40592 v08h  (Sino Biological)


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    Sino Biological 40592 v08h
    Comparison of seroconversion in patients with <t>COVID-19</t> and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with <t>SARS-CoV-2</t> and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS <t>2017–2019</t> (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.
    40592 V08h, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    40592 v08h - by Bioz Stars, 2021-02
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    Images

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

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

    Journal: JCI Insight

    doi: 10.1172/jci.insight.142386

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

    Techniques Used: Enzyme-linked Immunosorbent Assay

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

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

    2) Product Images from "Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation"

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

    Graphical illustrations of the COVID-19 related immunoassays that were performed with our microfluidic chemiluminescent ELISA platform, including (A) affinity evaluation of calibration antibodies, (B) detection of circulating anti-SARS-CoV-2 S1 IgG in serum samples, and (C) detection of SARS-CoV-2 antigens such as S1 and N protein.
    Figure Legend Snippet: Graphical illustrations of the COVID-19 related immunoassays that were performed with our microfluidic chemiluminescent ELISA platform, including (A) affinity evaluation of calibration antibodies, (B) detection of circulating anti-SARS-CoV-2 S1 IgG in serum samples, and (C) detection of SARS-CoV-2 antigens such as S1 and N protein.

    Techniques Used: Chemiluminescent ELISA

    3) Product Images from "High-Accuracy Multiplexed SARS-CoV-2 Antibody Assay with Avidity and Saliva Capability on a Nano-Plasmonic Platform"

    Article Title: High-Accuracy Multiplexed SARS-CoV-2 Antibody Assay with Avidity and Saliva Capability on a Nano-Plasmonic Platform

    Journal: bioRxiv

    doi: 10.1101/2020.06.16.155580

    Detection of SARS-CoV-2 antibodies in human saliva. (a) A confocal fluorescence image of IgG signals in the saliva of 4 recovered COVID-19 patients (denoted as P1-P4) and 11 healthy controls (denoted as P5-P15) and a 10 4 times diluted serum of a PCR-confirmed COVID-19 patient as a reference (denoted as ‘Ref’). Saliva was collected by a simple spitting method as shown in the schematic. (b) Median fluorescence intensity (MFI) signals of anti-S1 and anti-RBD IgG measured in the saliva samples and PCR-positive COVID-19 serum reference with background signals subtracted. The error bars indicate one standard deviation away from the mean.
    Figure Legend Snippet: Detection of SARS-CoV-2 antibodies in human saliva. (a) A confocal fluorescence image of IgG signals in the saliva of 4 recovered COVID-19 patients (denoted as P1-P4) and 11 healthy controls (denoted as P5-P15) and a 10 4 times diluted serum of a PCR-confirmed COVID-19 patient as a reference (denoted as ‘Ref’). Saliva was collected by a simple spitting method as shown in the schematic. (b) Median fluorescence intensity (MFI) signals of anti-S1 and anti-RBD IgG measured in the saliva samples and PCR-positive COVID-19 serum reference with background signals subtracted. The error bars indicate one standard deviation away from the mean.

    Techniques Used: Fluorescence, Polymerase Chain Reaction, Standard Deviation

    Antibody avidity against SARS-CoV-2 antigens. (a) Avidity of anti-S1 IgG and anti-RBD IgG measured in IgG-positive, PCR-confirmed COVID-19 patient sera collected 6-45 days post symptom onset. The serum of PAMF-065 showed unusually high avidity for anti-S1 IgG while being negative for anti-RBD IgG. (b) Upper panel: Fluorescence images of IgG-only channel showing PAMF-065 serum sample with high anti-S1 IgG level with and without urea treatment, hence high avidity. It showed negligible anti-RBD IgG. Lower panel: Fluorescence images showing another patient serum tested, PAMF-011, with much reduced anti-S1 IgG level after urea treatment, indicating low avidity. Low avidity was observed for all samples except PAMF-065. (c) Anti-S1 IgG median fluorescence intensity (MFI) signals of the PAMF-065 sample with and without urea treatment. The error bars indicate one standard deviation away from the mean.
    Figure Legend Snippet: Antibody avidity against SARS-CoV-2 antigens. (a) Avidity of anti-S1 IgG and anti-RBD IgG measured in IgG-positive, PCR-confirmed COVID-19 patient sera collected 6-45 days post symptom onset. The serum of PAMF-065 showed unusually high avidity for anti-S1 IgG while being negative for anti-RBD IgG. (b) Upper panel: Fluorescence images of IgG-only channel showing PAMF-065 serum sample with high anti-S1 IgG level with and without urea treatment, hence high avidity. It showed negligible anti-RBD IgG. Lower panel: Fluorescence images showing another patient serum tested, PAMF-011, with much reduced anti-S1 IgG level after urea treatment, indicating low avidity. Low avidity was observed for all samples except PAMF-065. (c) Anti-S1 IgG median fluorescence intensity (MFI) signals of the PAMF-065 sample with and without urea treatment. The error bars indicate one standard deviation away from the mean.

