using a one-tailed paired t test in infected animals, ** p = 0.006 and 0.002 at d2 and d4 relative to d0 in infused animals, pDCs; ** p = 0.005 at d2 relative to d0 using a one-tailed paired t test in infected animals, * p = 0.01 at d2 relative to d0 using a one-tailed paired t test in infused animals, mDCs; * p = 0.02 at d2 relative to d0 using a one-tailed paired t test in infected animals). C Serum chemokines monocyte chemoattractant protein (MCP)-1, interferon gamma induced protein (IP)-10, and interferon induced T-cell alpha chemoattractant (I-TAC) (MCP-1; ** p = 0.001 for infected and ** p = 0.005 for infused at d2 relative to d0 using a one-tailed paired t test, IP-10; * p = 0.03 for infected and *** p = 0.0008 for infused at d2 relative to d0 using a one-tailed paired t test, ITAC; *** p = 0.0005 for infected and *** p = 0.0007 for infused at d2 relative to d0 using a one-tailed paired t test). D Correlation of innate immune cells against chemokines, and interleukin (IL)-10 vs IL-6 (two-tailed Pearson test p values shown, 95% confidence bands of the best fit line are shown).

    Techniques Used: Infection, One-tailed Test, Two Tailed Test

    CD4 T fh cells targeting spike (S) and nucleocapsid (N) are generated in lymphoid tissue following SARS-CoV-2 infection. A Representative gating strategy to identify SARS-CoV-2-specific CD4 T cells following stimulation with peptide megapools; membrane (M), open reading frame non-structural proteins (ORF-nsp) and Phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Iono) Fluorochromes used were CD3-A700, Dead-APC-Cy7, CD8-BV510, CD4-BV650, CD95-BUV737, CXCR5-PE, PD-1-Pe Cy7, CD25-APC, OX40-BV786, IFNG-Pe-Cy7, TNFa-A488, IL-17-BV421, IL-21-APC. B Scatter plot showing Activation-induced marker (AIM) + CD4 subsets. Dashed line represents undetectable responses assigned a value of 0.01% C Gating strategy to identify cytokine profiles (interferon (IFN)γ, interleukin (IL)-2, tumor necrosis factor (TNF)a, interleukin (IL)-17, interleukin (IL)-21) of CXCR5 + , CXCR5-, and CD8 + CD95 + T cells) in spleen following stimulation. D Pie chart shows T-cell polyfunctionality.
    Figure Legend Snippet: CD4 T fh cells targeting spike (S) and nucleocapsid (N) are generated in lymphoid tissue following SARS-CoV-2 infection. A Representative gating strategy to identify SARS-CoV-2-specific CD4 T cells following stimulation with peptide megapools; membrane (M), open reading frame non-structural proteins (ORF-nsp) and Phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Iono) Fluorochromes used were CD3-A700, Dead-APC-Cy7, CD8-BV510, CD4-BV650, CD95-BUV737, CXCR5-PE, PD-1-Pe Cy7, CD25-APC, OX40-BV786, IFNG-Pe-Cy7, TNFa-A488, IL-17-BV421, IL-21-APC. B Scatter plot showing Activation-induced marker (AIM) + CD4 subsets. Dashed line represents undetectable responses assigned a value of 0.01% C Gating strategy to identify cytokine profiles (interferon (IFN)γ, interleukin (IL)-2, tumor necrosis factor (TNF)a, interleukin (IL)-17, interleukin (IL)-21) of CXCR5 + , CXCR5-, and CD8 + CD95 + T cells) in spleen following stimulation. D Pie chart shows T-cell polyfunctionality.

    Techniques Used: Generated, Infection, Activation Assay, Marker

    SARS-CoV-2 infection increases the number CD4 T follicular helper cells in peripheral blood. A Representative gating strategy to capture CD4 T cells expressing Ki-67 and programmed death-1 (PD-1) in whole blood. Fluorochromes used were CD3-A700, CD20/Dead-APC-Cy7, CD8-BUV 805, CD4-BV650, CD95-BUV737, CXCR5-PE, PD-1-Pe Cy7, Ki-67-A488, CXCR3-BV786, CCR6-PECF594, CCR4-BV605, SLAM-A488, CX3CR1-PECF594, CD28-Pe-Cy7, CCR7-BV711, ICOS-BV786. B Kinetics show frequency and absolute counts of Ki-67 + PD-1 + CD4 T follicular helper cells (T fh ) cells (% of T fh cells; * p = 0.01 at d4 and d7 relative to d0 for infected and ** p = 0.002 at d7 relative to d0 for infused using a one-tailed paired t test, absolute T fh cell counts; ** p = 0.003 at d4 and ** p = 0.0086 at d7 relative to d0 for infected and ** p = 0.003 at d7 relative to d0 for infused using a one-tailed paired t test. Data are from a real-time longitudinal staining of whole blood performed a single time) C correlation plots of Ki-67 + CD8 T cells against Ki-67 + CD4 subsets, and viral(v)RNA (all day 7) (two-tailed Pearson test p values shown. 95% confidence bands of the best fit line are shown) D t-distributed stochastic neighbor embedding (tSNE) plot based on flow cytometry data of CD4 Ki-67 + events at Day 7 from infected (16,197 events) and infected + infused animals (22,406 events); dot plot shows frequency of Ki-67 + CD4 T-cell subsets. ( E – F ) Histograms and median fluorescence intensity (MFI) dot plots illustrate relative expression of signaling lymphocyte activation molecule (SLAM), CX3C chemokine receptor 1 (CX3CR1), CD28, and C-C chemokine receptor type 7(CCR7) within four different populations identified at Day 7 in peripheral blood mononuclear cells (PBMCs, n = 7). Unique symbols identify animals in each of the experimental groups.
    Figure Legend Snippet: SARS-CoV-2 infection increases the number CD4 T follicular helper cells in peripheral blood. A Representative gating strategy to capture CD4 T cells expressing Ki-67 and programmed death-1 (PD-1) in whole blood. Fluorochromes used were CD3-A700, CD20/Dead-APC-Cy7, CD8-BUV 805, CD4-BV650, CD95-BUV737, CXCR5-PE, PD-1-Pe Cy7, Ki-67-A488, CXCR3-BV786, CCR6-PECF594, CCR4-BV605, SLAM-A488, CX3CR1-PECF594, CD28-Pe-Cy7, CCR7-BV711, ICOS-BV786. B Kinetics show frequency and absolute counts of Ki-67 + PD-1 + CD4 T follicular helper cells (T fh ) cells (% of T fh cells; * p = 0.01 at d4 and d7 relative to d0 for infected and ** p = 0.002 at d7 relative to d0 for infused using a one-tailed paired t test, absolute T fh cell counts; ** p = 0.003 at d4 and ** p = 0.0086 at d7 relative to d0 for infected and ** p = 0.003 at d7 relative to d0 for infused using a one-tailed paired t test. Data are from a real-time longitudinal staining of whole blood performed a single time) C correlation plots of Ki-67 + CD8 T cells against Ki-67 + CD4 subsets, and viral(v)RNA (all day 7) (two-tailed Pearson test p values shown. 95% confidence bands of the best fit line are shown) D t-distributed stochastic neighbor embedding (tSNE) plot based on flow cytometry data of CD4 Ki-67 + events at Day 7 from infected (16,197 events) and infected + infused animals (22,406 events); dot plot shows frequency of Ki-67 + CD4 T-cell subsets. ( E – F ) Histograms and median fluorescence intensity (MFI) dot plots illustrate relative expression of signaling lymphocyte activation molecule (SLAM), CX3C chemokine receptor 1 (CX3CR1), CD28, and C-C chemokine receptor type 7(CCR7) within four different populations identified at Day 7 in peripheral blood mononuclear cells (PBMCs, n = 7). Unique symbols identify animals in each of the experimental groups.

    Techniques Used: Infection, Expressing, One-tailed Test, Staining, Two Tailed Test, Flow Cytometry, Fluorescence, Activation Assay

    32) 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

    33) Product Images from "Discovery of human ACE2 variants with altered recognition by the SARS-CoV-2 spike protein"

    Article Title: Discovery of human ACE2 variants with altered recognition by the SARS-CoV-2 spike protein

    Journal: bioRxiv

    doi: 10.1101/2020.09.17.301861

    Large-scale mutagenesis of ACE2’s peptidase domain. (a) Analysis of how amino acid substitutions across positions 18-615 affect binding of the SARS-CoV-2 spike protein. The plot quantifies the importance of each site by taking the mean of the absolute value of all mutation effects observed at that site. The grey line represents the mean absolute value of the mutation effect and the blue line shows the moving average to highlight general regions of ACE2 that are important for binding. Key structural landmarks are highlighted with shaded regions along the length of the sequence. (b) Mutation effect heat maps for three different regions of ACE2. Red denotes mutations that increase ACE2 spike binding; blue denotes reduced binding. Overall, we observe the effects of 3571 amino acid substitutions across 597 positions in ACE2’s peptidase domain. (c) The mean absolute mutation effect mapped onto the three-dimensional ACE2 structure (PDB ID: 6LZG). Residues near the spike interface are important for binding, in addition to many sites located on the distal lobe of the protein domain. (d) The most important region of ACE2 structure is composed of a tightly packed cluster of residues located over 30 Å from the spike interface.
    Figure Legend Snippet: Large-scale mutagenesis of ACE2’s peptidase domain. (a) Analysis of how amino acid substitutions across positions 18-615 affect binding of the SARS-CoV-2 spike protein. The plot quantifies the importance of each site by taking the mean of the absolute value of all mutation effects observed at that site. The grey line represents the mean absolute value of the mutation effect and the blue line shows the moving average to highlight general regions of ACE2 that are important for binding. Key structural landmarks are highlighted with shaded regions along the length of the sequence. (b) Mutation effect heat maps for three different regions of ACE2. Red denotes mutations that increase ACE2 spike binding; blue denotes reduced binding. Overall, we observe the effects of 3571 amino acid substitutions across 597 positions in ACE2’s peptidase domain. (c) The mean absolute mutation effect mapped onto the three-dimensional ACE2 structure (PDB ID: 6LZG). Residues near the spike interface are important for binding, in addition to many sites located on the distal lobe of the protein domain. (d) The most important region of ACE2 structure is composed of a tightly packed cluster of residues located over 30 Å from the spike interface.

