recombinant sars cov 2 s protein  (Sino Biological)


Bioz Verified Symbol Sino Biological is a verified supplier
Bioz Manufacturer Symbol Sino Biological manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Name:
    SARS CoV 2 2019 nCoV Nucleocapsid His Recombinant Protein
    Description:
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Nucleocapsid Protein YP 009724397 2 Met1 Ala419 335Gly Ala was expressed with a polyhistidine tag at the N terminus
    Catalog Number:
    40588-v07e
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus NP Protein 2019-nCoV, coronavirus Nucleocapsid Protein 2019-nCoV, coronavirus Nucleoprotein Protein 2019-nCoV, cov np Protein 2019-nCoV, ncov NP Protein 2019-nCoV, NCP-CoV Nucleocapsid Protein 2019-nCoV, novel coronavirus NP Protein 2019-nCoV, novel coronavirus Nucleocapsid Protein 2019-nCoV, novel coronavirus Nucleoprotein Protein 2019-nCoV, np Protein 2019-nCoV, nucleocapsid Protein 2019-nCoV, Nucleoprotein Protein 2019-nCoV
    Host:
    E. coli
    Buy from Supplier


    Structured Review

    Sino Biological recombinant sars cov 2 s protein
    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 <t>SARS-CoV-2</t> 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.
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Nucleocapsid Protein YP 009724397 2 Met1 Ala419 335Gly Ala was expressed with a polyhistidine tag at the N terminus
    https://www.bioz.com/result/recombinant sars cov 2 s protein/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant sars cov 2 s protein - by Bioz Stars, 2021-03
    94/100 stars

    Images

    1) 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

    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

    2) 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

    3) Product Images from "Viral presence and immunopathology in patients with lethal COVID-19: a prospective autopsy cohort study"

    Article Title: Viral presence and immunopathology in patients with lethal COVID-19: a prospective autopsy cohort study

    Journal: The Lancet. Microbe

    doi: 10.1016/S2666-5247(20)30144-0

    SARS-CoV-2 tropism Two antibodies against SARS-CoV-2 nucleocapsid protein were used to detect infected cells. Staining of the same cell type by both antibodies was considered as positive immunoreactivity. (A) Median disease course per organ group with immunoreactivity for SARS-CoV-2. Error bars indicate the range. Adipose tissue consisted of mesocolic fat or omental fat (or both). The appendix (p 7) ) shows SARS-CoV-2 positivity per organ per patient. (B) Stain against SARS-CoV-2 in the lung of a patient with mainly exudative diffuse alveolar damage and a disease course of 5 days. Immunoreactive cells were abundant ( > 10% infected cells per high-power field). Infected cells were pneumocytes along the alveolar walls, stromal cells in the septae, endothelial cells in the small blood vessels, and alveolar macrophages. (C) Stain against SARS-CoV-2 in the lung later in the disease course (patient with a disease course of 22 days) revealed only scattered immunoreactive cells, conceivably pneumocytes. (D) Stain against SARS-CoV-2 in the lung later in the disease course (patient with a disease course of 31 days) also showed immunopositivity in a respiratory cell lining a bronchiole. (E) Stain against SARS-CoV-2 in the lung early in the disease course (patient with a disease course of 5 days [also represented in part B]) showed immunopositive endothelial cells in septal capillaries. (F) Stain against SARS-CoV-2 in the kidney (patient with a disease course of 24 days) revealed immunoreactivity of the distal tubular epithelial cells. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2.
    Figure Legend Snippet: SARS-CoV-2 tropism Two antibodies against SARS-CoV-2 nucleocapsid protein were used to detect infected cells. Staining of the same cell type by both antibodies was considered as positive immunoreactivity. (A) Median disease course per organ group with immunoreactivity for SARS-CoV-2. Error bars indicate the range. Adipose tissue consisted of mesocolic fat or omental fat (or both). The appendix (p 7) ) shows SARS-CoV-2 positivity per organ per patient. (B) Stain against SARS-CoV-2 in the lung of a patient with mainly exudative diffuse alveolar damage and a disease course of 5 days. Immunoreactive cells were abundant ( > 10% infected cells per high-power field). Infected cells were pneumocytes along the alveolar walls, stromal cells in the septae, endothelial cells in the small blood vessels, and alveolar macrophages. (C) Stain against SARS-CoV-2 in the lung later in the disease course (patient with a disease course of 22 days) revealed only scattered immunoreactive cells, conceivably pneumocytes. (D) Stain against SARS-CoV-2 in the lung later in the disease course (patient with a disease course of 31 days) also showed immunopositivity in a respiratory cell lining a bronchiole. (E) Stain against SARS-CoV-2 in the lung early in the disease course (patient with a disease course of 5 days [also represented in part B]) showed immunopositive endothelial cells in septal capillaries. (F) Stain against SARS-CoV-2 in the kidney (patient with a disease course of 24 days) revealed immunoreactivity of the distal tubular epithelial cells. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2.

    Techniques Used: Infection, Staining

    4) 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

    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

    5) 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

    6) Product Images from "A single dose of an adenovirus-vectored vaccine provides protection against SARS-CoV-2 challenge"

    Article Title: A single dose of an adenovirus-vectored vaccine provides protection against SARS-CoV-2 challenge

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17972-1

    Adenovirus-based vaccine design and immunogenicity in mice. a Schematic of SARS-CoV-2 S immunogens. b Western blot of transgene expression from (1) S original , (2) SP tPA add S mature , (3) SP original add S optimized , (4) SP tPA add S optimized , and (5) an empty plasmid transfected in HEK293 cells. BALB/c mice ( n = 10 per group) received a single immunization with different doses of Ad5-nCoV or Ad5 vector by the IM or IN route. c – h , Humoral immune responses were assessed at weeks 0, 2, 4, 6 and 8 following vaccination by S-specific ELISA c , f , SARS-CoV-2 NAb titration (MN 50 ) d , g and SARS-CoV-2 PNAb titration e , h with n = 10 biologically independent animals per group. Data represent the individual titre of each animal and the connecting lines reflect the geometric means of the titres. i , j , Cellular immune responses were assessed at day 14 following vaccination in the 5 × 10 8 VP dose groups by intracellular cytokine staining assays with n = 10 biologically independent animals per group. Data are presented as mean ± s.e.m. Statistical significance was determined by Kruskal–Wallis ANOVA with Dunn’s multiple comparisons tests. S = spike protein, SP = signal peptide, tPA = tissue plasminogen activator. Dotted line = the limit of detection. Source data are provided as a Source Data file.
    Figure Legend Snippet: Adenovirus-based vaccine design and immunogenicity in mice. a Schematic of SARS-CoV-2 S immunogens. b Western blot of transgene expression from (1) S original , (2) SP tPA add S mature , (3) SP original add S optimized , (4) SP tPA add S optimized , and (5) an empty plasmid transfected in HEK293 cells. BALB/c mice ( n = 10 per group) received a single immunization with different doses of Ad5-nCoV or Ad5 vector by the IM or IN route. c – h , Humoral immune responses were assessed at weeks 0, 2, 4, 6 and 8 following vaccination by S-specific ELISA c , f , SARS-CoV-2 NAb titration (MN 50 ) d , g and SARS-CoV-2 PNAb titration e , h with n = 10 biologically independent animals per group. Data represent the individual titre of each animal and the connecting lines reflect the geometric means of the titres. i , j , Cellular immune responses were assessed at day 14 following vaccination in the 5 × 10 8 VP dose groups by intracellular cytokine staining assays with n = 10 biologically independent animals per group. Data are presented as mean ± s.e.m. Statistical significance was determined by Kruskal–Wallis ANOVA with Dunn’s multiple comparisons tests. S = spike protein, SP = signal peptide, tPA = tissue plasminogen activator. Dotted line = the limit of detection. Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay, Western Blot, Expressing, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Titration, Staining

