rbd proteins  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike RBD Recombinant Protein
    Description:
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed
    Catalog Number:
    40592-VNAH
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus spike Protein 2019-nCoV, cov spike Protein 2019-nCoV, ncov RBD Protein 2019-nCoV, ncov s1 Protein 2019-nCoV, ncov s2 Protein 2019-nCoV, ncov spike Protein 2019-nCoV, NCP-CoV RBD Protein 2019-nCoV, NCP-CoV s1 Protein 2019-nCoV, NCP-CoV s2 Protein 2019-nCoV, NCP-CoV Spike Protein 2019-nCoV, novel coronavirus RBD Protein 2019-nCoV, novel coronavirus s1 Protein 2019-nCoV, novel coronavirus s2 Protein 2019-nCoV, novel coronavirus spike Protein 2019-nCoV, RBD Protein 2019-nCoV, S1 Protein 2019-nCoV, S2 Protein 2019-nCoV, Spike RBD Protein 2019-nCoV
    Host:
    HEK293 Cells
    Buy from Supplier


    Structured Review

    Sino Biological rbd proteins
    Isolation of <t>RBD-specific</t> memory B cells using flow cytometry. A. The heatmap depicts the specificity of convalescent patients’ plasma against S1 and RBD from SARS-CoV-2, SARS-CoV and MERS-CoV, measured by <t>ELISA.</t> Serial dilutions of plasma samples were performed to test the reactivity of antibodies in plasma. The plasma of healthy donors was used as the control. Data were shown with the mean of representative experiments. B. Gating strategy for SARS-CoV-2 RBD-specific IgG + B cells in PBMCs of the convalescent patients. Living CD19+ IgD − IgG + cells were gated, and cells with positive SARS-CoV-2 RBD staining were selected for single-cell sorting. C. FACS analysis of RBD-specific memory B cells in CD19 + IgD − IgG + memory B cells from PBMCs of three batch convalescent patients. Plots show CD19 + IgD − IgG + RBD+ populations using gating strategy described in B .
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed
    https://www.bioz.com/result/rbd proteins/product/Sino Biological
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    rbd proteins - by Bioz Stars, 2021-07
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    Images

    1) Product Images from "A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD"

    Article Title: A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD

    Journal: bioRxiv

    doi: 10.1101/2020.08.19.253369

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

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

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

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

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

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

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

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

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

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

    2) Product Images from "Kinetics of viral load and antibody response in relation to COVID-19 severity"

    Article Title: Kinetics of viral load and antibody response in relation to COVID-19 severity

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI138759

    Kinetics of IgM and IgG responses against SARS-CoV-2 in different tissues. Urine ( A ), sputum ( B ), feces ( C ), BALF, and pleural effusion ( D ) specimens from patients with COVID-19 were detected for the presence of IgM and IgG antibodies against the N protein of SARS-CoV-2. Positive (PC) and negative (NC) controls provided by detection kit were included to ensure test validity. Plasma from 48 HDs was also included.
    Figure Legend Snippet: Kinetics of IgM and IgG responses against SARS-CoV-2 in different tissues. Urine ( A ), sputum ( B ), feces ( C ), BALF, and pleural effusion ( D ) specimens from patients with COVID-19 were detected for the presence of IgM and IgG antibodies against the N protein of SARS-CoV-2. Positive (PC) and negative (NC) controls provided by detection kit were included to ensure test validity. Plasma from 48 HDs was also included.

    Techniques Used:

    Kinetics of IgM and IgG responses against SARS-CoV-2 in severely and mildly ill patients. IgM ( A ) and IgG ( B ) antibody responses against the N protein of SARS-CoV-2 in plasma were detected. Serial plasma samples were collected from 12 severely ill and 11 mildly ill patients infected with SARS-CoV-2. Forty-eight plasma samples previously collected from healthy volunteer donors in 2017–2018 were used as a healthy donor group (HD). Positive (PC) and negative (NC) controls provided by detection kit were included to ensure test validity.
    Figure Legend Snippet: Kinetics of IgM and IgG responses against SARS-CoV-2 in severely and mildly ill patients. IgM ( A ) and IgG ( B ) antibody responses against the N protein of SARS-CoV-2 in plasma were detected. Serial plasma samples were collected from 12 severely ill and 11 mildly ill patients infected with SARS-CoV-2. Forty-eight plasma samples previously collected from healthy volunteer donors in 2017–2018 were used as a healthy donor group (HD). Positive (PC) and negative (NC) controls provided by detection kit were included to ensure test validity.

    Techniques Used: Infection

    IgG cross-reactivity analysis between the other 6 human CoVs and SARS-CoV-2. Spike (S) and nucleoprotein (N) of the other 6 human CoVs were used as coated target antigens to establish an in-house ELISA to detect IgG antibody for HCoV-229E ( A ), HCoV-NL63 ( B ), HCoV-HKU1 ( C ), HCoV-OC43 ( D ), SARS-CoV ( E ), and MERS-CoV ( F ). Plasma from 96 HDs and 23 SARS-CoV-2–infected patients were used ( A – F ). Severe indicates a severely ill patient with COVID-19; mild indicates a mildly ill patient with COVID-19; HD indicates healthy donors. Plasma samples from 18 SARS-convalescent ( E ) and 12 MERS-convalescent ( F ) patients were used as controls, respectively. A Student’s t test was used to analyze differences in mean values between groups ( A – F ). Experiments for each virus were independently carried out. Multiple comparisons following 1-way ANOVA and Kruskal-Wallis test were performed for statistical analysis. Bonferroni’s correction was used to avoid inflation of experiment-wise Type I error. In A – D , a difference was considered statistically significant when the P value was lower than 0.0167 (0.05/3); * P ≤ 0.0167, ** P ≤ 0.0033, *** P ≤ 0.00033, **** P ≤ 0.000033. In E and F , a difference was considered statistically significant when the P value was lower than 0.0083 (0.05/6); † P ≤ 0.0083, †† P ≤ 0.0017, ‡ P ≤ 0.00017, ‡‡ P ≤0.000017.
    Figure Legend Snippet: IgG cross-reactivity analysis between the other 6 human CoVs and SARS-CoV-2. Spike (S) and nucleoprotein (N) of the other 6 human CoVs were used as coated target antigens to establish an in-house ELISA to detect IgG antibody for HCoV-229E ( A ), HCoV-NL63 ( B ), HCoV-HKU1 ( C ), HCoV-OC43 ( D ), SARS-CoV ( E ), and MERS-CoV ( F ). Plasma from 96 HDs and 23 SARS-CoV-2–infected patients were used ( A – F ). Severe indicates a severely ill patient with COVID-19; mild indicates a mildly ill patient with COVID-19; HD indicates healthy donors. Plasma samples from 18 SARS-convalescent ( E ) and 12 MERS-convalescent ( F ) patients were used as controls, respectively. A Student’s t test was used to analyze differences in mean values between groups ( A – F ). Experiments for each virus were independently carried out. Multiple comparisons following 1-way ANOVA and Kruskal-Wallis test were performed for statistical analysis. Bonferroni’s correction was used to avoid inflation of experiment-wise Type I error. In A – D , a difference was considered statistically significant when the P value was lower than 0.0167 (0.05/3); * P ≤ 0.0167, ** P ≤ 0.0033, *** P ≤ 0.00033, **** P ≤ 0.000033. In E and F , a difference was considered statistically significant when the P value was lower than 0.0083 (0.05/6); † P ≤ 0.0083, †† P ≤ 0.0017, ‡ P ≤ 0.00017, ‡‡ P ≤0.000017.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Infection

    IgG antibody response against different SARS-CoV-2 proteins or fragments. Plasma samples collected at different time points after admission were used for IgG detection in different protein-coated ELISAs: S (1209 aa) ( A ), S1 (681 aa) ( B ), RBD (457 aa) ( C ), S2 (539 aa) ( D ), and N (430 aa) ( E ). Eleven plasma samples from HDs were used as controls. The correlations among IgG levels against different viral proteins were analyzed and summarized. Pearson’s correlation coefficient was used to assess the relationship among antiviral IgG levels of different proteins ( F ). A Student’s t test was used to analyze differences in mean values between groups A – E . A P value less than 0.05 was considered to be statistically significant. ** P ≤ 0.01.
    Figure Legend Snippet: IgG antibody response against different SARS-CoV-2 proteins or fragments. Plasma samples collected at different time points after admission were used for IgG detection in different protein-coated ELISAs: S (1209 aa) ( A ), S1 (681 aa) ( B ), RBD (457 aa) ( C ), S2 (539 aa) ( D ), and N (430 aa) ( E ). Eleven plasma samples from HDs were used as controls. The correlations among IgG levels against different viral proteins were analyzed and summarized. Pearson’s correlation coefficient was used to assess the relationship among antiviral IgG levels of different proteins ( F ). A Student’s t test was used to analyze differences in mean values between groups A – E . A P value less than 0.05 was considered to be statistically significant. ** P ≤ 0.01.

    Techniques Used:

    Neutralizing and cross-protection of antibody response against SARS-CoV-2 in severely and mildly ill patients. Serial plasma samples were collected from severely ill ( A ) and mildly ill ( B ) patients infected with SARS-CoV-2, and used for authentic SARS-CoV2 neutralizing test FRNT 50 to evaluate kinetics of neutralizing antibodies in SARS-CoV-2 infected patients. Plasma samples collected 3 weeks after onset were used to compare cross-neutralizing antibodies between severely ill and mildly ill patients with SARS-CoV-2 and SARS-CoV–convalescent patients using SARS-CoV-2 pseudotype ( C ) and authentic virus ( D ) at a fixed dilution (1:40). A Student’s t test was used to analyze differences in mean values between groups. Experiments for each virus were independently carried out. Multiple comparisons following 1-way ANOVA and Kruskal-Wallis tests were performed for statistical analysis. Bonferroni’s correction was used to avoid inflation of experiment-wise Type I error. There were a total of 10 pairwise comparisons among 5 groups. Hence, a difference was considered statistically significant when the P value was lower than 0.005 (0.05/10). **** P ≤ 0.0001 ( C and D ). Pearson’s correlation coefficient was used to assess the relationship between neutralizing titer and S- and N-specific IgG levels ( E and F ); viral loads of respiratory specimens ( G ) were analyzed.
    Figure Legend Snippet: Neutralizing and cross-protection of antibody response against SARS-CoV-2 in severely and mildly ill patients. Serial plasma samples were collected from severely ill ( A ) and mildly ill ( B ) patients infected with SARS-CoV-2, and used for authentic SARS-CoV2 neutralizing test FRNT 50 to evaluate kinetics of neutralizing antibodies in SARS-CoV-2 infected patients. Plasma samples collected 3 weeks after onset were used to compare cross-neutralizing antibodies between severely ill and mildly ill patients with SARS-CoV-2 and SARS-CoV–convalescent patients using SARS-CoV-2 pseudotype ( C ) and authentic virus ( D ) at a fixed dilution (1:40). A Student’s t test was used to analyze differences in mean values between groups. Experiments for each virus were independently carried out. Multiple comparisons following 1-way ANOVA and Kruskal-Wallis tests were performed for statistical analysis. Bonferroni’s correction was used to avoid inflation of experiment-wise Type I error. There were a total of 10 pairwise comparisons among 5 groups. Hence, a difference was considered statistically significant when the P value was lower than 0.005 (0.05/10). **** P ≤ 0.0001 ( C and D ). Pearson’s correlation coefficient was used to assess the relationship between neutralizing titer and S- and N-specific IgG levels ( E and F ); viral loads of respiratory specimens ( G ) were analyzed.

    Techniques Used: Infection

    Temporal profile of serial viral load from different tissue samples. Viral loads in patients in the ICU (PT1–PT12) and patients with mild disease (PT13–PT23) as measured by nasal swabs ( A ), pharyngeal swabs ( B ), sputum ( C ), feces ( D ), urine ( E ), and blood ( F ). The x axis indicates the number of days after onset, the y axis indicates patient numbers. Heatmap of Ct values of viral loads were shown. A Ct value less than 37 indicates the presence of SARS-CoV-2 nucleic acid in the sample. Each square represents 1 sample detected and the gray squares indicate that the sample was viral nucleotide acid–negative.
    Figure Legend Snippet: Temporal profile of serial viral load from different tissue samples. Viral loads in patients in the ICU (PT1–PT12) and patients with mild disease (PT13–PT23) as measured by nasal swabs ( A ), pharyngeal swabs ( B ), sputum ( C ), feces ( D ), urine ( E ), and blood ( F ). The x axis indicates the number of days after onset, the y axis indicates patient numbers. Heatmap of Ct values of viral loads were shown. A Ct value less than 37 indicates the presence of SARS-CoV-2 nucleic acid in the sample. Each square represents 1 sample detected and the gray squares indicate that the sample was viral nucleotide acid–negative.

    Techniques Used:

    3) Product Images from "Rapid isolation and immune profiling of SARS-CoV-2 specific memory B cell in convalescent COVID-19 patients via LIBRA-seq"

    Article Title: Rapid isolation and immune profiling of SARS-CoV-2 specific memory B cell in convalescent COVID-19 patients via LIBRA-seq

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-021-00610-7

    Characteristics of SARS-CoV-2 reactive mAbs. SARS-CoV-2 antigen specificity as predicted was validated by ELISA for a subset of monoclonal antibodies to SARS-CoV-2. Data are represented as mean ± SD. Data are representative of two independent experiments. a SARS-CoV-2 RBD reactive mAbs, ( b ) SARS-CoV-2 NP reactive mAbs. c Maximum-likelihood phylogenetic tree of dominant clonotypes and antigen labeled antibodies’ heavy chains. Unrooted phylogenetic tree depicting the genetic relationships among all VH genes of antigen labeled antibodies. Branch lengths were scaled so that sequence relatedness can be readily assessed. Hvdj sequences with the same VH gene usage are shown in the same color at the clades. Hvdj sequences with the same antigen-labeled quantity are shown in the same color at the branch tips (red, blue and green means high, median and low antigen-labeled quantity respectively, and black means none). d Neutralization of C2767P3S and C14646P3S mAbs against pseudotyped SARS-CoV-2 virus in Huh-7 cells. Influenza relative mAbs CR9114 was used as a negative control. Data are represented as mean ± SEM. Data are representative of two independent experiments. e C2767P3S mAb was tested using the plaque reduction neutralization assay. DMEM was used as a negative control. Data are representative of two independent experiments. f Neutralization of C2767P3S mAb against live SARS-CoV-2 virus in Vero E6 cells. DMEM was used as a negative control. Data are represented as mean ± SEM. Data are representative of two independent experiments. g C14646P3S mAb was tested using the plaque reduction neutralization assay. DMEM was used as a negative control. Data are representative of two independent experiments. h Neutralization of C14646P3S mAb against live SARS-CoV-2 virus in Vero E6 cells. DMEM was used as a negative control. Data are represented as mean ± SEM. Data are representative of two independent experiments
    Figure Legend Snippet: Characteristics of SARS-CoV-2 reactive mAbs. SARS-CoV-2 antigen specificity as predicted was validated by ELISA for a subset of monoclonal antibodies to SARS-CoV-2. Data are represented as mean ± SD. Data are representative of two independent experiments. a SARS-CoV-2 RBD reactive mAbs, ( b ) SARS-CoV-2 NP reactive mAbs. c Maximum-likelihood phylogenetic tree of dominant clonotypes and antigen labeled antibodies’ heavy chains. Unrooted phylogenetic tree depicting the genetic relationships among all VH genes of antigen labeled antibodies. Branch lengths were scaled so that sequence relatedness can be readily assessed. Hvdj sequences with the same VH gene usage are shown in the same color at the clades. Hvdj sequences with the same antigen-labeled quantity are shown in the same color at the branch tips (red, blue and green means high, median and low antigen-labeled quantity respectively, and black means none). d Neutralization of C2767P3S and C14646P3S mAbs against pseudotyped SARS-CoV-2 virus in Huh-7 cells. Influenza relative mAbs CR9114 was used as a negative control. Data are represented as mean ± SEM. Data are representative of two independent experiments. e C2767P3S mAb was tested using the plaque reduction neutralization assay. DMEM was used as a negative control. Data are representative of two independent experiments. f Neutralization of C2767P3S mAb against live SARS-CoV-2 virus in Vero E6 cells. DMEM was used as a negative control. Data are represented as mean ± SEM. Data are representative of two independent experiments. g C14646P3S mAb was tested using the plaque reduction neutralization assay. DMEM was used as a negative control. Data are representative of two independent experiments. h Neutralization of C14646P3S mAb against live SARS-CoV-2 virus in Vero E6 cells. DMEM was used as a negative control. Data are represented as mean ± SEM. Data are representative of two independent experiments

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

    4) 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

    5) Product Images from "Cross-reactive antibody response between SARS-CoV-2 and SARS-CoV infections"

    Article Title: Cross-reactive antibody response between SARS-CoV-2 and SARS-CoV infections

    Journal: bioRxiv

    doi: 10.1101/2020.03.15.993097

    Human serological responses to SARS-CoV-2. (A) Schematic diagram of the SARS-CoV-2 spike protein. Locations of secretion signal peptide (SP), N-terminal domain (NTD), receptor-binding domain (RBD), S1/S2 cleavage site, fusion peptide (FP), S2’ cleavage site, internal fusion peptide (IFP), heptad repeat 1 (HR1), heptad repeat 1 (HR2), transmembrane domain (TM), and cytoplasmic domain (CP) are indicated. Regions corresponding to the S1, S2, S2’ subunits, and ectodomain are also indicated. (B) Binding of plasma from healthy donors and SARS-CoV-2 infected patients to SARS-CoV-2 spike protein, SARS-CoV-2 RBD protein, SARS-CoV-2 S2 subunit, SARS-CoV spike protein and SARS-CoV RBD protein were measured by ELISA. The mean OD 450 values calculated after testing each plasma sample in triplicate are shown. (C) Neutralization activities of plasma from SARS-CoV-2 infected patients to SARS-CoV-2 and SARS-CoV viruses were measured. Dashed line represents the lower detection limit. Black lines indicate mean +/- standard deviation. (B-C) Grey: plasma samples from healthy donors. Orange: plasma samples from SARS-CoV-2-infected patients. Blue: plasma samples from SARS-CoV-infected patients.
    Figure Legend Snippet: Human serological responses to SARS-CoV-2. (A) Schematic diagram of the SARS-CoV-2 spike protein. Locations of secretion signal peptide (SP), N-terminal domain (NTD), receptor-binding domain (RBD), S1/S2 cleavage site, fusion peptide (FP), S2’ cleavage site, internal fusion peptide (IFP), heptad repeat 1 (HR1), heptad repeat 1 (HR2), transmembrane domain (TM), and cytoplasmic domain (CP) are indicated. Regions corresponding to the S1, S2, S2’ subunits, and ectodomain are also indicated. (B) Binding of plasma from healthy donors and SARS-CoV-2 infected patients to SARS-CoV-2 spike protein, SARS-CoV-2 RBD protein, SARS-CoV-2 S2 subunit, SARS-CoV spike protein and SARS-CoV RBD protein were measured by ELISA. The mean OD 450 values calculated after testing each plasma sample in triplicate are shown. (C) Neutralization activities of plasma from SARS-CoV-2 infected patients to SARS-CoV-2 and SARS-CoV viruses were measured. Dashed line represents the lower detection limit. Black lines indicate mean +/- standard deviation. (B-C) Grey: plasma samples from healthy donors. Orange: plasma samples from SARS-CoV-2-infected patients. Blue: plasma samples from SARS-CoV-infected patients.

