sars cov 2 2019 ncov spike rbd antibody rabbit pab  (Sino Biological)


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    Structured Review

    Sino Biological sars cov 2 2019 ncov spike rbd antibody rabbit pab
    ELISA ( x -axis) vs. LFRET ( y -axis) results by disease severity. ( a ) Anti-NP IgA ELISA vs. anti-NP LFRET (N = 81, R = 0.25). ( b ) anti-NP IgG ELISA vs. anti-NP LFRET (N = 129, R = 0.62). ( c ) anti-NP IgM ELISA vs. anti-NP LFRET (N = 81, R = 0.13). ( d ) anti-SP IgA ELISA vs. anti-SP LFRET (N = 129, R = 0.53). ( e ) anti-SP IgG ELISA vs. anti-SP LFRET (N = 129, R = 0.62). ( f ) anti-SP IgM ELISA vs. anti-SP LFRET (N = 81, R = 0.56). Color of the dot indicates <t>SARS-CoV-2</t> PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal and vertical black lines indicate LFRET and ELISA cutoffs. On the x -axis, ELISA absorbance on a logarithmic scale and on the y -axis, LFRET signal on a logarithmic scale. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. R = Pearson’s correlation coefficient.
    Sars Cov 2 2019 Ncov Spike Rbd Antibody Rabbit Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2"

    Article Title: A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

    Journal: Viruses

    doi: 10.3390/v13020143

    ELISA ( x -axis) vs. LFRET ( y -axis) results by disease severity. ( a ) Anti-NP IgA ELISA vs. anti-NP LFRET (N = 81, R = 0.25). ( b ) anti-NP IgG ELISA vs. anti-NP LFRET (N = 129, R = 0.62). ( c ) anti-NP IgM ELISA vs. anti-NP LFRET (N = 81, R = 0.13). ( d ) anti-SP IgA ELISA vs. anti-SP LFRET (N = 129, R = 0.53). ( e ) anti-SP IgG ELISA vs. anti-SP LFRET (N = 129, R = 0.62). ( f ) anti-SP IgM ELISA vs. anti-SP LFRET (N = 81, R = 0.56). Color of the dot indicates SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal and vertical black lines indicate LFRET and ELISA cutoffs. On the x -axis, ELISA absorbance on a logarithmic scale and on the y -axis, LFRET signal on a logarithmic scale. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. R = Pearson’s correlation coefficient.
    Figure Legend Snippet: ELISA ( x -axis) vs. LFRET ( y -axis) results by disease severity. ( a ) Anti-NP IgA ELISA vs. anti-NP LFRET (N = 81, R = 0.25). ( b ) anti-NP IgG ELISA vs. anti-NP LFRET (N = 129, R = 0.62). ( c ) anti-NP IgM ELISA vs. anti-NP LFRET (N = 81, R = 0.13). ( d ) anti-SP IgA ELISA vs. anti-SP LFRET (N = 129, R = 0.53). ( e ) anti-SP IgG ELISA vs. anti-SP LFRET (N = 129, R = 0.62). ( f ) anti-SP IgM ELISA vs. anti-SP LFRET (N = 81, R = 0.56). Color of the dot indicates SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal and vertical black lines indicate LFRET and ELISA cutoffs. On the x -axis, ELISA absorbance on a logarithmic scale and on the y -axis, LFRET signal on a logarithmic scale. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. R = Pearson’s correlation coefficient.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Förster Resonance Energy Transfer

    Microneutralization vs. LFRET and ELISA. Microneutralization titers are on the x -axis and LFRET signal or ELISA absorbance on the y -axis. Logarithmic scale is used on both axes. ( a ) Microneutralization titer vs. anti-SP LFRET signal (N = 107, ρ = 0.87). ( b – d ) Microneutralization titer vs. anti-SP IgG, IgA and IgM ELISA (N = 107, 107 and 67, ρ = 0.68, 0.86 and 0.81). ( e ) Microneutralization titer vs. anti-NP LFRET signal (N = 107, ρ = 0.83). ( f – h ) Microneutralization titer vs. anti-NP IgG, IgA and IgM ELISA (N = 107, 67 and 67, ρ = 0.81, 0.69 and 0.61). Color of the dots indicate SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal black lines indicate LFRET/ELISA cutoffs. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. ρ = Spearman’s rank correlation coefficient.
    Figure Legend Snippet: Microneutralization vs. LFRET and ELISA. Microneutralization titers are on the x -axis and LFRET signal or ELISA absorbance on the y -axis. Logarithmic scale is used on both axes. ( a ) Microneutralization titer vs. anti-SP LFRET signal (N = 107, ρ = 0.87). ( b – d ) Microneutralization titer vs. anti-SP IgG, IgA and IgM ELISA (N = 107, 107 and 67, ρ = 0.68, 0.86 and 0.81). ( e ) Microneutralization titer vs. anti-NP LFRET signal (N = 107, ρ = 0.83). ( f – h ) Microneutralization titer vs. anti-NP IgG, IgA and IgM ELISA (N = 107, 67 and 67, ρ = 0.81, 0.69 and 0.61). Color of the dots indicate SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal black lines indicate LFRET/ELISA cutoffs. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. ρ = Spearman’s rank correlation coefficient.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Förster Resonance Energy Transfer

    Simplified protocol for SARS-CoV-2 NP and SP LFRET assay. Eu-NP/-SP = Europium-labeled nucleoprotein/spike glycoprotein. AF-L = Alexa Fluor™ 647 -labeled protein L. TR-FRET = time-resolved Förster resonance energy transfer. RT = room temperature. TBS+BSA (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.2% BSA) was used for all dilutions. On-plate dilutions were 5 nM Eu-NP/500 nM AF-L/serum 1/25 for anti-NP and 5 nM Eu-SP/250 nM AF-L/serum 1/100 for anti-SP LFRET. For further details see the prior publication [ 5 ].
    Figure Legend Snippet: Simplified protocol for SARS-CoV-2 NP and SP LFRET assay. Eu-NP/-SP = Europium-labeled nucleoprotein/spike glycoprotein. AF-L = Alexa Fluor™ 647 -labeled protein L. TR-FRET = time-resolved Förster resonance energy transfer. RT = room temperature. TBS+BSA (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.2% BSA) was used for all dilutions. On-plate dilutions were 5 nM Eu-NP/500 nM AF-L/serum 1/25 for anti-NP and 5 nM Eu-SP/250 nM AF-L/serum 1/100 for anti-SP LFRET. For further details see the prior publication [ 5 ].

