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
  • 96
    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
    Ag-specific proliferation of <t>SARS-CoV-2</t> 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
    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 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant sars cov 2 s protein - by Bioz Stars, 2021-06
    96/100 stars

    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) 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

    Related Articles

    Recombinant:

    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.

    Article Title: A Highly Immunogenic Measles Virus-based Th1-biased COVID-19 Vaccine
    Article Snippet: .. These animals were inoculated intraperitoneally (i.p.) with 1×105 TCID50 of recombinant vaccine viruses in 200 μl volume, or subcutaneously (s.c.) with 10 μg recombinant SARS-CoV-2 S protein (Sino Biological Europe, Eschborn, Germany) adjuvanted with 500 μg aluminium hydroxide (Alhydrogel adjuvant 2%, vac-alu-250, InvivoGen, San Diego, CA, USA) in 100 μl volume on days 0 and 28. ..

    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: 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: 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: 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: 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.

    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.

    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) .

    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. ..

    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.

    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.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Sino Biological sars cov 2 2019 ncov nucleocapsid his recombinant protein covid 19 nucleocapsid research
    GCG suppresses <t>SARS-CoV-2</t> replication. a , b Immunofluorescence analysis of N protein in A549-hACE2-Flag cells infected with SARS-CoV-2 for 24 h ( a ). The percentage of cells with N protein foci was quantified, n = 8 biologically independent samples, 20 randomly selected views were analyzed in each sample ( b ). Scale bar, 10 μm. c 3D images were obtained by Zeiss LSM 880 confocal microscope and reconstituted by Volocity 6.1.1 . d , e The inhibitory effect of GCG on the replication of SARS-CoV-2, n = 6 biologically independent samples ( d ). IC 50 was calculated, n = 5 biologically independent samples ( e ). The infection was performed after 1-h pretreatment of GCG. f , g Representative immunofluorescent images showed the inhibitory effect of GCG on SARS-CoV-2 N protein. 3D images were obtained by Zeiss LSM 880 confocal microscope and reconstituted by Volocity 6.1.1 ( f ). Violin plots showing foci of cells ( n = 50 biologically independent cells) from each group, lines within the plots, with 25th, 50th, and 75th percentiles marked ( g ). h Cells were infected with SARS-CoV-2 for 1 h followed by 24-h GCG treatment, n = 3 biologically independent samples. Representative images were shown. SARS-CoV-2 was used at an MOI of 1. Hoechst (blue), nuclear staining ( a , c , f ). Error bars, mean with s.d. ( b , d , e , g , h ). Two-tailed unpaired Student’s t -test, * P
    Sars Cov 2 2019 Ncov Nucleocapsid His Recombinant Protein Covid 19 Nucleocapsid Research, supplied by Sino Biological, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sars cov 2 2019 ncov nucleocapsid his recombinant protein covid 19 nucleocapsid research/product/Sino Biological
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 2019 ncov nucleocapsid his recombinant protein covid 19 nucleocapsid research - by Bioz Stars, 2021-06
    97/100 stars
      Buy from Supplier

    96
    Sino Biological recombinant sars cov 2 s protein
    Ag-specific proliferation of <t>SARS-CoV-2</t> 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
    Recombinant Sars Cov 2 S Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 96/100, 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 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant sars cov 2 s protein - by Bioz Stars, 2021-06
    96/100 stars
      Buy from Supplier

    Image Search Results


    GCG suppresses SARS-CoV-2 replication. a , b Immunofluorescence analysis of N protein in A549-hACE2-Flag cells infected with SARS-CoV-2 for 24 h ( a ). The percentage of cells with N protein foci was quantified, n = 8 biologically independent samples, 20 randomly selected views were analyzed in each sample ( b ). Scale bar, 10 μm. c 3D images were obtained by Zeiss LSM 880 confocal microscope and reconstituted by Volocity 6.1.1 . d , e The inhibitory effect of GCG on the replication of SARS-CoV-2, n = 6 biologically independent samples ( d ). IC 50 was calculated, n = 5 biologically independent samples ( e ). The infection was performed after 1-h pretreatment of GCG. f , g Representative immunofluorescent images showed the inhibitory effect of GCG on SARS-CoV-2 N protein. 3D images were obtained by Zeiss LSM 880 confocal microscope and reconstituted by Volocity 6.1.1 ( f ). Violin plots showing foci of cells ( n = 50 biologically independent cells) from each group, lines within the plots, with 25th, 50th, and 75th percentiles marked ( g ). h Cells were infected with SARS-CoV-2 for 1 h followed by 24-h GCG treatment, n = 3 biologically independent samples. Representative images were shown. SARS-CoV-2 was used at an MOI of 1. Hoechst (blue), nuclear staining ( a , c , f ). Error bars, mean with s.d. ( b , d , e , g , h ). Two-tailed unpaired Student’s t -test, * P