    7) Product Images from "Human Hematopoietic Stem, Progenitor, and Immune Cells Respond Ex Vivo to SARS-CoV-2 Spike Protein"

    Article Title: Human Hematopoietic Stem, Progenitor, and Immune Cells Respond Ex Vivo to SARS-CoV-2 Spike Protein

    Journal: Stem Cell Reviews and Reports

    doi: 10.1007/s12015-020-10056-z

    Cord blood HSCs/HPCs exhibit reduced colony forming capacity in the presence of SARS-CoV-2 S protein. ( a ) CD34+ enriched cells were plated at 100,000 cells/mL in media with stimulating growth factors (rhuTPO/rhuSCF/rhuFLT3L) and with 1 μg/mL recombinant S protein or PBS control and grown for 4 days in 5% O 2 and 5% CO 2 at 37 °C. Viable cells were counted using a hemocytometer and Trypan Blue viability stain. n = 5, stats: paired t-test, different symbols indicate the same cord blood unit in different treatment conditions. ( b-c ) CD34+ enriched cells were either taken for direct plating (unstimulated) or were grown for 24 h with stimulating growth factors (stimulated). 300 CD34+ enriched cells were plated in triplicate with the indicated doses of recombinant S protein or PBS control in 1% v /v methylcellulose with growth factors and serum and grown for 12 days in 5% O 2 and 5% CO 2 at 37 °C. CFU/1x10 6 cells were calculated. Stats: general linearized modeling including stimulated and unstimulated cells at varying doses in the same model followed by ANOVA with TukeyHSD post hoc tests. Significance codes indicate comparison of sample to 0 ng/mL (PBS) control in unstimulated cells. ( d-e ) 350 freshly harvested CD34+ cells were plated in triplicate in 1% v/v methylcellulose with serum and growth factors and with PBS control, 250 ng/mL recombinant S protein alone, 250 ng/mL S protein pre-incubated with 250 ng/mL SARS-CoV-2 neutralizing antibody (Antibody), or 250 ng/mL S protein with 250 ng/mL Angiotensin1–7 (Ang1–7) and grown for 12 days in 5% O 2 and 5% CO 2 at 37 °C. Total CFU/1x10 6 cells were calculated. Stats: general linearized modeling followed by ANOVA with TukeyHSD post hoc tests, matched symbols indicate the same cord blood unit in different treatment conditions. Shown are significance codes comparing all treatment levels to PBS control. * P
    Figure Legend Snippet: Cord blood HSCs/HPCs exhibit reduced colony forming capacity in the presence of SARS-CoV-2 S protein. ( a ) CD34+ enriched cells were plated at 100,000 cells/mL in media with stimulating growth factors (rhuTPO/rhuSCF/rhuFLT3L) and with 1 μg/mL recombinant S protein or PBS control and grown for 4 days in 5% O 2 and 5% CO 2 at 37 °C. Viable cells were counted using a hemocytometer and Trypan Blue viability stain. n = 5, stats: paired t-test, different symbols indicate the same cord blood unit in different treatment conditions. ( b-c ) CD34+ enriched cells were either taken for direct plating (unstimulated) or were grown for 24 h with stimulating growth factors (stimulated). 300 CD34+ enriched cells were plated in triplicate with the indicated doses of recombinant S protein or PBS control in 1% v /v methylcellulose with growth factors and serum and grown for 12 days in 5% O 2 and 5% CO 2 at 37 °C. CFU/1x10 6 cells were calculated. Stats: general linearized modeling including stimulated and unstimulated cells at varying doses in the same model followed by ANOVA with TukeyHSD post hoc tests. Significance codes indicate comparison of sample to 0 ng/mL (PBS) control in unstimulated cells. ( d-e ) 350 freshly harvested CD34+ cells were plated in triplicate in 1% v/v methylcellulose with serum and growth factors and with PBS control, 250 ng/mL recombinant S protein alone, 250 ng/mL S protein pre-incubated with 250 ng/mL SARS-CoV-2 neutralizing antibody (Antibody), or 250 ng/mL S protein with 250 ng/mL Angiotensin1–7 (Ang1–7) and grown for 12 days in 5% O 2 and 5% CO 2 at 37 °C. Total CFU/1x10 6 cells were calculated. Stats: general linearized modeling followed by ANOVA with TukeyHSD post hoc tests, matched symbols indicate the same cord blood unit in different treatment conditions. Shown are significance codes comparing all treatment levels to PBS control. * P

    Techniques Used: Recombinant, Staining, Incubation

    Cord blood HSCs/HPCs exhibit reduced expansion in the presence of SARS-CoV-2 S protein. ( a-h ) CD34+ enriched cells were plated at 100,000–200,000 cells/mL in media with stimulating growth factors and with PBS control, 1 μg/mL recombinant S protein alone, 1 μg/mL S protein pre-incubated with 1 μg/mL SARS-CoV-2 neutralizing antibody (Antibody), 1 μg/mL S protein pre-incubated with 1 μg/mL rhu ACE2, or 1 μg/mL S protein with 1 μg/mL Angiotensin1–7 (Ang1–7) and grown for 7 days in 5% O 2 and 5% CO 2 at 37 °C. ( a-d ). Cells were then analyzed by flow cytometry for the indicated cell populations and total cell numbers were calculated or ( e-h ) 350–500 CD34+ cells were plated in triplicate in 1% v/v methylcellulose with serum and growth factors and grown for 12 days in 5% O 2 and 5% CO 2 at 37 °C. Total CFU were calculated. n = 4/2 for ( a-e )/( f-h ), stats: generalized linear modelling followed by ANOVA with TukeyHSD post hoc tests, matched colors of points for 3a-e and matched symbols for points for 3f-h indicate the same cord blood unit in different treatment conditions. For ( f-h ) significance codes shown are for the comparison of the indicated treatment PBS control. * P
    Figure Legend Snippet: Cord blood HSCs/HPCs exhibit reduced expansion in the presence of SARS-CoV-2 S protein. ( a-h ) CD34+ enriched cells were plated at 100,000–200,000 cells/mL in media with stimulating growth factors and with PBS control, 1 μg/mL recombinant S protein alone, 1 μg/mL S protein pre-incubated with 1 μg/mL SARS-CoV-2 neutralizing antibody (Antibody), 1 μg/mL S protein pre-incubated with 1 μg/mL rhu ACE2, or 1 μg/mL S protein with 1 μg/mL Angiotensin1–7 (Ang1–7) and grown for 7 days in 5% O 2 and 5% CO 2 at 37 °C. ( a-d ). Cells were then analyzed by flow cytometry for the indicated cell populations and total cell numbers were calculated or ( e-h ) 350–500 CD34+ cells were plated in triplicate in 1% v/v methylcellulose with serum and growth factors and grown for 12 days in 5% O 2 and 5% CO 2 at 37 °C. Total CFU were calculated. n = 4/2 for ( a-e )/( f-h ), stats: generalized linear modelling followed by ANOVA with TukeyHSD post hoc tests, matched colors of points for 3a-e and matched symbols for points for 3f-h indicate the same cord blood unit in different treatment conditions. For ( f-h ) significance codes shown are for the comparison of the indicated treatment PBS control. * P