    Techniques Used: Labeling, Förster Resonance Energy Transfer

    2) Product Images from "Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response"

    Article Title: Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response

    Journal: bioRxiv

    doi: 10.1101/2020.06.27.175166

    Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.
    Figure Legend Snippet: Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.

    Techniques Used: Mouse Assay, Staining, Immunofluorescence, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Binding Assay

    Neutralizing activity against SARS-CoV-2. (A) Percent neutralization of SARS-CoV-2 virus expressing GFP. Serum collected from the naïve animals used as negative controls. (B) Neutralization titer against SARS-CoV-2 virus expressing GFP. (C, D) Correlations between neutralization titer and ELISA binding titer.
    Figure Legend Snippet: Neutralizing activity against SARS-CoV-2. (A) Percent neutralization of SARS-CoV-2 virus expressing GFP. Serum collected from the naïve animals used as negative controls. (B) Neutralization titer against SARS-CoV-2 virus expressing GFP. (C, D) Correlations between neutralization titer and ELISA binding titer.

    Techniques Used: Activity Assay, Neutralization, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay

    Analyzing SARS-CoV-2 RBD and S1 proteins affinities to human ACE2 (hACE2) proteins using biolayer interferometry (BLI). (A) Bio-Layer Interferometry sensograms of the binding of SARS-CoV-2 S1 and RBD proteins to immobilized Fc-human ACE2, after incubation of the analytes at 25°C for 0and 60 minutes. The traces represent BLI response curves for SARS-CoV-2 proteins serially diluted from 800nM to 12.5nM, as indicated. Dotted lines show raw response values, while bold solid lines show the fitted trace. Association and dissociation phases were monitored for 300s and 600s, respectively. The data was globally fit using a 1:1 binding model to estimate binding affinity. (B) Binding affinity specifications of S1 and RBD proteins against hu-ACE2.
    Figure Legend Snippet: Analyzing SARS-CoV-2 RBD and S1 proteins affinities to human ACE2 (hACE2) proteins using biolayer interferometry (BLI). (A) Bio-Layer Interferometry sensograms of the binding of SARS-CoV-2 S1 and RBD proteins to immobilized Fc-human ACE2, after incubation of the analytes at 25°C for 0and 60 minutes. The traces represent BLI response curves for SARS-CoV-2 proteins serially diluted from 800nM to 12.5nM, as indicated. Dotted lines show raw response values, while bold solid lines show the fitted trace. Association and dissociation phases were monitored for 300s and 600s, respectively. The data was globally fit using a 1:1 binding model to estimate binding affinity. (B) Binding affinity specifications of S1 and RBD proteins against hu-ACE2.

    Techniques Used: Binding Assay, Incubation

    Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.
    Figure Legend Snippet: Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.

    Techniques Used: Mouse Assay, Recombinant, Expressing, Binding Assay, Enzyme-linked Immunosorbent Assay, Luminex, Fluorescence

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

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

    Journal: JCI Insight

    doi: 10.1172/jci.insight.142386

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

    Techniques Used: Enzyme-linked Immunosorbent Assay

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

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

    Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.
    Figure Legend Snippet: Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.

    Techniques Used: Neutralization, Activity Assay, Binding Assay, Standard Deviation

    4) Product Images from "Mice immunized with the vaccine candidate HexaPro spike produce neutralizing antibodies against SARS-CoV-2"

    Article Title: Mice immunized with the vaccine candidate HexaPro spike produce neutralizing antibodies against SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2021.02.27.433054

    The recombinant SARS-CoV-2 HexaPro spike protein. (A) Schematic representation of the prefusion-stabilized SARS-CoV-2 HexaPro ectodomain showing the S1 and S2 subunits. Four additional proline substitutions from S-2P construct are indicated by the red arrows shown below the construct. (B) The HexaPro protein expressed in HEK293T cells was purified and characterized by SDS-PAGE (left), western blot using a commercial anti-RBD (middle), and western blot using pooled convalescence sera (right).
    Figure Legend Snippet: The recombinant SARS-CoV-2 HexaPro spike protein. (A) Schematic representation of the prefusion-stabilized SARS-CoV-2 HexaPro ectodomain showing the S1 and S2 subunits. Four additional proline substitutions from S-2P construct are indicated by the red arrows shown below the construct. (B) The HexaPro protein expressed in HEK293T cells was purified and characterized by SDS-PAGE (left), western blot using a commercial anti-RBD (middle), and western blot using pooled convalescence sera (right).

