sars cov 2 rbd  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike RBD Antibody Mouse PAb
    Description:
    Produced in mice immunized with purified recombinant SARS CoV 2 2019 nCoV Spike RBD Protein Catalog 40592 V08H YP 009724390 1 Arg319 Phe541
    Catalog Number:
    40592-MP01
    Price:
    None
    Category:
    Primary Antibody
    Reactivity:
    2019 nCoV
    Applications:
    ELISA
    Immunogen:
    Recombinant SARS-CoV-2 (2019-nCoV) Spike RBD-His Protein (Catalog#40592-V08H)
    Product Aliases:
    Anti-coronavirus spike Antibody, Anti-cov spike Antibody, Anti-ncov RBD Antibody, Anti-ncov s1 Antibody, Anti-ncov s2 Antibody, Anti-ncov spike Antibody, Anti-NCP-CoV RBD Antibody, Anti-NCP-CoV s1 Antibody, Anti-NCP-CoV s2 Antibody, Anti-NCP-CoV Spike Antibody, Anti-novel coronavirus RBD Antibody, Anti-novel coronavirus s1 Antibody, Anti-novel coronavirus s2 Antibody, Anti-novel coronavirus spike Antibody, Anti-RBD Antibody, Anti-S1 Antibody, Anti-S2 Antibody, Anti-Spike RBD Antibody
    Antibody Type:
    PAb
    Host:
    Mouse
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    Structured Review

    Sino Biological sars cov 2 rbd
    RU169 output clone diversity Using the <t>SARS-CoV-2</t> RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.
    Produced in mice immunized with purified recombinant SARS CoV 2 2019 nCoV Spike RBD Protein Catalog 40592 V08H YP 009724390 1 Arg319 Phe541
    https://www.bioz.com/result/sars cov 2 rbd/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 rbd - by Bioz Stars, 2021-04
    94/100 stars

    Images

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.07.27.224089

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

    Techniques Used: FACS, Selection, Produced, Clone Assay

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

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

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

    Techniques Used: Clone Assay, In Vitro

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

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

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

    Techniques Used: Binding Assay

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

    Techniques Used: Cytometry, Inhibition, Fluorescence, Labeling

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.06.15.153064

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

    Techniques Used: Polymerase Chain Reaction

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

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

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

    Techniques Used: Enzyme-linked Immunosorbent Assay

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

    Techniques Used: Incubation

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

    Techniques Used: Titration, Concentration Assay, Standard Deviation

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

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

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.0c01394

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

    Techniques Used: Selection

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

    Techniques Used: Flow Cytometry, Binding Assay

    4) Product Images from "CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells"

    Article Title: CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells

    Journal: bioRxiv

    doi: 10.1101/2021.01.14.426742

    S309-CAR-NK cells are superior to CR3022-CAR-NK cells. ( a ) Diagram of S309 and CR3022 neutralizing antibodies binding to different epitopes of the SARS-CoV-2 S protein. Both open and closed conformation states of SARS-CoV-2 S protein are shown. S309 binding site is indicated in magenta and CR3022 binding site is indicated in yellow. ( b ) Quantitative data of CD107a surface expression of both S309-CAR-NK-92MI and CR3022-CAR-NK-92MI. Both transient 293T-hACE2-RBD and stable A549-Spike cell lines were used as target cells. Error bars represent SEM from at least two independent experiments. ( c ) Comparison of killing activity of S309-CAR and CR3022-CAR using the 4-hour Cr 51 release assay. Effector cells were cocultured with Cr 51 -labeled target cells at 37°C for 4 hours. The assay was repeated for at least two times per target cell line. ( d ) Expanded S309-CAR-NK primary has increased killing activity against A549-Spike cells than primary CR3022-CAR-NK primary . Effector cells were blocked with anti-CD16 and anti-NKG2D prior to coculturing with A549-Spike target cells for 4 hours at 37°C. Data were pooled from three independent experiments. Unpaired Student’s t test was employed for all panels. ns p > 0.05, * p
    Figure Legend Snippet: S309-CAR-NK cells are superior to CR3022-CAR-NK cells. ( a ) Diagram of S309 and CR3022 neutralizing antibodies binding to different epitopes of the SARS-CoV-2 S protein. Both open and closed conformation states of SARS-CoV-2 S protein are shown. S309 binding site is indicated in magenta and CR3022 binding site is indicated in yellow. ( b ) Quantitative data of CD107a surface expression of both S309-CAR-NK-92MI and CR3022-CAR-NK-92MI. Both transient 293T-hACE2-RBD and stable A549-Spike cell lines were used as target cells. Error bars represent SEM from at least two independent experiments. ( c ) Comparison of killing activity of S309-CAR and CR3022-CAR using the 4-hour Cr 51 release assay. Effector cells were cocultured with Cr 51 -labeled target cells at 37°C for 4 hours. The assay was repeated for at least two times per target cell line. ( d ) Expanded S309-CAR-NK primary has increased killing activity against A549-Spike cells than primary CR3022-CAR-NK primary . Effector cells were blocked with anti-CD16 and anti-NKG2D prior to coculturing with A549-Spike target cells for 4 hours at 37°C. Data were pooled from three independent experiments. Unpaired Student’s t test was employed for all panels. ns p > 0.05, * p

    Techniques Used: Binding Assay, Expressing, Activity Assay, Release Assay, Labeling

    Increased CD107a surface expression and killing activity of S309-CAR-NK-92MI cells against 293T-hACE2-RBD and A549-Spike target cells. (a) Generation of transient 293T-hACE2-RBD and stable A549-Spike cell lines. 293T-hACE2 cells were transfected with RBD-containing plasmid for 48 hours. Transfected 293T-hACE2-RBD cells were then harvested. For the generation of A549-Spike, 293T cells were transfected with the retrovirus transfection system for 48 hours. The spike retrovirus was filtered and transduced into A549 cells for an additional 48-72 hours. ( b ) Representative dot plots showing the expressions of RBD or Spike in 293T-hACE2 or A549 cells, respectively. 293T-hACE2-RBD and A549-Spike cells were stained with anti-RBD and the expressions were confirmed by flow cytometry. The stable A549-Spike cell line was then sorted to achieve high levels of spike expression. ( c ) Quantitative data of CD107a surface expression assay of S309-CAR-NK against 293T-hACE2-RBD or A549-Spike cell lines. Briefly, S309-CAR-NK-92MI cells were cocultured with either 293T-hACE2-RBD cells, A549-Spike cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. Cells were then harvested and stained for CAR F(ab)2 domain [IgG (H+L)] and CD107a. Data represent mean ± SEM from two experiments. ( d ) 4-hour standard Cr 51 release assay of S309-CAR-NK-92MI and parental NK-92MI cells against various target cell lines. 293T-hACE2-RBD, A549-Spike, and HepG2 cell lines were used as target cells for S309-CAR-NK and NK-92MI. Experimental groups were performed in triplicates. Error bars represent mean ± SEM from at least two independent experiments. Unpaired Student’s t test was used for both panels ( c ) and ( d ). ns p > 0.05, * p
    Figure Legend Snippet: Increased CD107a surface expression and killing activity of S309-CAR-NK-92MI cells against 293T-hACE2-RBD and A549-Spike target cells. (a) Generation of transient 293T-hACE2-RBD and stable A549-Spike cell lines. 293T-hACE2 cells were transfected with RBD-containing plasmid for 48 hours. Transfected 293T-hACE2-RBD cells were then harvested. For the generation of A549-Spike, 293T cells were transfected with the retrovirus transfection system for 48 hours. The spike retrovirus was filtered and transduced into A549 cells for an additional 48-72 hours. ( b ) Representative dot plots showing the expressions of RBD or Spike in 293T-hACE2 or A549 cells, respectively. 293T-hACE2-RBD and A549-Spike cells were stained with anti-RBD and the expressions were confirmed by flow cytometry. The stable A549-Spike cell line was then sorted to achieve high levels of spike expression. ( c ) Quantitative data of CD107a surface expression assay of S309-CAR-NK against 293T-hACE2-RBD or A549-Spike cell lines. Briefly, S309-CAR-NK-92MI cells were cocultured with either 293T-hACE2-RBD cells, A549-Spike cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. Cells were then harvested and stained for CAR F(ab)2 domain [IgG (H+L)] and CD107a. Data represent mean ± SEM from two experiments. ( d ) 4-hour standard Cr 51 release assay of S309-CAR-NK-92MI and parental NK-92MI cells against various target cell lines. 293T-hACE2-RBD, A549-Spike, and HepG2 cell lines were used as target cells for S309-CAR-NK and NK-92MI. Experimental groups were performed in triplicates. Error bars represent mean ± SEM from at least two independent experiments. Unpaired Student’s t test was used for both panels ( c ) and ( d ). ns p > 0.05, * p

    Techniques Used: Expressing, Activity Assay, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Incubation, Release 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 "Identification of Human Single-Domain Antibodies against SARS-CoV-2"

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

    Journal: Cell Host & Microbe

    doi: 10.1016/j.chom.2020.04.023

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

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

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

    Techniques Used: Binding Assay

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.10.15.339838

    Sialic acid has modest effect on Spike binding and viral entry. A . Full-length proteins expressed on cells include wild-type Spike-protein [v1] and human ACE2 [v2] . N-glycosylation sites are indicated by lollipop. Fc-his soluble proteins encode for S1-subunit [v3] , RBD [v4] and soluble ACE2 [v5] . All constructs were co-expressed with fluorescent reporters separated by P2A. Note that the Fc-section also contains one N-glycosylation site. B . Western blot for purified Fc-proteins from HEK293T probed with anti-Fc, anti-RBD or anti-ACE2 Ab. CD44-Fc is positive control. C . Flow cytometry data showing S1-Fc (1.7µg/mL) and RBD-Fc (0.35µg/mL) binding to ACE2 expressed on HEK293T (middle panel). Spike expression enhances ACE2-Fc (1.4µg/mL) binding (bottom). D . Desialylation of Spike-protein expressed on 293T/S had minimal effect on ACE2-Fc (0.7µg/mL) binding. ACE2 desialylation on 293T/ACE2 increased binding of RBD-Fc (0.2µg/mL) and S1-Fc (1.7µg/mL) by 26-56% (paired experiments, * P
    Figure Legend Snippet: Sialic acid has modest effect on Spike binding and viral entry. A . Full-length proteins expressed on cells include wild-type Spike-protein [v1] and human ACE2 [v2] . N-glycosylation sites are indicated by lollipop. Fc-his soluble proteins encode for S1-subunit [v3] , RBD [v4] and soluble ACE2 [v5] . All constructs were co-expressed with fluorescent reporters separated by P2A. Note that the Fc-section also contains one N-glycosylation site. B . Western blot for purified Fc-proteins from HEK293T probed with anti-Fc, anti-RBD or anti-ACE2 Ab. CD44-Fc is positive control. C . Flow cytometry data showing S1-Fc (1.7µg/mL) and RBD-Fc (0.35µg/mL) binding to ACE2 expressed on HEK293T (middle panel). Spike expression enhances ACE2-Fc (1.4µg/mL) binding (bottom). D . Desialylation of Spike-protein expressed on 293T/S had minimal effect on ACE2-Fc (0.7µg/mL) binding. ACE2 desialylation on 293T/ACE2 increased binding of RBD-Fc (0.2µg/mL) and S1-Fc (1.7µg/mL) by 26-56% (paired experiments, * P

    Techniques Used: Binding Assay, Construct, Western Blot, Purification, Positive Control, Flow Cytometry, Expressing

    ACE2 glycosylation does not affect viral entry. A . Knocking out C1GALT1 and MGAT1 using CRISPR-Cas9 inhibits O- and N-glycan biosynthesis in HEK293Ts. B . Sanger sequencing results of isogenic 293T clones shows indels on all 3 alleles of C1GALT1 (‘[O] − 293T’) and single allele of MGAT1 (‘[N] − 293T’) knockout cells. Wild-type (WT) sequence is on the first line. Lower line shows base deletions (hyphen) and insertions (black fonts) for individual KOs. sgRNA target sequence is in red and protospacer adjacent motif is underlined. C . Increased VVA and reduced PHA-L binding confirm loss of O-linked glycans in [O] − 293Ts and N-glycans in [N] − 293Ts, respectively. D . Knocking out N-glycans on Spike protein reduced ACE2-Fc binding in cytometry based binding studies. Knocking out Spike O-glycans increased ACE-2 binding. E-F . Truncation of ACE2 N- and O-glycans did not affect either S1-Fc (panel E ) or RBD-Fc (panel F ) binding. G . ACE2 was transiently expressed on 293T, [O] − 293T and [N] − 293T cells. All pseudotyped virus efficiently entered ACE2 expressing cells. Virus was not titered for these runs, and thus comparison between viruses is not possible. * P
    Figure Legend Snippet: ACE2 glycosylation does not affect viral entry. A . Knocking out C1GALT1 and MGAT1 using CRISPR-Cas9 inhibits O- and N-glycan biosynthesis in HEK293Ts. B . Sanger sequencing results of isogenic 293T clones shows indels on all 3 alleles of C1GALT1 (‘[O] − 293T’) and single allele of MGAT1 (‘[N] − 293T’) knockout cells. Wild-type (WT) sequence is on the first line. Lower line shows base deletions (hyphen) and insertions (black fonts) for individual KOs. sgRNA target sequence is in red and protospacer adjacent motif is underlined. C . Increased VVA and reduced PHA-L binding confirm loss of O-linked glycans in [O] − 293Ts and N-glycans in [N] − 293Ts, respectively. D . Knocking out N-glycans on Spike protein reduced ACE2-Fc binding in cytometry based binding studies. Knocking out Spike O-glycans increased ACE-2 binding. E-F . Truncation of ACE2 N- and O-glycans did not affect either S1-Fc (panel E ) or RBD-Fc (panel F ) binding. G . ACE2 was transiently expressed on 293T, [O] − 293T and [N] − 293T cells. All pseudotyped virus efficiently entered ACE2 expressing cells. Virus was not titered for these runs, and thus comparison between viruses is not possible. * P