    Journal: Nature Communications

    Article Title: GCG inhibits SARS-CoV-2 replication by disrupting the liquid phase condensation of its nucleocapsid protein

    doi: 10.1038/s41467-021-22297-8

    Figure Lengend Snippet: GCG suppresses SARS-CoV-2 replication. a , b Immunofluorescence analysis of N protein in A549-hACE2-Flag cells infected with SARS-CoV-2 for 24 h ( a ). The percentage of cells with N protein foci was quantified, n = 8 biologically independent samples, 20 randomly selected views were analyzed in each sample ( b ). Scale bar, 10 μm. c 3D images were obtained by Zeiss LSM 880 confocal microscope and reconstituted by Volocity 6.1.1 . d , e The inhibitory effect of GCG on the replication of SARS-CoV-2, n = 6 biologically independent samples ( d ). IC 50 was calculated, n = 5 biologically independent samples ( e ). The infection was performed after 1-h pretreatment of GCG. f , g Representative immunofluorescent images showed the inhibitory effect of GCG on SARS-CoV-2 N protein. 3D images were obtained by Zeiss LSM 880 confocal microscope and reconstituted by Volocity 6.1.1 ( f ). Violin plots showing foci of cells ( n = 50 biologically independent cells) from each group, lines within the plots, with 25th, 50th, and 75th percentiles marked ( g ). h Cells were infected with SARS-CoV-2 for 1 h followed by 24-h GCG treatment, n = 3 biologically independent samples. Representative images were shown. SARS-CoV-2 was used at an MOI of 1. Hoechst (blue), nuclear staining ( a , c , f ). Error bars, mean with s.d. ( b , d , e , g , h ). Two-tailed unpaired Student’s t -test, * P

    Article Snippet: The recombinant N protein (40588-V08B) was from Sino-Biological.

    Techniques: Immunofluorescence, Infection, Microscopy, Staining, Two Tailed Test

    RNA triggers the LLPS of N protein. a Schematic drawing of SARS-CoV-2. b IDR scores of 29 proteins encoded by SARS-CoV-2 genome. FUS and mEGFP are positive and negative controls, respectively. IUPred2 and ANCHOR2 were used as prediction tools. c Time-lapse imaging of N-mEGFP protein (20 μM) in the presence of Cy5-labeled 60-nt vRNA (100 ng/μl), scale bar, 10 μm. d Representative fluorescent images of N-mEGFP-vRNA (60 nt) condensates fusion from a time-lapse movie, scale bar, 3 μm. e – g LLPS of N-mEGFP protein (20 μM) in the presence of indicated concentrations of 60-nt vRNA, scale bar, 10 μm ( e ). The partition coefficient of fluorescence intensity per droplet ( f ) and the partition coefficient of total fluorescence intensity in each view ( g ) were calculated. From left to right, n = 209, 1170, 1026, 1170 droplets ( f ) from 10 randomly selected views ( g ). h , i FRAP analysis of vRNA-induced liquid droplets of N-mEGFP protein, scale bar, 2 μm ( h ), and quantification of fluorescence intensity recovery of a photobleached N-mEGFP protein, n = 3 biologically independent experiments ( i ). The white dotted circle in h indicated the region of photobleaching. 20 μM N-mEGFP protein and 100 ng/μl 60-nt vRNA were used. Error bars, mean with s.d. ( f , g , i ). Two-tailed unpaired Student’s t -test ( f , g ), **** P