    Techniques Used: Recombinant, Construct, Purification, SDS Page, Western Blot

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

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

    Journal: bioRxiv

    doi: 10.1101/2021.03.07.434276

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

    Techniques Used: Infection, Negative Control

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

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

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

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

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

    Techniques Used: Expressing, Infection, Western Blot

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

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

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

    Techniques Used:

    6) Product Images from "Mice immunized with the vaccine candidate HexaPro spike produce neutralizing antibodies against SARS-CoV-2"

    Article Title: Mice immunized with the vaccine candidate HexaPro spike produce neutralizing antibodies against SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2021.02.27.433054

    The recombinant SARS-CoV-2 HexaPro spike protein. (A) Schematic representation of the prefusion-stabilized SARS-CoV-2 HexaPro ectodomain showing the S1 and S2 subunits. Four additional proline substitutions from S-2P construct are indicated by the red arrows shown below the construct. (B) The HexaPro protein expressed in HEK293T cells was purified and characterized by SDS-PAGE (left), western blot using a commercial anti-RBD (middle), and western blot using pooled convalescence sera (right).
    Figure Legend Snippet: The recombinant SARS-CoV-2 HexaPro spike protein. (A) Schematic representation of the prefusion-stabilized SARS-CoV-2 HexaPro ectodomain showing the S1 and S2 subunits. Four additional proline substitutions from S-2P construct are indicated by the red arrows shown below the construct. (B) The HexaPro protein expressed in HEK293T cells was purified and characterized by SDS-PAGE (left), western blot using a commercial anti-RBD (middle), and western blot using pooled convalescence sera (right).

    Techniques Used: Recombinant, Construct, Purification, SDS Page, Western Blot

    7) Product Images from "A novel viral protein translation mechanism reveals mitochondria as a target for antiviral drug development"

    Article Title: A novel viral protein translation mechanism reveals mitochondria as a target for antiviral drug development

    Journal: bioRxiv

    doi: 10.1101/2020.10.19.344713

    The possible effect of Transcription factors binding Leucine-specific tRNAs in viral protein translation (A) Genomic characteristics including histone modification and transcription factor binding status of tRNA TRL-TAA4-1 which matched with rare codon Leu-TTA was analysed using the UCSC database. (B) Overlap analysis of potential TRL-TAA regulatory transcription factors (TFs) and genes that increased in SARS-CoV-2 infection in A549-ACE2 and Calu3 cells using Venny2.1. (C) Heat map demonstrating the expression of 25 TFs in SARS-CoV-2-infected cell lines created using R studio. (D) Western Blot assay to assess the protein level of S after co-transfection with EGR1 or ATF2 in HEK-293 cells. (E). Differentially expressed genes after SARS-CoV-2 infection (GSE147507) were analyzed with R Studio. Pathway enrichment of highly expressed genes was mapped using Metascape (F) Distribution of genes including ACE2, TMPRSS2, ATF2 and identified TFs in human lung cells was created using public single cell sequence data( 18 ).
    Figure Legend Snippet: The possible effect of Transcription factors binding Leucine-specific tRNAs in viral protein translation (A) Genomic characteristics including histone modification and transcription factor binding status of tRNA TRL-TAA4-1 which matched with rare codon Leu-TTA was analysed using the UCSC database. (B) Overlap analysis of potential TRL-TAA regulatory transcription factors (TFs) and genes that increased in SARS-CoV-2 infection in A549-ACE2 and Calu3 cells using Venny2.1. (C) Heat map demonstrating the expression of 25 TFs in SARS-CoV-2-infected cell lines created using R studio. (D) Western Blot assay to assess the protein level of S after co-transfection with EGR1 or ATF2 in HEK-293 cells. (E). Differentially expressed genes after SARS-CoV-2 infection (GSE147507) were analyzed with R Studio. Pathway enrichment of highly expressed genes was mapped using Metascape (F) Distribution of genes including ACE2, TMPRSS2, ATF2 and identified TFs in human lung cells was created using public single cell sequence data( 18 ).

    Techniques Used: Binding Assay, Modification, Infection, Expressing, Western Blot, Cotransfection, Sequencing

    Rare codon bias can prevent translation of SARS-CoV-2 derived sequences (A) Protein expression of plasmids expressing SARS-CoV-2 S using the original or codon-optimized sequence after transfection into human A549 cells. Expression was evaluated using confocal microscopy, and an IRES-RFP sequence was included after the S open reading frame (ORF) for visualisation. (B) Western Blot assay was performed to detect the protein abundance of SARS-CoV-2-derived sequences including the four structural proteins (spike (S), envelope (E), membrane (M) and nucleocapsid (N)) and four accessory proteins (ORF3, ORF6, ORF7 and ORF8). The abbreviation ‘ns’ indicates non-specific. (C) Codon usage variability of selected SARS-CoV-2 ORFs was analysed with GCUA software using the human standard codon table as reference, and the figure was drawn using GraphPad Prism. (D) The number of rare codons (fraction
    Figure Legend Snippet: Rare codon bias can prevent translation of SARS-CoV-2 derived sequences (A) Protein expression of plasmids expressing SARS-CoV-2 S using the original or codon-optimized sequence after transfection into human A549 cells. Expression was evaluated using confocal microscopy, and an IRES-RFP sequence was included after the S open reading frame (ORF) for visualisation. (B) Western Blot assay was performed to detect the protein abundance of SARS-CoV-2-derived sequences including the four structural proteins (spike (S), envelope (E), membrane (M) and nucleocapsid (N)) and four accessory proteins (ORF3, ORF6, ORF7 and ORF8). The abbreviation ‘ns’ indicates non-specific. (C) Codon usage variability of selected SARS-CoV-2 ORFs was analysed with GCUA software using the human standard codon table as reference, and the figure was drawn using GraphPad Prism. (D) The number of rare codons (fraction

    Techniques Used: Derivative Assay, Expressing, Sequencing, Transfection, Confocal Microscopy, Western Blot, Software