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

    Mouse serological response to SARS-CoV-2 and SARS-CoV. (A-D) Binding of plasma from OC43-CoV-immunized mice, SARS-CoV-immunized mice, SARS-CoV-infected mice and mock-immunized mice against (A) SARS-CoV-2 spike protein, (B) SARS-CoV-2 RBD protein, (C) SARS-CoV spike protein and (D) SARS-CoV RBD protein were measured by ELISA. Since both SARS-CoV spike protein and SARS-CoV-2 spike contained a C-terminal foldon domain, binding of plasma from mice immunized with SARS-CoV spike protein plasma was not tested against spike proteins from SARS-CoV and SARS-CoV-2. The mean OD 450 values calculated after testing each plasma sample in triplicate are shown. (E-F) Neutralization activities of plasma from mice infected or immunized by SARS-CoV-2 or SARS-CoV to (E) SARS-CoV-2 virus or (F) SARS-CoV virus were measured. Dashed line represents the lower detection limit. Black lines indicate mean +/- standard deviation.
    Figure Legend Snippet: Mouse serological response to SARS-CoV-2 and SARS-CoV. (A-D) Binding of plasma from OC43-CoV-immunized mice, SARS-CoV-immunized mice, SARS-CoV-infected mice and mock-immunized mice against (A) SARS-CoV-2 spike protein, (B) SARS-CoV-2 RBD protein, (C) SARS-CoV spike protein and (D) SARS-CoV RBD protein were measured by ELISA. Since both SARS-CoV spike protein and SARS-CoV-2 spike contained a C-terminal foldon domain, binding of plasma from mice immunized with SARS-CoV spike protein plasma was not tested against spike proteins from SARS-CoV and SARS-CoV-2. The mean OD 450 values calculated after testing each plasma sample in triplicate are shown. (E-F) Neutralization activities of plasma from mice infected or immunized by SARS-CoV-2 or SARS-CoV to (E) SARS-CoV-2 virus or (F) SARS-CoV virus were measured. Dashed line represents the lower detection limit. Black lines indicate mean +/- standard deviation.

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

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

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

    Journal: bioRxiv

    doi: 10.1101/2021.01.19.427310

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

    Techniques Used: Mouse Assay

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

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

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

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

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

    Techniques Used: Mouse Assay

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

    Techniques Used: Neutralization, Infection

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

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

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

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

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

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

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

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

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

    Techniques Used: CRISPR, Binding Assay, Expressing

    7) Product Images from "Early cross-coronavirus reactive signatures of protective humoral immunity against COVID-19"

    Article Title: Early cross-coronavirus reactive signatures of protective humoral immunity against COVID-19

    Journal: bioRxiv

    doi: 10.1101/2021.05.11.443609

    SARS-CoV-2 S2-specific antibody functionality tracks with asymptomatic SARS-CoV-2 infection. (A) The whisker box plots show the overall humoral immune response to OC43 RBD-spike titers across a community based SARS-CoV-2 infection cohort divided by individuals that were asymptomatic (symptoms level 0) or experienced symptoms (symptoms level 1 or level 2, based on degree of symptoms) pre- and post-infection. (B) The bar graphs illustrate the SARS-CoV-2 specific humoral immune response across the RBD, S, S1, and S2 antigens across the same community based surveillance study divided by the degree of symptoms (symptoms levels). The dots show the scaled values of each sample. A two-sample Wilcox test was used to evaluate statistical differences across different epitopes for all the symptom categories. Significance corresponds to adjusted P-values. (* p
    Figure Legend Snippet: SARS-CoV-2 S2-specific antibody functionality tracks with asymptomatic SARS-CoV-2 infection. (A) The whisker box plots show the overall humoral immune response to OC43 RBD-spike titers across a community based SARS-CoV-2 infection cohort divided by individuals that were asymptomatic (symptoms level 0) or experienced symptoms (symptoms level 1 or level 2, based on degree of symptoms) pre- and post-infection. (B) The bar graphs illustrate the SARS-CoV-2 specific humoral immune response across the RBD, S, S1, and S2 antigens across the same community based surveillance study divided by the degree of symptoms (symptoms levels). The dots show the scaled values of each sample. A two-sample Wilcox test was used to evaluate statistical differences across different epitopes for all the symptom categories. Significance corresponds to adjusted P-values. (* p

    Techniques Used: Infection, Whisker Assay

    The temporal evolution of the human OC43 specific humoral immune response. (A) The whisker bar graphs show the distribution of human OC43 receptor binding domain (RBD)-specific antibody titers and OC43-specific antibody mediated Fc-receptor binding profiles across moderate, severe, and non-survivor COVID-19 groups over the study time course. The solid black line represents the median and box boundary (upper and bottom). (B-D) The volcano plots show the pairwise comparisons across the three COVID-19 severity groups, (A) individuals that passed away within 28 days (deceased) vs. severe survivors; (B) subjects who experienced moderate disease vs. severe survivors; (C) subjects who ultimately passed away (deceased) vs. subjects who developed moderate disease, including human OC43 RBD-specific humoral immune data. (E) The correlation heatmap shows the pairwise Spearman correlation matrices between OC43-specific and SARS-CoV-2 antibody levels across three COVID-19 severity groups (moderate, severe, and non-survivors) across the study time course. The correlation coefficients were shown only if statistically significant (adjust p -value
    Figure Legend Snippet: The temporal evolution of the human OC43 specific humoral immune response. (A) The whisker bar graphs show the distribution of human OC43 receptor binding domain (RBD)-specific antibody titers and OC43-specific antibody mediated Fc-receptor binding profiles across moderate, severe, and non-survivor COVID-19 groups over the study time course. The solid black line represents the median and box boundary (upper and bottom). (B-D) The volcano plots show the pairwise comparisons across the three COVID-19 severity groups, (A) individuals that passed away within 28 days (deceased) vs. severe survivors; (B) subjects who experienced moderate disease vs. severe survivors; (C) subjects who ultimately passed away (deceased) vs. subjects who developed moderate disease, including human OC43 RBD-specific humoral immune data. (E) The correlation heatmap shows the pairwise Spearman correlation matrices between OC43-specific and SARS-CoV-2 antibody levels across three COVID-19 severity groups (moderate, severe, and non-survivors) across the study time course. The correlation coefficients were shown only if statistically significant (adjust p -value

    Techniques Used: Whisker Assay, Binding Assay

    Evolution of early SARS-CoV-2 specific humoral immune responses following symptom onset across acutely ill COVID-19 patients. (A) The cartoon shows the study groups based on COVID-19 severity: 217 COVID-19-infected patients were sampled on days 0, 3, and 7 after admission to the hospital. Patients were classified into three groups based on the maximal acuity within 28 days of enrollment: Moderate: hospitalized that required supplemental oxygen ( n = 118). Severe: intubation, mechanical ventilation, and survival to 28 days ( n = 62). Deceased: death within 28 days ( n = 37). Based on the day of symptom onset, the samples were divided into four temporal groups: [0, 3), [3, 6), [6, 9), [9, 12). (B) The whisker plots show the distribution of antibody titers across moderate (blue), severe (yellow), and deceased (red) over the study time course. The solid black line represents the median, and the box boundary (upper and below) represents the first and third quartiles. The dots show the scaled values of each sample. A two-sample Wilcox test was used to evaluate statistical differences across groups for all the intervals and features. The P-values were corrected from multiple hypothesis testing using the Benjamini-Hochbery procedure per each interval. Significance corresponds to adjusted P-values. (* p
    Figure Legend Snippet: Evolution of early SARS-CoV-2 specific humoral immune responses following symptom onset across acutely ill COVID-19 patients. (A) The cartoon shows the study groups based on COVID-19 severity: 217 COVID-19-infected patients were sampled on days 0, 3, and 7 after admission to the hospital. Patients were classified into three groups based on the maximal acuity within 28 days of enrollment: Moderate: hospitalized that required supplemental oxygen ( n = 118). Severe: intubation, mechanical ventilation, and survival to 28 days ( n = 62). Deceased: death within 28 days ( n = 37). Based on the day of symptom onset, the samples were divided into four temporal groups: [0, 3), [3, 6), [6, 9), [9, 12). (B) The whisker plots show the distribution of antibody titers across moderate (blue), severe (yellow), and deceased (red) over the study time course. The solid black line represents the median, and the box boundary (upper and below) represents the first and third quartiles. The dots show the scaled values of each sample. A two-sample Wilcox test was used to evaluate statistical differences across groups for all the intervals and features. The P-values were corrected from multiple hypothesis testing using the Benjamini-Hochbery procedure per each interval. Significance corresponds to adjusted P-values. (* p

    Techniques Used: Infection, Whisker Assay

    8) Product Images from "Receptor-binding Domain Severe Acute Respiratory Syndrome Coronavirus 2-specific Antibodies in Human Milk From Mothers With Coronavirus Disease 2019 Polymerase Chain Reaction or With Symptoms Suggestive of Coronavirus Disease 2019"

    Article Title: Receptor-binding Domain Severe Acute Respiratory Syndrome Coronavirus 2-specific Antibodies in Human Milk From Mothers With Coronavirus Disease 2019 Polymerase Chain Reaction or With Symptoms Suggestive of Coronavirus Disease 2019

    Journal: Journal of Pediatric Gastroenterology and Nutrition

    doi: 10.1097/MPG.0000000000003158

    Comparison of titers (area under the curve [AUC]) in human milk antibodies specific to the receptor-binding domain (RBD) of SARS-CoV-2 between COVID-19 exposed mothers and unexposed mothers. (A) Secretory IgA (SIgA)/IgA; (B) secretory IgM (SIgM)/IgM; (C) IgG; (D) free secretory component (fSC). Mann-Whitney test was used to compare the two groups. Values are means ± SD, n = 16 for COVID-19 exposed mothers (n = 8 for mothers with confirmed COVID-19 PCR test and n = 8 for mothers with viral symptoms associated with COVID-19 infection) and n = 6 for unexposed mothers (control 2018). Mann-Whitney test was used to compare the two groups. Values are means ± SD. AUC was calculated using six serial dilutions (5× to 160×) in duplicate for each milk sample. COVID-19 = coronavirus disease 2019; IgA = immunoglobulin A; IgG = immunoglobulin G; IgM = immunoglobulin; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; SD = standard deviation.
    Figure Legend Snippet: Comparison of titers (area under the curve [AUC]) in human milk antibodies specific to the receptor-binding domain (RBD) of SARS-CoV-2 between COVID-19 exposed mothers and unexposed mothers. (A) Secretory IgA (SIgA)/IgA; (B) secretory IgM (SIgM)/IgM; (C) IgG; (D) free secretory component (fSC). Mann-Whitney test was used to compare the two groups. Values are means ± SD, n = 16 for COVID-19 exposed mothers (n = 8 for mothers with confirmed COVID-19 PCR test and n = 8 for mothers with viral symptoms associated with COVID-19 infection) and n = 6 for unexposed mothers (control 2018). Mann-Whitney test was used to compare the two groups. Values are means ± SD. AUC was calculated using six serial dilutions (5× to 160×) in duplicate for each milk sample. COVID-19 = coronavirus disease 2019; IgA = immunoglobulin A; IgG = immunoglobulin G; IgM = immunoglobulin; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; SD = standard deviation.

    Techniques Used: Binding Assay, MANN-WHITNEY, Polymerase Chain Reaction, Infection, Standard Deviation

    Comparison of titers (area under the curve [AUC]) in human milk antibodies specific to the receptor-binding domain (RBD) of SARS-CoV-2 between mothers with confirmed COVID-19 PCR test and mothers with viral symptoms without PCR testing. (A) Secretory IgA (SIgA)/IgA; (B) secretory IgM (SIgM)/IgM; (C) IgG; (D) free secretory component (fSC). Mann-Whitney test was used to compare the two groups. Values are means ± SD, n = 8 for mothers with confirmed COVID-19 PCR test and n = 8 for mothers with viral symptoms associated with COVID-19 infection. AUC was calculated using six serial dilutions (5× to 160×) in duplicate for each milk sample. COVID-19 = coronavirus disease 2019; IgA = immunoglobulin A; IgG = immunoglobulin G; IgM = immunoglobulin; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; SD = standard deviation.
    Figure Legend Snippet: Comparison of titers (area under the curve [AUC]) in human milk antibodies specific to the receptor-binding domain (RBD) of SARS-CoV-2 between mothers with confirmed COVID-19 PCR test and mothers with viral symptoms without PCR testing. (A) Secretory IgA (SIgA)/IgA; (B) secretory IgM (SIgM)/IgM; (C) IgG; (D) free secretory component (fSC). Mann-Whitney test was used to compare the two groups. Values are means ± SD, n = 8 for mothers with confirmed COVID-19 PCR test and n = 8 for mothers with viral symptoms associated with COVID-19 infection. AUC was calculated using six serial dilutions (5× to 160×) in duplicate for each milk sample. COVID-19 = coronavirus disease 2019; IgA = immunoglobulin A; IgG = immunoglobulin G; IgM = immunoglobulin; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; SD = standard deviation.

    Techniques Used: Binding Assay, Polymerase Chain Reaction, MANN-WHITNEY, Infection, Standard Deviation

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

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

    Journal: bioRxiv

    doi: 10.1101/2021.01.29.428890

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

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

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

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

    Journal: Science Translational Medicine

    doi: 10.1126/scitranslmed.abd6990

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

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

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

    Techniques Used: Recombinant

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

    Techniques Used: Recombinant, Mutagenesis, Incubation

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.05.21.107565

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

    Techniques Used: Infection

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

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

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

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

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

    Techniques Used: Infection

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

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

    12) Product Images from "Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of 2019-nCoV Spike pseudotyped virus"

    Article Title: Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of 2019-nCoV Spike pseudotyped virus

    Journal: Phytomedicine

    doi: 10.1016/j.phymed.2020.153333

    Effect of CQ and HCQ on the entrance of 2019-nCoV spike pseudotyped virus into ACE2 h cells. The experiments were repeat three times. Data are presented as mean ± S.D. * p
    Figure Legend Snippet: Effect of CQ and HCQ on the entrance of 2019-nCoV spike pseudotyped virus into ACE2 h cells. The experiments were repeat three times. Data are presented as mean ± S.D. * p

    Techniques Used:

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.09.08.280818

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

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

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

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

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2021.113008

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

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

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

    Techniques Used: Infection

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

    Techniques Used: Binding Assay, Inhibition, Whisker Assay

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

    Techniques Used:

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

    Techniques Used: Fluorescence, Conjugation Assay, Molecular Weight

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

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

    Journal: International Dairy Journal

    doi: 10.1016/j.idairyj.2021.105002

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

    Techniques Used: Enzyme-linked Immunosorbent Assay, Recombinant

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

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

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

    Techniques Used: Direct ELISA, Recombinant

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

    Techniques Used: Recombinant, Binding Assay

    16) Product Images from "Double Lock of a Potent Human Monoclonal Antibody against SARS-CoV-2"

    Article Title: Double Lock of a Potent Human Monoclonal Antibody against SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2020.11.24.393629

    Schematic diagram of SARS-CoV-2 S and the secondary structure of the RBD. Related to Figure 7 . (A) Overall topology of SARS-CoV-2 S. NTD: N-terminal domain; RBD: receptor-binding domain; RBM: receptor-binding motif; SD1: subdomain 1; SD2: subdomain 2; FP: fusion peptide; HR1: heptad repeat 1; HR2: heptad repeat 2; TM: transmembrane region; IC: intracellular domain. (B) Protein sequence and the secondary structure of SARS-CoV-2 RBD. The red three-pointed stars and blue rectangles mark the residues in SARS-CoV-2 S RBD that interact with HB27 and ACE2, respectively.
    Figure Legend Snippet: Schematic diagram of SARS-CoV-2 S and the secondary structure of the RBD. Related to Figure 7 . (A) Overall topology of SARS-CoV-2 S. NTD: N-terminal domain; RBD: receptor-binding domain; RBM: receptor-binding motif; SD1: subdomain 1; SD2: subdomain 2; FP: fusion peptide; HR1: heptad repeat 1; HR2: heptad repeat 2; TM: transmembrane region; IC: intracellular domain. (B) Protein sequence and the secondary structure of SARS-CoV-2 RBD. The red three-pointed stars and blue rectangles mark the residues in SARS-CoV-2 S RBD that interact with HB27 and ACE2, respectively.

    Techniques Used: Binding Assay, Sequencing

    HB27 potently binds and neutralizes SARS-CoV-2 wide type and mutant strain D614G. Related to Figure 7 . (A) The spike proteins of WT and D614G were transient expressed in 293T cells which were then examined for binding to HB27 by flow cytometry. (B) Neutralizing activities of HB27 against SARS-CoV-2 WT and D614G pseudoviruses (PSV).
    Figure Legend Snippet: HB27 potently binds and neutralizes SARS-CoV-2 wide type and mutant strain D614G. Related to Figure 7 . (A) The spike proteins of WT and D614G were transient expressed in 293T cells which were then examined for binding to HB27 by flow cytometry. (B) Neutralizing activities of HB27 against SARS-CoV-2 WT and D614G pseudoviruses (PSV).

    Techniques Used: Mutagenesis, Binding Assay, Flow Cytometry

    Prophylactic and therapeutic efficacy of HB27 in two SARS-CoV-2 susceptible mice models (A) Experimental design for therapeutic and prophylactic evaluations of HB27 in two SARS-CoV-2 susceptible mice models. Group of 6-to-8 week-old hACE2 mice and BALB/c mice were infected intranasally with 5×10 4 PFU of SARS-CoV-2 BetaCoV/Beijing/IME-BJ01/2020 or 1.6×10 4 PFU of MASCp6 as described previously, respectively. A dose of 20 mg/kg HB27 was injected intraperitoneally at 12 hours before infection (the prophylactic group, P) or at 2 hours after infection (the therapeutic group, T). PBS injections were used as control group. Then, the lung tissues of mice were collected at 3 and 5 dpi for virus titer, H E and Immunostaining. (B) and (C) Virus titers of lung and trachea tissues at 3 or 5 dpi in mouse model based on a SARS-CoV-2 mouse adapted strain MASCp6. The viral loads of the tissues were determined by qRT-PCR (*P
    Figure Legend Snippet: Prophylactic and therapeutic efficacy of HB27 in two SARS-CoV-2 susceptible mice models (A) Experimental design for therapeutic and prophylactic evaluations of HB27 in two SARS-CoV-2 susceptible mice models. Group of 6-to-8 week-old hACE2 mice and BALB/c mice were infected intranasally with 5×10 4 PFU of SARS-CoV-2 BetaCoV/Beijing/IME-BJ01/2020 or 1.6×10 4 PFU of MASCp6 as described previously, respectively. A dose of 20 mg/kg HB27 was injected intraperitoneally at 12 hours before infection (the prophylactic group, P) or at 2 hours after infection (the therapeutic group, T). PBS injections were used as control group. Then, the lung tissues of mice were collected at 3 and 5 dpi for virus titer, H E and Immunostaining. (B) and (C) Virus titers of lung and trachea tissues at 3 or 5 dpi in mouse model based on a SARS-CoV-2 mouse adapted strain MASCp6. The viral loads of the tissues were determined by qRT-PCR (*P

    Techniques Used: Mouse Assay, Infection, Injection, Immunostaining, Quantitative RT-PCR

    HB27 strongly binds various SARS-CoV-2 RBD mutants. Related to Figure 6 . (A) Sequence alignments of the mutated RBDs of circulating SARS-CoV-2 strains used in (A) and SARS-CoV. The genome sequences used in the alignments were downloaded from NCBI and GISAID with accession numbers: NC_045512.2 , EPI_ISL_406596, EPI_ISL_406595, EPI_ISL_413602, EPI_ISL_415605, EPI_ISL_408511, EPI_ISL_413522, EPI_ISL_415655, EPI_ISL_418055, EPI_ISL_416507, EPI_ISL_407071 and AY429078.1, respectively. The alignments were analyzed by Clustal W and BioEdit. (B) ELISA binding assays of HB27 with selected SARS-CoV-2 RBD mutants. SARS-CoV-2 RBD proteins with previously reported site mutations were examined for their binding abilities to HB27.
    Figure Legend Snippet: HB27 strongly binds various SARS-CoV-2 RBD mutants. Related to Figure 6 . (A) Sequence alignments of the mutated RBDs of circulating SARS-CoV-2 strains used in (A) and SARS-CoV. The genome sequences used in the alignments were downloaded from NCBI and GISAID with accession numbers: NC_045512.2 , EPI_ISL_406596, EPI_ISL_406595, EPI_ISL_413602, EPI_ISL_415605, EPI_ISL_408511, EPI_ISL_413522, EPI_ISL_415655, EPI_ISL_418055, EPI_ISL_416507, EPI_ISL_407071 and AY429078.1, respectively. The alignments were analyzed by Clustal W and BioEdit. (B) ELISA binding assays of HB27 with selected SARS-CoV-2 RBD mutants. SARS-CoV-2 RBD proteins with previously reported site mutations were examined for their binding abilities to HB27.