    Journal: Nature Communications

    Article Title: GCG inhibits SARS-CoV-2 replication by disrupting the liquid phase condensation of its nucleocapsid protein

    doi: 10.1038/s41467-021-22297-8

    Figure Lengend Snippet: RNA triggers the LLPS of N protein. a Schematic drawing of SARS-CoV-2. b IDR scores of 29 proteins encoded by SARS-CoV-2 genome. FUS and mEGFP are positive and negative controls, respectively. IUPred2 and ANCHOR2 were used as prediction tools. c Time-lapse imaging of N-mEGFP protein (20 μM) in the presence of Cy5-labeled 60-nt vRNA (100 ng/μl), scale bar, 10 μm. d Representative fluorescent images of N-mEGFP-vRNA (60 nt) condensates fusion from a time-lapse movie, scale bar, 3 μm. e – g LLPS of N-mEGFP protein (20 μM) in the presence of indicated concentrations of 60-nt vRNA, scale bar, 10 μm ( e ). The partition coefficient of fluorescence intensity per droplet ( f ) and the partition coefficient of total fluorescence intensity in each view ( g ) were calculated. From left to right, n = 209, 1170, 1026, 1170 droplets ( f ) from 10 randomly selected views ( g ). h , i FRAP analysis of vRNA-induced liquid droplets of N-mEGFP protein, scale bar, 2 μm ( h ), and quantification of fluorescence intensity recovery of a photobleached N-mEGFP protein, n = 3 biologically independent experiments ( i ). The white dotted circle in h indicated the region of photobleaching. 20 μM N-mEGFP protein and 100 ng/μl 60-nt vRNA were used. Error bars, mean with s.d. ( f , g , i ). Two-tailed unpaired Student’s t -test ( f , g ), **** P

    Article Snippet: The recombinant N protein (40588-V08B) was from Sino-Biological.

    Techniques: Imaging, Labeling, Fluorescence, Two Tailed Test

    NR203K/G204R gained greater ability to undergo RNA-induced LLPS. a Distribution of N gene variants among 100,849 SARS-CoV-2 genomes obtained from GISAID database. Colors indicated the nucleotide variability numbers from 100,849 genomes. The high-frequency trio-nucleotide polymorphism variant (GGG-to-AAC) is shown. b Coomassie brilliant blue-stained SDS-PAGE gel of purified variants of N-mEGFP protein. c – e LLPS of different N-mEGFP variants, in the presence of 50 ng/μl 60-nt vRNA ( c ). The partition coefficient of fluorescence intensity per droplet ( d ) and the partition coefficient of total fluorescence intensity in each view ( e ) were calculated. From left to right, n = 1232, 803, 897, 431 droplets ( d ) from 10 randomly selected views ( e ). f , g Time-lapse imaging of N R203/G204 -mEGFP and N R203K/G204R -mEGFP proteins (20 μM) in the presence of Cy5-labeled 60-nt vRNA (40 ng/μl) ( f ), and the partition coefficient ( n = 8 randomly selected views) of total fluorescence intensity in each view ( g ). Scale bars, 10 μm ( c , f ). Error bars, mean with s.d. ( d , e ) and mean with s.e.m. ( g ). Two-tailed unpaired Student’s t -test ( d , e ), **** P

    Journal: Nature Communications

    Article Title: GCG inhibits SARS-CoV-2 replication by disrupting the liquid phase condensation of its nucleocapsid protein

    doi: 10.1038/s41467-021-22297-8

    Figure Lengend Snippet: NR203K/G204R gained greater ability to undergo RNA-induced LLPS. a Distribution of N gene variants among 100,849 SARS-CoV-2 genomes obtained from GISAID database. Colors indicated the nucleotide variability numbers from 100,849 genomes. The high-frequency trio-nucleotide polymorphism variant (GGG-to-AAC) is shown. b Coomassie brilliant blue-stained SDS-PAGE gel of purified variants of N-mEGFP protein. c – e LLPS of different N-mEGFP variants, in the presence of 50 ng/μl 60-nt vRNA ( c ). The partition coefficient of fluorescence intensity per droplet ( d ) and the partition coefficient of total fluorescence intensity in each view ( e ) were calculated. From left to right, n = 1232, 803, 897, 431 droplets ( d ) from 10 randomly selected views ( e ). f , g Time-lapse imaging of N R203/G204 -mEGFP and N R203K/G204R -mEGFP proteins (20 μM) in the presence of Cy5-labeled 60-nt vRNA (40 ng/μl) ( f ), and the partition coefficient ( n = 8 randomly selected views) of total fluorescence intensity in each view ( g ). Scale bars, 10 μm ( c , f ). Error bars, mean with s.d. ( d , e ) and mean with s.e.m. ( g ). Two-tailed unpaired Student’s t -test ( d , e ), **** P

    Article Snippet: The recombinant N protein (40588-V08B) was from Sino-Biological.