    Mitochondrial localization is critical for the translation of SARS-CoV-2 S protein (A) The interaction network of proteins that bound with S was analyzed using Metascape. (B) The frequency of rare codons in SARS-CoV-2, S protein, human nuclear genome and the human mitochondrial genome using the Codon Usage Database. (C) The protein expression of S flanked by SARS-CoV-2 5’- and 3’-UTR sequences in HEK-293 cell lines. (D) The effect of EGR1 and ATF2 on S protein (plus SARS-CoV-2 5’- and 3’ UTS sequences) expression was analysed using Western blot after transfection into HEK-293 cells. Fold change was determined using Image J software. (E) The skeleton of recombinant plasmids encoding S with or without mitochondrial localisation signals (MLS) RnaseP or RMRP. (F) Protein expression of SARS-CoV-2 derived sequences including S, E and ORF8 and variants expressing MLS as analysed by Western blot after transfection into HEK-293 cells. (G) The effect of EGR1 and ATF2 on wild type spike with RMRP binding motif (MRP).
    Figure Legend Snippet: Mitochondrial localization is critical for the translation of SARS-CoV-2 S protein (A) The interaction network of proteins that bound with S was analyzed using Metascape. (B) The frequency of rare codons in SARS-CoV-2, S protein, human nuclear genome and the human mitochondrial genome using the Codon Usage Database. (C) The protein expression of S flanked by SARS-CoV-2 5’- and 3’-UTR sequences in HEK-293 cell lines. (D) The effect of EGR1 and ATF2 on S protein (plus SARS-CoV-2 5’- and 3’ UTS sequences) expression was analysed using Western blot after transfection into HEK-293 cells. Fold change was determined using Image J software. (E) The skeleton of recombinant plasmids encoding S with or without mitochondrial localisation signals (MLS) RnaseP or RMRP. (F) Protein expression of SARS-CoV-2 derived sequences including S, E and ORF8 and variants expressing MLS as analysed by Western blot after transfection into HEK-293 cells. (G) The effect of EGR1 and ATF2 on wild type spike with RMRP binding motif (MRP).

    Techniques Used: Expressing, Western Blot, Transfection, Software, Recombinant, Derivative Assay, Binding Assay

    8) Product Images from "Prunella vulgaris extract and suramin block SARS-coronavirus 2 virus Spike protein D614 and G614 variants mediated receptor association and virus entry in cell culture system"

    Article Title: Prunella vulgaris extract and suramin block SARS-coronavirus 2 virus Spike protein D614 and G614 variants mediated receptor association and virus entry in cell culture system

    Journal: bioRxiv

    doi: 10.1101/2020.08.28.270306

    SARS-CoV-2 SP-PVs’s infection in different cell lines and SARS-CoV-2 SP G614 variant exhibited stronger virus entry. A) 293T, 293T ACE2 and Vero-E6 cells were infected by equal amounts of SARS-CoV-2SP-, SARS-CoV-2SPΔC-pseudotyped viruses. At 48 hrs pi, the Gluc activity in supernatants was measured. B) the expression of SARS-CoV-2SP receptor, ACE2, in 293T, 293T ACE2 and Vero-E6 cells detected by WB with anti-ACE2 antibodies. C) The SPΔC G614 -GFP + PVs were produced with 293T cells and used to infect 293T ACE2 cells in 96-well plate After 48 hrs pi, GFP-positive cells (per well) were counted and photographed by fluorescence microscope (on the top of the panel). D) Detection of SARS-CoV-2 SPΔC, SPΔC G614 and HIV p24 protein expression in transfected 293T cells and viral particles by WB. E) Infectivity comparison of SPΔC-PVs and SPΔC G614 -PVs in 293T ACE2 cells. Equal amounts of SPΔC D614 -PVs and SPΔC G614 -PVs virions (adjusted by p24 level) were used to infect 293T ACE2 cells. At different days post-infection (pi), Gluc activity in supernatants was measured.
    Figure Legend Snippet: SARS-CoV-2 SP-PVs’s infection in different cell lines and SARS-CoV-2 SP G614 variant exhibited stronger virus entry. A) 293T, 293T ACE2 and Vero-E6 cells were infected by equal amounts of SARS-CoV-2SP-, SARS-CoV-2SPΔC-pseudotyped viruses. At 48 hrs pi, the Gluc activity in supernatants was measured. B) the expression of SARS-CoV-2SP receptor, ACE2, in 293T, 293T ACE2 and Vero-E6 cells detected by WB with anti-ACE2 antibodies. C) The SPΔC G614 -GFP + PVs were produced with 293T cells and used to infect 293T ACE2 cells in 96-well plate After 48 hrs pi, GFP-positive cells (per well) were counted and photographed by fluorescence microscope (on the top of the panel). D) Detection of SARS-CoV-2 SPΔC, SPΔC G614 and HIV p24 protein expression in transfected 293T cells and viral particles by WB. E) Infectivity comparison of SPΔC-PVs and SPΔC G614 -PVs in 293T ACE2 cells. Equal amounts of SPΔC D614 -PVs and SPΔC G614 -PVs virions (adjusted by p24 level) were used to infect 293T ACE2 cells. At different days post-infection (pi), Gluc activity in supernatants was measured.