    Techniques Used: CRISPR, Sequencing, Clone Assay, Knock-Out, Binding Assay, Cytometry, Expressing

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

    Techniques Used: Binding Assay, Expressing, Generated

    8) Product Images from "Development and effectiveness of Pseudotyped SARS-CoV-2 system as determined by neutralizing efficiency and entry inhibition test in vitro"

    Article Title: Development and effectiveness of Pseudotyped SARS-CoV-2 system as determined by neutralizing efficiency and entry inhibition test in vitro

    Journal: Biosafety and Health

    doi: 10.1016/j.bsheal.2020.08.004

    Identification of SARS-CoV-2 S protein expression and SARS-CoV-2 pseudotyped virus Construction and identification of S expressing plasmid. SARS-CoV-2 S protein gene was inserted in the pCDNA3.1 vector. Immunofluorescence assay for S protein expression in pcDNA3.1-SARS-CoV-2 S plasmid. The expression was determined using mouse pAb against SARS-CoV-2 S protein and convalescent serum samples from COVID-19 patients. Identification of S protein expression in SARS-CoV-2 pseudotyped virus by immunoblot assay. Bands corresponding to SARS-CoV-2 S and HIV-1 p24 proteins were detected at the same sample line in the gel.
    Figure Legend Snippet: Identification of SARS-CoV-2 S protein expression and SARS-CoV-2 pseudotyped virus Construction and identification of S expressing plasmid. SARS-CoV-2 S protein gene was inserted in the pCDNA3.1 vector. Immunofluorescence assay for S protein expression in pcDNA3.1-SARS-CoV-2 S plasmid. The expression was determined using mouse pAb against SARS-CoV-2 S protein and convalescent serum samples from COVID-19 patients. Identification of S protein expression in SARS-CoV-2 pseudotyped virus by immunoblot assay. Bands corresponding to SARS-CoV-2 S and HIV-1 p24 proteins were detected at the same sample line in the gel.

    Techniques Used: Expressing, Plasmid Preparation, Immunofluorescence

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

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

    Journal: bioRxiv

    doi: 10.1101/2021.03.07.434276

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

    Techniques Used: Infection, Negative Control

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

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

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

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

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

    Techniques Used: Expressing, Infection, Western Blot

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

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

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

    Techniques Used:

    10) 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

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.10.15.339838

    Sialic acid has modest effect on Spike binding and viral entry. A . Full-length proteins expressed on cells include wild-type Spike-protein [v1] and human ACE2 [v2] . N-glycosylation sites are indicated by lollipop. Fc-his soluble proteins encode for S1-subunit [v3] , RBD [v4] and soluble ACE2 [v5] . All constructs were co-expressed with fluorescent reporters separated by P2A. Note that the Fc-section also contains one N-glycosylation site. B . Western blot for purified Fc-proteins from HEK293T probed with anti-Fc, anti-RBD or anti-ACE2 Ab. CD44-Fc is positive control. C . Flow cytometry data showing S1-Fc (1.7µg/mL) and RBD-Fc (0.35µg/mL) binding to ACE2 expressed on HEK293T (middle panel). Spike expression enhances ACE2-Fc (1.4µg/mL) binding (bottom). D . Desialylation of Spike-protein expressed on 293T/S had minimal effect on ACE2-Fc (0.7µg/mL) binding. ACE2 desialylation on 293T/ACE2 increased binding of RBD-Fc (0.2µg/mL) and S1-Fc (1.7µg/mL) by 26-56% (paired experiments, * P
    Figure Legend Snippet: Sialic acid has modest effect on Spike binding and viral entry. A . Full-length proteins expressed on cells include wild-type Spike-protein [v1] and human ACE2 [v2] . N-glycosylation sites are indicated by lollipop. Fc-his soluble proteins encode for S1-subunit [v3] , RBD [v4] and soluble ACE2 [v5] . All constructs were co-expressed with fluorescent reporters separated by P2A. Note that the Fc-section also contains one N-glycosylation site. B . Western blot for purified Fc-proteins from HEK293T probed with anti-Fc, anti-RBD or anti-ACE2 Ab. CD44-Fc is positive control. C . Flow cytometry data showing S1-Fc (1.7µg/mL) and RBD-Fc (0.35µg/mL) binding to ACE2 expressed on HEK293T (middle panel). Spike expression enhances ACE2-Fc (1.4µg/mL) binding (bottom). D . Desialylation of Spike-protein expressed on 293T/S had minimal effect on ACE2-Fc (0.7µg/mL) binding. ACE2 desialylation on 293T/ACE2 increased binding of RBD-Fc (0.2µg/mL) and S1-Fc (1.7µg/mL) by 26-56% (paired experiments, * P

    Techniques Used: Binding Assay, Construct, Western Blot, Purification, Positive Control, Flow Cytometry, Expressing

    ACE2 glycosylation does not affect viral entry. A . Knocking out C1GALT1 and MGAT1 using CRISPR-Cas9 inhibits O- and N-glycan biosynthesis in HEK293Ts. B . Sanger sequencing results of isogenic 293T clones shows indels on all 3 alleles of C1GALT1 (‘[O] − 293T’) and single allele of MGAT1 (‘[N] − 293T’) knockout cells. Wild-type (WT) sequence is on the first line. Lower line shows base deletions (hyphen) and insertions (black fonts) for individual KOs. sgRNA target sequence is in red and protospacer adjacent motif is underlined. C . Increased VVA and reduced PHA-L binding confirm loss of O-linked glycans in [O] − 293Ts and N-glycans in [N] − 293Ts, respectively. D . Knocking out N-glycans on Spike protein reduced ACE2-Fc binding in cytometry based binding studies. Knocking out Spike O-glycans increased ACE-2 binding. E-F . Truncation of ACE2 N- and O-glycans did not affect either S1-Fc (panel E ) or RBD-Fc (panel F ) binding. G . ACE2 was transiently expressed on 293T, [O] − 293T and [N] − 293T cells. All pseudotyped virus efficiently entered ACE2 expressing cells. Virus was not titered for these runs, and thus comparison between viruses is not possible. * P
    Figure Legend Snippet: ACE2 glycosylation does not affect viral entry. A . Knocking out C1GALT1 and MGAT1 using CRISPR-Cas9 inhibits O- and N-glycan biosynthesis in HEK293Ts. B . Sanger sequencing results of isogenic 293T clones shows indels on all 3 alleles of C1GALT1 (‘[O] − 293T’) and single allele of MGAT1 (‘[N] − 293T’) knockout cells. Wild-type (WT) sequence is on the first line. Lower line shows base deletions (hyphen) and insertions (black fonts) for individual KOs. sgRNA target sequence is in red and protospacer adjacent motif is underlined. C . Increased VVA and reduced PHA-L binding confirm loss of O-linked glycans in [O] − 293Ts and N-glycans in [N] − 293Ts, respectively. D . Knocking out N-glycans on Spike protein reduced ACE2-Fc binding in cytometry based binding studies. Knocking out Spike O-glycans increased ACE-2 binding. E-F . Truncation of ACE2 N- and O-glycans did not affect either S1-Fc (panel E ) or RBD-Fc (panel F ) binding. G . ACE2 was transiently expressed on 293T, [O] − 293T and [N] − 293T cells. All pseudotyped virus efficiently entered ACE2 expressing cells. Virus was not titered for these runs, and thus comparison between viruses is not possible. * P

    Techniques Used: CRISPR, Sequencing, Clone Assay, Knock-Out, Binding Assay, Cytometry, Expressing

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

    Techniques Used: Binding Assay, Expressing, Generated

    12) Product Images from "The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles"

    Article Title: The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA120.016175

    The C-terminal moiety of S cytoplasmic tail is essential for M-mediated retention of SARS-CoV-2 S. A , alignment of sequences of the last amino acids of S of SARS-CoV-2 or mutated by deletion of the last 19 amino acids (SΔ19). The box represents the dibasic retrieval signal. B , representative confocal microscopy images of Vero E6 cells transfected with a plasmid encoding SΔ19 alone or SΔ19 combined with plasmids expressing E or M. The cis-Golgi was revealed with the anti-GM130 antibody ( green channel ), the S protein was revealed with the anti-SARS-CoV2 S1 antibody ( red channel ), and the nucleus was revealed with Hoechst ( blue channel ). The Manders’ coefficient M1 represents the fraction of S overlapping with GM130, and the M2 coefficients represent the fraction of GM130 overlapping with S. Scale bars of panels and zooms from squared area represent 10 μm and 2 μm, respectively. The S protein was also revealed on nonpermeabilized cells ( bottom ). C , representative pictures of syncytia detected in Vero E6 cells transfected with a plasmid encoding SΔ19 alone or SΔ19 combined with plasmids expressing E or M ( left ). Fusion index and number of nuclei per syncytia determined for the different conditions ( right ). The scale bar represents 40 μm. D , representative western blot analysis of 293T cells transfected with a plasmid encoding SΔ19 or SΔ19 combined with plasmids encoding E or M. The blots were revealed using an anti-S2 antibody. The arrows and bracket represent S0, S2, and S2 ∗ forms. E , quantification of indicated S forms from independent western blot as described in ( D ). F , Quantification of the percentage of S2 forms in the total (S2+S2 ∗ ) signal by quantitative western blot analysis as described in ( D ). The dots on the graphs represent results of independent experiments.
    Figure Legend Snippet: The C-terminal moiety of S cytoplasmic tail is essential for M-mediated retention of SARS-CoV-2 S. A , alignment of sequences of the last amino acids of S of SARS-CoV-2 or mutated by deletion of the last 19 amino acids (SΔ19). The box represents the dibasic retrieval signal. B , representative confocal microscopy images of Vero E6 cells transfected with a plasmid encoding SΔ19 alone or SΔ19 combined with plasmids expressing E or M. The cis-Golgi was revealed with the anti-GM130 antibody ( green channel ), the S protein was revealed with the anti-SARS-CoV2 S1 antibody ( red channel ), and the nucleus was revealed with Hoechst ( blue channel ). The Manders’ coefficient M1 represents the fraction of S overlapping with GM130, and the M2 coefficients represent the fraction of GM130 overlapping with S. Scale bars of panels and zooms from squared area represent 10 μm and 2 μm, respectively. The S protein was also revealed on nonpermeabilized cells ( bottom ). C , representative pictures of syncytia detected in Vero E6 cells transfected with a plasmid encoding SΔ19 alone or SΔ19 combined with plasmids expressing E or M ( left ). Fusion index and number of nuclei per syncytia determined for the different conditions ( right ). The scale bar represents 40 μm. D , representative western blot analysis of 293T cells transfected with a plasmid encoding SΔ19 or SΔ19 combined with plasmids encoding E or M. The blots were revealed using an anti-S2 antibody. The arrows and bracket represent S0, S2, and S2 ∗ forms. E , quantification of indicated S forms from independent western blot as described in ( D ). F , Quantification of the percentage of S2 forms in the total (S2+S2 ∗ ) signal by quantitative western blot analysis as described in ( D ). The dots on the graphs represent results of independent experiments.

    Techniques Used: Confocal Microscopy, Transfection, Plasmid Preparation, Expressing, Western Blot

    13) Product Images from "Oral delivery of SARS-CoV-2 DNA vaccines using attenuated Salmonella typhimurium as a carrier in rat"

    Article Title: Oral delivery of SARS-CoV-2 DNA vaccines using attenuated Salmonella typhimurium as a carrier in rat

    Journal: bioRxiv

    doi: 10.1101/2020.07.23.217174

    pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP plasmid map.
    Figure Legend Snippet: pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP plasmid map.