    Techniques: Variant Assay, Staining, SDS Page, Purification, Fluorescence, Imaging, Labeling, Two Tailed Test

    Antibody nCoV396 compromises SARS-CoV-2 N protein-induced complement hyperactivation. a Flow scheme of the SARS-CoV-2 N protein and nCoV396 influencing the protease activity of MASP-2 in the serum from donors. b Serum-01 to 06 are used to compare the MASP-2 activity to C2 of serum sample with normal C3 (Health-110,113,117, n = 3) and serum sample with abnormal C3 (Patient-81,123,130, n = 3), and the Michaelis–Menten curves of are presented as mean (three groups of above health serum alone with patient’s serum paired data are shown in Supplementary Fig. 8a ). c The Michaelis–Menten curve of N protein-induced excessive cleavage of C2 in the presence of recombinant MASP-2 in vitro. The reaction system without N protein, the increase of N protein concentration and negative control protein (ENL) expressed in E. coli are presenting. The Michaelis–Menten curve shows the effect of increasing the N protein concentration ( d ) and antibody concentration ( f ) on the substrate C2 cleavage of MAPS-2 in the Serum-07 and Serum-08. e A Hanes plot where C2 concentration/V0 is plotted against C2 concentration with the addition of 5 μM N protein. b – d , f All samples were performed in triplicates and mean were presented. g Five serum sample from biologically independent donors ( n = 5) with abnormal serologic C3 values. (Serum-08 to −12). And we used Michaelis–Menten equation to calculate the V max (with experimental data from Fig. 4f (Serum-08), Supplementary Fig. 5b (Serum-09 to −11), and Supplementary Fig. 7a (Serum-12)). Each sample was performed in triplicates and mean values ± SEM of V max are presented. Two-sided Kruskal–Wallis test with Dunnett’s multiple comparisons test was used for comparing the V max of groups. The significant reference is 0.05.

    Journal: Nature Communications

    Article Title: A SARS-CoV-2 antibody curbs viral nucleocapsid protein-induced complement hyperactivation

    doi: 10.1038/s41467-021-23036-9

    Figure Lengend Snippet: Antibody nCoV396 compromises SARS-CoV-2 N protein-induced complement hyperactivation. a Flow scheme of the SARS-CoV-2 N protein and nCoV396 influencing the protease activity of MASP-2 in the serum from donors. b Serum-01 to 06 are used to compare the MASP-2 activity to C2 of serum sample with normal C3 (Health-110,113,117, n = 3) and serum sample with abnormal C3 (Patient-81,123,130, n = 3), and the Michaelis–Menten curves of are presented as mean (three groups of above health serum alone with patient’s serum paired data are shown in Supplementary Fig. 8a ). c The Michaelis–Menten curve of N protein-induced excessive cleavage of C2 in the presence of recombinant MASP-2 in vitro. The reaction system without N protein, the increase of N protein concentration and negative control protein (ENL) expressed in E. coli are presenting. The Michaelis–Menten curve shows the effect of increasing the N protein concentration ( d ) and antibody concentration ( f ) on the substrate C2 cleavage of MAPS-2 in the Serum-07 and Serum-08. e A Hanes plot where C2 concentration/V0 is plotted against C2 concentration with the addition of 5 μM N protein. b – d , f All samples were performed in triplicates and mean were presented. g Five serum sample from biologically independent donors ( n = 5) with abnormal serologic C3 values. (Serum-08 to −12). And we used Michaelis–Menten equation to calculate the V max (with experimental data from Fig. 4f (Serum-08), Supplementary Fig. 5b (Serum-09 to −11), and Supplementary Fig. 7a (Serum-12)). Each sample was performed in triplicates and mean values ± SEM of V max are presented. Two-sided Kruskal–Wallis test with Dunnett’s multiple comparisons test was used for comparing the V max of groups. The significant reference is 0.05.

    Article Snippet: Recombinant SARS-CoV-2 full-length N protein with a CT 6x His tag (His tag, 40588-V08B) was purchased from Sino Biological.