    Techniques Used: Infection, Variant Assay, Activity Assay, Expressing, Western Blot, Produced, Fluorescence, Microscopy, Transfection

    SARS-CoV-2-SP-PV’s infection was efficiently blocked by CHPV and suramin. A) Images of the dried Prunella Vulgaris flowers and its water extract (CHPV). B) Dose -response anti-SARS-CoV-2 analysis by Gluc activity for CHPV or suramin. 293T ACE2 cells were infected by equal amounts of SARS-CoV-2SPΔC-pseudotyped viruses in the presence of different dose of CHPV or suramin. At 48 hrs pi, the Gluc activity in supernatants was measured. (% inhibition = 100 ⨯ [1 - (Gluc value in presence of drug)/(Gluc value in absence of drug)). C) Infection inhibition of CHPV or suramin on SARS-CoV-2-SPΔC G614 -PVs in 293T ACE2 cells. Equal amounts of SCoV-2-SPΔC G614 -PVs (adjusted by p24 level) were used to infect 293T ACE2 cells in presence of different concentrations of CHPV or suramin, in indicated at bottom of the panel. At 48 hrs pi, Gluc activity in supernatants was measured and present as % inhibition. Means ± SD were calculated from duplicate experiments. D) 293T ACE2 cells in 96-well plate were infected with SPΔC G614 -GFP + PVs. After 48 hrs pi, GFP-positive cells (per well) were counted (left panel) and photographed by fluorescence microscope (right panel, a. Without drugs; b. Without infection; c. In the presence of CHPV (100 μg/ml ) ; d. In the presence of suramin (100 μg/ml ) .
    Figure Legend Snippet: SARS-CoV-2-SP-PV’s infection was efficiently blocked by CHPV and suramin. A) Images of the dried Prunella Vulgaris flowers and its water extract (CHPV). B) Dose -response anti-SARS-CoV-2 analysis by Gluc activity for CHPV or suramin. 293T ACE2 cells were infected by equal amounts of SARS-CoV-2SPΔC-pseudotyped viruses in the presence of different dose of CHPV or suramin. At 48 hrs pi, the Gluc activity in supernatants was measured. (% inhibition = 100 ⨯ [1 - (Gluc value in presence of drug)/(Gluc value in absence of drug)). C) Infection inhibition of CHPV or suramin on SARS-CoV-2-SPΔC G614 -PVs in 293T ACE2 cells. Equal amounts of SCoV-2-SPΔC G614 -PVs (adjusted by p24 level) were used to infect 293T ACE2 cells in presence of different concentrations of CHPV or suramin, in indicated at bottom of the panel. At 48 hrs pi, Gluc activity in supernatants was measured and present as % inhibition. Means ± SD were calculated from duplicate experiments. D) 293T ACE2 cells in 96-well plate were infected with SPΔC G614 -GFP + PVs. After 48 hrs pi, GFP-positive cells (per well) were counted (left panel) and photographed by fluorescence microscope (right panel, a. Without drugs; b. Without infection; c. In the presence of CHPV (100 μg/ml ) ; d. In the presence of suramin (100 μg/ml ) .

    Techniques Used: Infection, Activity Assay, Inhibition, Fluorescence, Microscopy

    Characterization of the mechanisms of CHPV and suramin for their anti-SARS-COV-2-SP action. A) Time-dependent inhibition of SPΔC G614 -PVs infection mediated by CHPV or suramin. CHPV (100 μg/mL) or suramin (100 μg/mL) was added at 1 hr prior to infection, during infection (0 hr), and at 1 hr, and 3 hr pi. The positive controls (PC) were 293T ACE2 cells infected with SPΔC G614 -PVs in the absence of compounds. At 3 hrs pi, all of the cell cultures were replaced with fresh DMEM and cultured for 48 hrs. Then, the Gluc activity was monitored in the supernatant, and the data are shown as a percentage of inhibition (%). B) inhibitory effect of CHPV or suramin on SARS-CoV2-SP/ACE2 binding by ELISA as described in materials and methods. nAB: anti-COVID-19 neutralizing antibody (SAD-S35). The results are the mean ± SD of duplicate samples, and the data are representative of results obtained in two independent experiments.
    Figure Legend Snippet: Characterization of the mechanisms of CHPV and suramin for their anti-SARS-COV-2-SP action. A) Time-dependent inhibition of SPΔC G614 -PVs infection mediated by CHPV or suramin. CHPV (100 μg/mL) or suramin (100 μg/mL) was added at 1 hr prior to infection, during infection (0 hr), and at 1 hr, and 3 hr pi. The positive controls (PC) were 293T ACE2 cells infected with SPΔC G614 -PVs in the absence of compounds. At 3 hrs pi, all of the cell cultures were replaced with fresh DMEM and cultured for 48 hrs. Then, the Gluc activity was monitored in the supernatant, and the data are shown as a percentage of inhibition (%). B) inhibitory effect of CHPV or suramin on SARS-CoV2-SP/ACE2 binding by ELISA as described in materials and methods. nAB: anti-COVID-19 neutralizing antibody (SAD-S35). The results are the mean ± SD of duplicate samples, and the data are representative of results obtained in two independent experiments.

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

    Generation of a SARS-COV2-SP-pseudotyped lentiviruse particles (SCoV-2-SP-PVs). A) Schematic representation of SARS-CoV-2SP, SARS-CoV-2SPΔC, and SARS-CoV-2SP G614 ΔC expressing plasmids. B) Schematic representation of plasmids and and procedures for production of SARS-COV2-SP-pseudotyped lentivirus particles (SCoV-2-SP-PVs). C) Detection of SARS-CoV-2 SPs and HIV p24 protein expression in transfected 293T cells and viral particles by Western blot (WB) with anti-SP or anti-p24 antibodies. D) Different amounts of SCoV-2-SP-PVs and SCoV-2-SPΔC-PVs virions (adjusted by p24) were used to infect 293T ACE2 cells. At different time intervels, the Gaussia Luciferase activity (Gluc) (left panel) and PVs-associated p24 (at 72 hrs) in supernatants was measured.
    Figure Legend Snippet: Generation of a SARS-COV2-SP-pseudotyped lentiviruse particles (SCoV-2-SP-PVs). A) Schematic representation of SARS-CoV-2SP, SARS-CoV-2SPΔC, and SARS-CoV-2SP G614 ΔC expressing plasmids. B) Schematic representation of plasmids and and procedures for production of SARS-COV2-SP-pseudotyped lentivirus particles (SCoV-2-SP-PVs). C) Detection of SARS-CoV-2 SPs and HIV p24 protein expression in transfected 293T cells and viral particles by Western blot (WB) with anti-SP or anti-p24 antibodies. D) Different amounts of SCoV-2-SP-PVs and SCoV-2-SPΔC-PVs virions (adjusted by p24) were used to infect 293T ACE2 cells. At different time intervels, the Gaussia Luciferase activity (Gluc) (left panel) and PVs-associated p24 (at 72 hrs) in supernatants was measured.