    Techniques Used: Plasmid Preparation

    Micrographs of 293T cells transfected with pSARS-CoV-2-S (X 100). (A1, A2) 293T cells transfected with pSARS-CoV-2-S (pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP) at 48 hours after transfection. A1 fluorescence micrograph with GFP expression in cells and light micrograph with the same visual field as A2. (B) 293T cells transfected with pSARS-CoV-2-S at 48 hours after transfection. The SARS-CoV-2-S protein showed about 141 kDa.
    Figure Legend Snippet: Micrographs of 293T cells transfected with pSARS-CoV-2-S (X 100). (A1, A2) 293T cells transfected with pSARS-CoV-2-S (pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP) at 48 hours after transfection. A1 fluorescence micrograph with GFP expression in cells and light micrograph with the same visual field as A2. (B) 293T cells transfected with pSARS-CoV-2-S at 48 hours after transfection. The SARS-CoV-2-S protein showed about 141 kDa.

    Techniques Used: Transfection, Fluorescence, Expressing

    Humoral responses to SARS-CoV-2-S protein antigen in the rat after immunizationon day 0, day 14, and day 28 with Salmonella carrying the control vector or pSARS-CoV-2-S (as described in the methods). (A) After immunization with the control vector, test SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent test mean OD450 nm values (mean±SD) for each of 9 rats, or (B) After immunization with the pSARS-CoV-2-S vector, SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent mean OD450 nm values (3 times measurement, mean±SD) for each of 9 rats.
    Figure Legend Snippet: Humoral responses to SARS-CoV-2-S protein antigen in the rat after immunizationon day 0, day 14, and day 28 with Salmonella carrying the control vector or pSARS-CoV-2-S (as described in the methods). (A) After immunization with the control vector, test SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent test mean OD450 nm values (mean±SD) for each of 9 rats, or (B) After immunization with the pSARS-CoV-2-S vector, SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent mean OD450 nm values (3 times measurement, mean±SD) for each of 9 rats.

    Techniques Used: Plasmid Preparation, Binding Assay

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

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

    Journal: eLife

    doi: 10.7554/eLife.61552

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

    Techniques Used: Binding Assay, Expressing, Generated

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.07.29.227595

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

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

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

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

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

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

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

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

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

    Techniques Used: Expressing, Western Blot

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.08.28.244269

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    Journal: eLife

    doi: 10.7554/eLife.61552

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

    Techniques Used: Binding Assay, Expressing, Generated

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.10.15.339838

    Sialic acid has modest effect on Spike binding and viral entry. A . Full-length proteins expressed on cells include wild-type Spike-protein [v1] and human ACE2 [v2] . N-glycosylation sites are indicated by lollipop. Fc-his soluble proteins encode for S1-subunit [v3] , RBD [v4] and soluble ACE2 [v5] . All constructs were co-expressed with fluorescent reporters separated by P2A. Note that the Fc-section also contains one N-glycosylation site. B . Western blot for purified Fc-proteins from HEK293T probed with anti-Fc, anti-RBD or anti-ACE2 Ab. CD44-Fc is positive control. C . Flow cytometry data showing S1-Fc (1.7µg/mL) and RBD-Fc (0.35µg/mL) binding to ACE2 expressed on HEK293T (middle panel). Spike expression enhances ACE2-Fc (1.4µg/mL) binding (bottom). D . Desialylation of Spike-protein expressed on 293T/S had minimal effect on ACE2-Fc (0.7µg/mL) binding. ACE2 desialylation on 293T/ACE2 increased binding of RBD-Fc (0.2µg/mL) and S1-Fc (1.7µg/mL) by 26-56% (paired experiments, * P
    Figure Legend Snippet: Sialic acid has modest effect on Spike binding and viral entry. A . Full-length proteins expressed on cells include wild-type Spike-protein [v1] and human ACE2 [v2] . N-glycosylation sites are indicated by lollipop. Fc-his soluble proteins encode for S1-subunit [v3] , RBD [v4] and soluble ACE2 [v5] . All constructs were co-expressed with fluorescent reporters separated by P2A. Note that the Fc-section also contains one N-glycosylation site. B . Western blot for purified Fc-proteins from HEK293T probed with anti-Fc, anti-RBD or anti-ACE2 Ab. CD44-Fc is positive control. C . Flow cytometry data showing S1-Fc (1.7µg/mL) and RBD-Fc (0.35µg/mL) binding to ACE2 expressed on HEK293T (middle panel). Spike expression enhances ACE2-Fc (1.4µg/mL) binding (bottom). D . Desialylation of Spike-protein expressed on 293T/S had minimal effect on ACE2-Fc (0.7µg/mL) binding. ACE2 desialylation on 293T/ACE2 increased binding of RBD-Fc (0.2µg/mL) and S1-Fc (1.7µg/mL) by 26-56% (paired experiments, * P

    Techniques Used: Binding Assay, Construct, Western Blot, Purification, Positive Control, Flow Cytometry, Expressing

    ACE2 glycosylation does not affect viral entry. A . Knocking out C1GALT1 and MGAT1 using CRISPR-Cas9 inhibits O- and N-glycan biosynthesis in HEK293Ts. B . Sanger sequencing results of isogenic 293T clones shows indels on all 3 alleles of C1GALT1 (‘[O] − 293T’) and single allele of MGAT1 (‘[N] − 293T’) knockout cells. Wild-type (WT) sequence is on the first line. Lower line shows base deletions (hyphen) and insertions (black fonts) for individual KOs. sgRNA target sequence is in red and protospacer adjacent motif is underlined. C . Increased VVA and reduced PHA-L binding confirm loss of O-linked glycans in [O] − 293Ts and N-glycans in [N] − 293Ts, respectively. D . Knocking out N-glycans on Spike protein reduced ACE2-Fc binding in cytometry based binding studies. Knocking out Spike O-glycans increased ACE-2 binding. E-F . Truncation of ACE2 N- and O-glycans did not affect either S1-Fc (panel E ) or RBD-Fc (panel F ) binding. G . ACE2 was transiently expressed on 293T, [O] − 293T and [N] − 293T cells. All pseudotyped virus efficiently entered ACE2 expressing cells. Virus was not titered for these runs, and thus comparison between viruses is not possible. * P
    Figure Legend Snippet: ACE2 glycosylation does not affect viral entry. A . Knocking out C1GALT1 and MGAT1 using CRISPR-Cas9 inhibits O- and N-glycan biosynthesis in HEK293Ts. B . Sanger sequencing results of isogenic 293T clones shows indels on all 3 alleles of C1GALT1 (‘[O] − 293T’) and single allele of MGAT1 (‘[N] − 293T’) knockout cells. Wild-type (WT) sequence is on the first line. Lower line shows base deletions (hyphen) and insertions (black fonts) for individual KOs. sgRNA target sequence is in red and protospacer adjacent motif is underlined. C . Increased VVA and reduced PHA-L binding confirm loss of O-linked glycans in [O] − 293Ts and N-glycans in [N] − 293Ts, respectively. D . Knocking out N-glycans on Spike protein reduced ACE2-Fc binding in cytometry based binding studies. Knocking out Spike O-glycans increased ACE-2 binding. E-F . Truncation of ACE2 N- and O-glycans did not affect either S1-Fc (panel E ) or RBD-Fc (panel F ) binding. G . ACE2 was transiently expressed on 293T, [O] − 293T and [N] − 293T cells. All pseudotyped virus efficiently entered ACE2 expressing cells. Virus was not titered for these runs, and thus comparison between viruses is not possible. * P

    Techniques Used: CRISPR, Sequencing, Clone Assay, Knock-Out, Binding Assay, Cytometry, Expressing

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

    Techniques Used: Binding Assay, Expressing, Generated

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

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

    Journal: Emerging Microbes & Infections

    doi: 10.1080/22221751.2020.1815589

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

    Techniques Used: Infection, Recombinant, Fluorescence

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

    Techniques Used: Neutralization

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

    Techniques Used: Expressing, Infection, Fluorescence

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

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

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

    Techniques Used:

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

    Techniques Used: Recombinant, Infection, Produced, Fluorescence

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

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

    Journal: eLife

    doi: 10.7554/eLife.61552

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

    Techniques Used: Binding Assay, Expressing, Generated

    21) Product Images from "Low-Dose Ad26.COV2.S Protection Against SARS-CoV-2 Challenge in Rhesus Macaques"

    Article Title: Low-Dose Ad26.COV2.S Protection Against SARS-CoV-2 Challenge in Rhesus Macaques

    Journal: bioRxiv

    doi: 10.1101/2021.01.27.428380

    Histopathologic scoring of lung lesions in all lobes in vaccinated and sham animals following SARS-CoV-2 challenge. Scoring was performed independently by two blinded, veterinary pathologists. Lesions reported included 1) inflammation interstitial/septal thickening 2) infiltrate, macrophage 3) alveolar infiltrate, mononuclear 4) perivascular infiltrate, macrophage 5) bronchiolar type II pneumocyte hyperplasia 6) BALT hyperplasia 7) inflammation, bronchiolar/peribronchiolar infiltrate 8) neutrophils, bronchiolar/alveolar and 9) infiltrate, eosinophils. Lesions such as focal fibrosis and syncytia were reported but not included in scoring. Edema, alveolar flooding was excluded from scoring since animals received terminal bronchoalveolar lavages. Each feature assessed was assigned a score of 0= no significant findings; 1=minimal; 2= mild; 3=moderate; 4=marked/severe. Eight representative samples from cranial, middle, and caudal lung lobes from the left and right lungs were evaluated from each animal and were scored independently. Scores were added for all lesions across all lung lobes for each animal for a maximum possible score of 288 for each monkey. Lungs evaluated were inflated/suffused with 10% formalin. Horizontal lines reflect median values. Solid black circles indicate animals that showed no virus in BAL and NS following challenge, open black circles indicate animals that showed virus in NS but not BAL following challenge, and open red circles indicate animals that show virus in both BAL and NS following challenge.
    Figure Legend Snippet: Histopathologic scoring of lung lesions in all lobes in vaccinated and sham animals following SARS-CoV-2 challenge. Scoring was performed independently by two blinded, veterinary pathologists. Lesions reported included 1) inflammation interstitial/septal thickening 2) infiltrate, macrophage 3) alveolar infiltrate, mononuclear 4) perivascular infiltrate, macrophage 5) bronchiolar type II pneumocyte hyperplasia 6) BALT hyperplasia 7) inflammation, bronchiolar/peribronchiolar infiltrate 8) neutrophils, bronchiolar/alveolar and 9) infiltrate, eosinophils. Lesions such as focal fibrosis and syncytia were reported but not included in scoring. Edema, alveolar flooding was excluded from scoring since animals received terminal bronchoalveolar lavages. Each feature assessed was assigned a score of 0= no significant findings; 1=minimal; 2= mild; 3=moderate; 4=marked/severe. Eight representative samples from cranial, middle, and caudal lung lobes from the left and right lungs were evaluated from each animal and were scored independently. Scores were added for all lesions across all lung lobes for each animal for a maximum possible score of 288 for each monkey. Lungs evaluated were inflated/suffused with 10% formalin. Horizontal lines reflect median values. Solid black circles indicate animals that showed no virus in BAL and NS following challenge, open black circles indicate animals that showed virus in NS but not BAL following challenge, and open red circles indicate animals that show virus in both BAL and NS following challenge.

    Techniques Used:

    Comparative pathology in vaccinated and unvaccinated animals following SARS-CoV-2 challenge. Representative pathology from animals vaccinated with ( A-C ) 1×10 11 vp Ad26.COV2.S, ( D-F ) 2×10 9 vp Ad26.COV2.S, or ( H-I ) sham on day 10 following SARS-CoV-2 challenge. ( A, D, G ) representative H E histopathology. ( B, E, H ) immunohistochemistry for Iba-1 (macrophages). ( C, G, I ) showing immunohistochemistry for CD3 (T-lymphocytes). Animals that received a high vaccine dose had minimal evidence of SARS CoV-2 pathology ( AC ). Animals that received the lowest vaccine dose showed focal pathology ( D-F ) characterized by increased alveolar macrophages, focal interstitial septal thickening and aggregates of macrophages. Sham vaccinated animals had locally extensive moderate interstitial pneumonia ( G ) characterized by extensive macrophage infiltrates ( H ) and expansion of perivascular and interstitial CD3 T lymphocytes ( I ). Scale bars = 100 microns.
    Figure Legend Snippet: Comparative pathology in vaccinated and unvaccinated animals following SARS-CoV-2 challenge. Representative pathology from animals vaccinated with ( A-C ) 1×10 11 vp Ad26.COV2.S, ( D-F ) 2×10 9 vp Ad26.COV2.S, or ( H-I ) sham on day 10 following SARS-CoV-2 challenge. ( A, D, G ) representative H E histopathology. ( B, E, H ) immunohistochemistry for Iba-1 (macrophages). ( C, G, I ) showing immunohistochemistry for CD3 (T-lymphocytes). Animals that received a high vaccine dose had minimal evidence of SARS CoV-2 pathology ( AC ). Animals that received the lowest vaccine dose showed focal pathology ( D-F ) characterized by increased alveolar macrophages, focal interstitial septal thickening and aggregates of macrophages. Sham vaccinated animals had locally extensive moderate interstitial pneumonia ( G ) characterized by extensive macrophage infiltrates ( H ) and expansion of perivascular and interstitial CD3 T lymphocytes ( I ). Scale bars = 100 microns.