    Techniques Used: Recombinant, Incubation, Flow Cytometry

    Peripheral blood cells respond ex vivo to exposure to SARS-CoV-2 S protein. ( a-d ) Low density PB was incubated for 2 h with 1 μg/mL SARS-CoV-2 S recombinant S protein or PBS control. ( a ) Representative histogram from 1 PB sample treated with S protein or PBS showing CD14 expression of forward and side scatter gated monocytes and ( b ) mean fluorescence intensities for CD14 staining. n = 4, stats: paired t-test, different colors of points indicate PBs from the same donor. ( c ) Representative contour plot from 1 PB sample treated with S protein or PBS showing CD14 expression of forward and side scatter gated monocytes, the gating strategy for CD14hi cells and ( d ) difference in CD14hi monocytes represented as a fold-change of S protein treated cells relative to PBS treated cells. n = 4, stats: paired t-test performed on total numbers of CD14hi monocytes. ( e-g ) Low density PB was incubated for 18 h with serum in the presence of 1 μg/mL SARS-CoV-2 S recombinant S protein or PBS control. ( e ) Representative contour plot from 1 PB sample showing all cells excluding debris and the monocyte gate used. ( f ) Mean values for forward scatter (FSC) and ( g ) side scatter (SSC) of monocytes from PB samples after incubation with S protein or PBS. n = 5, stats = paired t-test, different colors of points indicate PBs from the same donor. FACS = fluorescence activated flow cytometry. *P
    Figure Legend Snippet: Peripheral blood cells respond ex vivo to exposure to SARS-CoV-2 S protein. ( a-d ) Low density PB was incubated for 2 h with 1 μg/mL SARS-CoV-2 S recombinant S protein or PBS control. ( a ) Representative histogram from 1 PB sample treated with S protein or PBS showing CD14 expression of forward and side scatter gated monocytes and ( b ) mean fluorescence intensities for CD14 staining. n = 4, stats: paired t-test, different colors of points indicate PBs from the same donor. ( c ) Representative contour plot from 1 PB sample treated with S protein or PBS showing CD14 expression of forward and side scatter gated monocytes, the gating strategy for CD14hi cells and ( d ) difference in CD14hi monocytes represented as a fold-change of S protein treated cells relative to PBS treated cells. n = 4, stats: paired t-test performed on total numbers of CD14hi monocytes. ( e-g ) Low density PB was incubated for 18 h with serum in the presence of 1 μg/mL SARS-CoV-2 S recombinant S protein or PBS control. ( e ) Representative contour plot from 1 PB sample showing all cells excluding debris and the monocyte gate used. ( f ) Mean values for forward scatter (FSC) and ( g ) side scatter (SSC) of monocytes from PB samples after incubation with S protein or PBS. n = 5, stats = paired t-test, different colors of points indicate PBs from the same donor. FACS = fluorescence activated flow cytometry. *P

    Techniques Used: Ex Vivo, Incubation, Recombinant, Expressing, Fluorescence, Staining, FACS, Flow Cytometry

    8) 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

    9) Product Images from "A Highly Immunogenic Measles Virus-based Th1-biased COVID-19 Vaccine"

    Article Title: A Highly Immunogenic Measles Virus-based Th1-biased COVID-19 Vaccine

    Journal: bioRxiv

    doi: 10.1101/2020.07.11.198291

    Ag-specific proliferation of SARS-CoV-2 S-specific T cells. Proliferation assay using splenocytes of mice vaccinated on days 0 and 28 with indicated viruses, isolated 21 days after boost immunization, after co-culture with DC2.4 dendritic cell line transgenic for SARS-CoV-2 S (SARS2-S) or untransduced parental DC2.4 (untr.). Depicted are the percentages of ( A ) CD4 + or ( B ) CD8 + T cells with low CFSE staining, indicating proliferation in the samples. To analyze cellular α-MeV responses, splenocytes were stimulated with 10 μg/ml MeV bulk antigens or were left unstimulated (medium). The reactivity of splenocytes was confirmed by concanavalin A (ConA) treatment (10 μg/ml). Results for splenocytes of vaccinated mice are displayed individually and the trend between paired unstimulated and re-stimulated samples is outlined (n = 2-4). One-tailed Mann-Whitney t-test. *, p
    Figure Legend Snippet: Ag-specific proliferation of SARS-CoV-2 S-specific T cells. Proliferation assay using splenocytes of mice vaccinated on days 0 and 28 with indicated viruses, isolated 21 days after boost immunization, after co-culture with DC2.4 dendritic cell line transgenic for SARS-CoV-2 S (SARS2-S) or untransduced parental DC2.4 (untr.). Depicted are the percentages of ( A ) CD4 + or ( B ) CD8 + T cells with low CFSE staining, indicating proliferation in the samples. To analyze cellular α-MeV responses, splenocytes were stimulated with 10 μg/ml MeV bulk antigens or were left unstimulated (medium). The reactivity of splenocytes was confirmed by concanavalin A (ConA) treatment (10 μg/ml). Results for splenocytes of vaccinated mice are displayed individually and the trend between paired unstimulated and re-stimulated samples is outlined (n = 2-4). One-tailed Mann-Whitney t-test. *, p

    Techniques Used: Proliferation Assay, Mouse Assay, Isolation, Co-Culture Assay, Transgenic Assay, Staining, One-tailed Test, MANN-WHITNEY

    Antigen-specific killing activity of SARS-CoV-2 S-specific T cells. Killing assay using splenocytes of mice vaccinated on days 0 and 28 isolated 21 days after the second immunization. Splenocytes were co-cultured with DC2.4 ( A ) or with antigen-presenting DC2.4-SARS2-S ( B ) cells or for 6 days. Activated CTLs were then co-cultured with EL-4 green -SARS2-S target cells (Antigen) and EL-4 red control cells (NC) at indicated E:T ratios for 4 h. Ratio of living target to non-target cells (Antigen:NC) was determined by flow cytometry. Depicted are means and standard deviation of each group (open diamonds, MeV vac2 -SARS2-S(H); filled circles, mock; filled squares, MV vac2 -ATU(P); grey triangles: S protein + Alum) (n = 3 - 5). For statistical analysis of grouped ELISpot data, two-way ANOVA analysis was applied with paired Tukey’s Multi comparison test used as post hoc test. ****, p
    Figure Legend Snippet: Antigen-specific killing activity of SARS-CoV-2 S-specific T cells. Killing assay using splenocytes of mice vaccinated on days 0 and 28 isolated 21 days after the second immunization. Splenocytes were co-cultured with DC2.4 ( A ) or with antigen-presenting DC2.4-SARS2-S ( B ) cells or for 6 days. Activated CTLs were then co-cultured with EL-4 green -SARS2-S target cells (Antigen) and EL-4 red control cells (NC) at indicated E:T ratios for 4 h. Ratio of living target to non-target cells (Antigen:NC) was determined by flow cytometry. Depicted are means and standard deviation of each group (open diamonds, MeV vac2 -SARS2-S(H); filled circles, mock; filled squares, MV vac2 -ATU(P); grey triangles: S protein + Alum) (n = 3 - 5). For statistical analysis of grouped ELISpot data, two-way ANOVA analysis was applied with paired Tukey’s Multi comparison test used as post hoc test. ****, p

    Techniques Used: Activity Assay, Mouse Assay, Isolation, Cell Culture, Flow Cytometry, Standard Deviation, Enzyme-linked Immunospot