    Techniques Used: Polymerase Chain Reaction, Fluorescence, Standard Deviation

    Correlation of antibodies against two SARS-CoV-2 antigens. (a) Correlation plot of anti-S1 IgG level (y-axis) and anti-RBD IgG level (x-axis) measured in PCR-confirmed COVID-19 patient sera. The dashed line was drawn to have a slope of 1. The upper left inset shows the scanned image of the IgG-only channel in a patient serum labeled as PAMF-065, which displayed high signal on the S1 antigen but not on the RBD antigen. The lower right inset shows the scanned image of IgG levels of a sample labeled as PAMF-011, displaying about equal IgG signals against S1 and RBD. (b) Correlation plot of anti-S1 IgM level (y-axis) and anti-RBD IgM level (x-axis) measured in COVID-19 patient sera. The dashed line was drawn to have a slope of 1.
    Figure Legend Snippet: Correlation of antibodies against two SARS-CoV-2 antigens. (a) Correlation plot of anti-S1 IgG level (y-axis) and anti-RBD IgG level (x-axis) measured in PCR-confirmed COVID-19 patient sera. The dashed line was drawn to have a slope of 1. The upper left inset shows the scanned image of the IgG-only channel in a patient serum labeled as PAMF-065, which displayed high signal on the S1 antigen but not on the RBD antigen. The lower right inset shows the scanned image of IgG levels of a sample labeled as PAMF-011, displaying about equal IgG signals against S1 and RBD. (b) Correlation plot of anti-S1 IgM level (y-axis) and anti-RBD IgM level (x-axis) measured in COVID-19 patient sera. The dashed line was drawn to have a slope of 1.

    Techniques Used: Polymerase Chain Reaction, Labeling

    A nano-plasmonic platform for SARS-CoV-2 antibody testing. (a) An overlay of confocal fluorescence scanned images of IgG (green) and IgM (red) channels acquired after testing 16 serum samples in 16 isolated wells (square-shaped regions). Yellowish-green colored spots correspond to the presence of both IgG and IgM in the sample. The lower right schematic drawing shows the printing layout of S1 (in green) and RBD (in blue) antigens and human IgG control spots (in white) in each well. The BSA-biotin spots (in red) are always labeled by a streptavidin dye in the IgM fluorescence channel to serve as an intrawell signal normalizer. (b) Box plots of IgG levels detected in PCR-negative COVID-19 or presumptive negative (‘Healthy’) and PCR-positive (‘PCR+’) COVID-19 samples with the cutoff indicated as a dashed red line. (c) The same as (b) except for IgM. (d) ROC curve for pGOLD SARS-CoV-2 IgG/IgM assay based on 384 negative and 62 PCR-positive COVID-19 serum, which was used to establish IgG and IgM cutoffs. (e) ROC curve for pGOLD SARS-CoV-2 IgG/IgM assay based on 384 negative and PCR-positive COVID-19 serum samples collected 15-45 days post symptom onset.
    Figure Legend Snippet: A nano-plasmonic platform for SARS-CoV-2 antibody testing. (a) An overlay of confocal fluorescence scanned images of IgG (green) and IgM (red) channels acquired after testing 16 serum samples in 16 isolated wells (square-shaped regions). Yellowish-green colored spots correspond to the presence of both IgG and IgM in the sample. The lower right schematic drawing shows the printing layout of S1 (in green) and RBD (in blue) antigens and human IgG control spots (in white) in each well. The BSA-biotin spots (in red) are always labeled by a streptavidin dye in the IgM fluorescence channel to serve as an intrawell signal normalizer. (b) Box plots of IgG levels detected in PCR-negative COVID-19 or presumptive negative (‘Healthy’) and PCR-positive (‘PCR+’) COVID-19 samples with the cutoff indicated as a dashed red line. (c) The same as (b) except for IgM. (d) ROC curve for pGOLD SARS-CoV-2 IgG/IgM assay based on 384 negative and 62 PCR-positive COVID-19 serum, which was used to establish IgG and IgM cutoffs. (e) ROC curve for pGOLD SARS-CoV-2 IgG/IgM assay based on 384 negative and PCR-positive COVID-19 serum samples collected 15-45 days post symptom onset.