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

    Structural basis for neutralization of SARS-CoV-2 by HB27 (A) Orthogonal views of the clashes between HB27 Fabs and ACE2 upon binding to SARS-CoV-2 S trimer. The SARS-CoV-2 S trimer is presented as ribbon diagrams and translucent molecular surfaces with three monomers colored in cyan, yellow and violet, respectively. The three copies of HB27 Fabs are rendered as molecular surfaces colored the same as in Figure 6 . The superposed ACE2 is presented as green ribbon diagrams as well as translucent molecular surface. Insets are close-up views of the clashes between ACE2 and HB27 upon binding to SARS-CoV-2 RBD. (B) Orthogonal views of the structure of HB27 Fab-A, Fab-B and Fab-C complexed with SARS-CoV-2 RBD. The S1 subunits of SARS-CoV-2 S trimer are rendered as cyan, yellow and violet surfaces and the S2 subunits are rendered as gray surfaces. See also Figures S6 and S7 .
    Figure Legend Snippet: Structural basis for neutralization of SARS-CoV-2 by HB27 (A) Orthogonal views of the clashes between HB27 Fabs and ACE2 upon binding to SARS-CoV-2 S trimer. The SARS-CoV-2 S trimer is presented as ribbon diagrams and translucent molecular surfaces with three monomers colored in cyan, yellow and violet, respectively. The three copies of HB27 Fabs are rendered as molecular surfaces colored the same as in Figure 6 . The superposed ACE2 is presented as green ribbon diagrams as well as translucent molecular surface. Insets are close-up views of the clashes between ACE2 and HB27 upon binding to SARS-CoV-2 RBD. (B) Orthogonal views of the structure of HB27 Fab-A, Fab-B and Fab-C complexed with SARS-CoV-2 RBD. The S1 subunits of SARS-CoV-2 S trimer are rendered as cyan, yellow and violet surfaces and the S2 subunits are rendered as gray surfaces. See also Figures S6 and S7 .

    Techniques Used: Neutralization, Binding Assay

    Characterization of SARS-CoV-2 and HB27, and cryo-EM maps and atomic models of SARS-CoV-2 S and HB27 complex. Related to Figure 6 . (A) Gel filtration of SARS-CoV-2 S trimer. (B) SDS-PAGE analysis of the SARS-CoV-2 S trimer, the HB27 IgG and the Fab fragment. (C) The gold-standard Fourier Shell Correlation (FSC) curves of the final cryo-EM maps of the SARS-CoV-2 S trimer-HB27 Fabs complex and of the binding interface. (D) Local resolution evaluations of the cryo-EM maps of SARS-CoV-2 S trimer complexed with three HB27 Fabs and the binding interface using ResMap ( Kucukelbir et al., 2014 ) are shown. (E) Cryo-EM map of SARS-CoV-2 S trimer complexed with three HB27 Fabs. (F) Cryo-EM map of the binding interface between SARS-CoV-2 RBD and one HB27 Fab. The color scheme is the same as in Figure 6 . The magnified panels illustrate both maps (mesh) and related atomic models. Residues are shown as sticks,
    Figure Legend Snippet: Characterization of SARS-CoV-2 and HB27, and cryo-EM maps and atomic models of SARS-CoV-2 S and HB27 complex. Related to Figure 6 . (A) Gel filtration of SARS-CoV-2 S trimer. (B) SDS-PAGE analysis of the SARS-CoV-2 S trimer, the HB27 IgG and the Fab fragment. (C) The gold-standard Fourier Shell Correlation (FSC) curves of the final cryo-EM maps of the SARS-CoV-2 S trimer-HB27 Fabs complex and of the binding interface. (D) Local resolution evaluations of the cryo-EM maps of SARS-CoV-2 S trimer complexed with three HB27 Fabs and the binding interface using ResMap ( Kucukelbir et al., 2014 ) are shown. (E) Cryo-EM map of SARS-CoV-2 S trimer complexed with three HB27 Fabs. (F) Cryo-EM map of the binding interface between SARS-CoV-2 RBD and one HB27 Fab. The color scheme is the same as in Figure 6 . The magnified panels illustrate both maps (mesh) and related atomic models. Residues are shown as sticks,

    Techniques Used: Filtration, SDS Page, Binding Assay

    HB27 potently competes with ACE2 for binding to SARS-CoV-2 RBD. Related to Figure 4 . (A) HB27 was demonstrated to compete with recombinant ACE2 for binding to SARS-CoV-2 RBD with an EC 50 value of 0.5 nM by the enzyme-linked immunosorbent assay (ELISA). (B) BIAcore SPR kinetic profile of SARS-CoV-2 S trimer and HB27. The binding affinity K D (equilibrium dissociation constant, K D = Kd/Ka, where Kd and Ka represent the dissociation rate constant and association rate constant, respectively) values were obtained using a series of HB27 concentrations and fitted in a global mode in each sensorgram. (C) BIAcore SPR kinetic profiles of SARS-CoV-2 RBD (left panel) and S trimer (right panel) with ACE2. The binding affinity K D (equilibrium dissociation constant, K D = Kd/Ka, where Kd and Ka represent the dissociation rate constant and association rate constant, respectively) values were obtained using a series of HB27 concentrations and fitted in a global mode in each sensorgram. (D) Competition of HB27 for SARS-CoV-2 RBD binding to 293T cells expressing GFP-tagged ACE2 as detected by immunofluorescence assay, scale bar, 100 μm. Anti-H7N9 mAb was used as an isotype control. (E) Competition of HB27 for ACE2-Fc-Apc binding to 293T cells expressing GFP-tagged SARS-CoV-2-Spike as detected by immunofluorescence assay. Anti-H7N9 mAb was used as an isotype control.
    Figure Legend Snippet: HB27 potently competes with ACE2 for binding to SARS-CoV-2 RBD. Related to Figure 4 . (A) HB27 was demonstrated to compete with recombinant ACE2 for binding to SARS-CoV-2 RBD with an EC 50 value of 0.5 nM by the enzyme-linked immunosorbent assay (ELISA). (B) BIAcore SPR kinetic profile of SARS-CoV-2 S trimer and HB27. The binding affinity K D (equilibrium dissociation constant, K D = Kd/Ka, where Kd and Ka represent the dissociation rate constant and association rate constant, respectively) values were obtained using a series of HB27 concentrations and fitted in a global mode in each sensorgram. (C) BIAcore SPR kinetic profiles of SARS-CoV-2 RBD (left panel) and S trimer (right panel) with ACE2. The binding affinity K D (equilibrium dissociation constant, K D = Kd/Ka, where Kd and Ka represent the dissociation rate constant and association rate constant, respectively) values were obtained using a series of HB27 concentrations and fitted in a global mode in each sensorgram. (D) Competition of HB27 for SARS-CoV-2 RBD binding to 293T cells expressing GFP-tagged ACE2 as detected by immunofluorescence assay, scale bar, 100 μm. Anti-H7N9 mAb was used as an isotype control. (E) Competition of HB27 for ACE2-Fc-Apc binding to 293T cells expressing GFP-tagged SARS-CoV-2-Spike as detected by immunofluorescence assay. Anti-H7N9 mAb was used as an isotype control.

    Techniques Used: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, SPR Assay, Expressing, Immunofluorescence

    HB27 blocks the interactions of SARS-CoV-2 with ACE2 (A) BIAcore SPR kinetics showing the competitive binding of HB27 and ACE2 to SARS-CoV-2 S trimer. For both panels, SARS-CoV-2 S protein was immobilized onto the sensor chips. In the upper panel, HB27 was first injected, followed by ACE2, whereas in the lower panel, ACE2 was injected first and then HB27. The control groups are as shown by the curves. (B) Blocking of SARS-CoV-2 RBD binding to 293T-ACE2 cells by HB27 (upper panel). Recombinant SARS-CoV-2 RBD protein and serially diluted HB27 were incubated with ACE2 expressing 293T cells (293T-ACE2) and tested for binding of HB27 to 293T-ACE2 cells. Competitive binding of HB27 and ACE2 to SARS-CoV-2-S cells (lower panel). Recombinant ACE2 and serially diluted HB27 were incubated with 293T cells expressing SARS-CoV-2 S (SARS-CoV-2-S) and tested for binding of HB27 to SARS-CoV-2-S cells. BSA was used as a negative control (NC). (C) Amount of virus on the cell surface, as detected by RT-PCR, when exposed to HB27 prior to (upper panel) and after (lower panel) the virus was allowed to attach to cells. Values are mean ± SD. Experiments were repeated in triplicate. See also Figure S2 .
    Figure Legend Snippet: HB27 blocks the interactions of SARS-CoV-2 with ACE2 (A) BIAcore SPR kinetics showing the competitive binding of HB27 and ACE2 to SARS-CoV-2 S trimer. For both panels, SARS-CoV-2 S protein was immobilized onto the sensor chips. In the upper panel, HB27 was first injected, followed by ACE2, whereas in the lower panel, ACE2 was injected first and then HB27. The control groups are as shown by the curves. (B) Blocking of SARS-CoV-2 RBD binding to 293T-ACE2 cells by HB27 (upper panel). Recombinant SARS-CoV-2 RBD protein and serially diluted HB27 were incubated with ACE2 expressing 293T cells (293T-ACE2) and tested for binding of HB27 to 293T-ACE2 cells. Competitive binding of HB27 and ACE2 to SARS-CoV-2-S cells (lower panel). Recombinant ACE2 and serially diluted HB27 were incubated with 293T cells expressing SARS-CoV-2 S (SARS-CoV-2-S) and tested for binding of HB27 to SARS-CoV-2-S cells. BSA was used as a negative control (NC). (C) Amount of virus on the cell surface, as detected by RT-PCR, when exposed to HB27 prior to (upper panel) and after (lower panel) the virus was allowed to attach to cells. Values are mean ± SD. Experiments were repeated in triplicate. See also Figure S2 .

    Techniques Used: SPR Assay, Binding Assay, Injection, Blocking Assay, Recombinant, Incubation, Expressing, Negative Control, Reverse Transcription Polymerase Chain Reaction

    HB27 is a SARS-CoV-2-specific antibody of high potency (A) Analysis of affinity of HB27 (left panel) and HB27 Fab fragments (right panel) for SARS-CoV-2 RBD. Biotinylated SARS-CoV-2 RBD protein was loaded on Octet SA sensor and tested for real-time association and dissociation from HB27 IgG and HB27 Fab fragments, respectively. (B) and (C) Analysis of affinity of HB27 for SARS-CoV RBD and MERS-CoV RBD, respectively. (D) and (E) Neutralizing activity of HB27 against SARS-CoV-2 and SARS-CoV pseudoviruses (PSV), respectively. Serially diluted HB27 titres were added to test neutralizing activity against SARS-CoV-2 and SARS-CoV PSV. (F) In vitro neutralization activity of HB27 against SARS-CoV-2 by plaque reduction neutralization test (PRNT) in Vero cells. Neutralizing activities are represented as mean ± SD. Experiments were performed in duplicates See also Figure S1 .
    Figure Legend Snippet: HB27 is a SARS-CoV-2-specific antibody of high potency (A) Analysis of affinity of HB27 (left panel) and HB27 Fab fragments (right panel) for SARS-CoV-2 RBD. Biotinylated SARS-CoV-2 RBD protein was loaded on Octet SA sensor and tested for real-time association and dissociation from HB27 IgG and HB27 Fab fragments, respectively. (B) and (C) Analysis of affinity of HB27 for SARS-CoV RBD and MERS-CoV RBD, respectively. (D) and (E) Neutralizing activity of HB27 against SARS-CoV-2 and SARS-CoV pseudoviruses (PSV), respectively. Serially diluted HB27 titres were added to test neutralizing activity against SARS-CoV-2 and SARS-CoV PSV. (F) In vitro neutralization activity of HB27 against SARS-CoV-2 by plaque reduction neutralization test (PRNT) in Vero cells. Neutralizing activities are represented as mean ± SD. Experiments were performed in duplicates See also Figure S1 .

    Techniques Used: Activity Assay, In Vitro, Neutralization, Plaque Reduction Neutralization Test

    Murine antibody mhB27 strongly binds SARS-CoV-2 RBD and neutralizes SARS-CoV-2 PSV. Related to Figure 1 . (A) Binding assay of mhB27 to SARS-CoV-2 RBD. mhB27 was serial diluted and tested its ability to bind to SARS-CoV-2 RBD by ELISA. (B) Neutralizing activities of mhB27 against SARS-CoV-2 pseudoviruses (PSV).
    Figure Legend Snippet: Murine antibody mhB27 strongly binds SARS-CoV-2 RBD and neutralizes SARS-CoV-2 PSV. Related to Figure 1 . (A) Binding assay of mhB27 to SARS-CoV-2 RBD. mhB27 was serial diluted and tested its ability to bind to SARS-CoV-2 RBD by ELISA. (B) Neutralizing activities of mhB27 against SARS-CoV-2 pseudoviruses (PSV).

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

    HB27 inhibits SARS-CoV-2 membrane fusion. (A) HB27 had potent neutralization activities when exposed to virus before or after attachment to Huh7 cells. Values are mean ± SD. Experiments were repeated in triplicate. (B) HB27 inhibits S protein-mediated cell-cell fusion. 293T cells were transfected with SARS-CoV-2 S-GFP protein, co-cultured with Vero E6 cells in the absence or presence of 100 μg/mL H014 or HB27 or anti-influenza H7N9 antibody (isotype control). No Ab: in the absence of antibodies. Images were taken after 48 h. Cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min and stained for nuclei with 4,6-diamidino-2-phenylindole (DAPI). (C) HB27 inhibits SARS-CoV-2-mediated cell-cell fusion. Huh7 cells were infected with 100 PFU of SARS-CoV-2 for 1 h at 4°C and washed for 3 times. After that cells were further cultured in the presence of a series of concentrations (0, 4, 20 and 100 nM) of HB27, or 100 nM of H014 at 37 °C for 48 h. Images were taken after 48 h. Cells were fixed with 4% (w/v) PFA for 20 min and incubated with anti-SARS-CoV-2 S protein antibody and stained for nuclei with DAPI. Scale bar equals 200 μm. (D) HB27 blocks receptor-mediated fusion of SARS-CoV-2 with liposomes. Liposomes were loaded with self-quenching concentrations of the fluorescent dye calcein. Perturbation of the bilayer causes the release of calcein resulting in dilution and a consequent increase in its fluorescence. Fusion of SARS-CoV-2 with liposomes occurred in the presence of both ACE2 and trypsin and a series of HB27 concentrations were used to inhibit the fusion. 10% Triton X-100 treatment was used to achieve 100% calcein leakage. All data shown are representative of three independent experiments.
    Figure Legend Snippet: HB27 inhibits SARS-CoV-2 membrane fusion. (A) HB27 had potent neutralization activities when exposed to virus before or after attachment to Huh7 cells. Values are mean ± SD. Experiments were repeated in triplicate. (B) HB27 inhibits S protein-mediated cell-cell fusion. 293T cells were transfected with SARS-CoV-2 S-GFP protein, co-cultured with Vero E6 cells in the absence or presence of 100 μg/mL H014 or HB27 or anti-influenza H7N9 antibody (isotype control). No Ab: in the absence of antibodies. Images were taken after 48 h. Cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min and stained for nuclei with 4,6-diamidino-2-phenylindole (DAPI). (C) HB27 inhibits SARS-CoV-2-mediated cell-cell fusion. Huh7 cells were infected with 100 PFU of SARS-CoV-2 for 1 h at 4°C and washed for 3 times. After that cells were further cultured in the presence of a series of concentrations (0, 4, 20 and 100 nM) of HB27, or 100 nM of H014 at 37 °C for 48 h. Images were taken after 48 h. Cells were fixed with 4% (w/v) PFA for 20 min and incubated with anti-SARS-CoV-2 S protein antibody and stained for nuclei with DAPI. Scale bar equals 200 μm. (D) HB27 blocks receptor-mediated fusion of SARS-CoV-2 with liposomes. Liposomes were loaded with self-quenching concentrations of the fluorescent dye calcein. Perturbation of the bilayer causes the release of calcein resulting in dilution and a consequent increase in its fluorescence. Fusion of SARS-CoV-2 with liposomes occurred in the presence of both ACE2 and trypsin and a series of HB27 concentrations were used to inhibit the fusion. 10% Triton X-100 treatment was used to achieve 100% calcein leakage. All data shown are representative of three independent experiments.

    Techniques Used: Neutralization, Transfection, Cell Culture, Staining, Infection, Incubation, Fluorescence

    Flowchart of Cryo-EM data processing of SARS-CoV-2 S trimer and HB27 complex. Related to Figure 6 .
    Figure Legend Snippet: Flowchart of Cryo-EM data processing of SARS-CoV-2 S trimer and HB27 complex. Related to Figure 6 .

    Techniques Used:

    Structure and interaction of the SARS-CoV-2 S trimer with HB27. (A) Orthogonal views of SARS-CoV-2 S trimer in complex with three copies of HB27 Fab. (B) Individual views of the three monomers each complexed with one HB27 Fab. (A) and (B) The S trimer and HB27 are rendered as molecular surfaces. Three monomers of the S trimer are colored in yellow, cyan and violet, respectively. The HB27 light and heavy chains are colored in hotpink and purpleblue, respectively. RBD: receptor binding domain. NTD: N-terminal domain. S2: the S2 subunit. (C) S1 subunits of the three monomers from SARS-CoV-2 S trimer complexed with HB27 are superposed; HB27 Fabs are not shown. All domains are presented as ribbon diagrams. Three RBD domains are colored in yellow, cyan and violet, respectively. SD1: subdomain 1. SD2: subdomain 2. (D) Cartoon representations of the structure of SARS-CoV-2 RBD in complex with HB27. The RBD is cyan, and the light and heavy chains of HB27 are hotpink and purpleblue, respectively. Residues constituting the HB27 epitope and the RBM are drawn as spheres and colored in green and blue, respectively. The overlapped residues between the HB27 epitope and the RBM are colored in red. The CDRs involved in the interactions with the RBD are labelled. CDR: complementary determining region. RBM: receptor binding motif. (E) Residues in SARS-CoV-2 RBD comprising the HB27 epitope and RBM are labeled. The RBD is rendered as cyan surface. Blue, green and red mark the HB27 epitope, the RBM and overlapped residues of them both, respectively. (F) Hydrophobic interactions between SARS-CoV-2 RBD and HB27. The RBD is shown as cyan ribbon diagrams, and the residues of which involved in hydrophobic interactions with HB27 are shown as side chains and labeled, the four dark orange circles mark the positions of four glycine residues. The HB27 light and heavy chain are rendered as light pink and pale blue molecular surfaces, respectively, of which the residues involved in the hydrophobic interactions with the RBD are highlighted in hotpink and purpleblue and labeled. (G) A few key interactions between SARS-CoV-2 RBD and the HB27 heavy (left) and light chain (right). Hydrogen bonds are presented as dashed lines. See also Figures S3 , S4 and S5 . Tables S2 and S3 .
    Figure Legend Snippet: Structure and interaction of the SARS-CoV-2 S trimer with HB27. (A) Orthogonal views of SARS-CoV-2 S trimer in complex with three copies of HB27 Fab. (B) Individual views of the three monomers each complexed with one HB27 Fab. (A) and (B) The S trimer and HB27 are rendered as molecular surfaces. Three monomers of the S trimer are colored in yellow, cyan and violet, respectively. The HB27 light and heavy chains are colored in hotpink and purpleblue, respectively. RBD: receptor binding domain. NTD: N-terminal domain. S2: the S2 subunit. (C) S1 subunits of the three monomers from SARS-CoV-2 S trimer complexed with HB27 are superposed; HB27 Fabs are not shown. All domains are presented as ribbon diagrams. Three RBD domains are colored in yellow, cyan and violet, respectively. SD1: subdomain 1. SD2: subdomain 2. (D) Cartoon representations of the structure of SARS-CoV-2 RBD in complex with HB27. The RBD is cyan, and the light and heavy chains of HB27 are hotpink and purpleblue, respectively. Residues constituting the HB27 epitope and the RBM are drawn as spheres and colored in green and blue, respectively. The overlapped residues between the HB27 epitope and the RBM are colored in red. The CDRs involved in the interactions with the RBD are labelled. CDR: complementary determining region. RBM: receptor binding motif. (E) Residues in SARS-CoV-2 RBD comprising the HB27 epitope and RBM are labeled. The RBD is rendered as cyan surface. Blue, green and red mark the HB27 epitope, the RBM and overlapped residues of them both, respectively. (F) Hydrophobic interactions between SARS-CoV-2 RBD and HB27. The RBD is shown as cyan ribbon diagrams, and the residues of which involved in hydrophobic interactions with HB27 are shown as side chains and labeled, the four dark orange circles mark the positions of four glycine residues. The HB27 light and heavy chain are rendered as light pink and pale blue molecular surfaces, respectively, of which the residues involved in the hydrophobic interactions with the RBD are highlighted in hotpink and purpleblue and labeled. (G) A few key interactions between SARS-CoV-2 RBD and the HB27 heavy (left) and light chain (right). Hydrogen bonds are presented as dashed lines. See also Figures S3 , S4 and S5 . Tables S2 and S3 .