    Techniques Used: Histopathology, Immunohistochemistry

    Protective efficacy following SARS-CoV-2 challenge. Rhesus macaques were challenged by the intranasal and intratracheal routes with 1.0×10 5 TCID50 SARS-CoV-2. ( A ) Peak log 10 sgRNA copies/ml (limit of quantification 50 copies/ml) were assessed in bronchoalveolar lavage (BAL) following challenge. ( B ) Peak log 10 sgRNA copies/swab (limit of quantification 50 copies/swab) were assessed in nasal swabs (NS) following challenge. Horizontal lines reflect geometric mean values. Solid black circles indicate animals that showed no virus in BAL and NS following challenge, open black circles indicate animals that showed virus in NS but not BAL following challenge, and open red circles indicate animals that show virus in both BAL and NS following challenge.
    Figure Legend Snippet: Protective efficacy following SARS-CoV-2 challenge. Rhesus macaques were challenged by the intranasal and intratracheal routes with 1.0×10 5 TCID50 SARS-CoV-2. ( A ) Peak log 10 sgRNA copies/ml (limit of quantification 50 copies/ml) were assessed in bronchoalveolar lavage (BAL) following challenge. ( B ) Peak log 10 sgRNA copies/swab (limit of quantification 50 copies/swab) were assessed in nasal swabs (NS) following challenge. Horizontal lines reflect geometric mean values. Solid black circles indicate animals that showed no virus in BAL and NS following challenge, open black circles indicate animals that showed virus in NS but not BAL following challenge, and open red circles indicate animals that show virus in both BAL and NS following challenge.

    Techniques Used:

    22) 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

    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

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.10.15.339838

    Sialic acid has modest effect on Spike binding and viral entry. A . Full-length proteins expressed on cells include wild-type Spike-protein [v1] and human ACE2 [v2] . N-glycosylation sites are indicated by lollipop. Fc-his soluble proteins encode for S1-subunit [v3] , RBD [v4] and soluble ACE2 [v5] . All constructs were co-expressed with fluorescent reporters separated by P2A. Note that the Fc-section also contains one N-glycosylation site. B . Western blot for purified Fc-proteins from HEK293T probed with anti-Fc, anti-RBD or anti-ACE2 Ab. CD44-Fc is positive control. C . Flow cytometry data showing S1-Fc (1.7µg/mL) and RBD-Fc (0.35µg/mL) binding to ACE2 expressed on HEK293T (middle panel). Spike expression enhances ACE2-Fc (1.4µg/mL) binding (bottom). D . Desialylation of Spike-protein expressed on 293T/S had minimal effect on ACE2-Fc (0.7µg/mL) binding. ACE2 desialylation on 293T/ACE2 increased binding of RBD-Fc (0.2µg/mL) and S1-Fc (1.7µg/mL) by 26-56% (paired experiments, * P
    Figure Legend Snippet: Sialic acid has modest effect on Spike binding and viral entry. A . Full-length proteins expressed on cells include wild-type Spike-protein [v1] and human ACE2 [v2] . N-glycosylation sites are indicated by lollipop. Fc-his soluble proteins encode for S1-subunit [v3] , RBD [v4] and soluble ACE2 [v5] . All constructs were co-expressed with fluorescent reporters separated by P2A. Note that the Fc-section also contains one N-glycosylation site. B . Western blot for purified Fc-proteins from HEK293T probed with anti-Fc, anti-RBD or anti-ACE2 Ab. CD44-Fc is positive control. C . Flow cytometry data showing S1-Fc (1.7µg/mL) and RBD-Fc (0.35µg/mL) binding to ACE2 expressed on HEK293T (middle panel). Spike expression enhances ACE2-Fc (1.4µg/mL) binding (bottom). D . Desialylation of Spike-protein expressed on 293T/S had minimal effect on ACE2-Fc (0.7µg/mL) binding. ACE2 desialylation on 293T/ACE2 increased binding of RBD-Fc (0.2µg/mL) and S1-Fc (1.7µg/mL) by 26-56% (paired experiments, * P

    Techniques Used: Binding Assay, Construct, Western Blot, Purification, Positive Control, Flow Cytometry, Expressing

    ACE2 glycosylation does not affect viral entry. A . Knocking out C1GALT1 and MGAT1 using CRISPR-Cas9 inhibits O- and N-glycan biosynthesis in HEK293Ts. B . Sanger sequencing results of isogenic 293T clones shows indels on all 3 alleles of C1GALT1 (‘[O] − 293T’) and single allele of MGAT1 (‘[N] − 293T’) knockout cells. Wild-type (WT) sequence is on the first line. Lower line shows base deletions (hyphen) and insertions (black fonts) for individual KOs. sgRNA target sequence is in red and protospacer adjacent motif is underlined. C . Increased VVA and reduced PHA-L binding confirm loss of O-linked glycans in [O] − 293Ts and N-glycans in [N] − 293Ts, respectively. D . Knocking out N-glycans on Spike protein reduced ACE2-Fc binding in cytometry based binding studies. Knocking out Spike O-glycans increased ACE-2 binding. E-F . Truncation of ACE2 N- and O-glycans did not affect either S1-Fc (panel E ) or RBD-Fc (panel F ) binding. G . ACE2 was transiently expressed on 293T, [O] − 293T and [N] − 293T cells. All pseudotyped virus efficiently entered ACE2 expressing cells. Virus was not titered for these runs, and thus comparison between viruses is not possible. * P
    Figure Legend Snippet: ACE2 glycosylation does not affect viral entry. A . Knocking out C1GALT1 and MGAT1 using CRISPR-Cas9 inhibits O- and N-glycan biosynthesis in HEK293Ts. B . Sanger sequencing results of isogenic 293T clones shows indels on all 3 alleles of C1GALT1 (‘[O] − 293T’) and single allele of MGAT1 (‘[N] − 293T’) knockout cells. Wild-type (WT) sequence is on the first line. Lower line shows base deletions (hyphen) and insertions (black fonts) for individual KOs. sgRNA target sequence is in red and protospacer adjacent motif is underlined. C . Increased VVA and reduced PHA-L binding confirm loss of O-linked glycans in [O] − 293Ts and N-glycans in [N] − 293Ts, respectively. D . Knocking out N-glycans on Spike protein reduced ACE2-Fc binding in cytometry based binding studies. Knocking out Spike O-glycans increased ACE-2 binding. E-F . Truncation of ACE2 N- and O-glycans did not affect either S1-Fc (panel E ) or RBD-Fc (panel F ) binding. G . ACE2 was transiently expressed on 293T, [O] − 293T and [N] − 293T cells. All pseudotyped virus efficiently entered ACE2 expressing cells. Virus was not titered for these runs, and thus comparison between viruses is not possible. * P

    Techniques Used: CRISPR, Sequencing, Clone Assay, Knock-Out, Binding Assay, Cytometry, Expressing

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

    Techniques Used: Binding Assay, Expressing, Generated

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.12.11.421008

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

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

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

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

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

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

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

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

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

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

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.11.16.385849

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

    Techniques Used: Expressing, Transduction

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

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

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

    Techniques Used: Expressing, Incubation

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

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

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

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

    26) Product Images from "The SARS-CoV-2 Envelope and Membrane proteins modulate maturation and retention of the Spike protein, allowing optimal formation of VLPs in presence of Nucleoprotein"

    Article Title: The SARS-CoV-2 Envelope and Membrane proteins modulate maturation and retention of the Spike protein, allowing optimal formation of VLPs in presence of Nucleoprotein

    Journal: bioRxiv

    doi: 10.1101/2020.08.24.260901

    Expression of SARS-CoV-2 E and M induced the retention of S thus preventing cell-cell fusion mediated by S. (A) Representative confocal microscopy images of VeroE6 cells infected or transfected with a plasmid encoding S alone or S combined with plasmids expressing M, E or N. The cis-Golgi was revealed with the anti-GM130 antibody (green channel), the S protein was revealed with the anti-SARS-CoV2 S1 antibody (red channel) and the nucleus was revealed with Hoechst. Scale bars of panels and zooms from squared area represent 10μm and 2μm, respectively. (B) The Manders’ coefficient M1 represents the fraction of S overlapping with GM130, and the M2 represents the fraction of GM130 overlapping with S. (C) Representative pictures of cell-cell fusion assay on VeroE6 cells transfected with a plasmid encoding S alone or S combined with plasmids expressing M, E or N. (D) Fusion index (left) and number of nuclei per syncytia (right) of the different conditions as described in (C).
    Figure Legend Snippet: Expression of SARS-CoV-2 E and M induced the retention of S thus preventing cell-cell fusion mediated by S. (A) Representative confocal microscopy images of VeroE6 cells infected or transfected with a plasmid encoding S alone or S combined with plasmids expressing M, E or N. The cis-Golgi was revealed with the anti-GM130 antibody (green channel), the S protein was revealed with the anti-SARS-CoV2 S1 antibody (red channel) and the nucleus was revealed with Hoechst. Scale bars of panels and zooms from squared area represent 10μm and 2μm, respectively. (B) The Manders’ coefficient M1 represents the fraction of S overlapping with GM130, and the M2 represents the fraction of GM130 overlapping with S. (C) Representative pictures of cell-cell fusion assay on VeroE6 cells transfected with a plasmid encoding S alone or S combined with plasmids expressing M, E or N. (D) Fusion index (left) and number of nuclei per syncytia (right) of the different conditions as described in (C).

    Techniques Used: Expressing, Confocal Microscopy, Infection, Transfection, Plasmid Preparation, Cell-Cell Fusion Assay

    The C-terminal moiety of S cytoplasmic tail is essential for M-mediated retention of SARS-CoV-2 S. (A) Alignment of sequences of the last amino-acids of S of SARS-CoV-2 or mutated by deletion of the last 19 amino acids (SΔ19). (B) Representative confocal microscopy images of VeroE6 cells transfected with a plasmid encoding SΔ19 alone or SΔ19 combined with plasmids expressing M or E. The cis-Golgi was revealed with the anti-GM130 antibody (green channel), the S protein was revealed with the anti-SARS-CoV2 S1 antibody (red channel) and the nucleus was revealed with Hoechst. The Manders’ coefficient M1 represents the fraction of S overlapping with GM130, and the M2 represents the fraction of GM130 overlapping with S. Scale bars of panels and zooms from squared area represent 10μm and 2μm, respectively. (C) Representative Western Blot of 293T transfected with a plasmid encoding SΔ19 or SΔ19 combined with plasmids encoding E or M. The arrows represent S0, S2 and S2* forms. (D) Quantification of S form of independent western blot as described in (C). (E) Cells lysates of 293T cells transfected with a plasmid encoding SΔ19 alone or SΔ19 combined with plasmids expressing E or M and N were treated with PNGase F to remove glycans. The arrows represent S0, S2 and S2* forms. (F) Representative pictures of cell-cell fusion assays on VeroE6 cells transfected with a plasmid encoding SΔ19 alone or SΔ19 combined with plasmids expressing M or E (left). Fusion index and number of nuclei per syncytia of the different conditions (right).
    Figure Legend Snippet: The C-terminal moiety of S cytoplasmic tail is essential for M-mediated retention of SARS-CoV-2 S. (A) Alignment of sequences of the last amino-acids of S of SARS-CoV-2 or mutated by deletion of the last 19 amino acids (SΔ19). (B) Representative confocal microscopy images of VeroE6 cells transfected with a plasmid encoding SΔ19 alone or SΔ19 combined with plasmids expressing M or E. The cis-Golgi was revealed with the anti-GM130 antibody (green channel), the S protein was revealed with the anti-SARS-CoV2 S1 antibody (red channel) and the nucleus was revealed with Hoechst. The Manders’ coefficient M1 represents the fraction of S overlapping with GM130, and the M2 represents the fraction of GM130 overlapping with S. Scale bars of panels and zooms from squared area represent 10μm and 2μm, respectively. (C) Representative Western Blot of 293T transfected with a plasmid encoding SΔ19 or SΔ19 combined with plasmids encoding E or M. The arrows represent S0, S2 and S2* forms. (D) Quantification of S form of independent western blot as described in (C). (E) Cells lysates of 293T cells transfected with a plasmid encoding SΔ19 alone or SΔ19 combined with plasmids expressing E or M and N were treated with PNGase F to remove glycans. The arrows represent S0, S2 and S2* forms. (F) Representative pictures of cell-cell fusion assays on VeroE6 cells transfected with a plasmid encoding SΔ19 alone or SΔ19 combined with plasmids expressing M or E (left). Fusion index and number of nuclei per syncytia of the different conditions (right).