    Techniques Used: Expressing, Transfection, Western Blot, Luciferase, Activity Assay

    Inhibitory effect of CHPV and Suramin on SARS-CoV-2 infection-induced cytopathic effects. Vero cells were infected with a wild type SARS-CoV-2 virus (hCoV-19/Canada/ON-VIDO-01/2020) in the presence or absence of different concentrations of CHPV and Suramin. After 72 hrs pi., the SARS-CoV-2 infection-induced cytopathic effects in Vero cells were monitored. Error bars represent variation between triplicate samples, and the data of (A) and (B) are representative of results obtained in two independent experiments.
    Figure Legend Snippet: Inhibitory effect of CHPV and Suramin on SARS-CoV-2 infection-induced cytopathic effects. Vero cells were infected with a wild type SARS-CoV-2 virus (hCoV-19/Canada/ON-VIDO-01/2020) in the presence or absence of different concentrations of CHPV and Suramin. After 72 hrs pi., the SARS-CoV-2 infection-induced cytopathic effects in Vero cells were monitored. Error bars represent variation between triplicate samples, and the data of (A) and (B) are representative of results obtained in two independent experiments.

    Techniques Used: Infection

    9) Product Images from "Native-like SARS-CoV-2 Spike Glycoprotein Expressed by ChAdOx1 nCoV-19/AZD1222 Vaccine"

    Article Title: Native-like SARS-CoV-2 Spike Glycoprotein Expressed by ChAdOx1 nCoV-19/AZD1222 Vaccine

    Journal: ACS Central Science

    doi: 10.1021/acscentsci.1c00080

    Site-specific glycan processing of SARS-CoV-2 S upon infection with ChAdOx1 nCoV-19. (A) Western blot analysis of SARS-CoV-2 spike proteins, using anti-S1 and anti-S1+S2 antibodies. Lane 1 = Protein pellet from 293F cell lysates infected with ChAdOx1 nCoV-19. Lane 2 = Reduced protein pellet from 293F infected with ChAdOx1 nCoV-19. Lane 3 = 2P-stabilized SARS-CoV-2 S protein. The white boxes correspond to gel bands that were excised for mass spectrometric analysis. (B) Site-specific N-linked glycosylation of SARS-CoV-2 S0 and S1/S2 glycoproteins. The bar graphs represent the relative quantities of digested glycopeptides possessing the identifiers of oligomannose/hybrid-type glycans (green), complex-type glycans (pink), unoccupied PNGs (gray), or not determined (N.D.) at each N-linked glycan sequon on the S protein, listed from N to C terminus. (C) Glycosylated model of the cleaved (S1/S2) SARS-CoV-2 spike. The pie charts summarize the mass spectrometric analysis of the oligomannose/hybrid (green), complex (pink), or unoccupied (gray) N-linked glycan populations. Representative glycans are modeled onto the prefusion structure of trimeric SARS-CoV-2 S glycoprotein (PDB ID: 6VSB), 3 with one RBD in the “up” conformation. The modeled glycans are colored according to oligomannose/hybrid-type glycan content with glycan sites labeled in green (80–100%), orange (30–79%), pink (0–29%), or gray (not detected).
    Figure Legend Snippet: Site-specific glycan processing of SARS-CoV-2 S upon infection with ChAdOx1 nCoV-19. (A) Western blot analysis of SARS-CoV-2 spike proteins, using anti-S1 and anti-S1+S2 antibodies. Lane 1 = Protein pellet from 293F cell lysates infected with ChAdOx1 nCoV-19. Lane 2 = Reduced protein pellet from 293F infected with ChAdOx1 nCoV-19. Lane 3 = 2P-stabilized SARS-CoV-2 S protein. The white boxes correspond to gel bands that were excised for mass spectrometric analysis. (B) Site-specific N-linked glycosylation of SARS-CoV-2 S0 and S1/S2 glycoproteins. The bar graphs represent the relative quantities of digested glycopeptides possessing the identifiers of oligomannose/hybrid-type glycans (green), complex-type glycans (pink), unoccupied PNGs (gray), or not determined (N.D.) at each N-linked glycan sequon on the S protein, listed from N to C terminus. (C) Glycosylated model of the cleaved (S1/S2) SARS-CoV-2 spike. The pie charts summarize the mass spectrometric analysis of the oligomannose/hybrid (green), complex (pink), or unoccupied (gray) N-linked glycan populations. Representative glycans are modeled onto the prefusion structure of trimeric SARS-CoV-2 S glycoprotein (PDB ID: 6VSB), 3 with one RBD in the “up” conformation. The modeled glycans are colored according to oligomannose/hybrid-type glycan content with glycan sites labeled in green (80–100%), orange (30–79%), pink (0–29%), or gray (not detected).