    Techniques Used: Fluorescence, Isolation, Labeling, Polymerase Chain Reaction

    Highly sensitive and specific SARS-CoV-2 antibody test. (a) Percentages of samples with IgG/IgM antibody status combinations according to days from symptom onset to sample collection date in a range from 0-7, 8-14, and 15-45 days. (b) Box plots of IgG levels detected in four groups of serum samples indicated on the x-axis with the cutoff displayed as a dashed red line. ‘PCR+’ denotes serum samples from patients who tested positive by PCR for COVID-19 and ‘PCR-’ denotes those who tested negative. ‘Pre-pand.’ corresponds to pre-pandemic collected samples. ‘Cross R.’ corresponds to samples from patients with other diseases for cross-reactivity evaluation. (c) The same as (b) except for IgM.
    Figure Legend Snippet: Highly sensitive and specific SARS-CoV-2 antibody test. (a) Percentages of samples with IgG/IgM antibody status combinations according to days from symptom onset to sample collection date in a range from 0-7, 8-14, and 15-45 days. (b) Box plots of IgG levels detected in four groups of serum samples indicated on the x-axis with the cutoff displayed as a dashed red line. ‘PCR+’ denotes serum samples from patients who tested positive by PCR for COVID-19 and ‘PCR-’ denotes those who tested negative. ‘Pre-pand.’ corresponds to pre-pandemic collected samples. ‘Cross R.’ corresponds to samples from patients with other diseases for cross-reactivity evaluation. (c) The same as (b) except for IgM.

    Techniques Used: Polymerase Chain Reaction

    Related Articles

    Microarray:

    Article Title: High-Accuracy Multiplexed SARS-CoV-2 Antibody Assay with Avidity and Saliva Capability on a Nano-Plasmonic Platform
    Article Snippet: .. Multiplexed SARS-CoV-2 microarray printing on pGOLD slidesEach pGOLD slide (Nirmidas Biotech Inc.) was printed with two SARS-CoV-2 antigens, namely the spike protein S1 subunit (S1) and S1 containing the receptor binding domain (RBD), using a GeSiM Nano-Plotter 2.1 at the following concentrations: 60 μg/mL for S1 (40591-V08H, Sino Biological Inc.) and 25 μg/mL for RBD (40592-V08H, Sino Biological Inc.). .. On the same biochip, 7.5 μg/mL human IgG and 50 μg/mL BSA-biotin (Thermo Fisher Scientific) were also printed to serve as a printing control and “intra-well signal normalizer”, respectively.

    Binding Assay:

    Article Title: High-Accuracy Multiplexed SARS-CoV-2 Antibody Assay with Avidity and Saliva Capability on a Nano-Plasmonic Platform
    Article Snippet: .. Multiplexed SARS-CoV-2 microarray printing on pGOLD slidesEach pGOLD slide (Nirmidas Biotech Inc.) was printed with two SARS-CoV-2 antigens, namely the spike protein S1 subunit (S1) and S1 containing the receptor binding domain (RBD), using a GeSiM Nano-Plotter 2.1 at the following concentrations: 60 μg/mL for S1 (40591-V08H, Sino Biological Inc.) and 25 μg/mL for RBD (40592-V08H, Sino Biological Inc.). .. On the same biochip, 7.5 μg/mL human IgG and 50 μg/mL BSA-biotin (Thermo Fisher Scientific) were also printed to serve as a printing control and “intra-well signal normalizer”, respectively.

    Article Title: Methylene Blue Inhibits the SARS-CoV-2 Spike–ACE2 Protein-Protein Interaction–a Mechanism that can Contribute to its Antiviral Activity Against COVID-19
    Article Snippet: Suramin ( > 99%; cat. no. 1472) was from Tocris Bioscience (Biotechne, Minneapolis, MN, United States). .. ACE2-Fc and SARS-CoV-2 S1 or RBD with His tag proteins used in the binding assays were obtained from Sino Biological (Wayne, PA, United States); catalog no. 10108-H05H, 40591-V08H, and 40592-V08H). .. Binding inhibition assays were performed in a 96-well cell-free format similar to the one described before ( ; ; ; ).