    Techniques Used: Confocal Microscopy, Transfection, Plasmid Preparation, Expressing, Western Blot

    27) Product Images from "Neutralizing Human Antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 Isolated from a Human Synthetic Fab Phage Display Library"

    Article Title: Neutralizing Human Antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 Isolated from a Human Synthetic Fab Phage Display Library

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22041913

    Characterization of human anti-SARS-2 RBD Fabs. ( a ) Soluble ELISA of ten serially diluted human anti-SARS-2 RBD Fabs on immobilized SARS-2 RBD surfaces to measure their apparent affinities ( EC 50 , nM). ( b ) Schematic drawings of a competitive ELISA of human anti-SARS-2 RBD Fabs between the SARS-2 RBD and ACE2 protein (left) or ACE2-overexpressed cells (right). ( c ) Competitive ELISA of human anti-SARS-2 RBD Fabs antagonizing the interaction between ACE2 and the SARS-CoV-2 RBD. ( d ) Competitive flow cytometry analysis of human anti-SARS-2 RBD Fabs antagonizing the interaction between ACE2 on cells and the SARS-CoV-2 RBD (tagged with mouse Fc (mFc)). Arrows indicate potentially neutralizing clones. mFc-PE: anti-mouse PE (phycoerythrin) conjugate; MFI: mean fluorescence intensity; n.s: not significant ( p > 0.05); NC: negative control. * and **: p
    Figure Legend Snippet: Characterization of human anti-SARS-2 RBD Fabs. ( a ) Soluble ELISA of ten serially diluted human anti-SARS-2 RBD Fabs on immobilized SARS-2 RBD surfaces to measure their apparent affinities ( EC 50 , nM). ( b ) Schematic drawings of a competitive ELISA of human anti-SARS-2 RBD Fabs between the SARS-2 RBD and ACE2 protein (left) or ACE2-overexpressed cells (right). ( c ) Competitive ELISA of human anti-SARS-2 RBD Fabs antagonizing the interaction between ACE2 and the SARS-CoV-2 RBD. ( d ) Competitive flow cytometry analysis of human anti-SARS-2 RBD Fabs antagonizing the interaction between ACE2 on cells and the SARS-CoV-2 RBD (tagged with mouse Fc (mFc)). Arrows indicate potentially neutralizing clones. mFc-PE: anti-mouse PE (phycoerythrin) conjugate; MFI: mean fluorescence intensity; n.s: not significant ( p > 0.05); NC: negative control. * and **: p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Competitive ELISA, Flow Cytometry, Clone Assay, Fluorescence, Negative Control

    Characterization of anti-SARS-2 RBD immunoglobulin Gs (IgGs). ( a ) Binding analysis of five human anti-SARS-2 RBD IgGs—C12 (IgG), H1 (IgG), C2 (IgG), D12 (IgG), and F7 (IgG)—to the SARS-2 RBD and its variants (top) and the SARS-CoV-2 S1 (D614G) and other coronavirus S1 proteins (bottom), respectively. ( b ) Soluble ELISA of five serially diluted human anti-SARS-2 RBD IgGs on immobilized SARS-2 RBD surfaces to measure their apparent affinities ( EC 50 , nM). ( c ) ELISA detection for five human anti-SARS-2 RBD IgGs blocking the binding of the ACE2 protein with the SARS-CoV-2 RBD (top) and analysis of the flow cytometry for the blocking effect between the SARS-CoV-2 RBD and an ACE2-overexpressed cell (bottom). ( d ) Size-exclusion chromatography analysis of five human anti-SARS-2 RBD IgGs. The positions of the molecular mass markers, shown as kDa, on the retention time x -axis are indicated above the peaks. The data are presented as the mean ± standard error (SEM). MFI: mean fluorescence intensity; NC: negative control; *, **, and ***: p
    Figure Legend Snippet: Characterization of anti-SARS-2 RBD immunoglobulin Gs (IgGs). ( a ) Binding analysis of five human anti-SARS-2 RBD IgGs—C12 (IgG), H1 (IgG), C2 (IgG), D12 (IgG), and F7 (IgG)—to the SARS-2 RBD and its variants (top) and the SARS-CoV-2 S1 (D614G) and other coronavirus S1 proteins (bottom), respectively. ( b ) Soluble ELISA of five serially diluted human anti-SARS-2 RBD IgGs on immobilized SARS-2 RBD surfaces to measure their apparent affinities ( EC 50 , nM). ( c ) ELISA detection for five human anti-SARS-2 RBD IgGs blocking the binding of the ACE2 protein with the SARS-CoV-2 RBD (top) and analysis of the flow cytometry for the blocking effect between the SARS-CoV-2 RBD and an ACE2-overexpressed cell (bottom). ( d ) Size-exclusion chromatography analysis of five human anti-SARS-2 RBD IgGs. The positions of the molecular mass markers, shown as kDa, on the retention time x -axis are indicated above the peaks. The data are presented as the mean ± standard error (SEM). MFI: mean fluorescence intensity; NC: negative control; *, **, and ***: p

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Flow Cytometry, Size-exclusion Chromatography, Fluorescence, Negative Control

    Panning of the phage-displayed synthetic Fab library on an immobilized SARS-2 receptor-binding domain (RBD). ( a ) Monitoring of the phage titers over four rounds (R2–R5) of panning. Black and gray bars indicate the ratio of the phage output to the input titers, presented as a percentage (%), from panning on immobilized SARS-2 RBD (black, SARS-2 RBD (+)) and non-immobilized SARS-2 RBD (gray, SARS-2 RBD (−)) surfaces. The ratio of the output to the input (%) = (phage output titer ÷ phage input titer) × 100. ( b ) Phage ELISA performed on the immobilized SARS-2 RBD surfaces using each panning library phage. ( c ) Frequency of ten Fab phage clones selected in the third and fourth rounds (left) and the distribution of HCDR3 lengths (right). The selection frequency of a unique clone (%) = (number of unique clones ÷ total number of phage ELISA positives) × 100. ( d ) Monoclonal ELISA of ten Fab phage clones against the SARS-2 RBD (red) and SARS-2 S1 protein (green). AA: amino acid residue; NC: negative control.
    Figure Legend Snippet: Panning of the phage-displayed synthetic Fab library on an immobilized SARS-2 receptor-binding domain (RBD). ( a ) Monitoring of the phage titers over four rounds (R2–R5) of panning. Black and gray bars indicate the ratio of the phage output to the input titers, presented as a percentage (%), from panning on immobilized SARS-2 RBD (black, SARS-2 RBD (+)) and non-immobilized SARS-2 RBD (gray, SARS-2 RBD (−)) surfaces. The ratio of the output to the input (%) = (phage output titer ÷ phage input titer) × 100. ( b ) Phage ELISA performed on the immobilized SARS-2 RBD surfaces using each panning library phage. ( c ) Frequency of ten Fab phage clones selected in the third and fourth rounds (left) and the distribution of HCDR3 lengths (right). The selection frequency of a unique clone (%) = (number of unique clones ÷ total number of phage ELISA positives) × 100. ( d ) Monoclonal ELISA of ten Fab phage clones against the SARS-2 RBD (red) and SARS-2 S1 protein (green). AA: amino acid residue; NC: negative control.

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

    In vitro neutralization assay of human anti-SARS-2 RBD IgGs. Pseudo-typed virus-based neutralization ( a ) and a neutralization assay using authentic SARS-CoV-2 ( b ). ( c ) Correlation in neutralization potencies between pseudo-typed virus- and authentic virus-based assays. ( d ) Correlation between affinities of anti-SARS-2 RBD IgGs and their neutralization potencies for the authentic virus. The data are showed as the mean ± standard error (SEM).
    Figure Legend Snippet: In vitro neutralization assay of human anti-SARS-2 RBD IgGs. Pseudo-typed virus-based neutralization ( a ) and a neutralization assay using authentic SARS-CoV-2 ( b ). ( c ) Correlation in neutralization potencies between pseudo-typed virus- and authentic virus-based assays. ( d ) Correlation between affinities of anti-SARS-2 RBD IgGs and their neutralization potencies for the authentic virus. The data are showed as the mean ± standard error (SEM).

    Techniques Used: In Vitro, Neutralization

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

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

    Journal: eLife

    doi: 10.7554/eLife.61552

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

    Techniques Used: Binding Assay, Expressing, Generated

    29) Product Images from "A longitudinal study of SARS-CoV-2-infected patients reveals a high correlation between neutralizing antibodies and COVID-19 severity"

    Article Title: A longitudinal study of SARS-CoV-2-infected patients reveals a high correlation between neutralizing antibodies and COVID-19 severity

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/s41423-020-00588-2

    Serum neutralization of SARS-CoV-2 correlates with anti-S antibodies. A Correlation between the percentage of neutralization of SARS-CoV-2pp (left) or ID50 of live virus (right) with seroconversion measured by anti-N antibodies. B Same as A with anti-S antibodies. C Anti-S (left) and anti-N (right) IgG value distributions in the three groups of patients
    Figure Legend Snippet: Serum neutralization of SARS-CoV-2 correlates with anti-S antibodies. A Correlation between the percentage of neutralization of SARS-CoV-2pp (left) or ID50 of live virus (right) with seroconversion measured by anti-N antibodies. B Same as A with anti-S antibodies. C Anti-S (left) and anti-N (right) IgG value distributions in the three groups of patients

    Techniques Used: Neutralization

    Sera from patients infected by endemic coronaviruses have no cross-neutralizing activity against SARS-CoV-2. A Characteristics of samples from patients infected with other coronaviruses. B Seroconversion assessed by anti-N (left) and anti-S (middle) SARS-CoV-2 or neutralization measured by SARS-CoV-2pp (right). For neutralization assays, a commercial anti-S antibody was used as a positive control (control+) and five prepandemic serum samples were used as a negative control (control−)
    Figure Legend Snippet: Sera from patients infected by endemic coronaviruses have no cross-neutralizing activity against SARS-CoV-2. A Characteristics of samples from patients infected with other coronaviruses. B Seroconversion assessed by anti-N (left) and anti-S (middle) SARS-CoV-2 or neutralization measured by SARS-CoV-2pp (right). For neutralization assays, a commercial anti-S antibody was used as a positive control (control+) and five prepandemic serum samples were used as a negative control (control−)

    Techniques Used: Infection, Activity Assay, Neutralization, Positive Control, Negative Control

    Serum neutralization of SARS-CoV-2 correlates with the hospitalization units of COVID-19 patients. A Correlation between the ID50 of live virus, as plotted as number of dilutions (twofold dilutions starting from serum diluted at 1/10) with the percentage of neutralization of SARS-CoV-2pp for all tested samples. B Number of patients classified in the indicated groups according to the percentage of neutralization. In white: samples that induced a percentage of neutralization below 90%. C Comparison of the percentage of neutralization with SARS-CoV-2pp (left) or ID50 with live virus (right) for each patient classified according to the hospitalization unit. For patients with serial serum samples, the sera collected at the time closest to twenty days post onset of symptoms were chosen. In light green are asymptomatic patients (ASY) among the EOC patients. D Percentage of neutralization according to the severity of symptoms in HOS patients. The cutoff for neutralization (35%) was set using the mean neutralization of a 1/100 dilution of negative sera + 2 SD 58 for SARS-CoV-2pp. For the wild-type virus, the cutoff for the ID50 was set at the 1/10 dilution (first tested dilution), as all negative sera were below this threshold
    Figure Legend Snippet: Serum neutralization of SARS-CoV-2 correlates with the hospitalization units of COVID-19 patients. A Correlation between the ID50 of live virus, as plotted as number of dilutions (twofold dilutions starting from serum diluted at 1/10) with the percentage of neutralization of SARS-CoV-2pp for all tested samples. B Number of patients classified in the indicated groups according to the percentage of neutralization. In white: samples that induced a percentage of neutralization below 90%. C Comparison of the percentage of neutralization with SARS-CoV-2pp (left) or ID50 with live virus (right) for each patient classified according to the hospitalization unit. For patients with serial serum samples, the sera collected at the time closest to twenty days post onset of symptoms were chosen. In light green are asymptomatic patients (ASY) among the EOC patients. D Percentage of neutralization according to the severity of symptoms in HOS patients. The cutoff for neutralization (35%) was set using the mean neutralization of a 1/100 dilution of negative sera + 2 SD 58 for SARS-CoV-2pp. For the wild-type virus, the cutoff for the ID50 was set at the 1/10 dilution (first tested dilution), as all negative sera were below this threshold

    Techniques Used: Neutralization

    The residue at position 614 of SARS-CoV-2 spike does not influence the activity of nAbs. Percentage of neutralization of SARS-CoV-2pp using the spike protein with either a G or D at position 614
    Figure Legend Snippet: The residue at position 614 of SARS-CoV-2 spike does not influence the activity of nAbs. Percentage of neutralization of SARS-CoV-2pp using the spike protein with either a G or D at position 614

    Techniques Used: Activity Assay, Neutralization

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

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

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112643

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

    Techniques Used: Polymerase Chain Reaction

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

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

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

    Techniques Used: Titration, Concentration Assay, Standard Deviation

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

    Techniques Used: Incubation

    31) 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

    BNT162b2-elicited antibody responses in mice. BALB/c mice (n=8 per group) were immunised intramuscularly (IM) with 0.2, 1 or 5 μg of BNT162b2 or buffer. a , RBD-binding IgG responses in sera obtained 7, 14, 21 and 28 days after immunisation, determined by ELISA. For day 0, a pre-screening of randomised animals was performed (n=4). Geometric mean of each group is shown. b , Representative surface plasmon resonance sensorgram of the binding kinetics of recombinant S1 ( left ) and RBD ( right ) to immobilised mouse IgG from serum 28 days after immunisation with 5 μg BNT162b2 (n=8). Actual binding (dark blue) and the best fit of the data to a 1:1 binding model (thin line in black). c , Number of infected cells per well in pseudovirus neutralisation assays conducted with serially diluted mouse serum samples obtained 28 days after immunisation with BNT162b2 are shown (n=8 per group, see also Figure 2b ).
    Figure Legend Snippet: BNT162b2-elicited antibody responses in mice. BALB/c mice (n=8 per group) were immunised intramuscularly (IM) with 0.2, 1 or 5 μg of BNT162b2 or buffer. a , RBD-binding IgG responses in sera obtained 7, 14, 21 and 28 days after immunisation, determined by ELISA. For day 0, a pre-screening of randomised animals was performed (n=4). Geometric mean of each group is shown. b , Representative surface plasmon resonance sensorgram of the binding kinetics of recombinant S1 ( left ) and RBD ( right ) to immobilised mouse IgG from serum 28 days after immunisation with 5 μg BNT162b2 (n=8). Actual binding (dark blue) and the best fit of the data to a 1:1 binding model (thin line in black). c , Number of infected cells per well in pseudovirus neutralisation assays conducted with serially diluted mouse serum samples obtained 28 days after immunisation with BNT162b2 are shown (n=8 per group, see also Figure 2b ).