    Techniques Used: Infection, Western Blot, Labeling

    ChAdOx1 nCoV-19 produces membrane associated SARS-CoV-2 S glycoprotein in native conformations able to bind its host receptor, ACE2. (A) Schematic representation of the vaccine encoded SARS-CoV-2 S protein, showing the position of N-linked glycosylation amino acid sequons (NXS/T, where X ≠ P) as branches. Protein domains are illustrated: N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), and transmembrane domain (TM), with the additional tPA secretion signal at the N-terminus. (B) HeLa S3 cells were infected with ChAdOx1 nCoV-19 and incubated with recombinant ACE2, anti-ChAdOx1 nCoV-19 (derived from vaccinated mice), or a panel of human mAbs (Ab44, Ab45, Ab71, and Ab111, which recognize S2, RBD, trimeric S, and NTD, respectively) and compared to noninfected controls, analyzed by flow cytometry. (Left). Relative frequency of cells and AlexaFluor 488 fluorescence associated with antispike detection is plotted. Left, (blue) anti-ChAdOx1 nCoV-19; middle (red), ACE2; and right (shades of green) human mAbs. In dark gray cells infected with an irrelevant ChAdOx1 vaccine and in light gray noninfected cells are shown as a control. Experimental replicates were performed two times, and representative data are shown.
    Figure Legend Snippet: ChAdOx1 nCoV-19 produces membrane associated SARS-CoV-2 S glycoprotein in native conformations able to bind its host receptor, ACE2. (A) Schematic representation of the vaccine encoded SARS-CoV-2 S protein, showing the position of N-linked glycosylation amino acid sequons (NXS/T, where X ≠ P) as branches. Protein domains are illustrated: N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), and transmembrane domain (TM), with the additional tPA secretion signal at the N-terminus. (B) HeLa S3 cells were infected with ChAdOx1 nCoV-19 and incubated with recombinant ACE2, anti-ChAdOx1 nCoV-19 (derived from vaccinated mice), or a panel of human mAbs (Ab44, Ab45, Ab71, and Ab111, which recognize S2, RBD, trimeric S, and NTD, respectively) and compared to noninfected controls, analyzed by flow cytometry. (Left). Relative frequency of cells and AlexaFluor 488 fluorescence associated with antispike detection is plotted. Left, (blue) anti-ChAdOx1 nCoV-19; middle (red), ACE2; and right (shades of green) human mAbs. In dark gray cells infected with an irrelevant ChAdOx1 vaccine and in light gray noninfected cells are shown as a control. Experimental replicates were performed two times, and representative data are shown.

    Techniques Used: Binding Assay, Infection, Incubation, Recombinant, Derivative Assay, Mouse Assay, Flow Cytometry, Fluorescence

    CryoET and subtomogram average of ChAdOx1 nCoV-19 derived spike. (A) Tomographic slice of U2OS cell transduced with ChAdOx1 nCoV-19. The slice is 6.4 Å thick; PM = plasma membrane, scale bar = 100 nm. (B) Detailed view of the boxed area marked in (A). White arrowheads indicate spike proteins on the cell surface; scale bar = 50 nm. (C–E) Subtomogram average of ChAdOx1 nCoV-19 spikes at 11.6 Å resolution as indicated by Fourier-Shell correlation at 0.5 cutoff (C), shown from side view (D) and top view (E). SARS-CoV-2 atomic model (PDB 6ZB5) 29 is fitted for reference.
    Figure Legend Snippet: CryoET and subtomogram average of ChAdOx1 nCoV-19 derived spike. (A) Tomographic slice of U2OS cell transduced with ChAdOx1 nCoV-19. The slice is 6.4 Å thick; PM = plasma membrane, scale bar = 100 nm. (B) Detailed view of the boxed area marked in (A). White arrowheads indicate spike proteins on the cell surface; scale bar = 50 nm. (C–E) Subtomogram average of ChAdOx1 nCoV-19 spikes at 11.6 Å resolution as indicated by Fourier-Shell correlation at 0.5 cutoff (C), shown from side view (D) and top view (E). SARS-CoV-2 atomic model (PDB 6ZB5) 29 is fitted for reference.

    Techniques Used: Derivative Assay, Transduction

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

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

    Journal: bioRxiv

    doi: 10.1101/2021.03.07.434276

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

    Techniques Used: Infection, Negative Control

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

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

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

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

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

    Techniques Used: Expressing, Infection, Western Blot

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

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

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

    Techniques Used:

    11) Product Images from "MERS-CoV and SARS-CoV-2 replication can be inhibited by targeting the interaction between the viral spike protein and the nucleocapsid protein"

    Article Title: MERS-CoV and SARS-CoV-2 replication can be inhibited by targeting the interaction between the viral spike protein and the nucleocapsid protein

    Journal: Theranostics

    doi: 10.7150/thno.55647

    Effect of R-Spike CD-SARS-CoV peptides on the replication of SARS-CoV-2. (A and B) Vero cells (A) and Calu-3 cells (B) infected with SARS-CoV-2 (0.1 MOI) and then treated with PBS or 2 μM of cell-penetrating peptides (R-Spike CD-SARS-CoV-2 or R-CP-1) at 6 h after virus infection (n = 3). Supernatants of virus-infected cell cultures were collected at 24 h after virus infection. Virus replication was quantified by qRT-PCR analysis of the SARS-CoV-2 RdRP gene (left) and plaque formation assay (right). * p
    Figure Legend Snippet: Effect of R-Spike CD-SARS-CoV peptides on the replication of SARS-CoV-2. (A and B) Vero cells (A) and Calu-3 cells (B) infected with SARS-CoV-2 (0.1 MOI) and then treated with PBS or 2 μM of cell-penetrating peptides (R-Spike CD-SARS-CoV-2 or R-CP-1) at 6 h after virus infection (n = 3). Supernatants of virus-infected cell cultures were collected at 24 h after virus infection. Virus replication was quantified by qRT-PCR analysis of the SARS-CoV-2 RdRP gene (left) and plaque formation assay (right). * p