    Techniques Used: Mutagenesis, Binding Assay, Sequencing

    34) Product Images from "Rapid protection from COVID-19 in nonhuman primates vaccinated intramuscularly but not intranasally with a single dose of a recombinant vaccine"

    Article Title: Rapid protection from COVID-19 in nonhuman primates vaccinated intramuscularly but not intranasally with a single dose of a recombinant vaccine

    Journal: bioRxiv

    doi: 10.1101/2021.01.19.426885

    Histopathology and Immunohistochemistry of NHP lungs. (A) Pulmonary lesions depicting typical coronavirus respiratory pathology including locally extensive regions of bronchointerstitial pneumonia and proteinaceous fluid accumulation in adjacent alveoli (40x, H E). (B) Disseminated immunopathology with prominent perivascular lymphocytic cuffing and multifocal involvement at terminal airways (40x, H E). (C) IN vaccination shows pulmonary pathology characterized by a combination of interstitial pneumonia and immunopathology (40x, H E). (D) Foci of interstitial pneumonia are characterized by prominent type II pneumocyte hyperplasia, leukocyte infiltration and expansion of alveolar septa and accumulation of low numbers of macrophages, neutrophils and proteinaceous fluid in alveolar spaces (200x, H E). (E) Terminal airways and medium to small caliber blood vessels are cuffed by moderate numbers of lymphocytes with scattered eosinophils (200x, H E). (F) Foci of interstitial pneumonia show pronounced type II pneumocyte hyperplasia, thickening of alveolar septa by an infiltration of leukocytes and leukocyte spillover into adjacent alveolar spaces with moderate numbers of alveolar eosinophils noted and multifocal fibrin mats filling alveolar spaces (200x, H E). (G) Low numbers of type I pneumocytes in regions lacking pathology are immunoreactive for SARS-CoV-2 antibody (200x, immunohistochemistry (IHC)). (H) SARS-CoV-2-specific immunoreactivity was not observed in evaluated sections of the IM vaccinated group (200x, IHC). (I) Low numbers of type I pneumocytes and alveolar macrophages are immunoreactive for SARS-CoV-2 in select foci of interstitial pneumonia (200x, IHC).
    Figure Legend Snippet: Histopathology and Immunohistochemistry of NHP lungs. (A) Pulmonary lesions depicting typical coronavirus respiratory pathology including locally extensive regions of bronchointerstitial pneumonia and proteinaceous fluid accumulation in adjacent alveoli (40x, H E). (B) Disseminated immunopathology with prominent perivascular lymphocytic cuffing and multifocal involvement at terminal airways (40x, H E). (C) IN vaccination shows pulmonary pathology characterized by a combination of interstitial pneumonia and immunopathology (40x, H E). (D) Foci of interstitial pneumonia are characterized by prominent type II pneumocyte hyperplasia, leukocyte infiltration and expansion of alveolar septa and accumulation of low numbers of macrophages, neutrophils and proteinaceous fluid in alveolar spaces (200x, H E). (E) Terminal airways and medium to small caliber blood vessels are cuffed by moderate numbers of lymphocytes with scattered eosinophils (200x, H E). (F) Foci of interstitial pneumonia show pronounced type II pneumocyte hyperplasia, thickening of alveolar septa by an infiltration of leukocytes and leukocyte spillover into adjacent alveolar spaces with moderate numbers of alveolar eosinophils noted and multifocal fibrin mats filling alveolar spaces (200x, H E). (G) Low numbers of type I pneumocytes in regions lacking pathology are immunoreactive for SARS-CoV-2 antibody (200x, immunohistochemistry (IHC)). (H) SARS-CoV-2-specific immunoreactivity was not observed in evaluated sections of the IM vaccinated group (200x, IHC). (I) Low numbers of type I pneumocytes and alveolar macrophages are immunoreactive for SARS-CoV-2 in select foci of interstitial pneumonia (200x, IHC).

    Techniques Used: Histopathology, Immunohistochemistry

    Schematic and characterization of VSV-based vaccines. (A) Schematic illustrating vaccine vector design. T7 promotor; N nucleoprotein; P phosphoprotein; M matrix protein; EBOV GP Ebola virus glycoprotein; L RNA-dependent RNA polymerase; SARS2-S SARS-CoV-2 S. (B) Western blot analysis of cell supernatant samples containing VSV vaccines probed for SARS-CoV-2 S (left), VSV M (middle) or EBOV GP (right). 1 VSV wildtype (VSVwt); 2 VSV-EBOV; 3 VSV-SARS2-EBOV. (C) Viral growth kinetics on VeroE6 cells. Geometric mean and SD are depicted. Results are not statistically significant. (D) Schematic outline of the rhesus macaque study.
    Figure Legend Snippet: Schematic and characterization of VSV-based vaccines. (A) Schematic illustrating vaccine vector design. T7 promotor; N nucleoprotein; P phosphoprotein; M matrix protein; EBOV GP Ebola virus glycoprotein; L RNA-dependent RNA polymerase; SARS2-S SARS-CoV-2 S. (B) Western blot analysis of cell supernatant samples containing VSV vaccines probed for SARS-CoV-2 S (left), VSV M (middle) or EBOV GP (right). 1 VSV wildtype (VSVwt); 2 VSV-EBOV; 3 VSV-SARS2-EBOV. (C) Viral growth kinetics on VeroE6 cells. Geometric mean and SD are depicted. Results are not statistically significant. (D) Schematic outline of the rhesus macaque study.

    Techniques Used: Plasmid Preparation, Western Blot

    Humoral immune responses in NHPs. Serum samples collected throughout the study from all NHPs were examined for (A) SARS-CoV-2 S-specific IgG, (B) SARS-CoV-2 S receptor binding domain (RBD)-specific IgG or (C) IgG subclasses specific to SARS-CoV-2 S by ELISA. (D) Neutralizing titers to SARS-CoV-2 were determined. (E) Bronchoalveolar lavage (BAL) samples were analyzed for SARS-CoV-2 S-specific IgG (S IgG) or IgA (S IgA), and SARS CoV-2 S RBD-specific IgG (RBD IgG) by ELISA. (A-D) Geometric mean and geometric standard deviation (SD) are depicted. (F) IgG subclasses specific to SARS-CoV-2 S in BAL samples were analyzed by ELISA. (E, F) Mean and SD are depicted. Statistical significance is indicated.
    Figure Legend Snippet: Humoral immune responses in NHPs. Serum samples collected throughout the study from all NHPs were examined for (A) SARS-CoV-2 S-specific IgG, (B) SARS-CoV-2 S receptor binding domain (RBD)-specific IgG or (C) IgG subclasses specific to SARS-CoV-2 S by ELISA. (D) Neutralizing titers to SARS-CoV-2 were determined. (E) Bronchoalveolar lavage (BAL) samples were analyzed for SARS-CoV-2 S-specific IgG (S IgG) or IgA (S IgA), and SARS CoV-2 S RBD-specific IgG (RBD IgG) by ELISA. (A-D) Geometric mean and geometric standard deviation (SD) are depicted. (F) IgG subclasses specific to SARS-CoV-2 S in BAL samples were analyzed by ELISA. (E, F) Mean and SD are depicted. Statistical significance is indicated.

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

    SARS-CoV-2 loads in vaccinated NHPs. Groups of 6 NHPs were IN or IM vaccinated with a single dose of VSV-SARS2-EBOV; 4 control animals received the VSV-EBOV. (A) Total SARS-CoV-2-specific RNA (left panel) and subgenomic (sg) RNA (right panel) in nasal swabs collected from NHPs. (B) Total SARS-CoV-2-specific RNA and sgRNA in bronchoalveolar lavage (BAL) samples collected on day 3. (C) Lung radiograph scores after challenge. Mean and standard deviation (SD) are shown. (D) Total SARS-CoV-2-specific RNA and sgRNA in lung samples collected on day 7. (A, B, D) Geometric mean and geometric SD are depicted. Statistical significance is indicated.
    Figure Legend Snippet: SARS-CoV-2 loads in vaccinated NHPs. Groups of 6 NHPs were IN or IM vaccinated with a single dose of VSV-SARS2-EBOV; 4 control animals received the VSV-EBOV. (A) Total SARS-CoV-2-specific RNA (left panel) and subgenomic (sg) RNA (right panel) in nasal swabs collected from NHPs. (B) Total SARS-CoV-2-specific RNA and sgRNA in bronchoalveolar lavage (BAL) samples collected on day 3. (C) Lung radiograph scores after challenge. Mean and standard deviation (SD) are shown. (D) Total SARS-CoV-2-specific RNA and sgRNA in lung samples collected on day 7. (A, B, D) Geometric mean and geometric SD are depicted. Statistical significance is indicated.