    Enzyme-linked Immunosorbent Assay:

    Article Title: Novel ACE2-Independent Carbohydrate-Binding of SARS-CoV-2 Spike Protein to Host Lectins and Lung Microbiota
    Article Snippet: For these three molecules, we used a concentration of 40μg/mL to coat the ELISA wells. .. Siglecs ELISA a solution of 50 µL of SARS-CoV-2 spike protein (2019-nCoV Spike RBD-His Recombinant Protein, Cat: 40592-V08H, expressed in HEK293 cells, purchased from Sinobiological) at 5 µg/mL, in PBS (10mM, pH=7.4), were used to coat the Nunc MaxiSorp plate 2h at 37°C. .. After discarding and washing (2×150µL) with Hanks’ Balanced Salt solution (Gibco™ HBSS) the wells were blocked with 200 µL of carbo-free blocking solution (Vector Laboratories, catalog No.NC9977573) at 37°C for 30 min.

    Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity
    Article Snippet: .. The stock S-RBD (2.5 μg/mL; 93.28 nM) was used to coat ELISA plates (Sino Biological 40592-V08H). .. The stock N-protein (1.25 μg/mL; 26.55 nM) was used to coat ELISA plates (Sino Biological 40588-V08B).

    Article Title: Single dose immunization with a COVID-19 DNA vaccine encoding a chimeric homodimeric protein targeting receptor binding domain (RBD) to antigen-presenting cells induces rapid, strong and long-lasting neutralizing IgG, Th1 dominated CD4+ T cells and strong CD8+ T cell responses in mice
    Article Snippet: Anti-RBD IgG ELISA The humoral immune response was evaluated in sera and bronchoalveolar lavages (BAL) collected at different time points (day 7, 14, 20, 28, 42, 56, 70, 90 or 99) after vaccination by an ELISA assay detecting total IgG specific for RBD from SARS-CoV2. .. ELISA plates (MaxiSorp Nunc-Immuno plates) were coated with 1 μg/ml recombinant RBD-His protein antigen (Cat. No. 40592-V08H, Sino Biological) in 1x D-PBS overnight at 4°C. ..

    Recombinant:

    Article Title: Novel ACE2-Independent Carbohydrate-Binding of SARS-CoV-2 Spike Protein to Host Lectins and Lung Microbiota
    Article Snippet: For these three molecules, we used a concentration of 40μg/mL to coat the ELISA wells. .. Siglecs ELISA a solution of 50 µL of SARS-CoV-2 spike protein (2019-nCoV Spike RBD-His Recombinant Protein, Cat: 40592-V08H, expressed in HEK293 cells, purchased from Sinobiological) at 5 µg/mL, in PBS (10mM, pH=7.4), were used to coat the Nunc MaxiSorp plate 2h at 37°C. .. After discarding and washing (2×150µL) with Hanks’ Balanced Salt solution (Gibco™ HBSS) the wells were blocked with 200 µL of carbo-free blocking solution (Vector Laboratories, catalog No.NC9977573) at 37°C for 30 min.

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation
    Article Snippet: The normal human serum (H4522-20ML), which was used as the dilution buffer and as one of the negative controls in IgG detection experiments, and the heat-inactivated normal human serum (H5667-20ML), which was used as another negative control in IgG detection experiments, were both purchased from Millipore Sigma. .. Human-cell-expressed SARS-CoV-2 Spike S1-His recombinant protein (40591-V08H), human-cell-expressed SARS-CoV-2 Spike RBD-His recombinant protein (40592-V08H) and insect-cell-expressed SARS-CoV Spike S1-His recombinant protein (40150-V08B1) were provided by Sino Biological. .. The recombinant CR3022 therapeutic antibody was purchased from Creative Biolabs (MRO-1214LC).