    Techniques: Activity Assay, Recombinant, In Vitro, Protein Concentration, Negative Control, Concentration Assay

    Complex structure of mAb nCoV396 with SARS-CoV-2 N-NTD. a Overall structure of the mAb nCoV396-SARS-CoV-2 N-NTD complex. The light chain (pink) and heavy chain (blue) of mAb nCoV396 are illustrated with the ribbon representation. SARS-CoV-2 N-NTD is illustrated with electrostatics surface, in which blue denotes a positive charge potential while red indicates a negative charge potential. b The N-NTD epitope recognized by mAb nCoV396. The interacting residues of N-NTD and nCoV396 are highlighted with the stick representation. Recognition of Q163 ( c ), K169 ( d ), and L167 ( e ) in N-NTD by mAb nCoV396. The dashed blue line represents hydrogen bonds. Hydrophobic interactions are illustrated with the dot representation. f Conformational changes of N-NTD upon mAb nCoV396 binding. The apo structure of N-NTD is colored with gray. Antibody-bound N-NTD is colored green. The N-terminus and C-terminus of the N-NTD are labeled with circles. mAb nCoV396 is illustrated with surface representation. All figures were prepared by Pymol.

    Journal: Nature Communications

    Article Title: A SARS-CoV-2 antibody curbs viral nucleocapsid protein-induced complement hyperactivation

    doi: 10.1038/s41467-021-23036-9

    Figure Lengend Snippet: Complex structure of mAb nCoV396 with SARS-CoV-2 N-NTD. a Overall structure of the mAb nCoV396-SARS-CoV-2 N-NTD complex. The light chain (pink) and heavy chain (blue) of mAb nCoV396 are illustrated with the ribbon representation. SARS-CoV-2 N-NTD is illustrated with electrostatics surface, in which blue denotes a positive charge potential while red indicates a negative charge potential. b The N-NTD epitope recognized by mAb nCoV396. The interacting residues of N-NTD and nCoV396 are highlighted with the stick representation. Recognition of Q163 ( c ), K169 ( d ), and L167 ( e ) in N-NTD by mAb nCoV396. The dashed blue line represents hydrogen bonds. Hydrophobic interactions are illustrated with the dot representation. f Conformational changes of N-NTD upon mAb nCoV396 binding. The apo structure of N-NTD is colored with gray. Antibody-bound N-NTD is colored green. The N-terminus and C-terminus of the N-NTD are labeled with circles. mAb nCoV396 is illustrated with surface representation. All figures were prepared by Pymol.

    Article Snippet: Recombinant SARS-CoV-2 full-length N protein with a CT 6x His tag (His tag, 40588-V08B) was purchased from Sino Biological.

    Techniques: Binding Assay, Labeling

    Acquisition and characterization of antibodies. Serum antibody titers of six SARS-CoV-2 convalescent patients and a healthy person (ZH0081, non-COVID-19) to the SARS-CoV-2 S ( a ) and N ( b ) proteins measured by ELISA. All samples were performed in triplicates and mean were presented. Sorting of single plasma cells ( c ) with CD38 and CD27 double-positive B cells. d To minimize false positives, each of the S1 and N proteins labeled with Phycoerythrin-canin7 (PE-Cy7) and Brilliant Violet (BV421) was used to sort antigen-specific memory B cells by FACS. e Percentage of different isotypes, VH and VL gene families of 32 isolated N-reactive antibodies. f Number of mutations in nucleotides and amino acids in VH and VL (Vκ and Vλ) of 32 N-reactive antibodies and eight S-reactive antibodies ( g ). Length of the 32 N-reactive antibodies ( h ) and eight S-reactive antibodies ( i ) in H-CDR3. f – i Data are presented as dot and mean values.

    Journal: Nature Communications

    Article Title: A SARS-CoV-2 antibody curbs viral nucleocapsid protein-induced complement hyperactivation

    doi: 10.1038/s41467-021-23036-9

    Figure Lengend Snippet: Acquisition and characterization of antibodies. Serum antibody titers of six SARS-CoV-2 convalescent patients and a healthy person (ZH0081, non-COVID-19) to the SARS-CoV-2 S ( a ) and N ( b ) proteins measured by ELISA. All samples were performed in triplicates and mean were presented. Sorting of single plasma cells ( c ) with CD38 and CD27 double-positive B cells. d To minimize false positives, each of the S1 and N proteins labeled with Phycoerythrin-canin7 (PE-Cy7) and Brilliant Violet (BV421) was used to sort antigen-specific memory B cells by FACS. e Percentage of different isotypes, VH and VL gene families of 32 isolated N-reactive antibodies. f Number of mutations in nucleotides and amino acids in VH and VL (Vκ and Vλ) of 32 N-reactive antibodies and eight S-reactive antibodies ( g ). Length of the 32 N-reactive antibodies ( h ) and eight S-reactive antibodies ( i ) in H-CDR3. f – i Data are presented as dot and mean values.