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

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

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

    32) 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:

    33) Product Images from "Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies"

    Article Title: Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/s41423-020-0458-z

    The antibody response induced by recombinant RBD of SARS-CoV and SARS-CoV-2 in mice. a Schematic of the vaccine regimen. Five C57BL/6 mice per group were immunized two times (2–3 weeks apart) intramuscularly with 25 µg of the SARS CoV-2 RBD-hFc or SARS CoV RBD-hFc protein in combination with quick adjuvant. Mice immunized without the RBD protein but with hIgG were included as controls. Mice were sacrificed on day 35 after immunization, and antisera were collected for subsequent tests. b Cross-reactivity of SARS-CoV-2-RBD- or SARS-CoV-RBD-specific mouse sera against the SARS-CoV RBD or SARS-CoV-2 RBD as determined by ELISA. Mouse antisera were serially diluted three-fold and tested for binding to the SARS-CoV RBD or SARS-CoV-2 RBD. The IgG antibody (Ab) titres of SARS-CoV-2 antisera (red), SARS-CoV antisera (blue) and control antisera (black) were calculated at the endpoint dilution that remained positively detectable for the SARS-CoV-2 RBD or SARS-CoV RBD. The data are presented as the mean A450 ± s.e.m. ( n = 5). c Cross-competition of SARS-CoV-2-RBD- or SARS-CoV-RBD-specific mouse sera and hACE2 with the SARS-CoV RBD or SARS-CoV-2 RBD as determined by ELISA. The data are presented as the mean blocking (%) ± s.e.m. ( n = 5). Fifty percent blocking antibody titres (BT 50 ) against the SARS-CoV pseudo-typed virus or SARS-CoV pseudo-typed virus were calculated. d Cross-neutralization of SARS-CoV-2-RBD- or SARS-CoV-RBD-specific mouse sera against SARS-CoV-2 or SARS-CoV pseudo-typed virus entry, measured by pseudo-typed virus neutralization assay. The data are presented as the mean neutralization (%) ± s.e.m. ( n = 5). Fifty percent neutralizing antibody titres (NT 50 ) against the SARS-CoV-2 or SARS-CoV pseudo-typed virus were calculated
    Figure Legend Snippet: The antibody response induced by recombinant RBD of SARS-CoV and SARS-CoV-2 in mice. a Schematic of the vaccine regimen. Five C57BL/6 mice per group were immunized two times (2–3 weeks apart) intramuscularly with 25 µg of the SARS CoV-2 RBD-hFc or SARS CoV RBD-hFc protein in combination with quick adjuvant. Mice immunized without the RBD protein but with hIgG were included as controls. Mice were sacrificed on day 35 after immunization, and antisera were collected for subsequent tests. b Cross-reactivity of SARS-CoV-2-RBD- or SARS-CoV-RBD-specific mouse sera against the SARS-CoV RBD or SARS-CoV-2 RBD as determined by ELISA. Mouse antisera were serially diluted three-fold and tested for binding to the SARS-CoV RBD or SARS-CoV-2 RBD. The IgG antibody (Ab) titres of SARS-CoV-2 antisera (red), SARS-CoV antisera (blue) and control antisera (black) were calculated at the endpoint dilution that remained positively detectable for the SARS-CoV-2 RBD or SARS-CoV RBD. The data are presented as the mean A450 ± s.e.m. ( n = 5). c Cross-competition of SARS-CoV-2-RBD- or SARS-CoV-RBD-specific mouse sera and hACE2 with the SARS-CoV RBD or SARS-CoV-2 RBD as determined by ELISA. The data are presented as the mean blocking (%) ± s.e.m. ( n = 5). Fifty percent blocking antibody titres (BT 50 ) against the SARS-CoV pseudo-typed virus or SARS-CoV pseudo-typed virus were calculated. d Cross-neutralization of SARS-CoV-2-RBD- or SARS-CoV-RBD-specific mouse sera against SARS-CoV-2 or SARS-CoV pseudo-typed virus entry, measured by pseudo-typed virus neutralization assay. The data are presented as the mean neutralization (%) ± s.e.m. ( n = 5). Fifty percent neutralizing antibody titres (NT 50 ) against the SARS-CoV-2 or SARS-CoV pseudo-typed virus were calculated

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

    Cross-reactivity of the RBD-targeting neutralizing mAbs against SARS-CoV and SARS-CoV-2. a Characteristics of the neutralizing mAbs against the SARS CoV-2 RBD and SARS CoV RBD. b , c Dose-dependent binding of SARS-CoV and SARS-CoV-2 mAbs to the SARS-CoV RBD ( b ) or SARS-CoV-2 RBD ( c ) as determined by ELISA. Isotype antibody was included as a control. Data are presented as the mean OD450 ± s.e.m. ( n = 2). d , e Dose-dependent competition of the SARS-CoV-2 or SARS-CoV mAbs and hACE2 with the SARS-CoV RBD ( d ) or SARS-CoV-2 RBD ( e ) as measured by ELISA. Data are presented as the mean OD450 ± s.e.m. ( n = 2). f IC 50 values were determined for a panel of mAbs neutralizing the SARS-CoV-2 or SARS-CoV pseudo-typed viruses. Representative data are shown
    Figure Legend Snippet: Cross-reactivity of the RBD-targeting neutralizing mAbs against SARS-CoV and SARS-CoV-2. a Characteristics of the neutralizing mAbs against the SARS CoV-2 RBD and SARS CoV RBD. b , c Dose-dependent binding of SARS-CoV and SARS-CoV-2 mAbs to the SARS-CoV RBD ( b ) or SARS-CoV-2 RBD ( c ) as determined by ELISA. Isotype antibody was included as a control. Data are presented as the mean OD450 ± s.e.m. ( n = 2). d , e Dose-dependent competition of the SARS-CoV-2 or SARS-CoV mAbs and hACE2 with the SARS-CoV RBD ( d ) or SARS-CoV-2 RBD ( e ) as measured by ELISA. Data are presented as the mean OD450 ± s.e.m. ( n = 2). f IC 50 values were determined for a panel of mAbs neutralizing the SARS-CoV-2 or SARS-CoV pseudo-typed viruses. Representative data are shown

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

    Single amino acid substitution mutagenesis of the SARS-CoV-2-RBD and SARS-CoV-RBD. a Sequence differences in the SARS-CoV and SARS-CoV-2 RBDs. RBM is in red. Previously, identified critical ACE2-binding residues are shaded in green. The conserved residues are marked with asterisks (*), the residues with similar properties between groups are marked with the colon symbol (:) and the residues with marginally similar properties are marked with the period symbol (.). b ACE2 binding with reciprocal amino acid substitutions in the SARS-CoV-2 RBD. Each value is calculated as the binding relative to that of the WT (%). The mean±S.E.M. of duplicate wells is shown for two independent experiments. The two red dotted lines represent 75% and 125% relative to the WT data, respectively. c , d Structural alignment of SARS-CoV-2-RBD and SARS-CoV-RBD binding with ACE2. The SARS-CoV-RBD complex (PDB ID: 2AJF) is superimposed on the SARS-CoV-2 RBD (PDB ID: 6lzj. grey: ACE2, wheat: SARS-CoV-2. Mutants that weaken the SARS-CoV-2 RBD binding with ACE2 are highlighted in cyan ( c ). The corresponding residues from SARS-CoV are indicated in green and are illustrated in detail ( c left). Mutants that enhance ACE2 binding are highlighted in magenta ( d ). e ACE2 binding with reciprocal amino acid substitutions in the SARS-CoV RBD. Each value is calculated as the binding relative to that of the WT (%). The mean ± S.E.M. of duplicate wells is shown in two independent experiments. The two red dotted lines represent 75 and 125% relative to the WT data, respectively. f Molecular docking of the SARS-CoV 2 RBD carrying the Q498Y mutant in complex with hACE2. Q498Y formed π-π stacking with Y41 in hACE2: left, Y498; right, Q498
    Figure Legend Snippet: Single amino acid substitution mutagenesis of the SARS-CoV-2-RBD and SARS-CoV-RBD. a Sequence differences in the SARS-CoV and SARS-CoV-2 RBDs. RBM is in red. Previously, identified critical ACE2-binding residues are shaded in green. The conserved residues are marked with asterisks (*), the residues with similar properties between groups are marked with the colon symbol (:) and the residues with marginally similar properties are marked with the period symbol (.). b ACE2 binding with reciprocal amino acid substitutions in the SARS-CoV-2 RBD. Each value is calculated as the binding relative to that of the WT (%). The mean±S.E.M. of duplicate wells is shown for two independent experiments. The two red dotted lines represent 75% and 125% relative to the WT data, respectively. c , d Structural alignment of SARS-CoV-2-RBD and SARS-CoV-RBD binding with ACE2. The SARS-CoV-RBD complex (PDB ID: 2AJF) is superimposed on the SARS-CoV-2 RBD (PDB ID: 6lzj. grey: ACE2, wheat: SARS-CoV-2. Mutants that weaken the SARS-CoV-2 RBD binding with ACE2 are highlighted in cyan ( c ). The corresponding residues from SARS-CoV are indicated in green and are illustrated in detail ( c left). Mutants that enhance ACE2 binding are highlighted in magenta ( d ). e ACE2 binding with reciprocal amino acid substitutions in the SARS-CoV RBD. Each value is calculated as the binding relative to that of the WT (%). The mean ± S.E.M. of duplicate wells is shown in two independent experiments. The two red dotted lines represent 75 and 125% relative to the WT data, respectively. f Molecular docking of the SARS-CoV 2 RBD carrying the Q498Y mutant in complex with hACE2. Q498Y formed π-π stacking with Y41 in hACE2: left, Y498; right, Q498