    Techniques Used: Standard Deviation

    Virus load in NHP tissue samples on day 7. (A) Total SARS-CoV-2-specific RNA (left panel) and subgenomic (sg) RNA (right panel) in lung samples collected from NHPs. RLU right lobe upper; RLM right lobe middle; RLL right lobe lower; LLU left lobe upper; LLM left lobe middle; LLL left lobe lower. (B) Total SARS-CoV-2-specific RNA in tissue samples (right panel) from NHPs. LN lymph node; R right; L left.
    Figure Legend Snippet: Virus load in NHP tissue samples on day 7. (A) Total SARS-CoV-2-specific RNA (left panel) and subgenomic (sg) RNA (right panel) in lung samples collected from NHPs. RLU right lobe upper; RLM right lobe middle; RLL right lobe lower; LLU left lobe upper; LLM left lobe middle; LLL left lobe lower. (B) Total SARS-CoV-2-specific RNA in tissue samples (right panel) from NHPs. LN lymph node; R right; L left.

    Techniques Used:

    BAL RNA-sequencing. (A) Venn diagram of differentially expressed genes (DEGs) expressed 3 days post challenge with SARS-CoV-2. Animals either received a control, intramuscular (IM) or intranasal (IN) vaccination. (B) Bubbleplot representing functional enrichment of DEGs shared by all infected groups at 3 days post challenge. Color intensity of each bubble represents the negative log of p-value and the relative size of each bubble represents the number of DEGs belonging to the specified Gene Ontology (GO) term. (C) Heatmap representing shared upregulated DEGs enriching to GO terms “lymphocyte activation” and “T cell differentiation in volved in the immune response.” Expression is represented as the normalized rpkm, where each column represents the median rpkm of the given group. Range of colors is based on scale and centered rpkm values of the represented DEGs. GO term network depicting functional enrichment of DEGs unique to (D) IN and (F) IM using Mediascape. Color-coded clustered nodes correspond to one GO term or KEGG pathway. Node size represents the number of DEGs associated with the indicated term or pathway. Gray lines represent shared interactions between terms/pathways, with density and number indicating the strength of connections between closely related terms/pathways. Heatmaps representing DEGs unique to (E) IN and (G) IM. Exemplar DEGs are annotated. Red represents upregulation, blue presents downregulation. Each column represents the median rpkm of the given group. For all heatmaps, range of colors is based on scale and centered rpkm values of the represented DEGs.
    Figure Legend Snippet: BAL RNA-sequencing. (A) Venn diagram of differentially expressed genes (DEGs) expressed 3 days post challenge with SARS-CoV-2. Animals either received a control, intramuscular (IM) or intranasal (IN) vaccination. (B) Bubbleplot representing functional enrichment of DEGs shared by all infected groups at 3 days post challenge. Color intensity of each bubble represents the negative log of p-value and the relative size of each bubble represents the number of DEGs belonging to the specified Gene Ontology (GO) term. (C) Heatmap representing shared upregulated DEGs enriching to GO terms “lymphocyte activation” and “T cell differentiation in volved in the immune response.” Expression is represented as the normalized rpkm, where each column represents the median rpkm of the given group. Range of colors is based on scale and centered rpkm values of the represented DEGs. GO term network depicting functional enrichment of DEGs unique to (D) IN and (F) IM using Mediascape. Color-coded clustered nodes correspond to one GO term or KEGG pathway. Node size represents the number of DEGs associated with the indicated term or pathway. Gray lines represent shared interactions between terms/pathways, with density and number indicating the strength of connections between closely related terms/pathways. Heatmaps representing DEGs unique to (E) IN and (G) IM. Exemplar DEGs are annotated. Red represents upregulation, blue presents downregulation. Each column represents the median rpkm of the given group. For all heatmaps, range of colors is based on scale and centered rpkm values of the represented DEGs.

    Techniques Used: RNA Sequencing Assay, Functional Assay, Infection, Activation Assay, Cell Differentiation, Expressing

    Lung RNA-Sequencing. (A) Venn diagram of differentially expressed genes (DEGs) expressed 3 days post challenge with SARS-CoV-2. Animals either received a control, intramuscular (IM) or intranasal (IN) vaccination. (B) Bubbleplot representing functional enrichment of DEGs shared by all infected groups at 7 days post challenge. Color intensity of each bubble represents the negative log of p-value and the relative size of each bubble represents the number of DEGs belonging to the specified Gene Ontology (GO) term. Heatmaps representing shared GO terms (C) “regulation of innate immune response”, “antigen processing and presentation of exogenous antigen” for downregulated DEGs; and (D) “regulated exocytosis”, “myeloid leukocyte activation” and “neutrophil degranulation” for upregulated DEGs. Expression is represented as the normalized rpkm, where each column represents the median rpkm of the given group. Range of colors is based on scale and centered rpkm values of the represented DEGs. GO term network depicting functional enrichment of DEGs unique to (E) IN and (G) IM using Mediascape. Color-coded clustered nodes correspond to one GO term or KEGG pathway. Node size represents the number of DEGs associated with the indicated term or pathway. Gray lines represent shared interactions between terms/pathways, with density and number indicating the strength of connections between closely related terms/pathways. Heatmaps representing DEGs unique to (F) IN and (H) IM. Exemplar DEGs are annotated. Red represents upregulation, blue presents downregulation. Each column represents the median rpkm of the given group. For all heatmaps, range of colors is based on scale and centered rpkm values of the represented DEGs.
    Figure Legend Snippet: Lung RNA-Sequencing. (A) Venn diagram of differentially expressed genes (DEGs) expressed 3 days post challenge with SARS-CoV-2. Animals either received a control, intramuscular (IM) or intranasal (IN) vaccination. (B) Bubbleplot representing functional enrichment of DEGs shared by all infected groups at 7 days post challenge. Color intensity of each bubble represents the negative log of p-value and the relative size of each bubble represents the number of DEGs belonging to the specified Gene Ontology (GO) term. Heatmaps representing shared GO terms (C) “regulation of innate immune response”, “antigen processing and presentation of exogenous antigen” for downregulated DEGs; and (D) “regulated exocytosis”, “myeloid leukocyte activation” and “neutrophil degranulation” for upregulated DEGs. Expression is represented as the normalized rpkm, where each column represents the median rpkm of the given group. Range of colors is based on scale and centered rpkm values of the represented DEGs. GO term network depicting functional enrichment of DEGs unique to (E) IN and (G) IM using Mediascape. Color-coded clustered nodes correspond to one GO term or KEGG pathway. Node size represents the number of DEGs associated with the indicated term or pathway. Gray lines represent shared interactions between terms/pathways, with density and number indicating the strength of connections between closely related terms/pathways. Heatmaps representing DEGs unique to (F) IN and (H) IM. Exemplar DEGs are annotated. Red represents upregulation, blue presents downregulation. Each column represents the median rpkm of the given group. For all heatmaps, range of colors is based on scale and centered rpkm values of the represented DEGs.

    Techniques Used: RNA Sequencing Assay, Functional Assay, Infection, Activation Assay, Expressing

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.07.14.201616

    Manipulation of cellular heparan sulfate decreases infection of native SARS-CoV-2 virus. A , Flow cytometry analysis of SARS-CoV-2 infected (Red) or uninfected (Black) Vero cells stained with antibodies against SARS-CoV-2 nucleocapsid and spike protein. B, SARS-CoV-2 infection of Vero cells performed in the absence and presence of HSase, or with incubation with different concentrations of UFH. The extent of infection was analyzed by flow cytometry as in panel A. The graph shows a composite of five separate experiments performed in triplicate. The mean data from the individual experiments are colorized to allow for separate visualization C, Same data as in B , but with each experimental repeat normalized to the Mock infection. D , SARS-CoV-2 infection of Hep3B mutants altered in HS biosynthesis enzymes. Cells were infected for 1hr and incubated 48hrs, allowing for new virus to be formed. The resulting titers were determined by plaque assays on Vero E6 cells. Average values with standard error mean are shown, along with the individual data points. Statistical analysis by one-way ANOVA ( B, C ) or unpaired t-test ( D ); ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001)
    Figure Legend Snippet: Manipulation of cellular heparan sulfate decreases infection of native SARS-CoV-2 virus. A , Flow cytometry analysis of SARS-CoV-2 infected (Red) or uninfected (Black) Vero cells stained with antibodies against SARS-CoV-2 nucleocapsid and spike protein. B, SARS-CoV-2 infection of Vero cells performed in the absence and presence of HSase, or with incubation with different concentrations of UFH. The extent of infection was analyzed by flow cytometry as in panel A. The graph shows a composite of five separate experiments performed in triplicate. The mean data from the individual experiments are colorized to allow for separate visualization C, Same data as in B , but with each experimental repeat normalized to the Mock infection. D , SARS-CoV-2 infection of Hep3B mutants altered in HS biosynthesis enzymes. Cells were infected for 1hr and incubated 48hrs, allowing for new virus to be formed. The resulting titers were determined by plaque assays on Vero E6 cells. Average values with standard error mean are shown, along with the individual data points. Statistical analysis by one-way ANOVA ( B, C ) or unpaired t-test ( D ); ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001)

    Techniques Used: Infection, Flow Cytometry, Staining, Incubation

    SARS-CoV-2 spike binds heparin through the RBD domain. A , Recombinant trimeric SARS-CoV-2 spike and RBD proteins were bound to heparin-Sepharose and eluted with a gradient of sodium chloride (broken line). B, Spike protein binds to immobilized unfractionated heparin. C, Binding of spike protein or ACE2 to heparin-BSA. Insert shows SARS-CoV-2 spike protein binding to heparin-BSA in the presence of ACE2. D, SARS-CoV-2 spike protein binding to immobilized recombinant ACE2 protein in the presence and absence of heparin or a heparin 16-mer. E, ACE2 binding to immobilized spike protein. F, ACE2 binding to spike protein immobilized on heparin-BSA. The broken line represents baseline binding. Statistical analysis was by one-way ANOVA. (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001).
    Figure Legend Snippet: SARS-CoV-2 spike binds heparin through the RBD domain. A , Recombinant trimeric SARS-CoV-2 spike and RBD proteins were bound to heparin-Sepharose and eluted with a gradient of sodium chloride (broken line). B, Spike protein binds to immobilized unfractionated heparin. C, Binding of spike protein or ACE2 to heparin-BSA. Insert shows SARS-CoV-2 spike protein binding to heparin-BSA in the presence of ACE2. D, SARS-CoV-2 spike protein binding to immobilized recombinant ACE2 protein in the presence and absence of heparin or a heparin 16-mer. E, ACE2 binding to immobilized spike protein. F, ACE2 binding to spike protein immobilized on heparin-BSA. The broken line represents baseline binding. Statistical analysis was by one-way ANOVA. (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001).