    Induction of a-SARS-CoV-2 S and a-MeV specific antibodies. Sera of mice vaccinated on days 0 and 28 with indicated viruses or Alum-adjuvanted S protein were sampled on day 0 (A, D, E, F), day 28 after prime- (B, E, H, K) and day 49 after boost-immunization (C, F, I, L) and analyzed for antibodies specific for SARS-CoV-2 S or MeV. Medium-inoculated mice served as mock. Pan-IgG binding to recombinant SARS-CoV S (A – C) or MeV bulk antigens (G – I) were determined by ELISA via the specific OD 450 nm value. Depicted are means and respective standard deviation of the mean (SEM) of each group (n = 5 - 6). Virus neutralizing titers (VNT) in vaccinated mice for SARS-CoV-2 (D - F) or MeV (J – L) were calculated as reciprocal of the highest dilution abolishing infectivity. ( M) SARS-CoV-2 VNT of 4 human Covid-19 reconvalescent sera. Dots represent single individuals; horizontal line represents mean per group. For statistical analysis of VNT data, one-way ANOVA was performed in combination with Tukey’s Multi comparison test to compare all pair means.
    Figure Legend Snippet: Induction of a-SARS-CoV-2 S and a-MeV specific antibodies. Sera of mice vaccinated on days 0 and 28 with indicated viruses or Alum-adjuvanted S protein were sampled on day 0 (A, D, E, F), day 28 after prime- (B, E, H, K) and day 49 after boost-immunization (C, F, I, L) and analyzed for antibodies specific for SARS-CoV-2 S or MeV. Medium-inoculated mice served as mock. Pan-IgG binding to recombinant SARS-CoV S (A – C) or MeV bulk antigens (G – I) were determined by ELISA via the specific OD 450 nm value. Depicted are means and respective standard deviation of the mean (SEM) of each group (n = 5 - 6). Virus neutralizing titers (VNT) in vaccinated mice for SARS-CoV-2 (D - F) or MeV (J – L) were calculated as reciprocal of the highest dilution abolishing infectivity. ( M) SARS-CoV-2 VNT of 4 human Covid-19 reconvalescent sera. Dots represent single individuals; horizontal line represents mean per group. For statistical analysis of VNT data, one-way ANOVA was performed in combination with Tukey’s Multi comparison test to compare all pair means.

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

    Immune bias of induced responses. To analyze skewing of immune responses towards Th1- or Th2-biased immunity ( A ) sera and ( B ) splenocytes of vaccinated mice depicted before were analyzed. ( A ) Sera of mice vaccinated on days 0 and 28 with MeV vac2 -SARS2-S(H) or Alum-adjuvanted S protein already shown in Fig. 2 were analysed for IgG1- or IgG2a-type antibodies specific for SARS-CoV-2 S. IgG1 (left panel) or IgG2a (right panel) binding to recombinant SARS-CoV S were determined by ELISA via the specific OD 450 nm value. Depicted are means and respective standard deviation of the mean (SEM) of each group (n = 5 - 6). ( B ) Splenocytes of the same mice already shown in Figs. 3 to 6 were analysed by multiplex cytokine analysis for secretion of typical marker cytokines in the supernatant after re-stimulation by co-culture with antigen-presenting DC2.4-SARS2-S cells. DC2.4 cells served as non-specific control stimulus. Dots represent individual animals, horizontal bars mean per group (n = 4 - 5). IFN-γ: upper limit of detection (ULOD): 2015.2 pg/mL; IL-6: ULOD: 3992,4 pg/mL; IL-17a lower limit of detection (LLOD): 0.473 pg/mL; IL-4 LLOD: 0.095 pg/mL; IL-5 LLOD: 0.685 pg/mL; IL-13 LLOD: 3.463 pg/mL. For statistical analysis of grouped multiplex data, two-way ANOVA analysis was applied with paired Tukey’s Multi comparison test as post hoc test. *, p
    Figure Legend Snippet: Immune bias of induced responses. To analyze skewing of immune responses towards Th1- or Th2-biased immunity ( A ) sera and ( B ) splenocytes of vaccinated mice depicted before were analyzed. ( A ) Sera of mice vaccinated on days 0 and 28 with MeV vac2 -SARS2-S(H) or Alum-adjuvanted S protein already shown in Fig. 2 were analysed for IgG1- or IgG2a-type antibodies specific for SARS-CoV-2 S. IgG1 (left panel) or IgG2a (right panel) binding to recombinant SARS-CoV S were determined by ELISA via the specific OD 450 nm value. Depicted are means and respective standard deviation of the mean (SEM) of each group (n = 5 - 6). ( B ) Splenocytes of the same mice already shown in Figs. 3 to 6 were analysed by multiplex cytokine analysis for secretion of typical marker cytokines in the supernatant after re-stimulation by co-culture with antigen-presenting DC2.4-SARS2-S cells. DC2.4 cells served as non-specific control stimulus. Dots represent individual animals, horizontal bars mean per group (n = 4 - 5). IFN-γ: upper limit of detection (ULOD): 2015.2 pg/mL; IL-6: ULOD: 3992,4 pg/mL; IL-17a lower limit of detection (LLOD): 0.473 pg/mL; IL-4 LLOD: 0.095 pg/mL; IL-5 LLOD: 0.685 pg/mL; IL-13 LLOD: 3.463 pg/mL. For statistical analysis of grouped multiplex data, two-way ANOVA analysis was applied with paired Tukey’s Multi comparison test as post hoc test. *, p

    Techniques Used: Mouse Assay, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Standard Deviation, Multiplex Assay, Marker, Co-Culture Assay

    Characterization of fusogenic phenotype of MeV vac2 -SARS2-S(P) and MeV vac2 -SARS2-S(H). (A ) Photographs of fusion activity of Vero cells infected at an MOI of 0.01 with MeV vac2 -SARS2-S(P) or MeVvac2-SARS2-S(H) encoding SARS-CoV-2 S in additional transcription units post P or post H, respectively, in direct comparison to MV vac2 -ATU(P) or MV vac2 -GFP(H) control vaccine viruses or MV NSe -GFP(N) hyperfusogenic oncolytic MeV. Representative picture of one out of three independent experiments. Scale bar represents 200 mm. ( B ) Cell fusion was quantified 30 h after infection. For statistical analysis, one-way ANOVA was performed in combination with Tukey’s Multi comparison test to compare all pair means. *, p
    Figure Legend Snippet: Characterization of fusogenic phenotype of MeV vac2 -SARS2-S(P) and MeV vac2 -SARS2-S(H). (A ) Photographs of fusion activity of Vero cells infected at an MOI of 0.01 with MeV vac2 -SARS2-S(P) or MeVvac2-SARS2-S(H) encoding SARS-CoV-2 S in additional transcription units post P or post H, respectively, in direct comparison to MV vac2 -ATU(P) or MV vac2 -GFP(H) control vaccine viruses or MV NSe -GFP(N) hyperfusogenic oncolytic MeV. Representative picture of one out of three independent experiments. Scale bar represents 200 mm. ( B ) Cell fusion was quantified 30 h after infection. For statistical analysis, one-way ANOVA was performed in combination with Tukey’s Multi comparison test to compare all pair means. *, p