    Techniques Used: Binding Assay, Labeling

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.07.07.191007

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

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

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

    Techniques Used: Infection, Expressing

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

    Techniques Used: Infection

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

    Techniques Used: Generated, Infection

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

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

    Journal: bioRxiv

    doi: 10.1101/2021.02.28.433291

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

    Techniques Used:

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

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

    Journal: Cell

    doi: 10.1016/j.cell.2020.04.031

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

    Techniques Used: SPR Assay, Binding Assay

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

    Techniques Used: Blocking Assay

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

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

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

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

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

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

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.06.26.174557

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

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

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

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

    21) Product Images from "A Murine CD8+ T Cell Epitope Identified in the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein"

    Article Title: A Murine CD8+ T Cell Epitope Identified in the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein

    Journal: Vaccines

    doi: 10.3390/vaccines9060641

    SARS-CoV-2 S 395– 404 increases H-2K b /D b -restricted IFN-γ production. ( A ) Splenocytes were harvested from SAS-adjuvanted SARS-CoV-2 RBD protein-immunized C57BL/6 mice ( n = 4) and stimulated with 8–11-mer peptides within the SARS-CoV-2 S 391–405 region. CD8 CTL activity was evaluated by enumerating IFN-γ SFUs via the ELISPOT assay. Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparison tests (F (27, 84) = 13.37, p
    Figure Legend Snippet: SARS-CoV-2 S 395– 404 increases H-2K b /D b -restricted IFN-γ production. ( A ) Splenocytes were harvested from SAS-adjuvanted SARS-CoV-2 RBD protein-immunized C57BL/6 mice ( n = 4) and stimulated with 8–11-mer peptides within the SARS-CoV-2 S 391–405 region. CD8 CTL activity was evaluated by enumerating IFN-γ SFUs via the ELISPOT assay. Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparison tests (F (27, 84) = 13.37, p

    Techniques Used: Mouse Assay, Activity Assay, Enzyme-linked Immunospot

    SARS-CoV-2 S 391–405 elicits IFN-γ production by CD8 T cells in splenocytes of SARS-CoV-2 S-RBD protein-immunized C57BL/6 mice. C57BL/6 mice ( n = 5) were immunized i.m. with RBD recombinant protein plus Sigma Adjuvant System (SAS) twice with a two-week interval. One week from the final immunization, cells were harvested from spleens of the immunized mice. ( A ) Splenocytes were simulated with 5 μg/mL of each RBD peptide or a peptide pool (each 2 μg/mL) for 2 days to evaluate CD8 CTL activity via an IFN-γ ELISPOT assay. Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparison test (F (43, 129) = 12.22, p
    Figure Legend Snippet: SARS-CoV-2 S 391–405 elicits IFN-γ production by CD8 T cells in splenocytes of SARS-CoV-2 S-RBD protein-immunized C57BL/6 mice. C57BL/6 mice ( n = 5) were immunized i.m. with RBD recombinant protein plus Sigma Adjuvant System (SAS) twice with a two-week interval. One week from the final immunization, cells were harvested from spleens of the immunized mice. ( A ) Splenocytes were simulated with 5 μg/mL of each RBD peptide or a peptide pool (each 2 μg/mL) for 2 days to evaluate CD8 CTL activity via an IFN-γ ELISPOT assay. Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparison test (F (43, 129) = 12.22, p

    Techniques Used: Mouse Assay, Recombinant, Activity Assay, Enzyme-linked Immunospot

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.03.28.013276

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

    Techniques Used: Recombinant, Sequencing, Binding Assay

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

    Techniques Used: Recombinant

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

    Techniques Used: Recombinant

    23) Product Images from "Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19"

    Article Title: Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19

    Journal: bioRxiv

    doi: 10.1101/2021.03.19.436183

    Neutralizing antibody titers and RBD binding antibodies of convalescent plasma and hCoV-2IG against various SARS-CoV-2 strains. (A) SARS-CoV-2 neutralizing antibody titers in CP, 2019-IVIG and hCoV-2IG preparations as determined by pseudovirus neutralization assay in 293-ACE2-TMPRSS2 cells with SARS-CoV-2 WA-1 strain, CA variant (B.1.429), UK variant (B.1.1.7), JP variant (P.1) or SA variant (B.1.351). PsVNA50 (50% neutralization titer) and PsVNA80 (80% neutralization titer) titers for control pre-pandemic 2019-IVIG (n=16), convalescent plasma (n =9) and hCoV-2IG (n = 6) were calculated with GraphPad prism version 8. Data show mean values + SEM for PsVNA50 and PsVNA80 titers for each of the 3 antibody groups against the SARS-CoV-2 WA-1, CA, UK, JP and SA variants. (B) End-point virus neutralization titers for six hCoV-2IG lots using wild type authentic SARS-CoV-2 WA-1, UK and SA virus strains in a classical BSL3 neutralization assay based on a plaque assay was performed as described in Materials and Methods. (C) Pearson two-tailed correlations are reported for the calculation of correlation of PRNT50 titers against wild-type SARS-CoV-2 strains (WA-1, UK or SA) and PsVNA50 titers against corresponding pseudovirions expressing either WA-1, UK or SA spike in pseudovirion neutralization assays for the six hCOV-2IG lots. (D) Antibody concentration (in mg/mL) required for each of the six hCoV-2IG batches to achieve 50% neutralization of SARS-CoV-2 WA-1, CA, UK, JP or SA variants in PsVNA. (E-F) Fold-decrease in PsVNA50 neutralization titers against emerging variant strain CA (B.1.429), UK (B.1.1.7), JP (P.1) and SA (B.1.351) for six hCoV-2IG lots (E) and nine CP lots (F) in comparison with SARS-CoV-2 WA-1 strain. The numbers above the group shows the mean fold-change for each variant. (G-H) Total antibody binding (Max RU) of 1mg/mL for the six batches of hCoV-2IG (hCoV-2IG-1 to hCoV-2IG-6) to purified WA-1 RBD (RBD-wt) and RBD mutants: RBD-K417N, RBD-N501Y and RBD-E484K by SPR (G). The numbers above the group show the mean antibody binding for each RBD. (H) Fold-decrease in antibody binding to mutants RBD-K417N, RBD-N501Y and RBD-E484K of hCoV-2IG in comparison with RBD-wt from WA-1 strain calculated from the data in Panel G. The numbers above the group shows the mean fold-change for each mutant RBD. All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variations for duplicate runs of SPR was
    Figure Legend Snippet: Neutralizing antibody titers and RBD binding antibodies of convalescent plasma and hCoV-2IG against various SARS-CoV-2 strains. (A) SARS-CoV-2 neutralizing antibody titers in CP, 2019-IVIG and hCoV-2IG preparations as determined by pseudovirus neutralization assay in 293-ACE2-TMPRSS2 cells with SARS-CoV-2 WA-1 strain, CA variant (B.1.429), UK variant (B.1.1.7), JP variant (P.1) or SA variant (B.1.351). PsVNA50 (50% neutralization titer) and PsVNA80 (80% neutralization titer) titers for control pre-pandemic 2019-IVIG (n=16), convalescent plasma (n =9) and hCoV-2IG (n = 6) were calculated with GraphPad prism version 8. Data show mean values + SEM for PsVNA50 and PsVNA80 titers for each of the 3 antibody groups against the SARS-CoV-2 WA-1, CA, UK, JP and SA variants. (B) End-point virus neutralization titers for six hCoV-2IG lots using wild type authentic SARS-CoV-2 WA-1, UK and SA virus strains in a classical BSL3 neutralization assay based on a plaque assay was performed as described in Materials and Methods. (C) Pearson two-tailed correlations are reported for the calculation of correlation of PRNT50 titers against wild-type SARS-CoV-2 strains (WA-1, UK or SA) and PsVNA50 titers against corresponding pseudovirions expressing either WA-1, UK or SA spike in pseudovirion neutralization assays for the six hCOV-2IG lots. (D) Antibody concentration (in mg/mL) required for each of the six hCoV-2IG batches to achieve 50% neutralization of SARS-CoV-2 WA-1, CA, UK, JP or SA variants in PsVNA. (E-F) Fold-decrease in PsVNA50 neutralization titers against emerging variant strain CA (B.1.429), UK (B.1.1.7), JP (P.1) and SA (B.1.351) for six hCoV-2IG lots (E) and nine CP lots (F) in comparison with SARS-CoV-2 WA-1 strain. The numbers above the group shows the mean fold-change for each variant. (G-H) Total antibody binding (Max RU) of 1mg/mL for the six batches of hCoV-2IG (hCoV-2IG-1 to hCoV-2IG-6) to purified WA-1 RBD (RBD-wt) and RBD mutants: RBD-K417N, RBD-N501Y and RBD-E484K by SPR (G). The numbers above the group show the mean antibody binding for each RBD. (H) Fold-decrease in antibody binding to mutants RBD-K417N, RBD-N501Y and RBD-E484K of hCoV-2IG in comparison with RBD-wt from WA-1 strain calculated from the data in Panel G. The numbers above the group shows the mean fold-change for each mutant RBD. All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variations for duplicate runs of SPR was

    Techniques Used: Binding Assay, Neutralization, Variant Assay, Plaque Assay, Two Tailed Test, Expressing, Concentration Assay, Purification, SPR Assay, Mutagenesis

    SARS-CoV-2 spike antibody epitope repertoires recognized by hCoV-2IG. SARS-CoV-2 spike GFPDL analyses of IgG antibodies in six batches of hCoV-2IG. (A) Number of IgG bound phage clones selected using SARS-CoV-2 spike GFPDL on six lots of hCoV-2IG (hCoV-2IG-1 to hCoV-2IG-6). (B) Epitope repertoires of IgG antibody in hCoV-2IG batches and their alignment to the spike protein of SARS-CoV-2. Graphical distribution of representative clones with a frequency of > 2, obtained after affinity selection, are shown. The horizontal position and the length of the bars indicate the alignment of peptide sequence displayed on the selected phage clone to its homologous sequence in the SARS-CoV-2 spike. The thickness of each bar represents the frequency of repetitively isolated phage. Scale value is shown enclosed in a black box beneath the alignments. The GFPDL affinity selection data was performed in duplicate (two independent experiments by researcher in the lab, who was blinded to sample identity), and a similar number of phage clones and epitope repertoire was observed in both phage display analysis. (C) SPR binding of hCOV-2IG (n=6; in red), control pre-pandemic 2019-IVIG (n=16; in black) and convalescent plasma (n=9; in blue) with SARS-CoV-2 spike antigenic site peptides identified using GFPDL analysis in Fig. 1B . The amino acid designation is based on the SARS-CoV-2 spike protein sequence ( Fig. S1 ). Total antibody binding is represented in maximum resonance units (RU) in this figure for 10-fold serum dilution of CP, and 1mg/mL of 2019-IVIG or hCoV-2IG. The numbers above the peptides show the mean value for each respective group antibody binding to the peptide and is color-coded (6 hCOV-2IG in red, 16 2019-IVIG in black, and 9 CPs in blue). All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variations for duplicate runs of SPR was
    Figure Legend Snippet: SARS-CoV-2 spike antibody epitope repertoires recognized by hCoV-2IG. SARS-CoV-2 spike GFPDL analyses of IgG antibodies in six batches of hCoV-2IG. (A) Number of IgG bound phage clones selected using SARS-CoV-2 spike GFPDL on six lots of hCoV-2IG (hCoV-2IG-1 to hCoV-2IG-6). (B) Epitope repertoires of IgG antibody in hCoV-2IG batches and their alignment to the spike protein of SARS-CoV-2. Graphical distribution of representative clones with a frequency of > 2, obtained after affinity selection, are shown. The horizontal position and the length of the bars indicate the alignment of peptide sequence displayed on the selected phage clone to its homologous sequence in the SARS-CoV-2 spike. The thickness of each bar represents the frequency of repetitively isolated phage. Scale value is shown enclosed in a black box beneath the alignments. The GFPDL affinity selection data was performed in duplicate (two independent experiments by researcher in the lab, who was blinded to sample identity), and a similar number of phage clones and epitope repertoire was observed in both phage display analysis. (C) SPR binding of hCOV-2IG (n=6; in red), control pre-pandemic 2019-IVIG (n=16; in black) and convalescent plasma (n=9; in blue) with SARS-CoV-2 spike antigenic site peptides identified using GFPDL analysis in Fig. 1B . The amino acid designation is based on the SARS-CoV-2 spike protein sequence ( Fig. S1 ). Total antibody binding is represented in maximum resonance units (RU) in this figure for 10-fold serum dilution of CP, and 1mg/mL of 2019-IVIG or hCoV-2IG. The numbers above the peptides show the mean value for each respective group antibody binding to the peptide and is color-coded (6 hCOV-2IG in red, 16 2019-IVIG in black, and 9 CPs in blue). All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variations for duplicate runs of SPR was

    Techniques Used: Clone Assay, Selection, Sequencing, Isolation, SPR Assay, Binding Assay

    Multiple sequence alignment of Spike protein of SARS-CoV-2 variants. Multiple sequence alignment of various SARS-CoV-2 variants namely WA-1 strain (QII87782.1), CA variant (B.1.429, EPI_ISL_648527), UK variant (B.1.1.7, QQQ47833.1), JP variant (P.1, QRX39425.1), and SA variant (B.1.351, EPI_ISL_678597) was performed using MAFFT version 7 alignment tool ( https://mafft.cbrc.jp/alignment/software/ ). Mutations in any or all of the variants are indicated with a red outline around each of them. Various domains of the spike protein are also indicated namely S1, S2, RBD and FP domains.
    Figure Legend Snippet: Multiple sequence alignment of Spike protein of SARS-CoV-2 variants. Multiple sequence alignment of various SARS-CoV-2 variants namely WA-1 strain (QII87782.1), CA variant (B.1.429, EPI_ISL_648527), UK variant (B.1.1.7, QQQ47833.1), JP variant (P.1, QRX39425.1), and SA variant (B.1.351, EPI_ISL_678597) was performed using MAFFT version 7 alignment tool ( https://mafft.cbrc.jp/alignment/software/ ). Mutations in any or all of the variants are indicated with a red outline around each of them. Various domains of the spike protein are also indicated namely S1, S2, RBD and FP domains.

    Techniques Used: Sequencing, Variant Assay, Software

    SARS-CoV-2 epitope profile of six hCoV-2IG batches. Heat map of immunodominant sites (≥3 clonal frequency in at least one hCoV-2IG lot) on the SARS-CoV-2 spike recognized by IgG antibodies in six hCoV-2IG lots identified using GFPDL analyses. The immunodominant sites on the left indicate amino acid residue of the antigenic sites in the spike protein. Color scale on the right represents range of percentage of clonal occurrences (frequency) of each site. Heat map was generated using R package.
    Figure Legend Snippet: SARS-CoV-2 epitope profile of six hCoV-2IG batches. Heat map of immunodominant sites (≥3 clonal frequency in at least one hCoV-2IG lot) on the SARS-CoV-2 spike recognized by IgG antibodies in six hCoV-2IG lots identified using GFPDL analyses. The immunodominant sites on the left indicate amino acid residue of the antigenic sites in the spike protein. Color scale on the right represents range of percentage of clonal occurrences (frequency) of each site. Heat map was generated using R package.

    Techniques Used: Generated

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

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

    Journal: bioRxiv

    doi: 10.1101/2021.01.19.427310

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

    Techniques Used: Mouse Assay

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

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

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

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

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

    Techniques Used: Mouse Assay

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

    Techniques Used: Neutralization, Infection

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

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

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

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

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

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

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

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

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

    Techniques Used: CRISPR, Binding Assay, Expressing

    25) Product Images from "SARS-CoV-2 IgG detection in human oral fluids"

    Article Title: SARS-CoV-2 IgG detection in human oral fluids

    Journal: medRxiv

    doi: 10.1101/2021.07.07.21260121

    Scattergrams of Abbot Architect IgG anti-SARS-CoV-2 S/CO determined in sera versus test result determined from concomitantly collected and paired oral fluids analysed in: 1a) IgG Anti SARS-CoV-2 (RBD, indirect format) 1b) IgG Anti SARS-CoV-2 (Spike, capture format) and 1c) IgG Anti SARS-CoV-2 (NP, capture format). – All data log transformed. Data from children is shown in solid black dots and samples from staff (adults) are shown in grey circles; numbers are shown in graph. Dotted lines represent data trends in each assay.
    Figure Legend Snippet: Scattergrams of Abbot Architect IgG anti-SARS-CoV-2 S/CO determined in sera versus test result determined from concomitantly collected and paired oral fluids analysed in: 1a) IgG Anti SARS-CoV-2 (RBD, indirect format) 1b) IgG Anti SARS-CoV-2 (Spike, capture format) and 1c) IgG Anti SARS-CoV-2 (NP, capture format). – All data log transformed. Data from children is shown in solid black dots and samples from staff (adults) are shown in grey circles; numbers are shown in graph. Dotted lines represent data trends in each assay.

    Techniques Used: Transformation Assay

    Scattergrams of total IgG concentration (in mg/L) determined in oral fluids versus test result determined in: 1a) IgG Anti SARS-CoV-2 (RBD, indirect format) 1b) IgG Anti SARS-CoV-2 (Spike, capture format) and 1c) IgG Anti SARS-CoV-2 (NP, capture format). Data from children is shown in solid black dots and samples from staff (adults) are shown in grey circles. – All data log transformed. The detection limit for the total IgG determination is 15mg/ml – data points from samples with IgG > 15mg/L were excluded from graphs. Number of samples from children: n=619, number of samples from adults/staff: N=695. Dotted lines represent data trends in each assay, R2 values are given for each trend.
    Figure Legend Snippet: Scattergrams of total IgG concentration (in mg/L) determined in oral fluids versus test result determined in: 1a) IgG Anti SARS-CoV-2 (RBD, indirect format) 1b) IgG Anti SARS-CoV-2 (Spike, capture format) and 1c) IgG Anti SARS-CoV-2 (NP, capture format). Data from children is shown in solid black dots and samples from staff (adults) are shown in grey circles. – All data log transformed. The detection limit for the total IgG determination is 15mg/ml – data points from samples with IgG > 15mg/L were excluded from graphs. Number of samples from children: n=619, number of samples from adults/staff: N=695. Dotted lines represent data trends in each assay, R2 values are given for each trend.