    Techniques Used: Recombinant, Binding Assay, Protein Binding

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

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

    SARS-CoV-2 spike ectodomain protein binding to cells is differentially affected by HS from different organs and potently inhibited by heparinoids. A, LC-MS disaccharide analysis of HS isolated from human kidney, liver, tonsil, and lung tissue. B, Inhibition of binding of recombinant SARS-CoV-2 S RBD protein to H1299 cells, using tissue HS. Analysis by flow cytometry. C, inhibition of recombinant trimeric SARS-CoV-2 protein (20 μg/ml) binding to H1299 cells, using CHO HS, heparin, MST heparin, and split-glycol heparin. Analysis by flow cytometry. D, Similar analysis of A549 cells. Curve fitting was performed using non-linear fitting in Prism. IC 50 values are listed in Table 1 . Graphs shows representative experiments performed in technical duplicates or triplicates. (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001).
    Figure Legend Snippet: SARS-CoV-2 spike ectodomain protein binding to cells is differentially affected by HS from different organs and potently inhibited by heparinoids. A, LC-MS disaccharide analysis of HS isolated from human kidney, liver, tonsil, and lung tissue. B, Inhibition of binding of recombinant SARS-CoV-2 S RBD protein to H1299 cells, using tissue HS. Analysis by flow cytometry. C, inhibition of recombinant trimeric SARS-CoV-2 protein (20 μg/ml) binding to H1299 cells, using CHO HS, heparin, MST heparin, and split-glycol heparin. Analysis by flow cytometry. D, Similar analysis of A549 cells. Curve fitting was performed using non-linear fitting in Prism. IC 50 values are listed in Table 1 . Graphs shows representative experiments performed in technical duplicates or triplicates. (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001).

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

    SARS-CoV-2 spike ectodomain binding to cells is dependent on cellular HS. A, Titration of recombinant SARS-CoV-2 spike protein binding to human H1299 cells with and without treatment with a mix of heparin lyases I, II, and III (HSase). B, Recombinant SARS-CoV-2 spike protein staining (20 μg/ml) of H1299, A549 and Hep3B cells, with and without HSase treatment. C, SARS-CoV-2 S RBD protein binding (20 μg/ml) to H1299, A549 and Hep3B cells with and without HSase treatment. D, SARS-CoV-2 spike protein binding (20 μg/ml) to H1299 and A375 cells with and without HSase treatment. E, Anti-HS (F58-10E4) staining of H1299, A549, Hep3B and A375 cells with and without HSase-treatment. All values were obtained by flow cytometry. Graphs shows representative experiments performed in technical triplicate. The experiments were repeated at least three times. Statistical analysis by unpaired t-test (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001).
    Figure Legend Snippet: SARS-CoV-2 spike ectodomain binding to cells is dependent on cellular HS. A, Titration of recombinant SARS-CoV-2 spike protein binding to human H1299 cells with and without treatment with a mix of heparin lyases I, II, and III (HSase). B, Recombinant SARS-CoV-2 spike protein staining (20 μg/ml) of H1299, A549 and Hep3B cells, with and without HSase treatment. C, SARS-CoV-2 S RBD protein binding (20 μg/ml) to H1299, A549 and Hep3B cells with and without HSase treatment. D, SARS-CoV-2 spike protein binding (20 μg/ml) to H1299 and A375 cells with and without HSase treatment. E, Anti-HS (F58-10E4) staining of H1299, A549, Hep3B and A375 cells with and without HSase-treatment. All values were obtained by flow cytometry. Graphs shows representative experiments performed in technical triplicate. The experiments were repeated at least three times. Statistical analysis by unpaired t-test (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001).

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

    SARS-CoV-2 spike ectodomain protein binding to cellular heparan sulfate depends on sulfation. A, Binding of recombinant SARS-CoV-2 spike protein (20 μg/ml) to Hep3B mutants altered in HS biosynthesis enzymes. Specific enzymes that were mutated are listed along the x-axis. B, Binding of SARS-CoV-2 S RBD protein (20 μg/ml) Hep3B mutants. Binding was measured by flow cytometry. All experiments were repeated at least three times. Graphs shows representative experiments performed in technical triplicates. Statistical analysis by unpaired t-test. (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001).
    Figure Legend Snippet: SARS-CoV-2 spike ectodomain protein binding to cellular heparan sulfate depends on sulfation. A, Binding of recombinant SARS-CoV-2 spike protein (20 μg/ml) to Hep3B mutants altered in HS biosynthesis enzymes. Specific enzymes that were mutated are listed along the x-axis. B, Binding of SARS-CoV-2 S RBD protein (20 μg/ml) Hep3B mutants. Binding was measured by flow cytometry. All experiments were repeated at least three times. Graphs shows representative experiments performed in technical triplicates. Statistical analysis by unpaired t-test. (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001).

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

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

    Techniques Used: Binding Assay, Sequencing

    ACE2 and cellular heparan sulfate are both necessary for binding of SARS-CoV-2 spike ectodomain. A, Western blot shows overexpression of ACE2 in the A375 and A375 B4GALT7 -/- cells. A representative blot is shown. B, Binding of SARS-CoV-2 spike protein to cells with and without ACE2 overexpression. Note that binding is reduced in the mutants deficient in HS. C, Gene targeting of ACE2 in A549 using CRISPR/Cas9. The bars show spike binding to two independent ACE2 CRISPR/Cas9 knockout clones with and without HSase treatment. Note that binding depends on ACE2 expression and that residual binding depends in part on HS. All analyses were done by flow cytometry. The graphs show representative experiments performed in triplicate technical replicates. Statistical analysis by unpaired t-test. (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001).
    Figure Legend Snippet: ACE2 and cellular heparan sulfate are both necessary for binding of SARS-CoV-2 spike ectodomain. A, Western blot shows overexpression of ACE2 in the A375 and A375 B4GALT7 -/- cells. A representative blot is shown. B, Binding of SARS-CoV-2 spike protein to cells with and without ACE2 overexpression. Note that binding is reduced in the mutants deficient in HS. C, Gene targeting of ACE2 in A549 using CRISPR/Cas9. The bars show spike binding to two independent ACE2 CRISPR/Cas9 knockout clones with and without HSase treatment. Note that binding depends on ACE2 expression and that residual binding depends in part on HS. All analyses were done by flow cytometry. The graphs show representative experiments performed in triplicate technical replicates. Statistical analysis by unpaired t-test. (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001).

    Techniques Used: Binding Assay, Western Blot, Over Expression, CRISPR, Knock-Out, Clone Assay, Expressing, Flow Cytometry

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

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

    Journal: Cell

    doi: 10.1016/j.cell.2020.09.033

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

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

    ACE2 and Cellular Heparan Sulfate Are Both Necessary for Binding of SARS-CoV-2 Spike Ectodomain (A) Western blot shows overexpression of ACE2 in the A375 and A375 B4GALT7 −/− cells. A representative blot is shown. (B) Binding of SARS-CoV-2 spike protein to cells with and without ACE2 overexpression. Note that binding is reduced in the cells deficient in HS. (C) Gene targeting of ACE2 in A549 using CRISPR-Cas9. The bars show spike binding to two independent ACE2 CRISPR-Cas9 knockout clones with and without HSase treatment. Note that binding depends on ACE2 expression and that residual binding depends in part on HS. All analyses were done by flow cytometry. The graphs show representative experiments performed in triplicate technical replicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S5 .
    Figure Legend Snippet: ACE2 and Cellular Heparan Sulfate Are Both Necessary for Binding of SARS-CoV-2 Spike Ectodomain (A) Western blot shows overexpression of ACE2 in the A375 and A375 B4GALT7 −/− cells. A representative blot is shown. (B) Binding of SARS-CoV-2 spike protein to cells with and without ACE2 overexpression. Note that binding is reduced in the cells deficient in HS. (C) Gene targeting of ACE2 in A549 using CRISPR-Cas9. The bars show spike binding to two independent ACE2 CRISPR-Cas9 knockout clones with and without HSase treatment. Note that binding depends on ACE2 expression and that residual binding depends in part on HS. All analyses were done by flow cytometry. The graphs show representative experiments performed in triplicate technical replicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S5 .