    Article Title: Single dose immunization with a COVID-19 DNA vaccine encoding a chimeric homodimeric protein targeting receptor binding domain (RBD) to antigen-presenting cells induces rapid, strong and long-lasting neutralizing IgG, Th1 dominated CD4+ T cells and strong CD8+ T cell responses in mice
    Article Snippet: Anti-RBD IgG ELISA The humoral immune response was evaluated in sera and bronchoalveolar lavages (BAL) collected at different time points (day 7, 14, 20, 28, 42, 56, 70, 90 or 99) after vaccination by an ELISA assay detecting total IgG specific for RBD from SARS-CoV2. .. ELISA plates (MaxiSorp Nunc-Immuno plates) were coated with 1 μg/ml recombinant RBD-His protein antigen (Cat. No. 40592-V08H, Sino Biological) in 1x D-PBS overnight at 4°C. ..

    Article Title: Bcr-Abl tyrosine kinase inhibitor imatinib as a potential drug for COVID-19
    Article Snippet: Experiments were conducted using the advanced kinetics mode, at room temperature and a buffer system consisting of 1X Kinetics Buffer (FortéBio), 5% anhydrous dimethyl sulfoxide (DMSO; Sigma Aldrich). .. Recombinant His-tagged SARS-CoV-2 RBD protein (40592-V08H; Sino Biological) at a concentration of 10 µg/ml was loaded on Anti-Penta-HIS (HIS1K) Biosensors (FortéBio), followed by a washing step with assay buffer to block the unoccupied sensor surface. .. The association and dissociation profiles of imatinib (Sigma Aldrich) were measured at various concentrations (four-point serial dilutions from 6.25 µM to 0.78 µM).

    Concentration Assay:

    Article Title: Bcr-Abl tyrosine kinase inhibitor imatinib as a potential drug for COVID-19
    Article Snippet: Experiments were conducted using the advanced kinetics mode, at room temperature and a buffer system consisting of 1X Kinetics Buffer (FortéBio), 5% anhydrous dimethyl sulfoxide (DMSO; Sigma Aldrich). .. Recombinant His-tagged SARS-CoV-2 RBD protein (40592-V08H; Sino Biological) at a concentration of 10 µg/ml was loaded on Anti-Penta-HIS (HIS1K) Biosensors (FortéBio), followed by a washing step with assay buffer to block the unoccupied sensor surface. .. The association and dissociation profiles of imatinib (Sigma Aldrich) were measured at various concentrations (four-point serial dilutions from 6.25 µM to 0.78 µM).

    Blocking Assay:

    Article Title: Bcr-Abl tyrosine kinase inhibitor imatinib as a potential drug for COVID-19
    Article Snippet: Experiments were conducted using the advanced kinetics mode, at room temperature and a buffer system consisting of 1X Kinetics Buffer (FortéBio), 5% anhydrous dimethyl sulfoxide (DMSO; Sigma Aldrich). .. Recombinant His-tagged SARS-CoV-2 RBD protein (40592-V08H; Sino Biological) at a concentration of 10 µg/ml was loaded on Anti-Penta-HIS (HIS1K) Biosensors (FortéBio), followed by a washing step with assay buffer to block the unoccupied sensor surface. .. The association and dissociation profiles of imatinib (Sigma Aldrich) were measured at various concentrations (four-point serial dilutions from 6.25 µM to 0.78 µM).

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    Sino Biological sars cov 2 rbd
    The antibody response induced by recombinant RBD of SARS-CoV and <t>SARS-CoV-2</t> 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
    Sars Cov 2 Rbd, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological sars cov 2 2019 ncov spike rbd his recombinant protein covid 19 spike rbd research
    Comparison of seroconversion in patients with <t>COVID-19</t> and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with <t>SARS-CoV-2</t> and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS <t>2017–2019</t> (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.
    Sars Cov 2 2019 Ncov Spike Rbd His Recombinant Protein Covid 19 Spike Rbd Research, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    Journal: Cellular and Molecular Immunology

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

    doi: 10.1038/s41423-020-0458-z

    Figure Lengend 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

    Article Snippet: Enzyme-linked immunosorbent assay (ELISA)To confirm whether the antibodies recognized SARS-CoV RBD or SARS-CoV-2 RBD, 96-well microwell plates (Nunc) were coated with 50 ng/well recombinant SARS-CoV RBD hFc and SARS-CoV-2 RBD hFc in 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4 °C.

    Techniques: 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

    Journal: Cellular and Molecular Immunology

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

    doi: 10.1038/s41423-020-0458-z

    Figure Lengend 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

    Article Snippet: Enzyme-linked immunosorbent assay (ELISA)To confirm whether the antibodies recognized SARS-CoV RBD or SARS-CoV-2 RBD, 96-well microwell plates (Nunc) were coated with 50 ng/well recombinant SARS-CoV RBD hFc and SARS-CoV-2 RBD hFc in 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4 °C.