    Article Snippet: Recombinant SARS-CoV-2 full-length N protein with a CT 6x His tag (His tag, 40588-V08B) was purchased from Sino Biological.

    Techniques: Enzyme-linked Immunosorbent Assay, Labeling, FACS, Isolation

    Sequence coverage and proteotypic target peptide selection for development of a PRM assay for the SARS CoV-2 Spike protein and NP. (Top panel) Diagram of SARS CoV-2 recombinant spike glycoprotein showing the location of NTD, RBD, fusion peptide and heptad repeats 1 and 2, and the protease cleavage sites, His and Strep tags. The amino acid sequence is given below. Glycosylation sites are indicated in green. (Bottom panel) Diagram of SARS CoV-2 recombinant NP showing intrinsically disordered regions, RNA binding and dimerization regions. Phosphorylation sites (S) are indicated in yellow. Bold italics indicate sites where sequence coverage was not obtained. Peptides monitored in the spectral library are boxed, peptides selected for the final PRM assay are boxed and indicated in red text. Overall 97.1% of the spike protein and 77.2% of the NP sequence was obtained from the DDA analysis.

    Journal: Analytical Chemistry

    Article Title: Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein

    doi: 10.1021/acs.analchem.0c02288

    Figure Lengend Snippet: Sequence coverage and proteotypic target peptide selection for development of a PRM assay for the SARS CoV-2 Spike protein and NP. (Top panel) Diagram of SARS CoV-2 recombinant spike glycoprotein showing the location of NTD, RBD, fusion peptide and heptad repeats 1 and 2, and the protease cleavage sites, His and Strep tags. The amino acid sequence is given below. Glycosylation sites are indicated in green. (Bottom panel) Diagram of SARS CoV-2 recombinant NP showing intrinsically disordered regions, RNA binding and dimerization regions. Phosphorylation sites (S) are indicated in yellow. Bold italics indicate sites where sequence coverage was not obtained. Peptides monitored in the spectral library are boxed, peptides selected for the final PRM assay are boxed and indicated in red text. Overall 97.1% of the spike protein and 77.2% of the NP sequence was obtained from the DDA analysis.

    Article Snippet: SARS-CoV-2 nucleocapsid-His recombinant protein was purchased from Sino Biological (100 μg, 40588-V08B) The protein was difficult to dissolve and required treatment before S-trap preparation below with 40 μL dimethyl sulfoxide (276855—100 mL, Sigma), 126 μL 1% TFA, and 100 μL 1× S-Trap lysis buffer (5% SDS, 50 mM TEAB, pH adjusted to 7.55 using 12% phosphoric acid).

    Techniques: Sequencing, Selection, Recombinant, RNA Binding Assay

    Chromatograms and calibration curves for two best target peptides used in the PRM assay for SARS CoV-2 spike protein and nuceloprotein. The summed area under curve values for the top four transitions of each peptide were taken to generate calibration curves for quantitation. The right panels display chromatograms obtained for each of transitions shown in different colors for (A) DQVILLNK (NP) and (B) FQTLLALHR (S). Three technical replicates were run on two separate days. The chromatograms on the left of each panel show a low and high standard from the SARS CoV-2 S and NP in a mucin background. Calibration curves were constructed from the PRM data (top right) and zoomed in (bottom right) displaying mean values at the low end of the curve to show the LOD (left dotted line) and LOQ (right dotted line).

    Journal: Analytical Chemistry

    Article Title: Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein

    doi: 10.1021/acs.analchem.0c02288

    Figure Lengend Snippet: Chromatograms and calibration curves for two best target peptides used in the PRM assay for SARS CoV-2 spike protein and nuceloprotein. The summed area under curve values for the top four transitions of each peptide were taken to generate calibration curves for quantitation. The right panels display chromatograms obtained for each of transitions shown in different colors for (A) DQVILLNK (NP) and (B) FQTLLALHR (S). Three technical replicates were run on two separate days. The chromatograms on the left of each panel show a low and high standard from the SARS CoV-2 S and NP in a mucin background. Calibration curves were constructed from the PRM data (top right) and zoomed in (bottom right) displaying mean values at the low end of the curve to show the LOD (left dotted line) and LOQ (right dotted line).