    Techniques Used: Binding Assay, Western Blot, Over Expression, CRISPR, Knock-Out, Clone Assay, Expressing, Flow Cytometry

    SARS-CoV-2 Spike Binds Heparin through the RBD (A) Recombinant trimeric SARS-CoV-2 spike and RBD proteins were bound to heparin-Sepharose and eluted with a gradient of sodium chloride. (B) RBD protein from SARS-CoV-1 and SARS-CoV-2 binding to immobilized heparin-BSA. (C) Binding of spike protein or ACE2 to heparin-BSA. Insert shows SARS-CoV-2 spike protein binding to heparin-BSA in the presence of ACE2. (D) Binding of spike protein in the active RBD open (Mut7) and inactive RBD closed (Mut2) conformation to heparin-BSA. (E) Binding of ACE2 to spike protein in active RBD open (Mut7) and inactive RBD closed (Mut 2) conformation. (F) ACE2 binding to spike protein immobilized on heparin-BSA. The broken line represents background binding. Statistical analysis was by one-way ANOVA. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). (G and H) Negative stain-electron microscopy analysis of binding of heparin and ACE2 to spike protein. ACE2 binding to spike protein increases in the presence of heparin. 3D class averages of SARS-CoV-2 spike bound to zero, one, two, or three ACE2 (white, orange, blue, or gray) when complexed with and without a heparin dp20. The incubation was done for 15 min (G) or 60 min (H). The percentage of particles belonging to each class is shown in pie charts. See also Figure S3 .
    Figure Legend Snippet: SARS-CoV-2 Spike Binds Heparin through the RBD (A) Recombinant trimeric SARS-CoV-2 spike and RBD proteins were bound to heparin-Sepharose and eluted with a gradient of sodium chloride. (B) RBD protein from SARS-CoV-1 and SARS-CoV-2 binding to immobilized heparin-BSA. (C) Binding of spike protein or ACE2 to heparin-BSA. Insert shows SARS-CoV-2 spike protein binding to heparin-BSA in the presence of ACE2. (D) Binding of spike protein in the active RBD open (Mut7) and inactive RBD closed (Mut2) conformation to heparin-BSA. (E) Binding of ACE2 to spike protein in active RBD open (Mut7) and inactive RBD closed (Mut 2) conformation. (F) ACE2 binding to spike protein immobilized on heparin-BSA. The broken line represents background binding. Statistical analysis was by one-way ANOVA. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). (G and H) Negative stain-electron microscopy analysis of binding of heparin and ACE2 to spike protein. ACE2 binding to spike protein increases in the presence of heparin. 3D class averages of SARS-CoV-2 spike bound to zero, one, two, or three ACE2 (white, orange, blue, or gray) when complexed with and without a heparin dp20. The incubation was done for 15 min (G) or 60 min (H). The percentage of particles belonging to each class is shown in pie charts. See also Figure S3 .

    Techniques Used: Recombinant, Binding Assay, Protein Binding, Staining, Electron Microscopy, Incubation

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

    Techniques Used: Binding Assay, Flow Cytometry

    Infection of Hep3B by MERS Pseudovirus, Related to Figure 6 Infection of Hep3B cells with SARS-CoV-2 and MERS-CoV S protein pseudotyped viruses carrying luciferase with and without treatment with heparin lyases.
    Figure Legend Snippet: Infection of Hep3B by MERS Pseudovirus, Related to Figure 6 Infection of Hep3B cells with SARS-CoV-2 and MERS-CoV S protein pseudotyped viruses carrying luciferase with and without treatment with heparin lyases.

    Techniques Used: Infection, Luciferase

    Manipulation of Cellular Heparan Sulfate Decreases Infection of Authentic SARS-CoV-2 Virus (A,) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) Vero cells stained with antibodies against SARS-CoV-2 nucleocapsid and spike protein. (B) SARS-CoV-2 infection of Vero cells performed in the absence and presence of HSase, or with incubation with different concentrations of unfractionated heparin (UFH). The extent of infection was analyzed by flow cytometry as in (A). The graph shows a composite of five separate experiments, each performed in triplicate. The MOI was 0.5, but the extent of infection varied. The MOI in the experiment shown in maroon and blue was 0.2. The mean data from the individual experiments are colorized to allow for separate visualization (C) Same data as in (B), but with the experimental data normalized to the mock infection for each respective experiment. (D) SARS-CoV-2 infection of Hep3B mutants altered in HS biosynthesis enzymes. Cells were infected for 1 h and incubated 48 h, allowing for new virus to form. The resulting viral titers in the culture supernatants were determined by plaque assays on Vero E6 cells. Average values with standard error mean are shown, along with the individual data points. The experiment was initially optimized and then performed in triplicate. (E) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) human bronchial epithelial cells at an air-liquid interface stained with antibodies against SARS-CoV-2 nucleocapsid. (F) SARS-CoV-2 infection of human bronchial epithelial cells at an air-liquid interface was performed in the absence and presence of HSase, or with incubation UFH. The extent of infection was analyzed by flow cytometry. The graph shows a composite of three separate experiments, each performed in triplicate. The mean data from the individual experiments are colorized to allow for separate visualization. (G) Same data as in (F), but with each experimental dataset normalized to the uninfected control. Statistical analysis by one-way ANOVA (B, C, and G) or unpaired t test (D); ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S7 .
    Figure Legend Snippet: Manipulation of Cellular Heparan Sulfate Decreases Infection of Authentic SARS-CoV-2 Virus (A,) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) Vero cells stained with antibodies against SARS-CoV-2 nucleocapsid and spike protein. (B) SARS-CoV-2 infection of Vero cells performed in the absence and presence of HSase, or with incubation with different concentrations of unfractionated heparin (UFH). The extent of infection was analyzed by flow cytometry as in (A). The graph shows a composite of five separate experiments, each performed in triplicate. The MOI was 0.5, but the extent of infection varied. The MOI in the experiment shown in maroon and blue was 0.2. The mean data from the individual experiments are colorized to allow for separate visualization (C) Same data as in (B), but with the experimental data normalized to the mock infection for each respective experiment. (D) SARS-CoV-2 infection of Hep3B mutants altered in HS biosynthesis enzymes. Cells were infected for 1 h and incubated 48 h, allowing for new virus to form. The resulting viral titers in the culture supernatants were determined by plaque assays on Vero E6 cells. Average values with standard error mean are shown, along with the individual data points. The experiment was initially optimized and then performed in triplicate. (E) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) human bronchial epithelial cells at an air-liquid interface stained with antibodies against SARS-CoV-2 nucleocapsid. (F) SARS-CoV-2 infection of human bronchial epithelial cells at an air-liquid interface was performed in the absence and presence of HSase, or with incubation UFH. The extent of infection was analyzed by flow cytometry. The graph shows a composite of three separate experiments, each performed in triplicate. The mean data from the individual experiments are colorized to allow for separate visualization. (G) Same data as in (F), but with each experimental dataset normalized to the uninfected control. Statistical analysis by one-way ANOVA (B, C, and G) or unpaired t test (D); ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S7 .

    Techniques Used: Infection, Flow Cytometry, Staining, Incubation

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

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

    Analysis of Recombinant Spike Proteins and Receptor-Binding Domain, Related to Star Methods, Protein Production (A) SDS-PAGE gel of recombinant SARS-CoV-2 spike ectodomain protein produced in ExpiCho cells and commercial recombinant SARS-CoV-2 RBD. (B) Transmission electron micrographs of recombinant SARS-CoV-2 spike ectodomain protein. (C) Size exclusion chromatography of recombinant SARS-CoV-2 spike ectodomain protein on a Superose 6 column. (D) SDS-PAGE gel of recombinant SARS-CoV-2 RBD produced in ExpiHEK cells. (E) Size exclusion chromatography of recombinant SARS-CoV-2 RBD on a Superdex200 column.
    Figure Legend Snippet: Analysis of Recombinant Spike Proteins and Receptor-Binding Domain, Related to Star Methods, Protein Production (A) SDS-PAGE gel of recombinant SARS-CoV-2 spike ectodomain protein produced in ExpiCho cells and commercial recombinant SARS-CoV-2 RBD. (B) Transmission electron micrographs of recombinant SARS-CoV-2 spike ectodomain protein. (C) Size exclusion chromatography of recombinant SARS-CoV-2 spike ectodomain protein on a Superose 6 column. (D) SDS-PAGE gel of recombinant SARS-CoV-2 RBD produced in ExpiHEK cells. (E) Size exclusion chromatography of recombinant SARS-CoV-2 RBD on a Superdex200 column.

    Techniques Used: Recombinant, Binding Assay, SDS Page, Produced, Transmission Assay, Size-exclusion Chromatography

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

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

    Location of the Putative Heparin/HS Binding Site in the Spike Protein RBD from SARS-CoV-2, Related to Figure 1 PDB files 6VSB and 6M0J were used to model the spike protein. The residues colored pink on the three RBDs (444+509+346+354+356+357+355+466+ 347+348+349+353+450+448+451+352) make up a potential binding site for heparin and heparan sulfate.
    Figure Legend Snippet: Location of the Putative Heparin/HS Binding Site in the Spike Protein RBD from SARS-CoV-2, Related to Figure 1 PDB files 6VSB and 6M0J were used to model the spike protein. The residues colored pink on the three RBDs (444+509+346+354+356+357+355+466+ 347+348+349+353+450+448+451+352) make up a potential binding site for heparin and heparan sulfate.