    Techniques Used: Concentration Assay, Transformation Assay

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

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

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20642-x

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

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

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

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

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

    Techniques Used: Infection

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

    Techniques Used: Infection, Fluorescence

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

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

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

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

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

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

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

    Techniques Used: Generated, Infection, Activation Assay, Marker

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

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

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

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

    Journal: Science Translational Medicine

    doi: 10.1126/scitranslmed.abd6990

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

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

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

    Techniques Used: Recombinant

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

    Techniques Used: Recombinant, Mutagenesis, Incubation

    28) Product Images from "Sex differences in lung imaging and SARS-CoV-2 antibody responses in a COVID-19 golden Syrian hamster model"

    Article Title: Sex differences in lung imaging and SARS-CoV-2 antibody responses in a COVID-19 golden Syrian hamster model

    Journal: bioRxiv

    doi: 10.1101/2021.04.02.438292

    Antibody responses in the plasma of SARS-CoV-2 infected female hamsters were greater than males. Plasma samples were collected at different dpi and IgG antibody responses against whole inactivated SARS-CoV-2 virions (A); virus neutralizing antibody responses (B); and S-RBD-specific IgM (C), IgA (D), and IgG (E) antibodies were determined. Likewise, cross-reactive IgG antibodies against mutant S-RBDs (viz. N501Y, Y453F, N439K, and E484K) were evaluated in plasma at 28 dpi (F). Considering similar antibody responses at 6 and 7 dpi, values were presented together as 7 dpi. Data represent mean ± standard error of the mean from two independent experiments (n = 4-14/group/sex) and significant differences between groups are denoted by asterisks (*p
    Figure Legend Snippet: Antibody responses in the plasma of SARS-CoV-2 infected female hamsters were greater than males. Plasma samples were collected at different dpi and IgG antibody responses against whole inactivated SARS-CoV-2 virions (A); virus neutralizing antibody responses (B); and S-RBD-specific IgM (C), IgA (D), and IgG (E) antibodies were determined. Likewise, cross-reactive IgG antibodies against mutant S-RBDs (viz. N501Y, Y453F, N439K, and E484K) were evaluated in plasma at 28 dpi (F). Considering similar antibody responses at 6 and 7 dpi, values were presented together as 7 dpi. Data represent mean ± standard error of the mean from two independent experiments (n = 4-14/group/sex) and significant differences between groups are denoted by asterisks (*p

    Techniques Used: Infection, Mutagenesis

    SARS-CoV-2 infected male hamsters experience greater disease than females. To evaluate morbidity, the percent change in body mass from pre-inoculation was measured up to 28 dpi (A). Representative coronal, transverse, and sagittal chest CT from SARS-CoV-2-infected male and female animals are shown (B). Lung lesions (GGO, consolidation and air bronchogram) are marked by the dashed yellow lines. Maximum intensity projections (MIP) marking total (red) and diseased lung (yellow) for both males and females are shown (C). The CT score is higher in male versus female hamsters at 7 dpi (D). Weights are represented as mean ± standard error of the mean from two independent replications (n = 9-10/group), and significant differences between groups are denoted by asterisks (*p
    Figure Legend Snippet: SARS-CoV-2 infected male hamsters experience greater disease than females. To evaluate morbidity, the percent change in body mass from pre-inoculation was measured up to 28 dpi (A). Representative coronal, transverse, and sagittal chest CT from SARS-CoV-2-infected male and female animals are shown (B). Lung lesions (GGO, consolidation and air bronchogram) are marked by the dashed yellow lines. Maximum intensity projections (MIP) marking total (red) and diseased lung (yellow) for both males and females are shown (C). The CT score is higher in male versus female hamsters at 7 dpi (D). Weights are represented as mean ± standard error of the mean from two independent replications (n = 9-10/group), and significant differences between groups are denoted by asterisks (*p

    Techniques Used: Infection

    Cytokine responses in the lungs of SARS-CoV-2 infected male and female hamsters were comparable. Adult (8-10 weeks) male and female golden Syrian hamsters were infected with 10 5 TCID 50 of SARS-CoV-2. Subsets of animals were euthanized at different dpi and IL-1β (A), TNF-α (B), IL-6 (C), IFN-α (D), IFN-β (E), and IFN-γ (F) cytokine concentrations (pg/mg total protein) were determined in the lungs by ELISA. Mock-infected animal samples from different dpi were presented together as 0 dpi. Data represent mean ± standard error of the mean from one or two independent experiments (n = 2-6/group/sex) and were analyzed by two-way ANOVA (mixed-effects analysis) followed by Bonferroni’s multiple comparison test.
    Figure Legend Snippet: Cytokine responses in the lungs of SARS-CoV-2 infected male and female hamsters were comparable. Adult (8-10 weeks) male and female golden Syrian hamsters were infected with 10 5 TCID 50 of SARS-CoV-2. Subsets of animals were euthanized at different dpi and IL-1β (A), TNF-α (B), IL-6 (C), IFN-α (D), IFN-β (E), and IFN-γ (F) cytokine concentrations (pg/mg total protein) were determined in the lungs by ELISA. Mock-infected animal samples from different dpi were presented together as 0 dpi. Data represent mean ± standard error of the mean from one or two independent experiments (n = 2-6/group/sex) and were analyzed by two-way ANOVA (mixed-effects analysis) followed by Bonferroni’s multiple comparison test.

    Techniques Used: Infection, Enzyme-linked Immunosorbent Assay

    Antibody responses in the respiratory system of SARS-CoV-2 infected female hamsters were greater than males. Lung homogenates were prepared at different dpi and S-RBD-specific IgM (A), IgA (B), and IgG (C) antibodies were determined. Likewise, S-RBD-specific IgG antibodies were tested in the homogenates of nasal turbinates, trachea, and lungs at 28 dpi (D). Data represent mean ± standard error of the mean from one or two independent experiment(s) (n = 3-10/group) and significant differences between groups are denoted by asterisks (*p
    Figure Legend Snippet: Antibody responses in the respiratory system of SARS-CoV-2 infected female hamsters were greater than males. Lung homogenates were prepared at different dpi and S-RBD-specific IgM (A), IgA (B), and IgG (C) antibodies were determined. Likewise, S-RBD-specific IgG antibodies were tested in the homogenates of nasal turbinates, trachea, and lungs at 28 dpi (D). Data represent mean ± standard error of the mean from one or two independent experiment(s) (n = 3-10/group) and significant differences between groups are denoted by asterisks (*p

    Techniques Used: Infection

    Virus titers were comparable in the respiratory system of SARS-CoV-2 infected male and female hamsters. Adult (8-10 weeks) male and female golden Syrian hamsters were infected with 10 5 TCID 50 of SARS-CoV-2. Infectious virus titers in the homogenates of nasal turbinates (A), trachea (B), and lungs (C), were determined by TCID 50 assay on 2, 4, and 7 dpi. Likewise, virus RNA copies in 100ng of total RNA were tested in the lungs of infected hamsters at 2, 4, 7, 14 and 28 dpi (D). Data represent mean ± standard error of the mean from one or two experiment(s) (n = 3-5/group) and were analyzed by two-way ANOVA (mixed-effects analysis) followed by Bonferroni’s multiple comparison test.
    Figure Legend Snippet: Virus titers were comparable in the respiratory system of SARS-CoV-2 infected male and female hamsters. Adult (8-10 weeks) male and female golden Syrian hamsters were infected with 10 5 TCID 50 of SARS-CoV-2. Infectious virus titers in the homogenates of nasal turbinates (A), trachea (B), and lungs (C), were determined by TCID 50 assay on 2, 4, and 7 dpi. Likewise, virus RNA copies in 100ng of total RNA were tested in the lungs of infected hamsters at 2, 4, 7, 14 and 28 dpi (D). Data represent mean ± standard error of the mean from one or two experiment(s) (n = 3-5/group) and were analyzed by two-way ANOVA (mixed-effects analysis) followed by Bonferroni’s multiple comparison test.

    Techniques Used: Infection

    SARS-CoV-2 infected male hamsters treated with estradiol (E2) developed similar lung pathology as placebo-treated males. Male hamsters were treated with E2 capsules or placebo capsules prior to SARS-CoV-2 infection. Estrogen levels were quantified in plasma at 7 dpi (A). Change in body mass for E2-and placebo-treated males were quantified (B). CT score shows no difference between E2-treated males and placebo-treated males (C). Histopathology (H E) in a representative SARS-CoV-2-infected placebo-treated male and E2-treated male hamster lungs at 4X magnification are shown (D). The dashed yellow lines indicate lung lesions (GGO, consolidations and air bronchogram). E2 concentrations represented as mean ± standard error of the mean of two independent experiments (n=11-12/group) and significant differences between groups are denoted in asterisk (*p
    Figure Legend Snippet: SARS-CoV-2 infected male hamsters treated with estradiol (E2) developed similar lung pathology as placebo-treated males. Male hamsters were treated with E2 capsules or placebo capsules prior to SARS-CoV-2 infection. Estrogen levels were quantified in plasma at 7 dpi (A). Change in body mass for E2-and placebo-treated males were quantified (B). CT score shows no difference between E2-treated males and placebo-treated males (C). Histopathology (H E) in a representative SARS-CoV-2-infected placebo-treated male and E2-treated male hamster lungs at 4X magnification are shown (D). The dashed yellow lines indicate lung lesions (GGO, consolidations and air bronchogram). E2 concentrations represented as mean ± standard error of the mean of two independent experiments (n=11-12/group) and significant differences between groups are denoted in asterisk (*p

    Techniques Used: Infection, Histopathology

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.07.14.201616

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

    Techniques Used: Infection, Flow Cytometry, Staining, Incubation

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

    Techniques Used: Recombinant, Binding Assay, Protein Binding

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

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

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

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

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

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

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

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

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

    Techniques Used: Binding Assay, Sequencing

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

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

    30) Product Images from "Tackling the COVID-19 “cytokine storm” with microRNA mimics directly targeting the 3’UTR of pro-inflammatory mRNAs"

    Article Title: Tackling the COVID-19 “cytokine storm” with microRNA mimics directly targeting the 3’UTR of pro-inflammatory mRNAs

    Journal: Medical Hypotheses

    doi: 10.1016/j.mehy.2020.110415

    Evaluation of the hypothesis. Induction of IL-8 upregulation can be obtained by exposing cultured in vitro cell lines to the SARS-CoV-2 Spike protein (S-protein). Possible inhibition of IL-8 gene expression can be obtained by transfection of the cells with agomiR molecules (in the example agomiR-93-5p) able to interact with the 3’UTR sequence of IL-8 mRNA (as depicted in the boxed area). Effects on mRNA content and translation (see also Figure 2 for a scheme of the agomiR-mediated effects) can be analyzed by RT-qPCR and ELISA (or Bio-plex approaches).
    Figure Legend Snippet: Evaluation of the hypothesis. Induction of IL-8 upregulation can be obtained by exposing cultured in vitro cell lines to the SARS-CoV-2 Spike protein (S-protein). Possible inhibition of IL-8 gene expression can be obtained by transfection of the cells with agomiR molecules (in the example agomiR-93-5p) able to interact with the 3’UTR sequence of IL-8 mRNA (as depicted in the boxed area). Effects on mRNA content and translation (see also Figure 2 for a scheme of the agomiR-mediated effects) can be analyzed by RT-qPCR and ELISA (or Bio-plex approaches).

    Techniques Used: Cell Culture, In Vitro, Inhibition, Expressing, Transfection, Sequencing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Possible use of “miRNA therapeutics” for downregulation of SARS-CoV-2 induced IL-8 gene expression. The upregulation of the IL-8 gene, occurring through the NF-kB/STAT3 axis [3] might be strongly inhibited by transfection of premiRNA (agomiRNA) targeting the 3’UTR of IL-8 mRNA. This might lead to IL-8 mRNA degradation or inhibition of IL-8 translation and consequent release. Evidence supporting (a) SARS-CoV-2 mediated IL-8 transcription and (b) post transcriptional, miRNA dependent regulation of IL-8 production have been reported in several studies [3] , [4] , [34] , [35] , [36] .
    Figure Legend Snippet: Possible use of “miRNA therapeutics” for downregulation of SARS-CoV-2 induced IL-8 gene expression. The upregulation of the IL-8 gene, occurring through the NF-kB/STAT3 axis [3] might be strongly inhibited by transfection of premiRNA (agomiRNA) targeting the 3’UTR of IL-8 mRNA. This might lead to IL-8 mRNA degradation or inhibition of IL-8 translation and consequent release. Evidence supporting (a) SARS-CoV-2 mediated IL-8 transcription and (b) post transcriptional, miRNA dependent regulation of IL-8 production have been reported in several studies [3] , [4] , [34] , [35] , [36] .

    Techniques Used: Expressing, Transfection, Inhibition

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

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

    Journal: Cell

    doi: 10.1016/j.cell.2020.09.033

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

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

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

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

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

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

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

    Techniques Used: Binding Assay, Flow Cytometry

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

    Techniques Used: Infection, Luciferase

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

    Techniques Used: Infection, Flow Cytometry, Staining, Incubation

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

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

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

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

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

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

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

    Techniques Used: Binding Assay

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

    Techniques Used: Binding Assay, Sequencing

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

    Techniques Used: Binding Assay, Transmission Assay

    32) Product Images from "Cross-reactive neutralization of SARS-CoV-2 by serum antibodies from recovered SARS patients and immunized animals"

    Article Title: Cross-reactive neutralization of SARS-CoV-2 by serum antibodies from recovered SARS patients and immunized animals

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052126

    Neutralizing activity of convalescent sera from SARS patients against SARS-CoV and SARS-CoV-2 determined by single-cycle infection assay. (A) Neutralizing activity of convalescent patient sera was tested at a 1:20 dilution. Statistical significance was tested by two-way ANOVA with Dunnett posttest, indicating that all the sera significantly inhibited SARS-CoV and SARS-CoV-2 (p
    Figure Legend Snippet: Neutralizing activity of convalescent sera from SARS patients against SARS-CoV and SARS-CoV-2 determined by single-cycle infection assay. (A) Neutralizing activity of convalescent patient sera was tested at a 1:20 dilution. Statistical significance was tested by two-way ANOVA with Dunnett posttest, indicating that all the sera significantly inhibited SARS-CoV and SARS-CoV-2 (p

    Techniques Used: Activity Assay, Infection

    Sequence comparison between the RBDs of SARS-CoV and SARS-CoV-2. (A) RBD comparison of the palm civet SARS-CoV strain SZ16 and the human SARS-CoV-2 strain IPBCAMS-WH-01 (designated SARS2). (B) RBD comparison of the palm civet SARS-CoV strain SZ16 and the human SARS-CoV strain GD03T0013. Conservative and non-conservative mutations are marked in blue and red, respectively.
    Figure Legend Snippet: Sequence comparison between the RBDs of SARS-CoV and SARS-CoV-2. (A) RBD comparison of the palm civet SARS-CoV strain SZ16 and the human SARS-CoV-2 strain IPBCAMS-WH-01 (designated SARS2). (B) RBD comparison of the palm civet SARS-CoV strain SZ16 and the human SARS-CoV strain GD03T0013. Conservative and non-conservative mutations are marked in blue and red, respectively.

    Techniques Used: Sequencing

    Cross-reactive and neutralizing activities of antisera from mice immunized with a full-length S protein of SARS-CoV. (A) Binding activity of mouse antisera at a 1:100 dilution to SARS-CoV (S1 and RBD) and SARS-CoV-2 (S, S1, RBD, and S2) antigens was determined by ELISA. A healthy mouse serum was tested as control. (B) Neutralizing activity of mouse antisera at indicated dilutions against SARS-CoV, SARS-CoV-2, and VSV-G pseudoviruses was determined by a single-cycle infection assay. The experiments were performed in triplicates and repeated three times, and data are shown as means with standard deviations. Statistical significance was tested by two-way ANOVA with Dunnett posttest.
    Figure Legend Snippet: Cross-reactive and neutralizing activities of antisera from mice immunized with a full-length S protein of SARS-CoV. (A) Binding activity of mouse antisera at a 1:100 dilution to SARS-CoV (S1 and RBD) and SARS-CoV-2 (S, S1, RBD, and S2) antigens was determined by ELISA. A healthy mouse serum was tested as control. (B) Neutralizing activity of mouse antisera at indicated dilutions against SARS-CoV, SARS-CoV-2, and VSV-G pseudoviruses was determined by a single-cycle infection assay. The experiments were performed in triplicates and repeated three times, and data are shown as means with standard deviations. Statistical significance was tested by two-way ANOVA with Dunnett posttest.

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

    Cross-reactivity and neutralization of purified rabbit anti-RBD antibodies. (A) Binding titers of purified rabbit anti-RBD antibodies to SARS-CoV (RBD) and SARS-CoV-2 (S, RBD, and S2) antigens were determined by ELISA. A healthy rabbit serum was tested as control. (B) Neutralizing titers of purified rabbit anti-RBD antibodies on SARS-CoV, SARS-CoV-2, and VSV-G pseudoviruses was determined by a single-cycle infection assay. The experiments were done in triplicates and repeated three times, and data are shown as means with standard deviations.
    Figure Legend Snippet: Cross-reactivity and neutralization of purified rabbit anti-RBD antibodies. (A) Binding titers of purified rabbit anti-RBD antibodies to SARS-CoV (RBD) and SARS-CoV-2 (S, RBD, and S2) antigens were determined by ELISA. A healthy rabbit serum was tested as control. (B) Neutralizing titers of purified rabbit anti-RBD antibodies on SARS-CoV, SARS-CoV-2, and VSV-G pseudoviruses was determined by a single-cycle infection assay. The experiments were done in triplicates and repeated three times, and data are shown as means with standard deviations.

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

    Cross-reactive and neutralizing activities of antisera from mice immunized with RBD proteins of SARS-CoV. (A) Binding activity of mouse antisera at a 1:100 dilution to SARS-CoV (S1 and RBD) and SARS-CoV-2 (S protein and RBD) antigens was determined by ELISA. A healthy mouse serum was tested as control. (B) Neutralizing activity of mouse antisera at indicated dilutions against SARS-CoV, SARS-CoV-2, and VSV-G pseudoviruses was determined by a single-cycle infection assay. The experiments were performed in triplicates and repeated three times, and data are shown as means with standard deviations. Statistical significance was tested by two-way ANOVA with Dunnett posttest.
    Figure Legend Snippet: Cross-reactive and neutralizing activities of antisera from mice immunized with RBD proteins of SARS-CoV. (A) Binding activity of mouse antisera at a 1:100 dilution to SARS-CoV (S1 and RBD) and SARS-CoV-2 (S protein and RBD) antigens was determined by ELISA. A healthy mouse serum was tested as control. (B) Neutralizing activity of mouse antisera at indicated dilutions against SARS-CoV, SARS-CoV-2, and VSV-G pseudoviruses was determined by a single-cycle infection assay. The experiments were performed in triplicates and repeated three times, and data are shown as means with standard deviations. Statistical significance was tested by two-way ANOVA with Dunnett posttest.

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

    Cross-reactivity of convalescent sera from SARS-CoV infected patients with SARS-CoV-2 determined by ELISA. (A) Reactivity of sera from 20 recovered SARS-CoV patients (P01 to P20) with the nucleoprotein (N) of SARS-CoV-2 was measured by a commercial ELISA kit. (B) Reactivity of convalescent SARS sera with the recombinant S1 and RBD proteins of SARS-CoV. (C) Reactivity of convalescent SARS sera with the S ectodomain (designated S), S1, RBD, and S2 proteins of SARS-CoV-2. Serum samples from two healthy donors were used as negative control (Ctrl-1 and Ctrl-2). The experiments were performed with duplicate samples and repeated three times, and data are shown as means with standard deviations.
    Figure Legend Snippet: Cross-reactivity of convalescent sera from SARS-CoV infected patients with SARS-CoV-2 determined by ELISA. (A) Reactivity of sera from 20 recovered SARS-CoV patients (P01 to P20) with the nucleoprotein (N) of SARS-CoV-2 was measured by a commercial ELISA kit. (B) Reactivity of convalescent SARS sera with the recombinant S1 and RBD proteins of SARS-CoV. (C) Reactivity of convalescent SARS sera with the S ectodomain (designated S), S1, RBD, and S2 proteins of SARS-CoV-2. Serum samples from two healthy donors were used as negative control (Ctrl-1 and Ctrl-2). The experiments were performed with duplicate samples and repeated three times, and data are shown as means with standard deviations.

    Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Recombinant, Negative Control

    Cross-reactive and neutralizing activities of antisera from rabbits immunized with the RBD proteins of SARS-CoV. (A) Binding activity of rabbit antisera at a 1:100 dilution to SARS-CoV (S1 and RBD) and SARS-CoV-2 (S protein and RBD) antigens was determined by ELISA. A healthy rabbit serum was tested as control. (B) Neutralizing activity of rabbit antisera or control serum at indicated dilutions on SARS-CoV, SARS-CoV-2, and VSV-G pseudoviruses was determined by a single-cycle infection assay. The experiments were done in triplicates and repeated three times, and data are shown as means with standard deviations. Statistical significance was tested by two-way ANOVA with Dunnett posttest.
    Figure Legend Snippet: Cross-reactive and neutralizing activities of antisera from rabbits immunized with the RBD proteins of SARS-CoV. (A) Binding activity of rabbit antisera at a 1:100 dilution to SARS-CoV (S1 and RBD) and SARS-CoV-2 (S protein and RBD) antigens was determined by ELISA. A healthy rabbit serum was tested as control. (B) Neutralizing activity of rabbit antisera or control serum at indicated dilutions on SARS-CoV, SARS-CoV-2, and VSV-G pseudoviruses was determined by a single-cycle infection assay. The experiments were done in triplicates and repeated three times, and data are shown as means with standard deviations. Statistical significance was tested by two-way ANOVA with Dunnett posttest.

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

    Inhibition of purified rabbit anti-RBD antibodies on the binding of RBD to 293T/ACE2 cells. (A) Blocking activity of rabbit anti-RBD antibodies on the binding of SARS-CoV RBD (upper panel) or SARS-CoV-2 RBD (lower panel) to 293T/ACE2 cells was determined by flow cytometry. (B) Purified rabbit anti-RBD antibodies inhibited the RBD-ACE2 binding does-dependently. The experiments repeated three times, and data are shown as means with standard deviations. Statistical significance was tested by two-way ANOVA with Dunnett posttest.
    Figure Legend Snippet: Inhibition of purified rabbit anti-RBD antibodies on the binding of RBD to 293T/ACE2 cells. (A) Blocking activity of rabbit anti-RBD antibodies on the binding of SARS-CoV RBD (upper panel) or SARS-CoV-2 RBD (lower panel) to 293T/ACE2 cells was determined by flow cytometry. (B) Purified rabbit anti-RBD antibodies inhibited the RBD-ACE2 binding does-dependently. The experiments repeated three times, and data are shown as means with standard deviations. Statistical significance was tested by two-way ANOVA with Dunnett posttest.

    Techniques Used: Inhibition, Purification, Binding Assay, Blocking Assay, Activity Assay, Flow Cytometry

    33) Product Images from "Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies"

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    Journal: bioRxiv

    doi: 10.1101/2021.04.03.438258

    ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of SARS-CoV-2 S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.
    Figure Legend Snippet: ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of SARS-CoV-2 S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.

    Techniques Used: Over Expression, Activity Assay, Neutralization, Infection, Purification, Labeling, Protein Binding, Flow Cytometry, Quantitative RT-PCR, Luciferase

    SARS-CoV-2 live virus neutralization. HEK293T cells stably expressing ACE2, SIGLEC1, DC-SIGN or L-SIGN were infected with SARS-CoV-2 at MOI 0.02 in the presence of the indicated mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and positive cells were quantified.
    Figure Legend Snippet: SARS-CoV-2 live virus neutralization. HEK293T cells stably expressing ACE2, SIGLEC1, DC-SIGN or L-SIGN were infected with SARS-CoV-2 at MOI 0.02 in the presence of the indicated mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and positive cells were quantified.

    Techniques Used: Neutralization, Stable Transfection, Expressing, Infection

    RBM mAbs trigger the fusogenic rearrangmement of the S protein and promote membrane fusion. a, MAbs or soluble ACE2 were incubated for 1 hour with native-like soluble prefusion S trimer of SARS-CoV-2 to track by negative stain EM imaging the fusogenic rearrangement of soluble S trimers visible as rosettes. b , Cell-cell fusion of CHO cells expressing SARS-CoV-2 S (CHO-S) on the plasma membrane in the absence (upper panel) or presence of 5 μg/ml of S2E12 mAb (lower panel) as detected by immuno-fluorescence. Nuclei stained with Hoechst dye; cytoplasm stained with CellTracker Green. ( c ), CHO-S cell-cell fusion mediated by different spike-specific mAbs quantified as described in Methods. d , Structures of 11 Fab-RBD complexes related to mAbs used in (c) (RBD orientation is fixed) and of ACE2-RBD as determined by a combination of X-ray crystallography and cryo-EM analysis (PDBs, Extended Data Table 1 ). Shown in parentheses the RBD antigenic site as defined according to Piccoli et al. 3 e , Inhibition of S2E12-induced cell-cell fusion performed as in (c) by a fixed amount (15 μg/ml) of indicated mAbs. f , Trans-fusion of S-positive CHO cells with S-negative fluorescently-labelled CHO cells. Staining as in (b).
    Figure Legend Snippet: RBM mAbs trigger the fusogenic rearrangmement of the S protein and promote membrane fusion. a, MAbs or soluble ACE2 were incubated for 1 hour with native-like soluble prefusion S trimer of SARS-CoV-2 to track by negative stain EM imaging the fusogenic rearrangement of soluble S trimers visible as rosettes. b , Cell-cell fusion of CHO cells expressing SARS-CoV-2 S (CHO-S) on the plasma membrane in the absence (upper panel) or presence of 5 μg/ml of S2E12 mAb (lower panel) as detected by immuno-fluorescence. Nuclei stained with Hoechst dye; cytoplasm stained with CellTracker Green. ( c ), CHO-S cell-cell fusion mediated by different spike-specific mAbs quantified as described in Methods. d , Structures of 11 Fab-RBD complexes related to mAbs used in (c) (RBD orientation is fixed) and of ACE2-RBD as determined by a combination of X-ray crystallography and cryo-EM analysis (PDBs, Extended Data Table 1 ). Shown in parentheses the RBD antigenic site as defined according to Piccoli et al. 3 e , Inhibition of S2E12-induced cell-cell fusion performed as in (c) by a fixed amount (15 μg/ml) of indicated mAbs. f , Trans-fusion of S-positive CHO cells with S-negative fluorescently-labelled CHO cells. Staining as in (b).

    Techniques Used: Incubation, Staining, Imaging, Expressing, Fluorescence, Cryo-EM Sample Prep, Inhibition

    S309 or a cocktail of S309 and S2E12 provide robust in vivo protection against SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount of mAb(s) 48 hours before intra-nasal challenge with SARS-CoV-2. ( a-b ) Quantification of viral RNA in the lungs 4 days post-infection. ( c-d ) Quantification of replicating virus in lung homogenates harvested 4 days post infection using a TCID50 assay. ( e-f ) Histopathological score of the lung tissue was assessed 4 days post infection. ( g-h ) Efficacy plots based on the correlation between the level of serum antibody measured at the time of infection and the level of SARS-CoV2 (viral RNA) measured in lungs on day 4 after infection. The dotted lines represents EC50 and EC90 for viral reduction (EC90 of S309 alone vs S309+S2E12: 9 vs 11 μg/ml, respectively).
    Figure Legend Snippet: S309 or a cocktail of S309 and S2E12 provide robust in vivo protection against SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount of mAb(s) 48 hours before intra-nasal challenge with SARS-CoV-2. ( a-b ) Quantification of viral RNA in the lungs 4 days post-infection. ( c-d ) Quantification of replicating virus in lung homogenates harvested 4 days post infection using a TCID50 assay. ( e-f ) Histopathological score of the lung tissue was assessed 4 days post infection. ( g-h ) Efficacy plots based on the correlation between the level of serum antibody measured at the time of infection and the level of SARS-CoV2 (viral RNA) measured in lungs on day 4 after infection. The dotted lines represents EC50 and EC90 for viral reduction (EC90 of S309 alone vs S309+S2E12: 9 vs 11 μg/ml, respectively).

    Techniques Used: In Vivo, Injection, Infection, TCID50 Assay

    SIGLEC1, DC-SIGN and L-SIGN modulate neutralizing activity by different classes of antibodies. a-d , Neutralization of infection by authentic SARS-CoV-2 pre-incubated with indicated mAbs of HEK293T cell lines stably overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2. Infection was measured by immunostaining at 24 hours for the SARS-CoV-2 nucleoprotein. e , Summary of the mechanisms of action of different classes of spike-specific mAbs based on this and previous studies. *, mAb-mediated inhibition of fusion between CHO-spike cells and ACE2+ Vero-E6 cells; **, based on mAb-dependent activation of human FcγRs performed with a bioluminescent reporter assay as in 27 . æ , S2X58 binds to open RDB due to a confomational clash with neighboring NTD
    Figure Legend Snippet: SIGLEC1, DC-SIGN and L-SIGN modulate neutralizing activity by different classes of antibodies. a-d , Neutralization of infection by authentic SARS-CoV-2 pre-incubated with indicated mAbs of HEK293T cell lines stably overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2. Infection was measured by immunostaining at 24 hours for the SARS-CoV-2 nucleoprotein. e , Summary of the mechanisms of action of different classes of spike-specific mAbs based on this and previous studies. *, mAb-mediated inhibition of fusion between CHO-spike cells and ACE2+ Vero-E6 cells; **, based on mAb-dependent activation of human FcγRs performed with a bioluminescent reporter assay as in 27 . æ , S2X58 binds to open RDB due to a confomational clash with neighboring NTD

    Techniques Used: Activity Assay, Neutralization, Infection, Incubation, Stable Transfection, Immunostaining, Inhibition, Activation Assay, Reporter Assay

    Expression of auxiliary receptors in infected tissues and their role in mediating trans-infection in vitro a, Distribution and expression of ACE2, DC-SIGN, L-SIGN, and SIGLEC1 in the human lung cell atlas. b, Major cell types with detectable SARS-CoV-2 genome in bronchoalverolar lavage fluid and sputum of severe COVID-19 patients. Left panel shows a t-SNE embedding of single-cell gene expression profiles coloured by cell type and sized by viral load (logCPM); right panel, distribution plots by annotated cell type denote the cumulative fraction of cells (y-axis) with detected viral RNA per cell up to the corresponding logCPM value (x-axis). c, Left panel shows a heatmap matrix of counts for cells with detected transcripts for receptor gene(s) on x-axis by SARS-CoV-2 + cell type on y-axis (total n=3,085 cells from 8 subjects in Ren et al. 20 ); right panel, correlation of receptor transcript counts with SARS-CoV-2 RNA counts in macrophages and in secretory cells. Correlation is based on counts (before log transformation), from Ren et al. 22 . d, Trans-infection: HeLa cells transduced with DC-SIGN, L-SIGN or SIGLEC1 were incubated with VSV-SARS-CoV-2, extensively washed and co-cultured with Vero-E6-TMPRSS2 susceptible target cells. Shown is RLU in the presence or absence of target cells. e, Trans-infection performed as in (d). VSV-SARS-CoV-2 viral adsorption was performed in the presence or absence of an anti-SIGLEC1 blocking antibody.
    Figure Legend Snippet: Expression of auxiliary receptors in infected tissues and their role in mediating trans-infection in vitro a, Distribution and expression of ACE2, DC-SIGN, L-SIGN, and SIGLEC1 in the human lung cell atlas. b, Major cell types with detectable SARS-CoV-2 genome in bronchoalverolar lavage fluid and sputum of severe COVID-19 patients. Left panel shows a t-SNE embedding of single-cell gene expression profiles coloured by cell type and sized by viral load (logCPM); right panel, distribution plots by annotated cell type denote the cumulative fraction of cells (y-axis) with detected viral RNA per cell up to the corresponding logCPM value (x-axis). c, Left panel shows a heatmap matrix of counts for cells with detected transcripts for receptor gene(s) on x-axis by SARS-CoV-2 + cell type on y-axis (total n=3,085 cells from 8 subjects in Ren et al. 20 ); right panel, correlation of receptor transcript counts with SARS-CoV-2 RNA counts in macrophages and in secretory cells. Correlation is based on counts (before log transformation), from Ren et al. 22 . d, Trans-infection: HeLa cells transduced with DC-SIGN, L-SIGN or SIGLEC1 were incubated with VSV-SARS-CoV-2, extensively washed and co-cultured with Vero-E6-TMPRSS2 susceptible target cells. Shown is RLU in the presence or absence of target cells. e, Trans-infection performed as in (d). VSV-SARS-CoV-2 viral adsorption was performed in the presence or absence of an anti-SIGLEC1 blocking antibody.

    Techniques Used: Expressing, Infection, In Vitro, Transformation Assay, Transduction, Incubation, Cell Culture, Adsorption, Blocking Assay

    Characterization of SARS-CoV-2-susceptible cell lines. a , SARS-CoV-2 neutralization with 10 μg/ml of S309, S2E12 and S2X33 on Vero E6 or Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection and viral nucleocapsid protein was immunostained. b , Purified, fluorescently-labelled SARS-CoV-2 spike protein or RBD protein was incubated with the indicated cell lines and protein binding was quantified by flow cytometry. c , Correlation analysis between ACE2 transcript levels and maximum antibody neutralization in all SARS-CoV-2-susceptible cell lines.
    Figure Legend Snippet: Characterization of SARS-CoV-2-susceptible cell lines. a , SARS-CoV-2 neutralization with 10 μg/ml of S309, S2E12 and S2X33 on Vero E6 or Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection and viral nucleocapsid protein was immunostained. b , Purified, fluorescently-labelled SARS-CoV-2 spike protein or RBD protein was incubated with the indicated cell lines and protein binding was quantified by flow cytometry. c , Correlation analysis between ACE2 transcript levels and maximum antibody neutralization in all SARS-CoV-2-susceptible cell lines.

    Techniques Used: Neutralization, Infection, Purification, Incubation, Protein Binding, Flow Cytometry

    Role of host effector function in SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount (mg/kg) of hamster IgG2a S309 either wt or Fc silenced (S309-N297A). a , Quantification of viral RNA in the lung 4 days post infection. b, Quantification of replicating virus in the lung 4 days post infection. c, Histopathological score in the lung 4 days post infection. Control animals (white symbols) were injected with 4 mg/kg unrelated control isotype mAb. *, **, ***, **** p
    Figure Legend Snippet: Role of host effector function in SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount (mg/kg) of hamster IgG2a S309 either wt or Fc silenced (S309-N297A). a , Quantification of viral RNA in the lung 4 days post infection. b, Quantification of replicating virus in the lung 4 days post infection. c, Histopathological score in the lung 4 days post infection. Control animals (white symbols) were injected with 4 mg/kg unrelated control isotype mAb. *, **, ***, **** p

    Techniques Used: Injection, Infection

    HeLa cells expressing DC-SIGN are refractory to SARS-CoV-2 infection. 293T or HeLa cells stably expressing DC-SIGN were infected with SARS-CoV-2-Nluc at MOI0.04 in the presence of the indicated antibodies. Infection was analyzed by quantification of luminescent signal at 24 h post infection.
    Figure Legend Snippet: HeLa cells expressing DC-SIGN are refractory to SARS-CoV-2 infection. 293T or HeLa cells stably expressing DC-SIGN were infected with SARS-CoV-2-Nluc at MOI0.04 in the presence of the indicated antibodies. Infection was analyzed by quantification of luminescent signal at 24 h post infection.

    Techniques Used: Expressing, Infection, Stable Transfection

    Characterization of DC-SIGN, L-SIGN and SIGLEC-1 as SARS-CoV-2 attachment factors. a-b, Binding of antibodies targeting DC/-L-SIGN, DC-SIGN, SIGLEC1 or ACE2 on HEK293T stably over-expressing the respective attachment receptors was analyzed by flow cytometry (a) and immunofluorescence analysis (b). c, HEK293T cells over-expressing the respective attachment receptors were infected with VSV-SARS-COV-2 wildtype spike (grey bars) or spike bearing mutations of the B.1.1.7 variant (red bars). Luminescence was analyzed one day post infection.
    Figure Legend Snippet: Characterization of DC-SIGN, L-SIGN and SIGLEC-1 as SARS-CoV-2 attachment factors. a-b, Binding of antibodies targeting DC/-L-SIGN, DC-SIGN, SIGLEC1 or ACE2 on HEK293T stably over-expressing the respective attachment receptors was analyzed by flow cytometry (a) and immunofluorescence analysis (b). c, HEK293T cells over-expressing the respective attachment receptors were infected with VSV-SARS-COV-2 wildtype spike (grey bars) or spike bearing mutations of the B.1.1.7 variant (red bars). Luminescence was analyzed one day post infection.

    Techniques Used: Binding Assay, Stable Transfection, Expressing, Flow Cytometry, Immunofluorescence, Infection, Variant Assay

    DC-SIGN, L-SIGN and SIGLEC1 function as auxiliary receptors for SARS-CoV-2 infection. a, VSV-SARS-CoV-2 pseudovirus infection of HEK293T cells transfected to over-express ACE2 or a panel of selected lectins and published receptor candidates. b, Stable HEK293T cell lines overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2 were infected with authentic SARS-CoV-2 (MOI 0.1), fixed and immunostained at 24 hours for the SARS-CoV-2 nucleocapsid protein (red). c, HEK293T stable cell lines were infected with SARS-CoV-2-Nluc and luciferase levels were quantified at 24 hours. d, Stable cell lines were incubated with different concentrations of anti-SIGLEC1 mAb (clone 7-239) and infected with SARS-CoV-2-Nluc. e, HEK293T, HeLa and MRC5 cells were transiently transduced to overexpress DC-SIGN, L-SIGN, SIGLEC1 or ACE2 and infected with VSV-SARS-CoV-2 pseudovirus. f, Stable cell lines were treated with ACE2 siRNA followed by infection with VSV-SARS-CoV-2 pseudovirus four days post transfection. g, Stable cell lines were incubated with different concentrations of anti-ACE2 goat polyclonal antibodies and infected with VSV-SARS-CoV-2 pseudovirus.
    Figure Legend Snippet: DC-SIGN, L-SIGN and SIGLEC1 function as auxiliary receptors for SARS-CoV-2 infection. a, VSV-SARS-CoV-2 pseudovirus infection of HEK293T cells transfected to over-express ACE2 or a panel of selected lectins and published receptor candidates. b, Stable HEK293T cell lines overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2 were infected with authentic SARS-CoV-2 (MOI 0.1), fixed and immunostained at 24 hours for the SARS-CoV-2 nucleocapsid protein (red). c, HEK293T stable cell lines were infected with SARS-CoV-2-Nluc and luciferase levels were quantified at 24 hours. d, Stable cell lines were incubated with different concentrations of anti-SIGLEC1 mAb (clone 7-239) and infected with SARS-CoV-2-Nluc. e, HEK293T, HeLa and MRC5 cells were transiently transduced to overexpress DC-SIGN, L-SIGN, SIGLEC1 or ACE2 and infected with VSV-SARS-CoV-2 pseudovirus. f, Stable cell lines were treated with ACE2 siRNA followed by infection with VSV-SARS-CoV-2 pseudovirus four days post transfection. g, Stable cell lines were incubated with different concentrations of anti-ACE2 goat polyclonal antibodies and infected with VSV-SARS-CoV-2 pseudovirus.