    Techniques Used: Activity Assay, Infection

    Generation and in vitro characterization of MeV vac2 -SARS2-S(P) and MeV vac2 -SARS2-S(H). (A) Schematic depiction of full-length SARS-CoV-2 S and recombinant MeV vac2 genomes used for expression of this antigen (lower schemes). Antigen or antigen encoding genes are depicted in dark grey; MeV viral gene cassettes (in light grey) are annotated. MluI and AatII restriction sites used for cloning of antigen-genes into post P or post H ATU are highlighted (B) Immunoblot analysis of Vero cells infected at an MOI of 0.01 with MeV vac2 -SARS2-S(P), MeV vac2 -SARS2-S(H), or MV vac2 -ATU(P) (MV vac2 ) as depicted above lanes. Uninfected cells served as mock. Blots were probed using rabbit polyclonal anti-SARS spike antibody (upper blot) or mAb reactive against MeV-N (lower blot). Arrows indicate specific bands. ( C, D ) Growth kinetics of recombinant MeV on Vero cells infected at an MOI of 0.03 with MV vac2 -ATU(P) or MeV vac2 -SARS2-S encoding extra genes in post H or post P. Titers of samples prepared at indicated time points post infection were titrated on Vero cells. Means and standard deviations of three to five independent experiments are presented. ( E ) SARS-CoV-2 S protein expression in Vero cells was verified via immunoperoxidase monolayer assay. 50× magnification; scale bar, 500 μm.
    Figure Legend Snippet: Generation and in vitro characterization of MeV vac2 -SARS2-S(P) and MeV vac2 -SARS2-S(H). (A) Schematic depiction of full-length SARS-CoV-2 S and recombinant MeV vac2 genomes used for expression of this antigen (lower schemes). Antigen or antigen encoding genes are depicted in dark grey; MeV viral gene cassettes (in light grey) are annotated. MluI and AatII restriction sites used for cloning of antigen-genes into post P or post H ATU are highlighted (B) Immunoblot analysis of Vero cells infected at an MOI of 0.01 with MeV vac2 -SARS2-S(P), MeV vac2 -SARS2-S(H), or MV vac2 -ATU(P) (MV vac2 ) as depicted above lanes. Uninfected cells served as mock. Blots were probed using rabbit polyclonal anti-SARS spike antibody (upper blot) or mAb reactive against MeV-N (lower blot). Arrows indicate specific bands. ( C, D ) Growth kinetics of recombinant MeV on Vero cells infected at an MOI of 0.03 with MV vac2 -ATU(P) or MeV vac2 -SARS2-S encoding extra genes in post H or post P. Titers of samples prepared at indicated time points post infection were titrated on Vero cells. Means and standard deviations of three to five independent experiments are presented. ( E ) SARS-CoV-2 S protein expression in Vero cells was verified via immunoperoxidase monolayer assay. 50× magnification; scale bar, 500 μm.

    Techniques Used: In Vitro, Recombinant, Expressing, Clone Assay, Infection

    Expression of SARS-CoV-2 S protein in Vero and 293T cells. Photographic depiction of fusion activity in Vero or 293T cells 48 h after transfection with 1 µg of SARS-CoV-2 S expression plasmid of control DNA. One representative out of three independent experiments is shown. Scale bar represents 100 mm.
    Figure Legend Snippet: Expression of SARS-CoV-2 S protein in Vero and 293T cells. Photographic depiction of fusion activity in Vero or 293T cells 48 h after transfection with 1 µg of SARS-CoV-2 S expression plasmid of control DNA. One representative out of three independent experiments is shown. Scale bar represents 100 mm.

    Techniques Used: Expressing, Activity Assay, Transfection, Plasmid Preparation

    Secretion of IFN-γ after antigen-specific re-stimulation of splenocytes. IFN-γ ELISpot analysis using splenocytes of mice vaccinated on days 0 and 28 with indicated vaccines, isolated 21 days after boost immunization, and after co-culture with DC2.4 or JAWSII dendritic cell lines transgenic for SARS-CoV-2 S (SARS2-S) or untransduced controls (untr.). To analyze cellular responses directed against MeV, splenocytes were stimulated with 10 μg/mL MeV bulk antigens or were left unstimulated as controls (medium). The reactivity of splenocytes was confirmed by Concanavalin A (ConA) treatment (10 μg/mL). The number of cells per 1×10 6 splenocytes represent the amount of cells expressing IFN-γ upon re-stimulation. Dots represent individual animals, horizontal bars mean per group (n = 5 - 6). Samples above the upper detection limit (ULOD) were displayed as such. For statistical analysis of grouped ELISpot data, two-way ANOVA analysis was applied with paired Tukey’s Multi comparison test used as post hoc test. ns, not significant (p > 0.05); ****, p
    Figure Legend Snippet: Secretion of IFN-γ after antigen-specific re-stimulation of splenocytes. IFN-γ ELISpot analysis using splenocytes of mice vaccinated on days 0 and 28 with indicated vaccines, isolated 21 days after boost immunization, and after co-culture with DC2.4 or JAWSII dendritic cell lines transgenic for SARS-CoV-2 S (SARS2-S) or untransduced controls (untr.). To analyze cellular responses directed against MeV, splenocytes were stimulated with 10 μg/mL MeV bulk antigens or were left unstimulated as controls (medium). The reactivity of splenocytes was confirmed by Concanavalin A (ConA) treatment (10 μg/mL). The number of cells per 1×10 6 splenocytes represent the amount of cells expressing IFN-γ upon re-stimulation. Dots represent individual animals, horizontal bars mean per group (n = 5 - 6). Samples above the upper detection limit (ULOD) were displayed as such. For statistical analysis of grouped ELISpot data, two-way ANOVA analysis was applied with paired Tukey’s Multi comparison test used as post hoc test. ns, not significant (p > 0.05); ****, p

    Techniques Used: Enzyme-linked Immunospot, Mouse Assay, Isolation, Co-Culture Assay, Transgenic Assay, Expressing

    10) 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

    Related Articles

    Recombinant:

    Article Title: Stereotypic Neutralizing VH Clonotypes Against SARS-CoV-2 RBD in COVID-19 Patients and the Healthy Population
    Article Snippet: The genes encoding the selected scFv clones were cloned into a modified mammalian expression vector, containing the hIgG1 Fc regions (hFc) or hCκ at the C-terminus , , before being transfected and purified by affinity chromatography, as described above. .. ELISAFirst, 100 ng of each recombinant SARS-CoV-2 S (Sino Biological Inc.), S1 (Sino Biological Inc.), S2 (Sino Biological Inc.), NP (Sino Biological Inc.), RBD, RBD mutants, SARS-CoV RBD (Sino Biological Inc.), MERS-CoV S (Sino Biological Inc.), RBD (Sino Biological Inc.), S2 (Sino Biological Inc.) proteins were added to microtiter plates (Costar), in coating buffer (0.1 M sodium bicarbonate, pH 8.6). .. After incubation at 4°C, overnight, and blocking with 3% bovine serum albumin (BSA) in PBS, for 1 h at 37°C, serially diluted plasma (5-fold, 6 dilutions, starting from 1:100) or scFv-hFc (5-fold, 12 dilutions, starting from 1,000 or 500 nM) in blocking buffer was added to individual wells and incubated for 1, h at 37°C.

    Article Title: Stereotypic Neutralizing VH Clonotypes Against SARS-CoV-2 RBD in COVID-19 Patients and the Healthy Population
    Article Snippet: Absorbance was measured at 405 nm or 650 nm, using a microplate spectrophotometer (Multiskan GO; Thermo Scientific). .. Flow cytometry The recombinant SARS-CoV-2 S protein (200 nM), fused with a polyhistidine tag at the C-terminus (Sino Biological Inc.), was incubated with scFv-hFc fusion proteins at a final concentration of either 200 nM (equimolar) or 600 nM (morlar ratio of 1:3), in 50 μL of 1% (w/v) BSA in PBS, containing 0.02% (w/v) sodium azide (FACS buffer), at 37°C for 1 h. Irrelevant scFv-hFc or scFv-hCκ fusion proteins were used as negative controls. .. Vero E6 cells (ACE2+ ) were seeded into v-bottom 96-well plates (Corning, Corning, NY, USA), at a density of 1.5 × 105 cells per well.

    Article Title: Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals
    Article Snippet: Absorbance was measured at 405 or 650 nm, respectively, using a microplate spectrophotometer (Multiskan GO, Thermo Fisher Scientific Inc.). .. Flow cytometry The recombinant SARS-CoV-2 S protein (200 nM), fused with a HIS tag at the C terminus (Sino Biological Inc.), was incubated with scFv-hFc fusion proteins at a final concentration of either 200 nM (equimolar) or 600 nM (molar ratio of 1:3), in 50 μl of 1% (w/v) BSA in PBS, containing 0.02% (w/v) sodium azide [flow cytometry staining buffer (FCSB)], at 37°C for 1 hour. ..