    Article Snippet: SARS-CoV-2 nucleocapsid-His recombinant protein was purchased from Sino Biological (100 μg, 40588-V08B) The protein was difficult to dissolve and required treatment before S-trap preparation below with 40 μL dimethyl sulfoxide (276855—100 mL, Sigma), 126 μL 1% TFA, and 100 μL 1× S-Trap lysis buffer (5% SDS, 50 mM TEAB, pH adjusted to 7.55 using 12% phosphoric acid).

    Techniques: Quantitation Assay, Construct

    PRM assay results of mock (SARS-CoV-2 spiked) samples. Three biological replicates processed on different days and averaged from three technical replicates from each mock sample were evaluated using the calibration curves for the two best performing peptides (A) DQVILLNK and (B) FQTLLALHR. The samples represent the spiked-in amounts; low (3.125 μL) and high (12.5 μL) of inactivated SARS-CoV-2 virions into in vitro derived mucus. Tables below display the average calculated amol amounts obtained on each day along with the interday mean and % CV. The dotted line indicates the calculated LOD and the dashed line indicated the LOQ determined from the calibration curves generated for each peptide.

    Journal: Analytical Chemistry

    Article Title: Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein

    doi: 10.1021/acs.analchem.0c02288

    Figure Lengend Snippet: PRM assay results of mock (SARS-CoV-2 spiked) samples. Three biological replicates processed on different days and averaged from three technical replicates from each mock sample were evaluated using the calibration curves for the two best performing peptides (A) DQVILLNK and (B) FQTLLALHR. The samples represent the spiked-in amounts; low (3.125 μL) and high (12.5 μL) of inactivated SARS-CoV-2 virions into in vitro derived mucus. Tables below display the average calculated amol amounts obtained on each day along with the interday mean and % CV. The dotted line indicates the calculated LOD and the dashed line indicated the LOQ determined from the calibration curves generated for each peptide.

    Article Snippet: SARS-CoV-2 nucleocapsid-His recombinant protein was purchased from Sino Biological (100 μg, 40588-V08B) The protein was difficult to dissolve and required treatment before S-trap preparation below with 40 μL dimethyl sulfoxide (276855—100 mL, Sigma), 126 μL 1% TFA, and 100 μL 1× S-Trap lysis buffer (5% SDS, 50 mM TEAB, pH adjusted to 7.55 using 12% phosphoric acid).

    Techniques: In Vitro, Derivative Assay, Generated

    Schematic of the workflow used to develop a PRM assay for the detection and quantitation of SARS-CoV-2 spike and NP (A) PRM assay development was performed using recombinant SARS CoV-2 spike protein and NP. Proteotypic target peptides/transitions were selected to generate a spectral library in Skyline. (B) PRM assay was then used to quantitate the SARS-CoV-2 protein levels in a mock sample that was created by adding an inactivated virus sample to in vitro derived mucus.

    Journal: Analytical Chemistry

    Article Title: Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein

    doi: 10.1021/acs.analchem.0c02288

    Figure Lengend Snippet: Schematic of the workflow used to develop a PRM assay for the detection and quantitation of SARS-CoV-2 spike and NP (A) PRM assay development was performed using recombinant SARS CoV-2 spike protein and NP. Proteotypic target peptides/transitions were selected to generate a spectral library in Skyline. (B) PRM assay was then used to quantitate the SARS-CoV-2 protein levels in a mock sample that was created by adding an inactivated virus sample to in vitro derived mucus.

    Article Snippet: SARS-CoV-2 nucleocapsid-His recombinant protein was purchased from Sino Biological (100 μg, 40588-V08B) The protein was difficult to dissolve and required treatment before S-trap preparation below with 40 μL dimethyl sulfoxide (276855—100 mL, Sigma), 126 μL 1% TFA, and 100 μL 1× S-Trap lysis buffer (5% SDS, 50 mM TEAB, pH adjusted to 7.55 using 12% phosphoric acid).

    Techniques: Quantitation Assay, Recombinant, In Vitro, Derivative Assay

    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

    Journal: bioRxiv

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

    doi: 10.1101/2020.07.11.198291

    Figure Lengend 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

    Article Snippet: These animals were inoculated intraperitoneally (i.p.) with 1×105 TCID50 of recombinant vaccine viruses in 200 μl volume, or subcutaneously (s.c.) with 10 μg recombinant SARS-CoV-2 S protein (Sino Biological Europe, Eschborn, Germany) adjuvanted with 500 μg aluminium hydroxide (Alhydrogel adjuvant 2%, vac-alu-250, InvivoGen, San Diego, CA, USA) in 100 μl volume on days 0 and 28.