    Techniques Used: Mutagenesis, Sequencing, Binding Assay

    Both the SARS-CoV-2 RBD and SARS-CoV RBD bind to hACE2. a Receptor-dependent infection of SARS-CoV-2 and SARS-CoV pseudo-typed virus entry into hACE2 + 293 T cells. 293T cells stably expressing hACE2 were infected with SARS-CoV-2 or SARS-CoV pseudo-typed viruses, and the cells were harvested to detect the luciferase activity. Fold changes were calculated by comparison to the levels in the uninfected cells. VSV pseudo-typed viruses were included as controls. b Syncytia formation between S protein- and hACE2-expressing cells. 293T cells transfected with hACE2 plasmid were mixed at a 1:1 ratio with 293T cells transfected with plasmid encoding S protein from SARS-CoV-2 (bottom left) or SARS-CoV (bottom right). As controls, 293T cells transfected with an empty plasmid were either mixed at a 1:1 ratio with 293T cells transfected with the hACE2 plasmid (top row), S protein from SARS-CoV-2 (middle left) or SARS-CoV (middle right). Images were photographed at ×20 magnification. Representative images are shown. c Dose-dependent binding of the SARS-CoV-2 RBD to soluble hACE2 as determined by ELISA. The binding of both the SARS-CoV-2 RBD and SARS-CoV RBD with an Fc tag on hACE2 was tested. Human Fc was included as a control. Data are presented as the mean OD450 ± s.e.m. ( n = 2). d Binding profiles of the SARS-CoV-2 RBD and SARS-CoV RBD to the soluble hACE2 receptor measured by biolayer interferometry in an Octet RED96 instrument. The biotin-conjugated hACE2 protein was captured by streptavidin that was immobilized on a chip and tested for binding with gradient concentrations of the soluble RBD of S proteins from SARS CoV and SARS CoV-2. Binding kinetics were evaluated using a 1:1 Langmuir binding model by ForteBio Data Analysis 9.0 software
    Figure Legend Snippet: Both the SARS-CoV-2 RBD and SARS-CoV RBD bind to hACE2. a Receptor-dependent infection of SARS-CoV-2 and SARS-CoV pseudo-typed virus entry into hACE2 + 293 T cells. 293T cells stably expressing hACE2 were infected with SARS-CoV-2 or SARS-CoV pseudo-typed viruses, and the cells were harvested to detect the luciferase activity. Fold changes were calculated by comparison to the levels in the uninfected cells. VSV pseudo-typed viruses were included as controls. b Syncytia formation between S protein- and hACE2-expressing cells. 293T cells transfected with hACE2 plasmid were mixed at a 1:1 ratio with 293T cells transfected with plasmid encoding S protein from SARS-CoV-2 (bottom left) or SARS-CoV (bottom right). As controls, 293T cells transfected with an empty plasmid were either mixed at a 1:1 ratio with 293T cells transfected with the hACE2 plasmid (top row), S protein from SARS-CoV-2 (middle left) or SARS-CoV (middle right). Images were photographed at ×20 magnification. Representative images are shown. c Dose-dependent binding of the SARS-CoV-2 RBD to soluble hACE2 as determined by ELISA. The binding of both the SARS-CoV-2 RBD and SARS-CoV RBD with an Fc tag on hACE2 was tested. Human Fc was included as a control. Data are presented as the mean OD450 ± s.e.m. ( n = 2). d Binding profiles of the SARS-CoV-2 RBD and SARS-CoV RBD to the soluble hACE2 receptor measured by biolayer interferometry in an Octet RED96 instrument. The biotin-conjugated hACE2 protein was captured by streptavidin that was immobilized on a chip and tested for binding with gradient concentrations of the soluble RBD of S proteins from SARS CoV and SARS CoV-2. Binding kinetics were evaluated using a 1:1 Langmuir binding model by ForteBio Data Analysis 9.0 software

    Techniques Used: Infection, Stable Transfection, Expressing, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Binding Assay, Enzyme-linked Immunosorbent Assay, Chromatin Immunoprecipitation, Software

    Recognition pattern of mAbs to single amino acid substitute mutants of SARS-CoV or SARS-CoV-2 RBD. a Sequence conservation in the SARS-CoV and SARS-CoV-2 RBDs in a surface representation. Red, different; grey, identical. b Site mutagenesis scanning. The SARS-CoV and SARS-CoV-2 RBD mutant panel includes the reported antibody epitope positions and sequence changes within the RBMs. Relative binding to the wild-type: 0–25% presented in black; 25–50%, presented in dark grey; 50–75% presented in light grey; > 75%, presented in white. The results shown represent the mean percentage of binding signal for the mAbs bound to the mutants relative to that of the wild-type RBD in at least two independent experiments. c Interaction of Y484 and D480 in the SARS-CoV RBD with 80 R (PDB ID: 2ghw). Polar interactions are indicated by yellow dashed lines. d Interaction of Y484 and T487 in the SARS-CoV RBD with m396 (PDB ID: 2dd8). Yellow: heavy chain, cyan: light chain. The binding surface of m396 is shown by electrostatic surface representations. e The residues that are important for HA001 binding are on the interface of the ACE2 and RBD (PDB ID: 6VW1)
    Figure Legend Snippet: Recognition pattern of mAbs to single amino acid substitute mutants of SARS-CoV or SARS-CoV-2 RBD. a Sequence conservation in the SARS-CoV and SARS-CoV-2 RBDs in a surface representation. Red, different; grey, identical. b Site mutagenesis scanning. The SARS-CoV and SARS-CoV-2 RBD mutant panel includes the reported antibody epitope positions and sequence changes within the RBMs. Relative binding to the wild-type: 0–25% presented in black; 25–50%, presented in dark grey; 50–75% presented in light grey; > 75%, presented in white. The results shown represent the mean percentage of binding signal for the mAbs bound to the mutants relative to that of the wild-type RBD in at least two independent experiments. c Interaction of Y484 and D480 in the SARS-CoV RBD with 80 R (PDB ID: 2ghw). Polar interactions are indicated by yellow dashed lines. d Interaction of Y484 and T487 in the SARS-CoV RBD with m396 (PDB ID: 2dd8). Yellow: heavy chain, cyan: light chain. The binding surface of m396 is shown by electrostatic surface representations. e The residues that are important for HA001 binding are on the interface of the ACE2 and RBD (PDB ID: 6VW1)

    Techniques Used: Sequencing, Mutagenesis, Binding Assay

    34) Product Images from "Recombinant Fc-fusion vaccine of RBD induced protection against SARS-CoV-2 in non-human primate and mice"

    Article Title: Recombinant Fc-fusion vaccine of RBD induced protection against SARS-CoV-2 in non-human primate and mice

    Journal: bioRxiv

    doi: 10.1101/2020.11.29.402339

    Correlations of SARS-CoV-2 NAb titers (a), pseudovirus titers (b), and RBD protein specific IgG titers (c) prior to challenge with log peak mRNA copies/g in lungs following challenge in hACE2-Tg mice. Red lines reflect the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests.
    Figure Legend Snippet: Correlations of SARS-CoV-2 NAb titers (a), pseudovirus titers (b), and RBD protein specific IgG titers (c) prior to challenge with log peak mRNA copies/g in lungs following challenge in hACE2-Tg mice. Red lines reflect the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests.

    Techniques Used: Mouse Assay

    SARS-Cov2 RBD-Fc Vacc elicit strong humoral immunity in macaca fascicularis. a) Schematic diagram of immunization, sample collection and challenge schedule. b). Macaca fascicularis (n=5) were immunized on day 0, day 14, d28 with 20ug and 40ug doses of RBD-Fc Vacc or PBS and the serum were collected at the indicated time. The SARS-CoV-2 RBD specific IgG were examined by ELISA. c) Neutralizing antibodies were determined by microneutralization assay using the SARS-CoV-2 (NT50). d). Correlation analysis of antibody titers tested by ELISA and microneutralization assay. P and R values reflect two-sided Spearman rank-correlation tests. e) Serum cross neutralization against SARS-CoV-2 epidemic strains in RBD-Fc immunized macaca fascicularis. NT50 against the SARS-CoV-2 epidemic strains (BJ08, BJ05, BJ01) were performed using macaca fascicularis sera collected at 21 days post third immunization. Data are analyzed by one-way ANOVA with multiple comparison tests. (n.s., not significant).
    Figure Legend Snippet: SARS-Cov2 RBD-Fc Vacc elicit strong humoral immunity in macaca fascicularis. a) Schematic diagram of immunization, sample collection and challenge schedule. b). Macaca fascicularis (n=5) were immunized on day 0, day 14, d28 with 20ug and 40ug doses of RBD-Fc Vacc or PBS and the serum were collected at the indicated time. The SARS-CoV-2 RBD specific IgG were examined by ELISA. c) Neutralizing antibodies were determined by microneutralization assay using the SARS-CoV-2 (NT50). d). Correlation analysis of antibody titers tested by ELISA and microneutralization assay. P and R values reflect two-sided Spearman rank-correlation tests. e) Serum cross neutralization against SARS-CoV-2 epidemic strains in RBD-Fc immunized macaca fascicularis. NT50 against the SARS-CoV-2 epidemic strains (BJ08, BJ05, BJ01) were performed using macaca fascicularis sera collected at 21 days post third immunization. Data are analyzed by one-way ANOVA with multiple comparison tests. (n.s., not significant).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Microneutralization Assay, Neutralization

    Characterization of the cellular immune response for SARS-CoV-2 RBD-Fc vaccine. a) ELISPOT assay for IFN-γ in PBMCs from macaca fascicularis. Data are shown as mean ± SEM. Significance was calculated using unpaired t-test (n.s., not significant). b). SARS-CoV-2 RBD-specific IL-4 + T cells and TNF-a + T cells in CD4 + T and CD8 + T cells from macaca fascicularis PBMCs were detected by flow cytometry.
    Figure Legend Snippet: Characterization of the cellular immune response for SARS-CoV-2 RBD-Fc vaccine. a) ELISPOT assay for IFN-γ in PBMCs from macaca fascicularis. Data are shown as mean ± SEM. Significance was calculated using unpaired t-test (n.s., not significant). b). SARS-CoV-2 RBD-specific IL-4 + T cells and TNF-a + T cells in CD4 + T and CD8 + T cells from macaca fascicularis PBMCs were detected by flow cytometry.

    Techniques Used: Enzyme-linked Immunospot, Flow Cytometry

    SARS-Cov2 RBD-Fc Vacc elicit strong humoral immunity in mice. hACE2-Tg mice were immunized on day 0, day 14, d28 with 10ug and 20ug doses of RBD-Fc Vacc or adjuvant and the serum were collected at the indicated time. a) The SARS-CoV-2 RBD specific IgG were examined by ELISA. b c) Neutralizing antibodies were determined by microneutralization assay using both the SARS-CoV-2 (b) and pseudovirus (c).
    Figure Legend Snippet: SARS-Cov2 RBD-Fc Vacc elicit strong humoral immunity in mice. hACE2-Tg mice were immunized on day 0, day 14, d28 with 10ug and 20ug doses of RBD-Fc Vacc or adjuvant and the serum were collected at the indicated time. a) The SARS-CoV-2 RBD specific IgG were examined by ELISA. b c) Neutralizing antibodies were determined by microneutralization assay using both the SARS-CoV-2 (b) and pseudovirus (c).

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Microneutralization Assay

    SARS-Cov2 RBD-Fc vaccine Design. a) Two RBD domains are fused through Fc fragment to form the Y-shaped structure via protein structure prediction server version 3.0 (left panel). RBD-Fc protein expressed from CHO cells were identified by western blot under reduced and non-reduced condition using both anti-serum from COVID-19 recovered patients and commercial antibody (right panel). b) N-glycosylation and O-glycosylation sites identified using mass spectrum (up panel). The docking between ACE-2 and RBD-Fc predicted by ZDOCK server. An overview of the glycosylation sites illustrated based on the solved complex structure of SARS-CoV-2 RBD-Fc bound to ACE2 (PDB code: 1R42). The identified sites, colored red for N-glycosylation, purple for O-glycosylation are shown as spheres and labeled. The right panel (surface representation) was generated by rotating the structure in the Left panel (cartoon representation) around a vertical axis for about 90° (lower panel). c) The real-time binding profile between our purified RBD-Fc protein and ACE2 characterized by SPR Biacore. d e) Balb/C mice were immunized with RBD-his or RBD-Fc (10ug/mouse) in the presence of aluminum at d0 and d14. Two weeks post last vaccination the serum were collected ELISA assay shows the SARS-CoV-2 RBD-specific IgG titers (d). SARS-CoV-2 neutralization assay shows the NT50 (e).
    Figure Legend Snippet: SARS-Cov2 RBD-Fc vaccine Design. a) Two RBD domains are fused through Fc fragment to form the Y-shaped structure via protein structure prediction server version 3.0 (left panel). RBD-Fc protein expressed from CHO cells were identified by western blot under reduced and non-reduced condition using both anti-serum from COVID-19 recovered patients and commercial antibody (right panel). b) N-glycosylation and O-glycosylation sites identified using mass spectrum (up panel). The docking between ACE-2 and RBD-Fc predicted by ZDOCK server. An overview of the glycosylation sites illustrated based on the solved complex structure of SARS-CoV-2 RBD-Fc bound to ACE2 (PDB code: 1R42). The identified sites, colored red for N-glycosylation, purple for O-glycosylation are shown as spheres and labeled. The right panel (surface representation) was generated by rotating the structure in the Left panel (cartoon representation) around a vertical axis for about 90° (lower panel). c) The real-time binding profile between our purified RBD-Fc protein and ACE2 characterized by SPR Biacore. d e) Balb/C mice were immunized with RBD-his or RBD-Fc (10ug/mouse) in the presence of aluminum at d0 and d14. Two weeks post last vaccination the serum were collected ELISA assay shows the SARS-CoV-2 RBD-specific IgG titers (d). SARS-CoV-2 neutralization assay shows the NT50 (e).

    Techniques Used: Western Blot, Labeling, Generated, Binding Assay, Purification, SPR Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Neutralization

    Related Articles

    Selection:

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein
    Article Snippet: .. SELEX Procedures We performed the aptamer selection procedure for SARS-CoV-2 RBD in a manner similar to our previous work. .. The initial ssDNA library consisted of a 40-nt randomized region and two flanking regions as a PCR primer (5′- ATCCAGAGTGACGCAGCA - 40N - TGGACACGGTGGCTTAGT-3′).

    other:

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein
    Article Snippet: By retaining the main motif of the full-length aptamers, we optimized two aptamers (with K d values of 5.8 nM and 19.9 nM) against SARS-CoV-2 RBD.