    Article Title: A single dose of an adenovirus-vectored vaccine provides protection against SARS-CoV-2 challenge
    Article Snippet: .. ELISAFor SARS-CoV-2 S-specific IgG assays in mice, 96-well polystyrene high-binding microplates (Corning, USA) were coated with 2 μg/mL recombinant SARS-CoV-2 S protein purified from insect cells (Sino Biological, China) in carbonate-bicarbonate buffer pH 9.6, and the plates were incubated at 4 °C overnight. .. The plates were then blocked at 37 °C for 1 h with PBS pH 7.4 in 5% skim milk (blocking buffer) and washed with PBST.

    Article Title: Human Hematopoietic Stem, Progenitor, and Immune Cells Respond Ex Vivo to SARS-CoV-2 Spike Protein
    Article Snippet: To test whether direct effects of S protein on HPC colony formation capacity can be neutralized, 350 CD34+ enriched cells were plated in triplicate with 250 ng recombinant SARS-CoV-2 S protein alone, 250 ng SARS-CoV-2 antibody (Sino Biological 40,591-MM43, Beijing, China) alone, 250 ng soluble rhu ACE2 (Sigma Aldrich) alone, 250 ng Angiotensin1–7 (Sigma Aldrich) alone, 250 ng recombinant S protein pre-incubated at room temperature for 30 min with 250 ng SARS-CoV-2 antibody, 250 ng recombinant S protein pre-incubated at 37 °C for 30 min with 250 ng soluble rhu ACE2 (Sigma Aldrich), or 250 ng recombinant S protein with 250 ng Angiotensin1–7 (Sigma Aldrich). .. To test the whether the effects of S protein on HSC/HPC ex vivo expansion could be neutralized, 100,000 CD34+ enriched cells were plated in liquid culture media with growth factors and with 1 μg/mL recombinant SARS-CoV-2 S protein alone, 1 μg/mL SARS-CoV-2 antibody (Sino Biological 40,591-MM43, Beijing, China) alone, 1 μg/mL soluble rhu ACE2 (Sigma Aldrich) alone, 1 μg/mL Angiotensin1–7 (Sigma Aldrich) alone, 1 μg/mL recombinant S protein pre-incubated at room temperature for 30 min with 1 μg/mL SARS-CoV-2 antibody, 1 μg/mL recombinant S protein pre-incubated at 37 °C for 30 min with 1 μg/mL soluble rhu ACE2 (Sigma Aldrich), or 1 μg/mL recombinant S protein with 1 μg/mL Angiotensin1–7 (Sigma Aldrich). .. Cells were expanded for 7 days in a humidified 5% O2 , 5% CO2 , 37 °C incubator, then 1 × 106 cells were stained for flow cytometry analysis and 500 cells were plated in triplicate in methylcellulose for HPC CFU assay.

    Article Title: SARS-CoV-2 induces robust germinal center CD4 T follicular helper cell responses in rhesus macaques
    Article Snippet: .. Binding antibody multiplex assay (BAMA) for IgG and IgM antibodies to S1, S2 and N proteins A customized BAMA was developed to simultaneously measure antibodies to the following recombinant SARS-CoV-2 proteins (all from SinoBiologicals, Wayne, PA): S1 (#40591-V08H), S2 extracellular domain (#40590-V08B) and nucleocapsid (N; #40588-V08B). .. Briefly, proteins were dialyzed in PBS and conjugated to Bioplex Pro carboxylated magnetic beads (BioRad, Hercules, CA) .

    Article Title: Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals
    Article Snippet: Then, IgG2/4 was purified by affinity chromatography using MabSelect columns with the AKTA pure chromatography system (GE Healthcare) following the manufacturer’s protocol. .. Enzyme-linked immunosorbent assayOne hundred nanograms of each recombinant SARS-CoV-2 S (Sino Biological Inc.), S1 (Sino Biological Inc.), S1 D614G (Sino Biological Inc.), S2 (Sino Biological Inc.), nucleocapsid (N) (Sino Biological Inc.), RBD, RBD mutants, SARS-CoV RBD (Sino Biological Inc.), MERS-CoV S (Sino Biological Inc.), RBD (Sino Biological Inc.), and S2 (Sino Biological Inc.) proteins were added to microtiter plates (CoStar), in coating buffer (0.1 M sodium bicarbonate, pH 8.6). .. After incubation at 4°C overnight and blocking with 3% bovine serum albumin (BSA) in PBS, for 1 hour at 37°C, serially diluted plasma (fivefold, six dilutions, starting from 1:100) or scFv-hFc (fivefold, 12 dilutions, starting from 1000 or 500 nM) in blocking buffer was added to individual wells and incubated for 1 hour at 37°C.

    Flow Cytometry:

    Article Title: Stereotypic Neutralizing VH Clonotypes Against SARS-CoV-2 RBD in COVID-19 Patients and the Healthy Population
    Article Snippet: Absorbance was measured at 405 nm or 650 nm, using a microplate spectrophotometer (Multiskan GO; Thermo Scientific). .. Flow cytometry The recombinant SARS-CoV-2 S protein (200 nM), fused with a polyhistidine tag at the C-terminus (Sino Biological Inc.), was incubated with scFv-hFc fusion proteins at a final concentration of either 200 nM (equimolar) or 600 nM (morlar ratio of 1:3), in 50 μL of 1% (w/v) BSA in PBS, containing 0.02% (w/v) sodium azide (FACS buffer), at 37°C for 1 h. Irrelevant scFv-hFc or scFv-hCκ fusion proteins were used as negative controls. .. Vero E6 cells (ACE2+ ) were seeded into v-bottom 96-well plates (Corning, Corning, NY, USA), at a density of 1.5 × 105 cells per well.

    Article Title: Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals
    Article Snippet: Absorbance was measured at 405 or 650 nm, respectively, using a microplate spectrophotometer (Multiskan GO, Thermo Fisher Scientific Inc.). .. Flow cytometry The recombinant SARS-CoV-2 S protein (200 nM), fused with a HIS tag at the C terminus (Sino Biological Inc.), was incubated with scFv-hFc fusion proteins at a final concentration of either 200 nM (equimolar) or 600 nM (molar ratio of 1:3), in 50 μl of 1% (w/v) BSA in PBS, containing 0.02% (w/v) sodium azide [flow cytometry staining buffer (FCSB)], at 37°C for 1 hour. ..

    Incubation:

    Article Title: Stereotypic Neutralizing VH Clonotypes Against SARS-CoV-2 RBD in COVID-19 Patients and the Healthy Population
    Article Snippet: Absorbance was measured at 405 nm or 650 nm, using a microplate spectrophotometer (Multiskan GO; Thermo Scientific). .. Flow cytometry The recombinant SARS-CoV-2 S protein (200 nM), fused with a polyhistidine tag at the C-terminus (Sino Biological Inc.), was incubated with scFv-hFc fusion proteins at a final concentration of either 200 nM (equimolar) or 600 nM (morlar ratio of 1:3), in 50 μL of 1% (w/v) BSA in PBS, containing 0.02% (w/v) sodium azide (FACS buffer), at 37°C for 1 h. Irrelevant scFv-hFc or scFv-hCκ fusion proteins were used as negative controls. .. Vero E6 cells (ACE2+ ) were seeded into v-bottom 96-well plates (Corning, Corning, NY, USA), at a density of 1.5 × 105 cells per well.

    Article Title: Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals
    Article Snippet: Absorbance was measured at 405 or 650 nm, respectively, using a microplate spectrophotometer (Multiskan GO, Thermo Fisher Scientific Inc.). .. Flow cytometry The recombinant SARS-CoV-2 S protein (200 nM), fused with a HIS tag at the C terminus (Sino Biological Inc.), was incubated with scFv-hFc fusion proteins at a final concentration of either 200 nM (equimolar) or 600 nM (molar ratio of 1:3), in 50 μl of 1% (w/v) BSA in PBS, containing 0.02% (w/v) sodium azide [flow cytometry staining buffer (FCSB)], at 37°C for 1 hour. ..