    Techniques Used: Binding Assay

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

    Techniques Used: Binding Assay, Sequencing

    Binding of Spike Protein to Heparin and ACE2 and Electron Micrographs of the Spike-ACE2 Complexes, Related to Figure 2 (A) SARS-CoV-2 spike binding to immobilized heparin or BSA. (B) ACE2 binding to immobilized spike protein. (C) Transmission electron micrographs of stabilized spike protein treated with ACE2 and with or without dp20 for 15 min or 1 h. (D) 2D classes averages for each condition.
    Figure Legend Snippet: Binding of Spike Protein to Heparin and ACE2 and Electron Micrographs of the Spike-ACE2 Complexes, Related to Figure 2 (A) SARS-CoV-2 spike binding to immobilized heparin or BSA. (B) ACE2 binding to immobilized spike protein. (C) Transmission electron micrographs of stabilized spike protein treated with ACE2 and with or without dp20 for 15 min or 1 h. (D) 2D classes averages for each condition.

    Techniques Used: Binding Assay, Transmission Assay

    37) Product Images from "Immunization with the receptor–binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement"

    Article Title: Immunization with the receptor–binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement

    Journal: bioRxiv

    doi: 10.1101/2020.05.21.107565

    Treatment with the anti-RBD-Fc sera #1 inhibited SARS-CoV-2 infection-triggered CPE. VeroE6 cells were inoculated with mixtures of authentic SARS-CoV-2 and serially diluted anti-RBD-Fc sera #1. The cells were checked daily for CPE. Data presented are images taken at 48 hours post-infection. The test concentrations of the anti-RBD-Fc sera #1 are indicated.
    Figure Legend Snippet: Treatment with the anti-RBD-Fc sera #1 inhibited SARS-CoV-2 infection-triggered CPE. VeroE6 cells were inoculated with mixtures of authentic SARS-CoV-2 and serially diluted anti-RBD-Fc sera #1. The cells were checked daily for CPE. Data presented are images taken at 48 hours post-infection. The test concentrations of the anti-RBD-Fc sera #1 are indicated.

    Techniques Used: Infection

    Neutralization potency and breadth of the anti-RBD sera. ( A ) Anti-RBD sera neutralized SARS2-PV infection in vitro. The day-40 pooled sera were serially diluted and tested for neutralization of retrovirus pseudotyped with SARS-CoV-2 S protein. Data (means±SD) from three independent experiments are shown. ( B ) Anti-RBD sera cross-neutralized SARS-PV infection in vitro. The day-40 pooled sera were serially diluted and tested for neutralization of retrovirus pseudotyped with SARS-CoV S protein. Data (means±SD) from three independent experiments are shown. ( C ) Neutralization efficiency of the anti-RBD sera against authentic SARS-CoV-2 infection. Serially diluted anti-RBD sera were mixed with 200 PFU of live SARS-CoV-2 and then incubated for 1 hr at 37°C. The antisera/virus mixtures were added to pre-seeded VeroE6 cells, followed by incubation for three days. The cells were then analyzed for viral RNA copy number by qPCR analysis. Data are expressed as percentage of the viral RNA copy number of the treatment groups in relation to that of the virus-only control. Means ± SD of triplicate wells are shown. Significant differences between treatment groups and the virus-only control were calculated using student’s two-tail t test and shown as: ***, P
    Figure Legend Snippet: Neutralization potency and breadth of the anti-RBD sera. ( A ) Anti-RBD sera neutralized SARS2-PV infection in vitro. The day-40 pooled sera were serially diluted and tested for neutralization of retrovirus pseudotyped with SARS-CoV-2 S protein. Data (means±SD) from three independent experiments are shown. ( B ) Anti-RBD sera cross-neutralized SARS-PV infection in vitro. The day-40 pooled sera were serially diluted and tested for neutralization of retrovirus pseudotyped with SARS-CoV S protein. Data (means±SD) from three independent experiments are shown. ( C ) Neutralization efficiency of the anti-RBD sera against authentic SARS-CoV-2 infection. Serially diluted anti-RBD sera were mixed with 200 PFU of live SARS-CoV-2 and then incubated for 1 hr at 37°C. The antisera/virus mixtures were added to pre-seeded VeroE6 cells, followed by incubation for three days. The cells were then analyzed for viral RNA copy number by qPCR analysis. Data are expressed as percentage of the viral RNA copy number of the treatment groups in relation to that of the virus-only control. Means ± SD of triplicate wells are shown. Significant differences between treatment groups and the virus-only control were calculated using student’s two-tail t test and shown as: ***, P

    Techniques Used: Neutralization, Infection, In Vitro, Incubation, Real-time Polymerase Chain Reaction

    Assessment of the anti-RBD sera for potential ADE. ( A-C ) ADE assays with SARS2-PV as the inoculum. Serial dilutions of the anti-RBD or the control sera were incubated with SARS2-S pseudotyped retrovirus for 1 hour at 37 °C. The mixtures were added to ( A ) A20, ( B ) THP-1, or ( C ) K562 cell suspensions, followed by incubation at 37°C for three days. Infected cells were subjected to flow cytometry analysis. Data are expressed as percentage of the GFP-expressing cells in relation to the total cells counted. Means ± SD of triplicate wells are shown. ( D ) ADE assay with live SARS-CoV-2 virus as the inoculum. Serial dilutions of the anti-RBD or the control sera were mixed with the live SARS-CoV-2 virus and incubated for 1 hour at 37 °C. The mixtures were added to K562 cell suspensions, followed by incubation at 37 °C for three days. Infected cell cultures were subjected to RNA extraction and qPCR analysis. Data are expressed as percentage of the viral RNA copy number of the treatment groups in relation to that of the virus-only control. Means ± SD of triplicate wells are shown. Significant differences between the virus only (without antisera treatment) group and each of the antisera treatment groups were indicated: n.s., P > 0.05.
    Figure Legend Snippet: Assessment of the anti-RBD sera for potential ADE. ( A-C ) ADE assays with SARS2-PV as the inoculum. Serial dilutions of the anti-RBD or the control sera were incubated with SARS2-S pseudotyped retrovirus for 1 hour at 37 °C. The mixtures were added to ( A ) A20, ( B ) THP-1, or ( C ) K562 cell suspensions, followed by incubation at 37°C for three days. Infected cells were subjected to flow cytometry analysis. Data are expressed as percentage of the GFP-expressing cells in relation to the total cells counted. Means ± SD of triplicate wells are shown. ( D ) ADE assay with live SARS-CoV-2 virus as the inoculum. Serial dilutions of the anti-RBD or the control sera were mixed with the live SARS-CoV-2 virus and incubated for 1 hour at 37 °C. The mixtures were added to K562 cell suspensions, followed by incubation at 37 °C for three days. Infected cell cultures were subjected to RNA extraction and qPCR analysis. Data are expressed as percentage of the viral RNA copy number of the treatment groups in relation to that of the virus-only control. Means ± SD of triplicate wells are shown. Significant differences between the virus only (without antisera treatment) group and each of the antisera treatment groups were indicated: n.s., P > 0.05.

    Techniques Used: Incubation, Infection, Flow Cytometry, Expressing, RNA Extraction, Real-time Polymerase Chain Reaction

    Treatment with the anti-RBD sera inhibited SARS-CoV-2 infection-triggered CPE. VeroE6 cells were inoculated with mixtures of the authentic SARS-CoV-2 virus and serially diluted anti-RBD sera. The cells were checked daily for CPE. Data presented are images taken at 48 hours post-infection. The test concentrations of the anti-RBD sera are indicated.
    Figure Legend Snippet: Treatment with the anti-RBD sera inhibited SARS-CoV-2 infection-triggered CPE. VeroE6 cells were inoculated with mixtures of the authentic SARS-CoV-2 virus and serially diluted anti-RBD sera. The cells were checked daily for CPE. Data presented are images taken at 48 hours post-infection. The test concentrations of the anti-RBD sera are indicated.

    Techniques Used: Infection

    Immunization with recombinant RBD-Fc fusion protein potently elicited SARS-CoV-2 neutralizing antibodies in mice. ( A ) RBD-binding activities of the sera from the three RBD-Fc-immunized mice and the control (naïve) mouse. The sera were serially diluted and then analyzed by ELISA with recombinant SARS2-RBD protein as the coating antigen. Data shown are means and SD of triplicate wells. ( B ) Inhibitory effect of the anti-RBD-Fc sera on the RBD/ACE2 interaction. The anti-RBD-Fc sera #1 and the control sera were serially diluted and then subjected to ACE2 competition ELISA. Data shown are means and SD of triplicate wells. ( C ) Neutralization potency of the antisera against SARS-CoV-2 pseudovirus infection. The antisera were serially diluted and then evaluated for neutralization of SARS-CoV-2 spike-pseudotyped retrovirus. Results from three independent experiments are shown. ( D ) Neutralization potency of the antisera against authentic SARS-CoV-2 infection. Serially diluted antisera were subjected to neutralization assay with SARS-CoV-2 strain nCoV-SH01 as the challenge virus. Data shown are means and SD of triplicate wells. Significant differences were calculated using student’s two-tail t test and shown as: ***, P
    Figure Legend Snippet: Immunization with recombinant RBD-Fc fusion protein potently elicited SARS-CoV-2 neutralizing antibodies in mice. ( A ) RBD-binding activities of the sera from the three RBD-Fc-immunized mice and the control (naïve) mouse. The sera were serially diluted and then analyzed by ELISA with recombinant SARS2-RBD protein as the coating antigen. Data shown are means and SD of triplicate wells. ( B ) Inhibitory effect of the anti-RBD-Fc sera on the RBD/ACE2 interaction. The anti-RBD-Fc sera #1 and the control sera were serially diluted and then subjected to ACE2 competition ELISA. Data shown are means and SD of triplicate wells. ( C ) Neutralization potency of the antisera against SARS-CoV-2 pseudovirus infection. The antisera were serially diluted and then evaluated for neutralization of SARS-CoV-2 spike-pseudotyped retrovirus. Results from three independent experiments are shown. ( D ) Neutralization potency of the antisera against authentic SARS-CoV-2 infection. Serially diluted antisera were subjected to neutralization assay with SARS-CoV-2 strain nCoV-SH01 as the challenge virus. Data shown are means and SD of triplicate wells. Significant differences were calculated using student’s two-tail t test and shown as: ***, P