    Techniques: 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

    Journal: bioRxiv

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

    doi: 10.1101/2020.07.11.198291

    Figure Lengend 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

    Article Snippet: These animals were inoculated intraperitoneally (i.p.) with 1×105 TCID50 of recombinant vaccine viruses in 200 μl volume, or subcutaneously (s.c.) with 10 μg recombinant SARS-CoV-2 S protein (Sino Biological Europe, Eschborn, Germany) adjuvanted with 500 μg aluminium hydroxide (Alhydrogel adjuvant 2%, vac-alu-250, InvivoGen, San Diego, CA, USA) in 100 μl volume on days 0 and 28.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.07.11.198291

    Figure Lengend 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.

    Article Snippet: These animals were inoculated intraperitoneally (i.p.) with 1×105 TCID50 of recombinant vaccine viruses in 200 μl volume, or subcutaneously (s.c.) with 10 μg recombinant SARS-CoV-2 S protein (Sino Biological Europe, Eschborn, Germany) adjuvanted with 500 μg aluminium hydroxide (Alhydrogel adjuvant 2%, vac-alu-250, InvivoGen, San Diego, CA, USA) in 100 μl volume on days 0 and 28.

    Techniques: 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

    Journal: bioRxiv

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

    doi: 10.1101/2020.07.11.198291

    Figure Lengend 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

    Article Snippet: These animals were inoculated intraperitoneally (i.p.) with 1×105 TCID50 of recombinant vaccine viruses in 200 μl volume, or subcutaneously (s.c.) with 10 μg recombinant SARS-CoV-2 S protein (Sino Biological Europe, Eschborn, Germany) adjuvanted with 500 μg aluminium hydroxide (Alhydrogel adjuvant 2%, vac-alu-250, InvivoGen, San Diego, CA, USA) in 100 μl volume on days 0 and 28.

    Techniques: 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

    Journal: bioRxiv

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

    doi: 10.1101/2020.07.11.198291

    Figure Lengend 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

    Article Snippet: These animals were inoculated intraperitoneally (i.p.) with 1×105 TCID50 of recombinant vaccine viruses in 200 μl volume, or subcutaneously (s.c.) with 10 μg recombinant SARS-CoV-2 S protein (Sino Biological Europe, Eschborn, Germany) adjuvanted with 500 μg aluminium hydroxide (Alhydrogel adjuvant 2%, vac-alu-250, InvivoGen, San Diego, CA, USA) in 100 μl volume on days 0 and 28.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.07.11.198291

    Figure Lengend 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.

    Article Snippet: These animals were inoculated intraperitoneally (i.p.) with 1×105 TCID50 of recombinant vaccine viruses in 200 μl volume, or subcutaneously (s.c.) with 10 μg recombinant SARS-CoV-2 S protein (Sino Biological Europe, Eschborn, Germany) adjuvanted with 500 μg aluminium hydroxide (Alhydrogel adjuvant 2%, vac-alu-250, InvivoGen, San Diego, CA, USA) in 100 μl volume on days 0 and 28.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.07.11.198291

    Figure Lengend 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.

    Article Snippet: These animals were inoculated intraperitoneally (i.p.) with 1×105 TCID50 of recombinant vaccine viruses in 200 μl volume, or subcutaneously (s.c.) with 10 μg recombinant SARS-CoV-2 S protein (Sino Biological Europe, Eschborn, Germany) adjuvanted with 500 μg aluminium hydroxide (Alhydrogel adjuvant 2%, vac-alu-250, InvivoGen, San Diego, CA, USA) in 100 μl volume on days 0 and 28.

    Techniques: 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

    Journal: bioRxiv

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

    doi: 10.1101/2020.07.11.198291

    Figure Lengend 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

    Article Snippet: These animals were inoculated intraperitoneally (i.p.) with 1×105 TCID50 of recombinant vaccine viruses in 200 μl volume, or subcutaneously (s.c.) with 10 μg recombinant SARS-CoV-2 S protein (Sino Biological Europe, Eschborn, Germany) adjuvanted with 500 μg aluminium hydroxide (Alhydrogel adjuvant 2%, vac-alu-250, InvivoGen, San Diego, CA, USA) in 100 μl volume on days 0 and 28.

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