    Article Title: A single dose of an adenovirus-vectored vaccine provides protection against SARS-CoV-2 challenge
    Article Snippet: .. ELISAFor SARS-CoV-2 S-specific IgG assays in mice, 96-well polystyrene high-binding microplates (Corning, USA) were coated with 2 μg/mL recombinant SARS-CoV-2 S protein purified from insect cells (Sino Biological, China) in carbonate-bicarbonate buffer pH 9.6, and the plates were incubated at 4 °C overnight. .. The plates were then blocked at 37 °C for 1 h with PBS pH 7.4 in 5% skim milk (blocking buffer) and washed with PBST.

    Concentration Assay:

    Article Title: Stereotypic Neutralizing VH Clonotypes Against SARS-CoV-2 RBD in COVID-19 Patients and the Healthy Population
    Article Snippet: Absorbance was measured at 405 nm or 650 nm, using a microplate spectrophotometer (Multiskan GO; Thermo Scientific). .. Flow cytometry The recombinant SARS-CoV-2 S protein (200 nM), fused with a polyhistidine tag at the C-terminus (Sino Biological Inc.), was incubated with scFv-hFc fusion proteins at a final concentration of either 200 nM (equimolar) or 600 nM (morlar ratio of 1:3), in 50 μL of 1% (w/v) BSA in PBS, containing 0.02% (w/v) sodium azide (FACS buffer), at 37°C for 1 h. Irrelevant scFv-hFc or scFv-hCκ fusion proteins were used as negative controls. .. Vero E6 cells (ACE2+ ) were seeded into v-bottom 96-well plates (Corning, Corning, NY, USA), at a density of 1.5 × 105 cells per well.

    Article Title: Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals
    Article Snippet: Absorbance was measured at 405 or 650 nm, respectively, using a microplate spectrophotometer (Multiskan GO, Thermo Fisher Scientific Inc.). .. Flow cytometry The recombinant SARS-CoV-2 S protein (200 nM), fused with a HIS tag at the C terminus (Sino Biological Inc.), was incubated with scFv-hFc fusion proteins at a final concentration of either 200 nM (equimolar) or 600 nM (molar ratio of 1:3), in 50 μl of 1% (w/v) BSA in PBS, containing 0.02% (w/v) sodium azide [flow cytometry staining buffer (FCSB)], at 37°C for 1 hour. ..

    FACS:

    Article Title: Stereotypic Neutralizing VH Clonotypes Against SARS-CoV-2 RBD in COVID-19 Patients and the Healthy Population
    Article Snippet: Absorbance was measured at 405 nm or 650 nm, using a microplate spectrophotometer (Multiskan GO; Thermo Scientific). .. Flow cytometry The recombinant SARS-CoV-2 S protein (200 nM), fused with a polyhistidine tag at the C-terminus (Sino Biological Inc.), was incubated with scFv-hFc fusion proteins at a final concentration of either 200 nM (equimolar) or 600 nM (morlar ratio of 1:3), in 50 μL of 1% (w/v) BSA in PBS, containing 0.02% (w/v) sodium azide (FACS buffer), at 37°C for 1 h. Irrelevant scFv-hFc or scFv-hCκ fusion proteins were used as negative controls. .. Vero E6 cells (ACE2+ ) were seeded into v-bottom 96-well plates (Corning, Corning, NY, USA), at a density of 1.5 × 105 cells per well.

    Staining:

    Article Title: Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals
    Article Snippet: Absorbance was measured at 405 or 650 nm, respectively, using a microplate spectrophotometer (Multiskan GO, Thermo Fisher Scientific Inc.). .. Flow cytometry The recombinant SARS-CoV-2 S protein (200 nM), fused with a HIS tag at the C terminus (Sino Biological Inc.), was incubated with scFv-hFc fusion proteins at a final concentration of either 200 nM (equimolar) or 600 nM (molar ratio of 1:3), in 50 μl of 1% (w/v) BSA in PBS, containing 0.02% (w/v) sodium azide [flow cytometry staining buffer (FCSB)], at 37°C for 1 hour. ..

    Mouse Assay:

    Article Title: A single dose of an adenovirus-vectored vaccine provides protection against SARS-CoV-2 challenge
    Article Snippet: .. ELISAFor SARS-CoV-2 S-specific IgG assays in mice, 96-well polystyrene high-binding microplates (Corning, USA) were coated with 2 μg/mL recombinant SARS-CoV-2 S protein purified from insect cells (Sino Biological, China) in carbonate-bicarbonate buffer pH 9.6, and the plates were incubated at 4 °C overnight. .. The plates were then blocked at 37 °C for 1 h with PBS pH 7.4 in 5% skim milk (blocking buffer) and washed with PBST.

    Purification:

    Article Title: A single dose of an adenovirus-vectored vaccine provides protection against SARS-CoV-2 challenge
    Article Snippet: .. ELISAFor SARS-CoV-2 S-specific IgG assays in mice, 96-well polystyrene high-binding microplates (Corning, USA) were coated with 2 μg/mL recombinant SARS-CoV-2 S protein purified from insect cells (Sino Biological, China) in carbonate-bicarbonate buffer pH 9.6, and the plates were incubated at 4 °C overnight. .. The plates were then blocked at 37 °C for 1 h with PBS pH 7.4 in 5% skim milk (blocking buffer) and washed with PBST.

    Ex Vivo:

    Article Title: Human Hematopoietic Stem, Progenitor, and Immune Cells Respond Ex Vivo to SARS-CoV-2 Spike Protein
    Article Snippet: To test whether direct effects of S protein on HPC colony formation capacity can be neutralized, 350 CD34+ enriched cells were plated in triplicate with 250 ng recombinant SARS-CoV-2 S protein alone, 250 ng SARS-CoV-2 antibody (Sino Biological 40,591-MM43, Beijing, China) alone, 250 ng soluble rhu ACE2 (Sigma Aldrich) alone, 250 ng Angiotensin1–7 (Sigma Aldrich) alone, 250 ng recombinant S protein pre-incubated at room temperature for 30 min with 250 ng SARS-CoV-2 antibody, 250 ng recombinant S protein pre-incubated at 37 °C for 30 min with 250 ng soluble rhu ACE2 (Sigma Aldrich), or 250 ng recombinant S protein with 250 ng Angiotensin1–7 (Sigma Aldrich). .. To test the whether the effects of S protein on HSC/HPC ex vivo expansion could be neutralized, 100,000 CD34+ enriched cells were plated in liquid culture media with growth factors and with 1 μg/mL recombinant SARS-CoV-2 S protein alone, 1 μg/mL SARS-CoV-2 antibody (Sino Biological 40,591-MM43, Beijing, China) alone, 1 μg/mL soluble rhu ACE2 (Sigma Aldrich) alone, 1 μg/mL Angiotensin1–7 (Sigma Aldrich) alone, 1 μg/mL recombinant S protein pre-incubated at room temperature for 30 min with 1 μg/mL SARS-CoV-2 antibody, 1 μg/mL recombinant S protein pre-incubated at 37 °C for 30 min with 1 μg/mL soluble rhu ACE2 (Sigma Aldrich), or 1 μg/mL recombinant S protein with 1 μg/mL Angiotensin1–7 (Sigma Aldrich). .. Cells were expanded for 7 days in a humidified 5% O2 , 5% CO2 , 37 °C incubator, then 1 × 106 cells were stained for flow cytometry analysis and 500 cells were plated in triplicate in methylcellulose for HPC CFU assay.