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

    38) Product Images from "Acute SARS-CoV-2 Infection Impairs Dendritic Cell and T Cell Responses"

    Article Title: Acute SARS-CoV-2 Infection Impairs Dendritic Cell and T Cell Responses

    Journal: Immunity

    doi: 10.1016/j.immuni.2020.07.026

    Peripheral T Cells Display Functional Loss during Acute SARS-CoV-2 Infection (A) Frequencies of Ki67 + cells on CD4 and CD8 T cells were determined by flow cytometry. Fresh PBMCs from 13 APs and 9 CPs were collected at a median of 9 (range, 1–20 days) and 31 days (range, 23–54 days) after symptoms onset, respectively. Frequencies of CD38 + HLA-DR + and PD-1 + cells on CD4 T cells (left) and CD8 T cells (right) were also determined by flow cytometry. Samples of 17 APs and 20 CPs were collected at a median of 13 (range, 1–42 days) and 29.5 days (range, 21–54 days) after symptoms onset, respectively. Samples of 17 HDs were included as controls. Severe patients in the AP and CP groups were presented as black symbols. (B) Proliferation ability of T cells from COVID-19 patients was determined by flow cytometry. Fresh PBMCs from 6 APs (1 severe and 5 mild patients) and 6 mild CPs were obtained at a median of 12 (range, 2–25 days) and 32 days (range, 23–39 days) after symptoms onset, respectively. PBMCs were labeled with CFSE and then were cultured in the presence or absences of anti-CD3 and anti-CD28 mAbs for 3 days before the flow cytometry. PBMCs of 6 HDs were included as controls. Representative histograms (top left) and quantified results (top right) depict the CFSE profiles of CD4 and CD8 T cells, respectively. The presence of IFN-γ, TNF-α, and IL-2 in culture supernatants after anti-CD3/CD28 stimulation was also quantified by using the bead-based cytokine assays (bottom). (C) T cell responses to non-specific stimulation. Fresh PBMCs (same samples from Figure 3 B) were stimulated with PMA/Ionomycin activation cocktail in the presence of brefeldin A (BFA) for 6 h. Expression of IFN-γ and TNF-α in T cells were determined by intracellular cytokine staining analysis. Representative plots showing IFN-γ and TNF-α expression in CD4 and CD8 T cells (top). Frequencies of IFN-γ + and TNF-α + cells were gated on CD45RA − CCR7 + CM and CD45RA − CCR7 − EM CD4 T cells (middle), as well as on EM and CD45RA + CCR7 − (CD45RA + effector memory, EMRA) CD8 T cells (bottom). (D) Expression of granzyme B and perforin in unstimulated EM and EMRA CD8 T cells (same samples from Figure 3 B) was determined by intracellular staining. Representative plots (top) and quantified results (bottom) are shown. Each symbol represents an individual donor. Error bars indicate standard deviation. Statistics were generated by using one-way ANOVA followed by Tukey’s multiple comparisons test, Mann-Whitney test, and 2-tailed Student’s t test. ∗ p
    Figure Legend Snippet: Peripheral T Cells Display Functional Loss during Acute SARS-CoV-2 Infection (A) Frequencies of Ki67 + cells on CD4 and CD8 T cells were determined by flow cytometry. Fresh PBMCs from 13 APs and 9 CPs were collected at a median of 9 (range, 1–20 days) and 31 days (range, 23–54 days) after symptoms onset, respectively. Frequencies of CD38 + HLA-DR + and PD-1 + cells on CD4 T cells (left) and CD8 T cells (right) were also determined by flow cytometry. Samples of 17 APs and 20 CPs were collected at a median of 13 (range, 1–42 days) and 29.5 days (range, 21–54 days) after symptoms onset, respectively. Samples of 17 HDs were included as controls. Severe patients in the AP and CP groups were presented as black symbols. (B) Proliferation ability of T cells from COVID-19 patients was determined by flow cytometry. Fresh PBMCs from 6 APs (1 severe and 5 mild patients) and 6 mild CPs were obtained at a median of 12 (range, 2–25 days) and 32 days (range, 23–39 days) after symptoms onset, respectively. PBMCs were labeled with CFSE and then were cultured in the presence or absences of anti-CD3 and anti-CD28 mAbs for 3 days before the flow cytometry. PBMCs of 6 HDs were included as controls. Representative histograms (top left) and quantified results (top right) depict the CFSE profiles of CD4 and CD8 T cells, respectively. The presence of IFN-γ, TNF-α, and IL-2 in culture supernatants after anti-CD3/CD28 stimulation was also quantified by using the bead-based cytokine assays (bottom). (C) T cell responses to non-specific stimulation. Fresh PBMCs (same samples from Figure 3 B) were stimulated with PMA/Ionomycin activation cocktail in the presence of brefeldin A (BFA) for 6 h. Expression of IFN-γ and TNF-α in T cells were determined by intracellular cytokine staining analysis. Representative plots showing IFN-γ and TNF-α expression in CD4 and CD8 T cells (top). Frequencies of IFN-γ + and TNF-α + cells were gated on CD45RA − CCR7 + CM and CD45RA − CCR7 − EM CD4 T cells (middle), as well as on EM and CD45RA + CCR7 − (CD45RA + effector memory, EMRA) CD8 T cells (bottom). (D) Expression of granzyme B and perforin in unstimulated EM and EMRA CD8 T cells (same samples from Figure 3 B) was determined by intracellular staining. Representative plots (top) and quantified results (bottom) are shown. Each symbol represents an individual donor. Error bars indicate standard deviation. Statistics were generated by using one-way ANOVA followed by Tukey’s multiple comparisons test, Mann-Whitney test, and 2-tailed Student’s t test. ∗ p

    Techniques Used: Functional Assay, Infection, Flow Cytometry, Labeling, Cell Culture, Activation Assay, Expressing, Staining, Standard Deviation, Generated, MANN-WHITNEY

    Acute SARS-CoV-2 Infection Results in Broad Immune Cell Suppression Fresh PBMCs were isolated from acute patients (APs), convalescent patients (CPs), and healthy donors (HDs). (A) For analysis of lymphocyte subsets including T, B, and NK cells, samples of 17 APs and 25 CPs were collected at a median of 13 (range, 1–42 days) and 30 days (range, 21–54 days) after symptoms onset, respectively. (B) For analysis of myeloid cells including DCs, CD14 + + CD16 − monocytes, and M-MDSCs, samples of 17 APs and 29 CPs were collected at a median of 13 (range, 1–42 days) and 30 days (range, 21–54 days) after symptoms onset, respectively. Twenty HDs were included as controls. Cells were stained with different markers of immune cell populations and were subjected to flow cytometry analysis. Cumulative data show the cell frequencies. Each symbol represents an individual donor with a line indicating the mean of each group. Severe patients in both the AP and CP groups were presented as black symbols. Statistics were generated by using one-way ANOVA followed by Tukey’s multiple comparisons test and Mann-Whitney test. ∗ p
    Figure Legend Snippet: Acute SARS-CoV-2 Infection Results in Broad Immune Cell Suppression Fresh PBMCs were isolated from acute patients (APs), convalescent patients (CPs), and healthy donors (HDs). (A) For analysis of lymphocyte subsets including T, B, and NK cells, samples of 17 APs and 25 CPs were collected at a median of 13 (range, 1–42 days) and 30 days (range, 21–54 days) after symptoms onset, respectively. (B) For analysis of myeloid cells including DCs, CD14 + + CD16 − monocytes, and M-MDSCs, samples of 17 APs and 29 CPs were collected at a median of 13 (range, 1–42 days) and 30 days (range, 21–54 days) after symptoms onset, respectively. Twenty HDs were included as controls. Cells were stained with different markers of immune cell populations and were subjected to flow cytometry analysis. Cumulative data show the cell frequencies. Each symbol represents an individual donor with a line indicating the mean of each group. Severe patients in both the AP and CP groups were presented as black symbols. Statistics were generated by using one-way ANOVA followed by Tukey’s multiple comparisons test and Mann-Whitney test. ∗ p

    Techniques Used: Infection, Isolation, Staining, Flow Cytometry, Generated, MANN-WHITNEY

    39) 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

    40) 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:

    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.

    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).

    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. ..

    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
    The structure of CT-P59 Fab in complex with <t>SARS-CoV-2</t> 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.
    Sars Cov 2 Rbd, supplied by Sino Biological, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-20602-5

    Figure Lengend 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.

    Article Snippet: After several rounds of biopanning, scFv phages binding to SARS-CoV-2 RBD were identified by phage ELISA for further selection.

    Techniques:

    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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-20602-5

    Figure Lengend 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

    Article Snippet: After several rounds of biopanning, scFv phages binding to SARS-CoV-2 RBD were identified by phage ELISA for further selection.