    Techniques Used: Infection, Transfection, Stable Transfection, Luciferase, Incubation

    Data collection and processing of the S/S2X58 complex cryoEM datasets. a,b , Representative electron micrograph and 2D class averages of SARS-CoV-2 S in complex with the S2X58 Fab embedded in vitreous ice. Scale bar: 400 Å. c , Gold-standard Fourier shell correlation curves for the S2X58-bound SARS-CoV-2 S trimer in one RBD closed (black line) and three RBDs open conformations (gray line). The 0.143 cutoff is indicated by a horizontal dashed line. d, Local resolution maps calculated using cryoSPARC for the SARS-CoV-2 S/S2X58 Fab complex structure with one RBD closed and three RBDs open shown in two orthogonal orientations.
    Figure Legend Snippet: Data collection and processing of the S/S2X58 complex cryoEM datasets. a,b , Representative electron micrograph and 2D class averages of SARS-CoV-2 S in complex with the S2X58 Fab embedded in vitreous ice. Scale bar: 400 Å. c , Gold-standard Fourier shell correlation curves for the S2X58-bound SARS-CoV-2 S trimer in one RBD closed (black line) and three RBDs open conformations (gray line). The 0.143 cutoff is indicated by a horizontal dashed line. d, Local resolution maps calculated using cryoSPARC for the SARS-CoV-2 S/S2X58 Fab complex structure with one RBD closed and three RBDs open shown in two orthogonal orientations.

    Techniques Used:

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.05.21.107565

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

    Techniques Used: Infection

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

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

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

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

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

    Techniques Used: Infection

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

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

    35) Product Images from "Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of 2019-nCoV Spike pseudotyped virus"

    Article Title: Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of 2019-nCoV Spike pseudotyped virus

    Journal: Phytomedicine

    doi: 10.1016/j.phymed.2020.153333

    Effect of CQ and HCQ on the entrance of 2019-nCoV spike pseudotyped virus into ACE2 h cells. The experiments were repeat three times. Data are presented as mean ± S.D. * p
    Figure Legend Snippet: Effect of CQ and HCQ on the entrance of 2019-nCoV spike pseudotyped virus into ACE2 h cells. The experiments were repeat three times. Data are presented as mean ± S.D. * p

    Techniques Used:

    36) Product Images from "FN3-based monobodies selective for the receptor binding domain of the SARS-CoV-2 spike protein"

    Article Title: FN3-based monobodies selective for the receptor binding domain of the SARS-CoV-2 spike protein

    Journal: New Biotechnology

    doi: 10.1016/j.nbt.2021.01.010

    Isolation of four monobodies that bind the RBD of the SARS-CoV-2 virus by phage-display. (a) The 3D visualization of the fibronectin type III (FN3) domain (PDB: 1TTG) as shown in PyMOL, with the BC, DE, and FG loops labelled in different colors [ 66 ]. (b) Virions displaying the four monobody sequences (A, B, C, and D) were confirmed by ELISA to bind the RBD-Fc fusion protein and not to the Fc (negative control). Error bars represent standard error (SE) of triplicate measurements. (c) The amino acid sequences of the BC, DE, and FG loops within the four monobodies. Frequency represents the number of times a given monobody was identified among 9 confirmed binders. The complete primary structures of the four monobodies are shown in Suppl. Figure S3.
    Figure Legend Snippet: Isolation of four monobodies that bind the RBD of the SARS-CoV-2 virus by phage-display. (a) The 3D visualization of the fibronectin type III (FN3) domain (PDB: 1TTG) as shown in PyMOL, with the BC, DE, and FG loops labelled in different colors [ 66 ]. (b) Virions displaying the four monobody sequences (A, B, C, and D) were confirmed by ELISA to bind the RBD-Fc fusion protein and not to the Fc (negative control). Error bars represent standard error (SE) of triplicate measurements. (c) The amino acid sequences of the BC, DE, and FG loops within the four monobodies. Frequency represents the number of times a given monobody was identified among 9 confirmed binders. The complete primary structures of the four monobodies are shown in Suppl. Figure S3.

    Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay, Negative Control

    Specificity of anti-RBD monobodies. The four MBP-FN3 fusions were adsorbed on microtiter plate wells and incubated with chemically biotinylated SARS-CoV-1 and SARS-CoV-2 RBD proteins mixed with a bacterial cell lysate. Wells coated with MBP alone served as a negative control and wells coated with an anti-spike monoclonal antibody, clone CR3022 [ 35 ], which binds equally well to the RBDs of both SARS-CoV-1 and SARS-CoV-2, served as a positive control. Binding of SARS-CoV-1 and SARS-CoV-2 RBD-Fc fusion proteins was revealed with streptavidin-HRP. Error bars represent standard error (SE) of triplicate measurements.
    Figure Legend Snippet: Specificity of anti-RBD monobodies. The four MBP-FN3 fusions were adsorbed on microtiter plate wells and incubated with chemically biotinylated SARS-CoV-1 and SARS-CoV-2 RBD proteins mixed with a bacterial cell lysate. Wells coated with MBP alone served as a negative control and wells coated with an anti-spike monoclonal antibody, clone CR3022 [ 35 ], which binds equally well to the RBDs of both SARS-CoV-1 and SARS-CoV-2, served as a positive control. Binding of SARS-CoV-1 and SARS-CoV-2 RBD-Fc fusion proteins was revealed with streptavidin-HRP. Error bars represent standard error (SE) of triplicate measurements.

    Techniques Used: Incubation, Negative Control, Positive Control, Binding Assay

    Purification of RBD-Fc, ACE2-Fc, and spike protein. (a) Recombinant. SARS-CoV-2 spike RBD-Fc fusion protein. The predicted molecular weight (MW) is ∼ 65 kDa, when resolved by SDS-PAGE under reducing conditions with sized standards (MW shown in kDa); > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. (b) Recombinant ACE2-Fc fusion protein. The predicted MW is ∼110 kDa, when resolved by SDS-PAGE under reducing conditions, and judged to be > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. (c) Spike protein. The near full-length protein resolved as a doublet with a MW of ∼170 kDa under reducing conditions and was judged to be > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. The doublet bands are thought to differ in post-translational modifications. Composite image of two lanes from the same gel.
    Figure Legend Snippet: Purification of RBD-Fc, ACE2-Fc, and spike protein. (a) Recombinant. SARS-CoV-2 spike RBD-Fc fusion protein. The predicted molecular weight (MW) is ∼ 65 kDa, when resolved by SDS-PAGE under reducing conditions with sized standards (MW shown in kDa); > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. (b) Recombinant ACE2-Fc fusion protein. The predicted MW is ∼110 kDa, when resolved by SDS-PAGE under reducing conditions, and judged to be > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. (c) Spike protein. The near full-length protein resolved as a doublet with a MW of ∼170 kDa under reducing conditions and was judged to be > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. The doublet bands are thought to differ in post-translational modifications. Composite image of two lanes from the same gel.

    Techniques Used: Purification, Recombinant, Molecular Weight, SDS Page, Staining

    Detection of the SARS-CoV-2 RBD in a complex biological mixture. An E. coli cell lysate was mixed with various concentrations of SARS-CoV-2 RBD and added to microtiter wells coated with the FN3A-MBP fusion protein. After incubation and washing of the wells, the ectodomain of ACE2, conjugated to HRP, was added. Negative controls consisted of MBP in lieu of FN3A and Fc alone in lieu of RBD. Error bars represent SE of triplicate measurements.
    Figure Legend Snippet: Detection of the SARS-CoV-2 RBD in a complex biological mixture. An E. coli cell lysate was mixed with various concentrations of SARS-CoV-2 RBD and added to microtiter wells coated with the FN3A-MBP fusion protein. After incubation and washing of the wells, the ectodomain of ACE2, conjugated to HRP, was added. Negative controls consisted of MBP in lieu of FN3A and Fc alone in lieu of RBD. Error bars represent SE of triplicate measurements.

    Techniques Used: Incubation

    37) Product Images from "A traditional Chinese medicine formula NRICM101 to target COVID-19 through multiple pathways: A bedside-to-bench study"

    Article Title: A traditional Chinese medicine formula NRICM101 to target COVID-19 through multiple pathways: A bedside-to-bench study

    Journal: Biomedicine & Pharmacotherapy

    doi: 10.1016/j.biopha.2020.111037

    Simplified representation of NRICM101 targeting potential pathways of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Selected mechanisms of SARS-CoV-2 pathogenesis targeted by NRICM101: binding of viral spike protein to human angiotensin-converting enzyme 2 (ACE2), 3CL protease that facilitates SARS-CoV-2 replication, production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α.
    Figure Legend Snippet: Simplified representation of NRICM101 targeting potential pathways of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Selected mechanisms of SARS-CoV-2 pathogenesis targeted by NRICM101: binding of viral spike protein to human angiotensin-converting enzyme 2 (ACE2), 3CL protease that facilitates SARS-CoV-2 replication, production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α.

    Techniques Used: Infection, Binding Assay

    Pharmacological data of NRICM101. ( A ) Binding reactivity of the NRICM101 to spike RBD protein were determined by SPR. The serially diluted decoctions (1/5X, 1/10X, 1/20X, 1/40X, 1/80X, and 1/160X) were prepared in the PBS buffer as the analysts for analysis. ( B ) Interaction of spike RBD to the ACE2 was inhibited by serially diluted NRICM101 in the ACE2-spike protein inhibition ELISA. The inhibition percentage was determined according to the binding signal normalized to the interaction of spike RBD to the ACE2 without NRICM101 treatment. ( C ) NRICM101 inhibited SARS-CoV-2 3CL protease activity. Serial dilutions of the decoction were used to investigate NRICM101′s inhibitory activity against 3CL protease. ( D ) Anti-SARS-CoV-2 data of the immunofluorescent assay (IFA, upper) and plaque reduction neutralization test (PRNT, lower). ( E ) The data of CCK-8 cell viability and viral infection in IFA. ( F , G ) NRICM101 inhibited LPS-induced expression of IL-6 and TNF-α in murine alveolar macrophages. The data represented as mean ± SD from three independent experiments. 50 % inhibition concentration (IC 50 ) and 50 % cytotoxic concentration (CC 50 ) were calculated by Prism software. The red dots indicate 50 % inhibition; the data represented as mean ± SD from three independent experiments.
    Figure Legend Snippet: Pharmacological data of NRICM101. ( A ) Binding reactivity of the NRICM101 to spike RBD protein were determined by SPR. The serially diluted decoctions (1/5X, 1/10X, 1/20X, 1/40X, 1/80X, and 1/160X) were prepared in the PBS buffer as the analysts for analysis. ( B ) Interaction of spike RBD to the ACE2 was inhibited by serially diluted NRICM101 in the ACE2-spike protein inhibition ELISA. The inhibition percentage was determined according to the binding signal normalized to the interaction of spike RBD to the ACE2 without NRICM101 treatment. ( C ) NRICM101 inhibited SARS-CoV-2 3CL protease activity. Serial dilutions of the decoction were used to investigate NRICM101′s inhibitory activity against 3CL protease. ( D ) Anti-SARS-CoV-2 data of the immunofluorescent assay (IFA, upper) and plaque reduction neutralization test (PRNT, lower). ( E ) The data of CCK-8 cell viability and viral infection in IFA. ( F , G ) NRICM101 inhibited LPS-induced expression of IL-6 and TNF-α in murine alveolar macrophages. The data represented as mean ± SD from three independent experiments. 50 % inhibition concentration (IC 50 ) and 50 % cytotoxic concentration (CC 50 ) were calculated by Prism software. The red dots indicate 50 % inhibition; the data represented as mean ± SD from three independent experiments.

    Techniques Used: Binding Assay, SPR Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Activity Assay, Immunofluorescence, Plaque Reduction Neutralization Test, CCK-8 Assay, Infection, Expressing, Concentration Assay, Software

    Pharmacological data of single herbs of NRICM101. ( A ) Interaction of spike RBD to the ACE2 was determined by the ACE2-spike protein inhibition ELISA. ( B ) Inhibition of SARS-CoV-2 3CL protease activity. ( C ) Inhibition data of the immunofluorescent assay of HA and HC. ( D ) Plaque reduction neutralization test of HA and HC. ( E,F ) Inhibition data of LPS-induced expression of TNF-α and IL-6 in murine alveolar macrophages. The red dots indicate 50 % inhibition of 3CL protease activity. The data represented as mean ± SD from three independent experiments. 50 % inhibition concentration (IC 50 ) and 50 % cytotoxic concentration (CC 50 ) were calculated by Prism software.
    Figure Legend Snippet: Pharmacological data of single herbs of NRICM101. ( A ) Interaction of spike RBD to the ACE2 was determined by the ACE2-spike protein inhibition ELISA. ( B ) Inhibition of SARS-CoV-2 3CL protease activity. ( C ) Inhibition data of the immunofluorescent assay of HA and HC. ( D ) Plaque reduction neutralization test of HA and HC. ( E,F ) Inhibition data of LPS-induced expression of TNF-α and IL-6 in murine alveolar macrophages. The red dots indicate 50 % inhibition of 3CL protease activity. The data represented as mean ± SD from three independent experiments. 50 % inhibition concentration (IC 50 ) and 50 % cytotoxic concentration (CC 50 ) were calculated by Prism software.

    Techniques Used: Inhibition, Enzyme-linked Immunosorbent Assay, Activity Assay, Plaque Reduction Neutralization Test, Expressing, Concentration Assay, Software

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

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

    Journal: Immunity

    doi: 10.1016/j.immuni.2020.07.026

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

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

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

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

    39) Product Images from "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:

    40) Product Images from "Experimental infection of domestic dogs and cats with SARS-CoV-2: Pathogenesis, transmission, and response to reexposure in cats"

    Article Title: Experimental infection of domestic dogs and cats with SARS-CoV-2: Pathogenesis, transmission, and response to reexposure in cats

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.2013102117

    Inoculation and exposure with SARS-CoV-2 leads to nasal and oropharnygeal shedding in cats. SARS-CoV-2 virus is detected by plaque assay from ( A ) nasal and ( B ) oropharyngeal secretions of cats 1–5 DPI. Viral titers expressed as log 10 pfu/mL. Cats 1, 2, and 3 represent cohort 1. Cats 4, 5, 6, and 7 represent cohort 2. Cats 4 and 5 were euthanized on 5 DPI. Cats 6 and 7 were introduced to the infected cats in cohort 2 on 2 DPI.
    Figure Legend Snippet: Inoculation and exposure with SARS-CoV-2 leads to nasal and oropharnygeal shedding in cats. SARS-CoV-2 virus is detected by plaque assay from ( A ) nasal and ( B ) oropharyngeal secretions of cats 1–5 DPI. Viral titers expressed as log 10 pfu/mL. Cats 1, 2, and 3 represent cohort 1. Cats 4, 5, 6, and 7 represent cohort 2. Cats 4 and 5 were euthanized on 5 DPI. Cats 6 and 7 were introduced to the infected cats in cohort 2 on 2 DPI.

    Techniques Used: Plaque Assay, Infection

    Cats and dogs infected with SARS-CoV-2 rapidly develop antibodies against viral antigens. ( A ) Sera from cats with intranasal inoculation of SARS-CoV-2 ( n = 3, ‘EI’) or exposed to inoculated cats ( n = 2, ‘C’) were evaluated for seroreactivity to RBD, Spike, or NP for 30–42 d post exposure. IgG reactivity to Spike and RBD was evident at day 7, and all animals had clearly seroconverted by day 14. ( B ) IgM against RBD was transiently detected at low levels relative to IgG on days 7 and 14 post exposure in cats (experimentally inoculated animals, n = 3). Bars represent 1 SE of the mean. Dogs infected with SARS-CoV-2 seroconvert versus Spike and RBD antigen with lower reactivity than cats (C ). Sera tested on days indicated. IgG reactivity was evident by day 14 but plateaued and/or waned by day 42. Dashed lines indicate cut off values for seropositive diagnosis. Colors correspond to RBD (red), Spike (blue), or Nucleocapsid (green) ELISAs.
    Figure Legend Snippet: Cats and dogs infected with SARS-CoV-2 rapidly develop antibodies against viral antigens. ( A ) Sera from cats with intranasal inoculation of SARS-CoV-2 ( n = 3, ‘EI’) or exposed to inoculated cats ( n = 2, ‘C’) were evaluated for seroreactivity to RBD, Spike, or NP for 30–42 d post exposure. IgG reactivity to Spike and RBD was evident at day 7, and all animals had clearly seroconverted by day 14. ( B ) IgM against RBD was transiently detected at low levels relative to IgG on days 7 and 14 post exposure in cats (experimentally inoculated animals, n = 3). Bars represent 1 SE of the mean. Dogs infected with SARS-CoV-2 seroconvert versus Spike and RBD antigen with lower reactivity than cats (C ). Sera tested on days indicated. IgG reactivity was evident by day 14 but plateaued and/or waned by day 42. Dashed lines indicate cut off values for seropositive diagnosis. Colors correspond to RBD (red), Spike (blue), or Nucleocapsid (green) ELISAs.

    Techniques Used: Infection

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

    Enzyme-linked Immunosorbent Assay:

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

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    Binding Assay:

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    Derivative Assay:

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    Sino Biological rbd proteins
    Isolation of <t>RBD-specific</t> memory B cells using flow cytometry. A. The heatmap depicts the specificity of convalescent patients’ plasma against S1 and RBD from SARS-CoV-2, SARS-CoV and MERS-CoV, measured by <t>ELISA.</t> Serial dilutions of plasma samples were performed to test the reactivity of antibodies in plasma. The plasma of healthy donors was used as the control. Data were shown with the mean of representative experiments. B. Gating strategy for SARS-CoV-2 RBD-specific IgG + B cells in PBMCs of the convalescent patients. Living CD19+ IgD − IgG + cells were gated, and cells with positive SARS-CoV-2 RBD staining were selected for single-cell sorting. C. FACS analysis of RBD-specific memory B cells in CD19 + IgD − IgG + memory B cells from PBMCs of three batch convalescent patients. Plots show CD19 + IgD − IgG + RBD+ populations using gating strategy described in B .
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    Isolation of RBD-specific memory B cells using flow cytometry. A. The heatmap depicts the specificity of convalescent patients’ plasma against S1 and RBD from SARS-CoV-2, SARS-CoV and MERS-CoV, measured by ELISA. Serial dilutions of plasma samples were performed to test the reactivity of antibodies in plasma. The plasma of healthy donors was used as the control. Data were shown with the mean of representative experiments. B. Gating strategy for SARS-CoV-2 RBD-specific IgG + B cells in PBMCs of the convalescent patients. Living CD19+ IgD − IgG + cells were gated, and cells with positive SARS-CoV-2 RBD staining were selected for single-cell sorting. C. FACS analysis of RBD-specific memory B cells in CD19 + IgD − IgG + memory B cells from PBMCs of three batch convalescent patients. Plots show CD19 + IgD − IgG + RBD+ populations using gating strategy described in B .

    Journal: bioRxiv

    Article Title: A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD

    doi: 10.1101/2020.08.19.253369

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

    Article Snippet: ELISA binding assay and competitive ELISA2 μg/ml the recombinant S or RBD proteins derived from SARS-CoV-2, SARS-CoV, or MERS-CoV (Sino Biological, Beijing) were coated on 384-well plates (Corning) at 4°C overnight.

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

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

    Journal: bioRxiv

    Article Title: A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD

    doi: 10.1101/2020.08.19.253369

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

    Article Snippet: ELISA binding assay and competitive ELISA2 μg/ml the recombinant S or RBD proteins derived from SARS-CoV-2, SARS-CoV, or MERS-CoV (Sino Biological, Beijing) were coated on 384-well plates (Corning) at 4°C overnight.

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

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

    Journal: bioRxiv

    Article Title: A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD

    doi: 10.1101/2020.08.19.253369

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

    Article Snippet: ELISA binding assay and competitive ELISA2 μg/ml the recombinant S or RBD proteins derived from SARS-CoV-2, SARS-CoV, or MERS-CoV (Sino Biological, Beijing) were coated on 384-well plates (Corning) at 4°C overnight.