    Binding Assay:

    Article Title: SARS-CoV-2 induces robust germinal center CD4 T follicular helper cell responses in rhesus macaques
    Article Snippet: .. Binding antibody multiplex assay (BAMA) for IgG and IgM antibodies to S1, S2 and N proteins A customized BAMA was developed to simultaneously measure antibodies to the following recombinant SARS-CoV-2 proteins (all from SinoBiologicals, Wayne, PA): S1 (#40591-V08H), S2 extracellular domain (#40590-V08B) and nucleocapsid (N; #40588-V08B). .. Briefly, proteins were dialyzed in PBS and conjugated to Bioplex Pro carboxylated magnetic beads (BioRad, Hercules, CA) .

    Multiplex Assay:

    Article Title: SARS-CoV-2 induces robust germinal center CD4 T follicular helper cell responses in rhesus macaques
    Article Snippet: .. Binding antibody multiplex assay (BAMA) for IgG and IgM antibodies to S1, S2 and N proteins A customized BAMA was developed to simultaneously measure antibodies to the following recombinant SARS-CoV-2 proteins (all from SinoBiologicals, Wayne, PA): S1 (#40591-V08H), S2 extracellular domain (#40590-V08B) and nucleocapsid (N; #40588-V08B). .. Briefly, proteins were dialyzed in PBS and conjugated to Bioplex Pro carboxylated magnetic beads (BioRad, Hercules, CA) .

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Sino Biological recombinant sars cov 2 s protein
    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 <t>SARS-CoV-2</t> 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.
    Recombinant Sars Cov 2 S Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant sars cov 2 s protein/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant sars cov 2 s protein - by Bioz Stars, 2021-03
    94/100 stars
      Buy from Supplier

    Image Search Results


    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.

    Journal: Science Translational Medicine

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

    doi: 10.1126/scitranslmed.abd6990

    Figure Lengend 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.

    Article Snippet: Flow cytometry The recombinant SARS-CoV-2 S protein (200 nM), fused with a HIS tag at the C terminus (Sino Biological Inc.), was incubated with scFv-hFc fusion proteins at a final concentration of either 200 nM (equimolar) or 600 nM (molar ratio of 1:3), in 50 μl of 1% (w/v) BSA in PBS, containing 0.02% (w/v) sodium azide [flow cytometry staining buffer (FCSB)], at 37°C for 1 hour.

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

    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.

    Journal: Science Translational Medicine

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

    doi: 10.1126/scitranslmed.abd6990

    Figure Lengend 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.

    Article Snippet: Flow cytometry The recombinant SARS-CoV-2 S protein (200 nM), fused with a HIS tag at the C terminus (Sino Biological Inc.), was incubated with scFv-hFc fusion proteins at a final concentration of either 200 nM (equimolar) or 600 nM (molar ratio of 1:3), in 50 μl of 1% (w/v) BSA in PBS, containing 0.02% (w/v) sodium azide [flow cytometry staining buffer (FCSB)], at 37°C for 1 hour.

    Techniques: Recombinant, Mutagenesis, Incubation

    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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.06.26.174557

    Figure Lengend 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.

    Article Snippet: ELISAFirst, 100 ng of each recombinant SARS-CoV-2 S (Sino Biological Inc.), S1 (Sino Biological Inc.), S2 (Sino Biological Inc.), NP (Sino Biological Inc.), RBD, RBD mutants, SARS-CoV RBD (Sino Biological Inc.), MERS-CoV S (Sino Biological Inc.), RBD (Sino Biological Inc.), S2 (Sino Biological Inc.) proteins were added to microtiter plates (Costar), in coating buffer (0.1 M sodium bicarbonate, pH 8.6).

    Techniques: 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

    Journal: bioRxiv

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

    doi: 10.1101/2020.06.26.174557

    Figure Lengend 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

    Article Snippet: ELISAFirst, 100 ng of each recombinant SARS-CoV-2 S (Sino Biological Inc.), S1 (Sino Biological Inc.), S2 (Sino Biological Inc.), NP (Sino Biological Inc.), RBD, RBD mutants, SARS-CoV RBD (Sino Biological Inc.), MERS-CoV S (Sino Biological Inc.), RBD (Sino Biological Inc.), S2 (Sino Biological Inc.) proteins were added to microtiter plates (Costar), in coating buffer (0.1 M sodium bicarbonate, pH 8.6).

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

    SARS-CoV-2 tropism Two antibodies against SARS-CoV-2 nucleocapsid protein were used to detect infected cells. Staining of the same cell type by both antibodies was considered as positive immunoreactivity. (A) Median disease course per organ group with immunoreactivity for SARS-CoV-2. Error bars indicate the range. Adipose tissue consisted of mesocolic fat or omental fat (or both). The appendix (p 7) ) shows SARS-CoV-2 positivity per organ per patient. (B) Stain against SARS-CoV-2 in the lung of a patient with mainly exudative diffuse alveolar damage and a disease course of 5 days. Immunoreactive cells were abundant ( > 10% infected cells per high-power field). Infected cells were pneumocytes along the alveolar walls, stromal cells in the septae, endothelial cells in the small blood vessels, and alveolar macrophages. (C) Stain against SARS-CoV-2 in the lung later in the disease course (patient with a disease course of 22 days) revealed only scattered immunoreactive cells, conceivably pneumocytes. (D) Stain against SARS-CoV-2 in the lung later in the disease course (patient with a disease course of 31 days) also showed immunopositivity in a respiratory cell lining a bronchiole. (E) Stain against SARS-CoV-2 in the lung early in the disease course (patient with a disease course of 5 days [also represented in part B]) showed immunopositive endothelial cells in septal capillaries. (F) Stain against SARS-CoV-2 in the kidney (patient with a disease course of 24 days) revealed immunoreactivity of the distal tubular epithelial cells. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2.

    Journal: The Lancet. Microbe

    Article Title: Viral presence and immunopathology in patients with lethal COVID-19: a prospective autopsy cohort study

    doi: 10.1016/S2666-5247(20)30144-0

    Figure Lengend Snippet: SARS-CoV-2 tropism Two antibodies against SARS-CoV-2 nucleocapsid protein were used to detect infected cells. Staining of the same cell type by both antibodies was considered as positive immunoreactivity. (A) Median disease course per organ group with immunoreactivity for SARS-CoV-2. Error bars indicate the range. Adipose tissue consisted of mesocolic fat or omental fat (or both). The appendix (p 7) ) shows SARS-CoV-2 positivity per organ per patient. (B) Stain against SARS-CoV-2 in the lung of a patient with mainly exudative diffuse alveolar damage and a disease course of 5 days. Immunoreactive cells were abundant ( > 10% infected cells per high-power field). Infected cells were pneumocytes along the alveolar walls, stromal cells in the septae, endothelial cells in the small blood vessels, and alveolar macrophages. (C) Stain against SARS-CoV-2 in the lung later in the disease course (patient with a disease course of 22 days) revealed only scattered immunoreactive cells, conceivably pneumocytes. (D) Stain against SARS-CoV-2 in the lung later in the disease course (patient with a disease course of 31 days) also showed immunopositivity in a respiratory cell lining a bronchiole. (E) Stain against SARS-CoV-2 in the lung early in the disease course (patient with a disease course of 5 days [also represented in part B]) showed immunopositive endothelial cells in septal capillaries. (F) Stain against SARS-CoV-2 in the kidney (patient with a disease course of 24 days) revealed immunoreactivity of the distal tubular epithelial cells. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2.

    Article Snippet: Presence of SARS-CoV-2 was revealed by two different antibodies against the SARS-CoV-2 nucleocapsid protein: a non-commercial monoclonal mouse antibody and a polyclonal rabbit antibody (Sino Biological and Nanommune, Irvine, CA, USA).

    Techniques: Infection, Staining