    Article Title: Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients
    Article Snippet: Calculated limits of detection for these data were 43.3 ng/mL (SARS-CoV-2 S1 + S2 ECD), 40.7 ng/mL (SARS-CoV-2 S1), and 25.1 ng/mL (SARS-CoV-2 RBD).

    Inhibition:

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries
    Article Snippet: Streptavidin biosensors (ForteBio, Cat: 18-5019) were loaded with AviTag™-biotinylated scFv or biotinylated SARS-CoV-2 S1 trimer (Acro Biosystems, Cat: S1N-C82E8), blocked with biotin, washed in PBS, and then associated with protein ligand in PBS. .. ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay. .. Biotinylated soluble ACE2 protein (Acro Biosystems, Cat: AC2-H82E6) was bound to MyOne Streptavidin C1 Dynabeads (0.33 mg ACE2 per mL Dynabeads) for 30 minutes, then the beads were magnetically purified, washed, and blocked with free biotin.

    Blocking Assay:

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries
    Article Snippet: Streptavidin biosensors (ForteBio, Cat: 18-5019) were loaded with AviTag™-biotinylated scFv or biotinylated SARS-CoV-2 S1 trimer (Acro Biosystems, Cat: S1N-C82E8), blocked with biotin, washed in PBS, and then associated with protein ligand in PBS. .. ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay. .. Biotinylated soluble ACE2 protein (Acro Biosystems, Cat: AC2-H82E6) was bound to MyOne Streptavidin C1 Dynabeads (0.33 mg ACE2 per mL Dynabeads) for 30 minutes, then the beads were magnetically purified, washed, and blocked with free biotin.

    Cell Culture:

    Article Title: CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells
    Article Snippet: The spike protein expression was determined by flow cytometry by staining the transduced cells with anti-RBD antibody (SinoBiological) followed by a goat anti-rabbit fluorophore-conjugated secondary antibody. .. A549-Spike cells were cultured for a few days prior to sorting using anti-RBD. .. Sorted cells were cultured in DMEM supplemented with 10% (v/v) FBS, and 100 U/mL Penicillin-Streptomycin.

    Binding Assay:

    Article Title: Identification of Human Single-Domain Antibodies against SARS-CoV-2
    Article Snippet: The experiments included the following steps at 37°C: (1) equilibration (60 s); (2) activation of AR2G by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysucci-nimide (300 s); (3) immobilization of S1 protein onto sensors (100 s); (4) quenching with ethanolamine (300 s); (5) baseline in kinetics buffer (120 s); (6) association of antibodies for measurement of k on (300-600 s); and (7) dissociation of antibodies for measurement of k off (300-600 s). .. For measuring binding kinetics of single-domain antibodies with SARS-CoV-2 RBD, Avi-tagged recombinant RBD was biotinylated with the BirA biotinylation kit (Avidity), diluted in kinetics buffer and immobilized on streptavidin (SA) coated biosensors (Pall FortéBio) at ~50% of the sensor maximum binding capacity. ..

    Recombinant:

    Article Title: Identification of Human Single-Domain Antibodies against SARS-CoV-2
    Article Snippet: The experiments included the following steps at 37°C: (1) equilibration (60 s); (2) activation of AR2G by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysucci-nimide (300 s); (3) immobilization of S1 protein onto sensors (100 s); (4) quenching with ethanolamine (300 s); (5) baseline in kinetics buffer (120 s); (6) association of antibodies for measurement of k on (300-600 s); and (7) dissociation of antibodies for measurement of k off (300-600 s). .. For measuring binding kinetics of single-domain antibodies with SARS-CoV-2 RBD, Avi-tagged recombinant RBD was biotinylated with the BirA biotinylation kit (Avidity), diluted in kinetics buffer and immobilized on streptavidin (SA) coated biosensors (Pall FortéBio) at ~50% of the sensor maximum binding capacity. ..

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    Sino Biological sars cov 2 rbd
    RU169 output clone diversity Using the <t>SARS-CoV-2</t> RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.
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    RU169 output clone diversity Using the SARS-CoV-2 RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.

    Journal: bioRxiv

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

    doi: 10.1101/2020.07.27.224089

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

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

    Techniques: FACS, Selection, Produced, Clone Assay

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.07.27.224089

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

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

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

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.07.27.224089

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

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

    Techniques: Clone Assay, In Vitro

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.07.27.224089

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

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

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

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.07.27.224089

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

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

    Techniques: Binding Assay

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.07.27.224089

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

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

    Techniques: Cytometry, Inhibition, Fluorescence, Labeling

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.06.15.153064

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

    Article Snippet: Calculated limits of detection for these data were 43.3 ng/mL (SARS-CoV-2 S1 + S2 ECD), 40.7 ng/mL (SARS-CoV-2 S1), and 25.1 ng/mL (SARS-CoV-2 RBD).

    Techniques: Polymerase Chain Reaction

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.06.15.153064

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

    Article Snippet: Calculated limits of detection for these data were 43.3 ng/mL (SARS-CoV-2 S1 + S2 ECD), 40.7 ng/mL (SARS-CoV-2 S1), and 25.1 ng/mL (SARS-CoV-2 RBD).

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

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.06.15.153064

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

    Article Snippet: Calculated limits of detection for these data were 43.3 ng/mL (SARS-CoV-2 S1 + S2 ECD), 40.7 ng/mL (SARS-CoV-2 S1), and 25.1 ng/mL (SARS-CoV-2 RBD).

    Techniques: Enzyme-linked Immunosorbent Assay

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.06.15.153064

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

    Article Snippet: Calculated limits of detection for these data were 43.3 ng/mL (SARS-CoV-2 S1 + S2 ECD), 40.7 ng/mL (SARS-CoV-2 S1), and 25.1 ng/mL (SARS-CoV-2 RBD).

    Techniques: Incubation

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.06.15.153064

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

    Article Snippet: Calculated limits of detection for these data were 43.3 ng/mL (SARS-CoV-2 S1 + S2 ECD), 40.7 ng/mL (SARS-CoV-2 S1), and 25.1 ng/mL (SARS-CoV-2 RBD).

    Techniques: Titration, Concentration Assay, Standard Deviation

    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: SELEX Procedures We performed the aptamer selection procedure for SARS-CoV-2 RBD in a manner similar to our previous work.

    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: SELEX Procedures We performed the aptamer selection procedure for SARS-CoV-2 RBD in a manner similar to our previous work.

    Techniques: Flow Cytometry, Binding Assay

    S309-CAR-NK cells are superior to CR3022-CAR-NK cells. ( a ) Diagram of S309 and CR3022 neutralizing antibodies binding to different epitopes of the SARS-CoV-2 S protein. Both open and closed conformation states of SARS-CoV-2 S protein are shown. S309 binding site is indicated in magenta and CR3022 binding site is indicated in yellow. ( b ) Quantitative data of CD107a surface expression of both S309-CAR-NK-92MI and CR3022-CAR-NK-92MI. Both transient 293T-hACE2-RBD and stable A549-Spike cell lines were used as target cells. Error bars represent SEM from at least two independent experiments. ( c ) Comparison of killing activity of S309-CAR and CR3022-CAR using the 4-hour Cr 51 release assay. Effector cells were cocultured with Cr 51 -labeled target cells at 37°C for 4 hours. The assay was repeated for at least two times per target cell line. ( d ) Expanded S309-CAR-NK primary has increased killing activity against A549-Spike cells than primary CR3022-CAR-NK primary . Effector cells were blocked with anti-CD16 and anti-NKG2D prior to coculturing with A549-Spike target cells for 4 hours at 37°C. Data were pooled from three independent experiments. Unpaired Student’s t test was employed for all panels. ns p > 0.05, * p

    Journal: bioRxiv

    Article Title: CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells

    doi: 10.1101/2021.01.14.426742

    Figure Lengend Snippet: S309-CAR-NK cells are superior to CR3022-CAR-NK cells. ( a ) Diagram of S309 and CR3022 neutralizing antibodies binding to different epitopes of the SARS-CoV-2 S protein. Both open and closed conformation states of SARS-CoV-2 S protein are shown. S309 binding site is indicated in magenta and CR3022 binding site is indicated in yellow. ( b ) Quantitative data of CD107a surface expression of both S309-CAR-NK-92MI and CR3022-CAR-NK-92MI. Both transient 293T-hACE2-RBD and stable A549-Spike cell lines were used as target cells. Error bars represent SEM from at least two independent experiments. ( c ) Comparison of killing activity of S309-CAR and CR3022-CAR using the 4-hour Cr 51 release assay. Effector cells were cocultured with Cr 51 -labeled target cells at 37°C for 4 hours. The assay was repeated for at least two times per target cell line. ( d ) Expanded S309-CAR-NK primary has increased killing activity against A549-Spike cells than primary CR3022-CAR-NK primary . Effector cells were blocked with anti-CD16 and anti-NKG2D prior to coculturing with A549-Spike target cells for 4 hours at 37°C. Data were pooled from three independent experiments. Unpaired Student’s t test was employed for all panels. ns p > 0.05, * p

    Article Snippet: A549-Spike cells were cultured for a few days prior to sorting using anti-RBD.

    Techniques: Binding Assay, Expressing, Activity Assay, Release Assay, Labeling

    Increased CD107a surface expression and killing activity of S309-CAR-NK-92MI cells against 293T-hACE2-RBD and A549-Spike target cells. (a) Generation of transient 293T-hACE2-RBD and stable A549-Spike cell lines. 293T-hACE2 cells were transfected with RBD-containing plasmid for 48 hours. Transfected 293T-hACE2-RBD cells were then harvested. For the generation of A549-Spike, 293T cells were transfected with the retrovirus transfection system for 48 hours. The spike retrovirus was filtered and transduced into A549 cells for an additional 48-72 hours. ( b ) Representative dot plots showing the expressions of RBD or Spike in 293T-hACE2 or A549 cells, respectively. 293T-hACE2-RBD and A549-Spike cells were stained with anti-RBD and the expressions were confirmed by flow cytometry. The stable A549-Spike cell line was then sorted to achieve high levels of spike expression. ( c ) Quantitative data of CD107a surface expression assay of S309-CAR-NK against 293T-hACE2-RBD or A549-Spike cell lines. Briefly, S309-CAR-NK-92MI cells were cocultured with either 293T-hACE2-RBD cells, A549-Spike cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. Cells were then harvested and stained for CAR F(ab)2 domain [IgG (H+L)] and CD107a. Data represent mean ± SEM from two experiments. ( d ) 4-hour standard Cr 51 release assay of S309-CAR-NK-92MI and parental NK-92MI cells against various target cell lines. 293T-hACE2-RBD, A549-Spike, and HepG2 cell lines were used as target cells for S309-CAR-NK and NK-92MI. Experimental groups were performed in triplicates. Error bars represent mean ± SEM from at least two independent experiments. Unpaired Student’s t test was used for both panels ( c ) and ( d ). ns p > 0.05, * p

    Journal: bioRxiv

    Article Title: CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells

    doi: 10.1101/2021.01.14.426742

    Figure Lengend Snippet: Increased CD107a surface expression and killing activity of S309-CAR-NK-92MI cells against 293T-hACE2-RBD and A549-Spike target cells. (a) Generation of transient 293T-hACE2-RBD and stable A549-Spike cell lines. 293T-hACE2 cells were transfected with RBD-containing plasmid for 48 hours. Transfected 293T-hACE2-RBD cells were then harvested. For the generation of A549-Spike, 293T cells were transfected with the retrovirus transfection system for 48 hours. The spike retrovirus was filtered and transduced into A549 cells for an additional 48-72 hours. ( b ) Representative dot plots showing the expressions of RBD or Spike in 293T-hACE2 or A549 cells, respectively. 293T-hACE2-RBD and A549-Spike cells were stained with anti-RBD and the expressions were confirmed by flow cytometry. The stable A549-Spike cell line was then sorted to achieve high levels of spike expression. ( c ) Quantitative data of CD107a surface expression assay of S309-CAR-NK against 293T-hACE2-RBD or A549-Spike cell lines. Briefly, S309-CAR-NK-92MI cells were cocultured with either 293T-hACE2-RBD cells, A549-Spike cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. Cells were then harvested and stained for CAR F(ab)2 domain [IgG (H+L)] and CD107a. Data represent mean ± SEM from two experiments. ( d ) 4-hour standard Cr 51 release assay of S309-CAR-NK-92MI and parental NK-92MI cells against various target cell lines. 293T-hACE2-RBD, A549-Spike, and HepG2 cell lines were used as target cells for S309-CAR-NK and NK-92MI. Experimental groups were performed in triplicates. Error bars represent mean ± SEM from at least two independent experiments. Unpaired Student’s t test was used for both panels ( c ) and ( d ). ns p > 0.05, * p

    Article Snippet: A549-Spike cells were cultured for a few days prior to sorting using anti-RBD.

    Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Incubation, Release Assay