    Techniques: 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

    Journal: Cellular and Molecular Immunology

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

    doi: 10.1038/s41423-020-0458-z

    Figure Lengend 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

    Article Snippet: Enzyme-linked immunosorbent assay (ELISA)To confirm whether the antibodies recognized SARS-CoV RBD or SARS-CoV-2 RBD, 96-well microwell plates (Nunc) were coated with 50 ng/well recombinant SARS-CoV RBD hFc and SARS-CoV-2 RBD hFc in 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4 °C.

    Techniques: 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

    Journal: Cellular and Molecular Immunology

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

    doi: 10.1038/s41423-020-0458-z

    Figure Lengend 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

    Article Snippet: Enzyme-linked immunosorbent assay (ELISA)To confirm whether the antibodies recognized SARS-CoV RBD or SARS-CoV-2 RBD, 96-well microwell plates (Nunc) were coated with 50 ng/well recombinant SARS-CoV RBD hFc and SARS-CoV-2 RBD hFc in 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4 °C.

    Techniques: 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)

    Journal: Cellular and Molecular Immunology

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

    doi: 10.1038/s41423-020-0458-z

    Figure Lengend 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)

    Article Snippet: Enzyme-linked immunosorbent assay (ELISA)To confirm whether the antibodies recognized SARS-CoV RBD or SARS-CoV-2 RBD, 96-well microwell plates (Nunc) were coated with 50 ng/well recombinant SARS-CoV RBD hFc and SARS-CoV-2 RBD hFc in 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4 °C.

    Techniques: Sequencing, Mutagenesis, Binding Assay

    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.

    Journal: Cell Host & Microbe

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

    doi: 10.1016/j.chom.2020.04.023

    Figure Lengend 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.

    Article Snippet: 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.

    Techniques: 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

    Journal: Cell Host & Microbe

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

    doi: 10.1016/j.chom.2020.04.023

    Figure Lengend 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

    Article Snippet: 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.

    Techniques: Binding Assay

    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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.12.11.421008

    Figure Lengend 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.

    Article Snippet: Binding analysis of captured murine IgG antibodies to S1-His or RBD-His (Sino Biological Inc.) was performed using a multi-cycle kinetic method with concentrations ranging from 25 to 400 nM or 1.5625 to 50 nM, respectively.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.12.11.421008

    Figure Lengend 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.

    Article Snippet: Binding analysis of captured murine IgG antibodies to S1-His or RBD-His (Sino Biological Inc.) was performed using a multi-cycle kinetic method with concentrations ranging from 25 to 400 nM or 1.5625 to 50 nM, respectively.

    Techniques: 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 .

    Journal: bioRxiv

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

    doi: 10.1101/2020.12.11.421008

    Figure Lengend 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 .

    Article Snippet: Binding analysis of captured murine IgG antibodies to S1-His or RBD-His (Sino Biological Inc.) was performed using a multi-cycle kinetic method with concentrations ranging from 25 to 400 nM or 1.5625 to 50 nM, respectively.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.12.11.421008

    Figure Lengend 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.

    Article Snippet: Binding analysis of captured murine IgG antibodies to S1-His or RBD-His (Sino Biological Inc.) was performed using a multi-cycle kinetic method with concentrations ranging from 25 to 400 nM or 1.5625 to 50 nM, respectively.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.12.11.421008

    Figure Lengend 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.

    Article Snippet: Binding analysis of captured murine IgG antibodies to S1-His or RBD-His (Sino Biological Inc.) was performed using a multi-cycle kinetic method with concentrations ranging from 25 to 400 nM or 1.5625 to 50 nM, respectively.

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

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

    Journal: JCI Insight

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

    doi: 10.1172/jci.insight.142386

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

    Article Snippet: The stock S-RBD (2.5 μg/mL; 93.28 nM) was used to coat ELISA plates (Sino Biological 40592-V08H).

    Techniques: Enzyme-linked Immunosorbent Assay

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

    Journal: JCI Insight

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

    doi: 10.1172/jci.insight.142386

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

    Article Snippet: The stock S-RBD (2.5 μg/mL; 93.28 nM) was used to coat ELISA plates (Sino Biological 40592-V08H).

    Techniques: Binding Assay, Polymerase Chain Reaction, Negative Control