    Techniques: 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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-20602-5

    Figure Lengend 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

    Article Snippet: After several rounds of biopanning, scFv phages binding to SARS-CoV-2 RBD were identified by phage ELISA for further selection.

    Techniques: 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).

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-20602-5

    Figure Lengend 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).

    Article Snippet: After several rounds of biopanning, scFv phages binding to SARS-CoV-2 RBD were identified by phage ELISA for further selection.

    Techniques: 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.

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-20602-5

    Figure Lengend 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.

    Article Snippet: After several rounds of biopanning, scFv phages binding to SARS-CoV-2 RBD were identified by phage ELISA for further selection.

    Techniques: Infection, In Vitro, Standard Deviation

    Isolation of RBD-specific memory B cells using flow cytometry. A. The heatmap depicts the specificity of convalescent patients’ plasma against S1 and RBD from SARS-CoV-2, SARS-CoV and MERS-CoV, measured by ELISA. Serial dilutions of plasma samples were performed to test the reactivity of antibodies in plasma. The plasma of healthy donors was used as the control. Data were shown with the mean of representative experiments. B. Gating strategy for SARS-CoV-2 RBD-specific IgG + B cells in PBMCs of the convalescent patients. Living CD19+ IgD − IgG + cells were gated, and cells with positive SARS-CoV-2 RBD staining were selected for single-cell sorting. C. FACS analysis of RBD-specific memory B cells in CD19 + IgD − IgG + memory B cells from PBMCs of three batch convalescent patients. Plots show CD19 + IgD − IgG + RBD+ populations using gating strategy described in B .

    Journal: bioRxiv

    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

    doi: 10.1101/2020.08.19.253369

    Figure Lengend Snippet: Isolation of RBD-specific memory B cells using flow cytometry. A. The heatmap depicts the specificity of convalescent patients’ plasma against S1 and RBD from SARS-CoV-2, SARS-CoV and MERS-CoV, measured by ELISA. Serial dilutions of plasma samples were performed to test the reactivity of antibodies in plasma. The plasma of healthy donors was used as the control. Data were shown with the mean of representative experiments. B. Gating strategy for SARS-CoV-2 RBD-specific IgG + B cells in PBMCs of the convalescent patients. Living CD19+ IgD − IgG + cells were gated, and cells with positive SARS-CoV-2 RBD staining were selected for single-cell sorting. C. FACS analysis of RBD-specific memory B cells in CD19 + IgD − IgG + memory B cells from PBMCs of three batch convalescent patients. Plots show CD19 + IgD − IgG + RBD+ populations using gating strategy described in B .

    Article Snippet: 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.

    Techniques: Isolation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, FACS

    Identification of RBD specific monoclonal antibodies from convalescent COVID-19 patients. A. Screening of specific Abs against SARS-CoV-2 S1 and RBD. The heatmap reveals that the binding ability of 198 Ab supernatants produced by HEK239T cells transfected with linear Ab gene expression cassette. The mAbs rank as the screening sequence, and binding activity of mAbs against SARS-CoV-2 S1 and RBD were tested by ELISA. The brightness of blue represents the binding strength, which reflected the OD405 nm value tested by ELISA. The neutralizing activity of mAbs was discriminated according to the neutralizing value. Antibody-mediated blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus transfected into hACE2/ HEK293T cells were measured by values of relative light units (RUL). The Green columns indicate potential neutralization (neutralizing activity > 75%), while white indicate partial or not neutralization (neutralizing activity

    Journal: bioRxiv

    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

    doi: 10.1101/2020.08.19.253369

    Figure Lengend Snippet: Identification of RBD specific monoclonal antibodies from convalescent COVID-19 patients. A. Screening of specific Abs against SARS-CoV-2 S1 and RBD. The heatmap reveals that the binding ability of 198 Ab supernatants produced by HEK239T cells transfected with linear Ab gene expression cassette. The mAbs rank as the screening sequence, and binding activity of mAbs against SARS-CoV-2 S1 and RBD were tested by ELISA. The brightness of blue represents the binding strength, which reflected the OD405 nm value tested by ELISA. The neutralizing activity of mAbs was discriminated according to the neutralizing value. Antibody-mediated blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus transfected into hACE2/ HEK293T cells were measured by values of relative light units (RUL). The Green columns indicate potential neutralization (neutralizing activity > 75%), while white indicate partial or not neutralization (neutralizing activity

    Article Snippet: 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.

    Techniques: Binding Assay, Produced, Transfection, Expressing, Sequencing, Activity Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Luciferase, Neutralization

    The binding activity and inhibition of ACE2-RBD interaction of mAbs tested by ELISA and competitive ELISA. A. The OD 405 nm value refects a binding strength of purified mAbs to 1 μg/ml SARS-CoV-2 S1 or RBD. Plates were coated with recombinant S1 or RBD protein of SARS-CoV-2, then incubated with purified mAbs. A SARS specific mAb (CR3022) was set as the positive control. The blue dashed lines indicated the OD 405nm value of a negative sample. B. The inhibitory effect of purified mAbs against the interaction between SARS-CoV-2 RBD and hACE2 was tested via competitive ELISA analysis. Blocking efficacy was determined by comparing response units with and without prior antibody incubation. The green dashed lines indicated 50% inhibition on blocking the interaction ACE2 and RBD interaction.

    Journal: bioRxiv

    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

    doi: 10.1101/2020.08.19.253369

    Figure Lengend Snippet: The binding activity and inhibition of ACE2-RBD interaction of mAbs tested by ELISA and competitive ELISA. A. The OD 405 nm value refects a binding strength of purified mAbs to 1 μg/ml SARS-CoV-2 S1 or RBD. Plates were coated with recombinant S1 or RBD protein of SARS-CoV-2, then incubated with purified mAbs. A SARS specific mAb (CR3022) was set as the positive control. The blue dashed lines indicated the OD 405nm value of a negative sample. B. The inhibitory effect of purified mAbs against the interaction between SARS-CoV-2 RBD and hACE2 was tested via competitive ELISA analysis. Blocking efficacy was determined by comparing response units with and without prior antibody incubation. The green dashed lines indicated 50% inhibition on blocking the interaction ACE2 and RBD interaction.

    Article Snippet: 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.

    Techniques: Binding Assay, Activity Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Competitive ELISA, Purification, Recombinant, Incubation, Positive Control, Blocking Assay

    Schematic model depicting a rapid and efficient screening system of neutralizing Abs. Rapid neutralizing antibody screening workflows and timelines are shown, representing the multiple workflows conducted in parallel. PBMC were isolated from collected convalescent patients’ blood, and the RBD-specific memory B cells in the PBMCs were sorted as single-cell via flow-cytometric sorter (day 1). Then, the IgG heavy and light chains of monoclonal antibody genes were amplified by RT-PCR on the same day. 2 nd PCR products were cloned into linear expression cassettes on the second day. Antibodies were expressed by transient transfection with equal amounts of paired heavy and light chain linear expression cassettes in HEK293T cells and culture for two days. The cell supernatants in HEK293T cells were detected for the specificity of antibodies by ELISA in 384-well plates on the fourth day. The neutralizing activity of antibodies was detected with pseudovirus bearing SARS-CoV-2 S in 96-well plates on the sixth day. The potential neutralization antibody expression plasmids were transfected into Exi293F cells for large-scale production of Ab proteins. The cell supernatants in Exi293F cells were collected, and antibody proteins were purified by protein G. They were further measured for the binding ability and neutralizing activity via ELISA and competitive ELISA in vitro . Additionally, virus neutralization assay was performed. Created with Biorender.com.

    Journal: bioRxiv

    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

    doi: 10.1101/2020.08.19.253369

    Figure Lengend Snippet: Schematic model depicting a rapid and efficient screening system of neutralizing Abs. Rapid neutralizing antibody screening workflows and timelines are shown, representing the multiple workflows conducted in parallel. PBMC were isolated from collected convalescent patients’ blood, and the RBD-specific memory B cells in the PBMCs were sorted as single-cell via flow-cytometric sorter (day 1). Then, the IgG heavy and light chains of monoclonal antibody genes were amplified by RT-PCR on the same day. 2 nd PCR products were cloned into linear expression cassettes on the second day. Antibodies were expressed by transient transfection with equal amounts of paired heavy and light chain linear expression cassettes in HEK293T cells and culture for two days. The cell supernatants in HEK293T cells were detected for the specificity of antibodies by ELISA in 384-well plates on the fourth day. The neutralizing activity of antibodies was detected with pseudovirus bearing SARS-CoV-2 S in 96-well plates on the sixth day. The potential neutralization antibody expression plasmids were transfected into Exi293F cells for large-scale production of Ab proteins. The cell supernatants in Exi293F cells were collected, and antibody proteins were purified by protein G. They were further measured for the binding ability and neutralizing activity via ELISA and competitive ELISA in vitro . Additionally, virus neutralization assay was performed. Created with Biorender.com.

    Article Snippet: 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.

    Techniques: Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Clone Assay, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Activity Assay, Neutralization, Purification, Binding Assay, Competitive ELISA, In Vitro

    Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.

    Journal: Analytical Chemistry

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

    doi: 10.1021/acs.analchem.0c01394

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

    Article Snippet: 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.

    Techniques: 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.

    Journal: Analytical Chemistry

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

    doi: 10.1021/acs.analchem.0c01394

    Figure Lengend 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.

    Article Snippet: 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.

    Techniques: Flow Cytometry, Binding Assay