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

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

    Journal: bioRxiv

    Article Title: A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD

    doi: 10.1101/2020.08.19.253369

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

    Article Snippet: ELISA binding assay and competitive ELISA2 μg/ml the recombinant S or RBD proteins derived from SARS-CoV-2, SARS-CoV, or MERS-CoV (Sino Biological, Beijing) were coated on 384-well plates (Corning) at 4°C overnight.

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

    Kinetics of IgM and IgG responses against SARS-CoV-2 in different tissues. Urine ( A ), sputum ( B ), feces ( C ), BALF, and pleural effusion ( D ) specimens from patients with COVID-19 were detected for the presence of IgM and IgG antibodies against the N protein of SARS-CoV-2. Positive (PC) and negative (NC) controls provided by detection kit were included to ensure test validity. Plasma from 48 HDs was also included.

    Journal: The Journal of Clinical Investigation

    Article Title: Kinetics of viral load and antibody response in relation to COVID-19 severity

    doi: 10.1172/JCI138759

    Figure Lengend Snippet: Kinetics of IgM and IgG responses against SARS-CoV-2 in different tissues. Urine ( A ), sputum ( B ), feces ( C ), BALF, and pleural effusion ( D ) specimens from patients with COVID-19 were detected for the presence of IgM and IgG antibodies against the N protein of SARS-CoV-2. Positive (PC) and negative (NC) controls provided by detection kit were included to ensure test validity. Plasma from 48 HDs was also included.

    Article Snippet: To assess the antibody response to different SARS-CoV-2 proteins or different fragments of the spike protein, SARS-CoV-2 S (spike protein, 1203 aa), S1 (675 aa), S2 (533 aa), RBD (228 aa), and N (424 aa) proteins were obtained from Sino Biological Inc. and in-house ELISAs for detection of SARS-CoV-2–specific IgG antibody were established.

    Techniques:

    Kinetics of IgM and IgG responses against SARS-CoV-2 in severely and mildly ill patients. IgM ( A ) and IgG ( B ) antibody responses against the N protein of SARS-CoV-2 in plasma were detected. Serial plasma samples were collected from 12 severely ill and 11 mildly ill patients infected with SARS-CoV-2. Forty-eight plasma samples previously collected from healthy volunteer donors in 2017–2018 were used as a healthy donor group (HD). Positive (PC) and negative (NC) controls provided by detection kit were included to ensure test validity.

    Journal: The Journal of Clinical Investigation

    Article Title: Kinetics of viral load and antibody response in relation to COVID-19 severity

    doi: 10.1172/JCI138759

    Figure Lengend Snippet: Kinetics of IgM and IgG responses against SARS-CoV-2 in severely and mildly ill patients. IgM ( A ) and IgG ( B ) antibody responses against the N protein of SARS-CoV-2 in plasma were detected. Serial plasma samples were collected from 12 severely ill and 11 mildly ill patients infected with SARS-CoV-2. Forty-eight plasma samples previously collected from healthy volunteer donors in 2017–2018 were used as a healthy donor group (HD). Positive (PC) and negative (NC) controls provided by detection kit were included to ensure test validity.

    Article Snippet: To assess the antibody response to different SARS-CoV-2 proteins or different fragments of the spike protein, SARS-CoV-2 S (spike protein, 1203 aa), S1 (675 aa), S2 (533 aa), RBD (228 aa), and N (424 aa) proteins were obtained from Sino Biological Inc. and in-house ELISAs for detection of SARS-CoV-2–specific IgG antibody were established.

    Techniques: Infection

    IgG cross-reactivity analysis between the other 6 human CoVs and SARS-CoV-2. Spike (S) and nucleoprotein (N) of the other 6 human CoVs were used as coated target antigens to establish an in-house ELISA to detect IgG antibody for HCoV-229E ( A ), HCoV-NL63 ( B ), HCoV-HKU1 ( C ), HCoV-OC43 ( D ), SARS-CoV ( E ), and MERS-CoV ( F ). Plasma from 96 HDs and 23 SARS-CoV-2–infected patients were used ( A – F ). Severe indicates a severely ill patient with COVID-19; mild indicates a mildly ill patient with COVID-19; HD indicates healthy donors. Plasma samples from 18 SARS-convalescent ( E ) and 12 MERS-convalescent ( F ) patients were used as controls, respectively. A Student’s t test was used to analyze differences in mean values between groups ( A – F ). Experiments for each virus were independently carried out. Multiple comparisons following 1-way ANOVA and Kruskal-Wallis test were performed for statistical analysis. Bonferroni’s correction was used to avoid inflation of experiment-wise Type I error. In A – D , a difference was considered statistically significant when the P value was lower than 0.0167 (0.05/3); * P ≤ 0.0167, ** P ≤ 0.0033, *** P ≤ 0.00033, **** P ≤ 0.000033. In E and F , a difference was considered statistically significant when the P value was lower than 0.0083 (0.05/6); † P ≤ 0.0083, †† P ≤ 0.0017, ‡ P ≤ 0.00017, ‡‡ P ≤0.000017.

    Journal: The Journal of Clinical Investigation

    Article Title: Kinetics of viral load and antibody response in relation to COVID-19 severity

    doi: 10.1172/JCI138759

    Figure Lengend Snippet: IgG cross-reactivity analysis between the other 6 human CoVs and SARS-CoV-2. Spike (S) and nucleoprotein (N) of the other 6 human CoVs were used as coated target antigens to establish an in-house ELISA to detect IgG antibody for HCoV-229E ( A ), HCoV-NL63 ( B ), HCoV-HKU1 ( C ), HCoV-OC43 ( D ), SARS-CoV ( E ), and MERS-CoV ( F ). Plasma from 96 HDs and 23 SARS-CoV-2–infected patients were used ( A – F ). Severe indicates a severely ill patient with COVID-19; mild indicates a mildly ill patient with COVID-19; HD indicates healthy donors. Plasma samples from 18 SARS-convalescent ( E ) and 12 MERS-convalescent ( F ) patients were used as controls, respectively. A Student’s t test was used to analyze differences in mean values between groups ( A – F ). Experiments for each virus were independently carried out. Multiple comparisons following 1-way ANOVA and Kruskal-Wallis test were performed for statistical analysis. Bonferroni’s correction was used to avoid inflation of experiment-wise Type I error. In A – D , a difference was considered statistically significant when the P value was lower than 0.0167 (0.05/3); * P ≤ 0.0167, ** P ≤ 0.0033, *** P ≤ 0.00033, **** P ≤ 0.000033. In E and F , a difference was considered statistically significant when the P value was lower than 0.0083 (0.05/6); † P ≤ 0.0083, †† P ≤ 0.0017, ‡ P ≤ 0.00017, ‡‡ P ≤0.000017.

    Article Snippet: To assess the antibody response to different SARS-CoV-2 proteins or different fragments of the spike protein, SARS-CoV-2 S (spike protein, 1203 aa), S1 (675 aa), S2 (533 aa), RBD (228 aa), and N (424 aa) proteins were obtained from Sino Biological Inc. and in-house ELISAs for detection of SARS-CoV-2–specific IgG antibody were established.

    Techniques: Enzyme-linked Immunosorbent Assay, Infection

    IgG antibody response against different SARS-CoV-2 proteins or fragments. Plasma samples collected at different time points after admission were used for IgG detection in different protein-coated ELISAs: S (1209 aa) ( A ), S1 (681 aa) ( B ), RBD (457 aa) ( C ), S2 (539 aa) ( D ), and N (430 aa) ( E ). Eleven plasma samples from HDs were used as controls. The correlations among IgG levels against different viral proteins were analyzed and summarized. Pearson’s correlation coefficient was used to assess the relationship among antiviral IgG levels of different proteins ( F ). A Student’s t test was used to analyze differences in mean values between groups A – E . A P value less than 0.05 was considered to be statistically significant. ** P ≤ 0.01.

    Journal: The Journal of Clinical Investigation

    Article Title: Kinetics of viral load and antibody response in relation to COVID-19 severity

    doi: 10.1172/JCI138759

    Figure Lengend Snippet: IgG antibody response against different SARS-CoV-2 proteins or fragments. Plasma samples collected at different time points after admission were used for IgG detection in different protein-coated ELISAs: S (1209 aa) ( A ), S1 (681 aa) ( B ), RBD (457 aa) ( C ), S2 (539 aa) ( D ), and N (430 aa) ( E ). Eleven plasma samples from HDs were used as controls. The correlations among IgG levels against different viral proteins were analyzed and summarized. Pearson’s correlation coefficient was used to assess the relationship among antiviral IgG levels of different proteins ( F ). A Student’s t test was used to analyze differences in mean values between groups A – E . A P value less than 0.05 was considered to be statistically significant. ** P ≤ 0.01.

    Article Snippet: To assess the antibody response to different SARS-CoV-2 proteins or different fragments of the spike protein, SARS-CoV-2 S (spike protein, 1203 aa), S1 (675 aa), S2 (533 aa), RBD (228 aa), and N (424 aa) proteins were obtained from Sino Biological Inc. and in-house ELISAs for detection of SARS-CoV-2–specific IgG antibody were established.

    Techniques:

    Neutralizing and cross-protection of antibody response against SARS-CoV-2 in severely and mildly ill patients. Serial plasma samples were collected from severely ill ( A ) and mildly ill ( B ) patients infected with SARS-CoV-2, and used for authentic SARS-CoV2 neutralizing test FRNT 50 to evaluate kinetics of neutralizing antibodies in SARS-CoV-2 infected patients. Plasma samples collected 3 weeks after onset were used to compare cross-neutralizing antibodies between severely ill and mildly ill patients with SARS-CoV-2 and SARS-CoV–convalescent patients using SARS-CoV-2 pseudotype ( C ) and authentic virus ( D ) at a fixed dilution (1:40). A Student’s t test was used to analyze differences in mean values between groups. Experiments for each virus were independently carried out. Multiple comparisons following 1-way ANOVA and Kruskal-Wallis tests were performed for statistical analysis. Bonferroni’s correction was used to avoid inflation of experiment-wise Type I error. There were a total of 10 pairwise comparisons among 5 groups. Hence, a difference was considered statistically significant when the P value was lower than 0.005 (0.05/10). **** P ≤ 0.0001 ( C and D ). Pearson’s correlation coefficient was used to assess the relationship between neutralizing titer and S- and N-specific IgG levels ( E and F ); viral loads of respiratory specimens ( G ) were analyzed.

    Journal: The Journal of Clinical Investigation

    Article Title: Kinetics of viral load and antibody response in relation to COVID-19 severity

    doi: 10.1172/JCI138759

    Figure Lengend Snippet: Neutralizing and cross-protection of antibody response against SARS-CoV-2 in severely and mildly ill patients. Serial plasma samples were collected from severely ill ( A ) and mildly ill ( B ) patients infected with SARS-CoV-2, and used for authentic SARS-CoV2 neutralizing test FRNT 50 to evaluate kinetics of neutralizing antibodies in SARS-CoV-2 infected patients. Plasma samples collected 3 weeks after onset were used to compare cross-neutralizing antibodies between severely ill and mildly ill patients with SARS-CoV-2 and SARS-CoV–convalescent patients using SARS-CoV-2 pseudotype ( C ) and authentic virus ( D ) at a fixed dilution (1:40). A Student’s t test was used to analyze differences in mean values between groups. Experiments for each virus were independently carried out. Multiple comparisons following 1-way ANOVA and Kruskal-Wallis tests were performed for statistical analysis. Bonferroni’s correction was used to avoid inflation of experiment-wise Type I error. There were a total of 10 pairwise comparisons among 5 groups. Hence, a difference was considered statistically significant when the P value was lower than 0.005 (0.05/10). **** P ≤ 0.0001 ( C and D ). Pearson’s correlation coefficient was used to assess the relationship between neutralizing titer and S- and N-specific IgG levels ( E and F ); viral loads of respiratory specimens ( G ) were analyzed.

    Article Snippet: To assess the antibody response to different SARS-CoV-2 proteins or different fragments of the spike protein, SARS-CoV-2 S (spike protein, 1203 aa), S1 (675 aa), S2 (533 aa), RBD (228 aa), and N (424 aa) proteins were obtained from Sino Biological Inc. and in-house ELISAs for detection of SARS-CoV-2–specific IgG antibody were established.

    Techniques: Infection

    Temporal profile of serial viral load from different tissue samples. Viral loads in patients in the ICU (PT1–PT12) and patients with mild disease (PT13–PT23) as measured by nasal swabs ( A ), pharyngeal swabs ( B ), sputum ( C ), feces ( D ), urine ( E ), and blood ( F ). The x axis indicates the number of days after onset, the y axis indicates patient numbers. Heatmap of Ct values of viral loads were shown. A Ct value less than 37 indicates the presence of SARS-CoV-2 nucleic acid in the sample. Each square represents 1 sample detected and the gray squares indicate that the sample was viral nucleotide acid–negative.

    Journal: The Journal of Clinical Investigation

    Article Title: Kinetics of viral load and antibody response in relation to COVID-19 severity

    doi: 10.1172/JCI138759

    Figure Lengend Snippet: Temporal profile of serial viral load from different tissue samples. Viral loads in patients in the ICU (PT1–PT12) and patients with mild disease (PT13–PT23) as measured by nasal swabs ( A ), pharyngeal swabs ( B ), sputum ( C ), feces ( D ), urine ( E ), and blood ( F ). The x axis indicates the number of days after onset, the y axis indicates patient numbers. Heatmap of Ct values of viral loads were shown. A Ct value less than 37 indicates the presence of SARS-CoV-2 nucleic acid in the sample. Each square represents 1 sample detected and the gray squares indicate that the sample was viral nucleotide acid–negative.

    Article Snippet: To assess the antibody response to different SARS-CoV-2 proteins or different fragments of the spike protein, SARS-CoV-2 S (spike protein, 1203 aa), S1 (675 aa), S2 (533 aa), RBD (228 aa), and N (424 aa) proteins were obtained from Sino Biological Inc. and in-house ELISAs for detection of SARS-CoV-2–specific IgG antibody were established.

    Techniques:

    Characteristics of SARS-CoV-2 reactive mAbs. SARS-CoV-2 antigen specificity as predicted was validated by ELISA for a subset of monoclonal antibodies to SARS-CoV-2. Data are represented as mean ± SD. Data are representative of two independent experiments. a SARS-CoV-2 RBD reactive mAbs, ( b ) SARS-CoV-2 NP reactive mAbs. c Maximum-likelihood phylogenetic tree of dominant clonotypes and antigen labeled antibodies’ heavy chains. Unrooted phylogenetic tree depicting the genetic relationships among all VH genes of antigen labeled antibodies. Branch lengths were scaled so that sequence relatedness can be readily assessed. Hvdj sequences with the same VH gene usage are shown in the same color at the clades. Hvdj sequences with the same antigen-labeled quantity are shown in the same color at the branch tips (red, blue and green means high, median and low antigen-labeled quantity respectively, and black means none). d Neutralization of C2767P3S and C14646P3S mAbs against pseudotyped SARS-CoV-2 virus in Huh-7 cells. Influenza relative mAbs CR9114 was used as a negative control. Data are represented as mean ± SEM. Data are representative of two independent experiments. e C2767P3S mAb was tested using the plaque reduction neutralization assay. DMEM was used as a negative control. Data are representative of two independent experiments. f Neutralization of C2767P3S mAb against live SARS-CoV-2 virus in Vero E6 cells. DMEM was used as a negative control. Data are represented as mean ± SEM. Data are representative of two independent experiments. g C14646P3S mAb was tested using the plaque reduction neutralization assay. DMEM was used as a negative control. Data are representative of two independent experiments. h Neutralization of C14646P3S mAb against live SARS-CoV-2 virus in Vero E6 cells. DMEM was used as a negative control. Data are represented as mean ± SEM. Data are representative of two independent experiments

    Journal: Signal Transduction and Targeted Therapy

    Article Title: Rapid isolation and immune profiling of SARS-CoV-2 specific memory B cell in convalescent COVID-19 patients via LIBRA-seq

    doi: 10.1038/s41392-021-00610-7

    Figure Lengend Snippet: Characteristics of SARS-CoV-2 reactive mAbs. SARS-CoV-2 antigen specificity as predicted was validated by ELISA for a subset of monoclonal antibodies to SARS-CoV-2. Data are represented as mean ± SD. Data are representative of two independent experiments. a SARS-CoV-2 RBD reactive mAbs, ( b ) SARS-CoV-2 NP reactive mAbs. c Maximum-likelihood phylogenetic tree of dominant clonotypes and antigen labeled antibodies’ heavy chains. Unrooted phylogenetic tree depicting the genetic relationships among all VH genes of antigen labeled antibodies. Branch lengths were scaled so that sequence relatedness can be readily assessed. Hvdj sequences with the same VH gene usage are shown in the same color at the clades. Hvdj sequences with the same antigen-labeled quantity are shown in the same color at the branch tips (red, blue and green means high, median and low antigen-labeled quantity respectively, and black means none). d Neutralization of C2767P3S and C14646P3S mAbs against pseudotyped SARS-CoV-2 virus in Huh-7 cells. Influenza relative mAbs CR9114 was used as a negative control. Data are represented as mean ± SEM. Data are representative of two independent experiments. e C2767P3S mAb was tested using the plaque reduction neutralization assay. DMEM was used as a negative control. Data are representative of two independent experiments. f Neutralization of C2767P3S mAb against live SARS-CoV-2 virus in Vero E6 cells. DMEM was used as a negative control. Data are represented as mean ± SEM. Data are representative of two independent experiments. g C14646P3S mAb was tested using the plaque reduction neutralization assay. DMEM was used as a negative control. Data are representative of two independent experiments. h Neutralization of C14646P3S mAb against live SARS-CoV-2 virus in Vero E6 cells. DMEM was used as a negative control. Data are represented as mean ± SEM. Data are representative of two independent experiments

    Article Snippet: The SARS-CoV-2 S1 antigen protein was purchased from Sino Biological Inc.

    Techniques: Enzyme-linked Immunosorbent Assay, Labeling, Sequencing, Neutralization, Negative Control

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-20465-w

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

    Article Snippet: Recombinant proteins and antibodiesFor mouse immunization, recombinant SARS-CoV-2 RBD (residues R319 to F541) fused with a C-terminal mouse IgG1 Fc tag (RBD-mFc) was purchased from Sino Biological Inc (Beijing, China).

    Techniques: Labeling, Binding Assay

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-20465-w

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

    Article Snippet: Recombinant proteins and antibodiesFor mouse immunization, recombinant SARS-CoV-2 RBD (residues R319 to F541) fused with a C-terminal mouse IgG1 Fc tag (RBD-mFc) was purchased from Sino Biological Inc (Beijing, China).

    Techniques: 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.

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-20465-w

    Figure Lengend Snippet: 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.

    Article Snippet: Recombinant proteins and antibodiesFor mouse immunization, recombinant SARS-CoV-2 RBD (residues R319 to F541) fused with a C-terminal mouse IgG1 Fc tag (RBD-mFc) was purchased from Sino Biological Inc (Beijing, China).

    Techniques: 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.

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-20465-w

    Figure Lengend Snippet: 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.

    Article Snippet: Recombinant proteins and antibodiesFor mouse immunization, recombinant SARS-CoV-2 RBD (residues R319 to F541) fused with a C-terminal mouse IgG1 Fc tag (RBD-mFc) was purchased from Sino Biological Inc (Beijing, China).

    Techniques: 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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-20465-w

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

    Article Snippet: Recombinant proteins and antibodiesFor mouse immunization, recombinant SARS-CoV-2 RBD (residues R319 to F541) fused with a C-terminal mouse IgG1 Fc tag (RBD-mFc) was purchased from Sino Biological Inc (Beijing, China).

    Techniques: 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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-020-20465-w

    Figure Lengend Snippet: 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

    Article Snippet: Recombinant proteins and antibodiesFor mouse immunization, recombinant SARS-CoV-2 RBD (residues R319 to F541) fused with a C-terminal mouse IgG1 Fc tag (RBD-mFc) was purchased from Sino Biological Inc (Beijing, China).

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