    Techniques Used: Infection, Quantitative RT-PCR, Plaque Formation Assay

    Localization of MERS-CoV R-Spike CD in Vero cells and cytotoxicity of the cell-penetrating peptides. (A) Vero cells were cultured for 24 h and then incubated with R-Spike CD-MERS-CoV-Biotin peptide for 30 min in a 5% CO 2 incubator at 37 °C. The samples were fixed with 4% paraformaldehyde and permeabilized with 0.1% triton X-100. Cell-penetrated R-Spike CD-MERS-CoV-Biotin peptide was detected using Alexa Fluor-488-conjugated Streptavidin (Green) and a Carl Zeiss LSM710 microscope. Nuclei were stained with Hoechst 33258 (Blue). Scale bar, 10 µm. (B) Effect of cell-penetrating peptides on the growth of Vero cells and Calu-3 cells. Vero cells or Calu-3 cells were cultured with the indicated concentrations of cell-penetrating peptides for 3 days. The cells were incubated with CCK-8 solution, and then, soluble formazan was measured using a microplate reader. R-Spike CD-MERS-CoV, the peptide corresponding to the C-terminal domain of the MERS-CoV S protein conjugated with nine D-arginine residues at the N-terminus; R-Spike CD-MERS-CoV-Biotin, a biotinylated R-Spike CD-MERS-CoV peptide; R-Spike CD-SARS-CoV-2, the peptide corresponding to the C-terminal domain of the SARS-CoV-2 S protein conjugated with nine D-arginine residues at the N-terminus; R-CP-1, a nine D-arginine-conjugated control peptide.
    Figure Legend Snippet: Localization of MERS-CoV R-Spike CD in Vero cells and cytotoxicity of the cell-penetrating peptides. (A) Vero cells were cultured for 24 h and then incubated with R-Spike CD-MERS-CoV-Biotin peptide for 30 min in a 5% CO 2 incubator at 37 °C. The samples were fixed with 4% paraformaldehyde and permeabilized with 0.1% triton X-100. Cell-penetrated R-Spike CD-MERS-CoV-Biotin peptide was detected using Alexa Fluor-488-conjugated Streptavidin (Green) and a Carl Zeiss LSM710 microscope. Nuclei were stained with Hoechst 33258 (Blue). Scale bar, 10 µm. (B) Effect of cell-penetrating peptides on the growth of Vero cells and Calu-3 cells. Vero cells or Calu-3 cells were cultured with the indicated concentrations of cell-penetrating peptides for 3 days. The cells were incubated with CCK-8 solution, and then, soluble formazan was measured using a microplate reader. R-Spike CD-MERS-CoV, the peptide corresponding to the C-terminal domain of the MERS-CoV S protein conjugated with nine D-arginine residues at the N-terminus; R-Spike CD-MERS-CoV-Biotin, a biotinylated R-Spike CD-MERS-CoV peptide; R-Spike CD-SARS-CoV-2, the peptide corresponding to the C-terminal domain of the SARS-CoV-2 S protein conjugated with nine D-arginine residues at the N-terminus; R-CP-1, a nine D-arginine-conjugated control peptide.

    Techniques Used: Cell Culture, Incubation, Microscopy, Staining, CCK-8 Assay

    Interaction of SARS-CoV-2 S protein with N protein and effects of R-Spike CD-SARS-CoV-2 on production of SARS-CoV-2 proteins. (A) Interaction of SARS-CoV-2 S protein with N protein. Lysates were prepared from uninfected and SARS-CoV-2 (0.1 MOI)-infected Vero cells. The lysates were immunoprecipitated with anti-SARS-CoV-2 S mAb (left). The immunocomplexes were subjected to western blotting with anti-SARS-CoV-2 S mAb or anti-SARS-CoV-2 N Ab. The cell lysates were analyzed by western blotting with the indicated antibodies (right). Anti-S Ab, anti-SARS-CoV-2 S Ab. Anti-N mAb, anti-SARS-CoV-2 N mAb. (B-E) Effects of R-Spike CD-SARS-CoV-2 on production of SARS-CoV-2 proteins. Vero cells (B and C) and Calu-3 cells (D and E) were infected with SARS-CoV-2 (0.1 MOI) and then treated with PBS or 2 μM of cell-penetrating peptides (R-Spike CD-SARS-CoV-2 or R-CP-1) at 6 h after virus infection (n = 3) in DMEM medium containing 2% FBS. The cells were cultured for 48 h and then analyzed by confocal microscopy after staining with anti-SARS-CoV-2 S Ab (B and D) or anti-SARS-CoV-2 N mAb (C and E) and then, Alexa Fluor 488-conjugated secondary antibody. Scale bar, 20 μm. These results are representative of two independent experiments.
    Figure Legend Snippet: Interaction of SARS-CoV-2 S protein with N protein and effects of R-Spike CD-SARS-CoV-2 on production of SARS-CoV-2 proteins. (A) Interaction of SARS-CoV-2 S protein with N protein. Lysates were prepared from uninfected and SARS-CoV-2 (0.1 MOI)-infected Vero cells. The lysates were immunoprecipitated with anti-SARS-CoV-2 S mAb (left). The immunocomplexes were subjected to western blotting with anti-SARS-CoV-2 S mAb or anti-SARS-CoV-2 N Ab. The cell lysates were analyzed by western blotting with the indicated antibodies (right). Anti-S Ab, anti-SARS-CoV-2 S Ab. Anti-N mAb, anti-SARS-CoV-2 N mAb. (B-E) Effects of R-Spike CD-SARS-CoV-2 on production of SARS-CoV-2 proteins. Vero cells (B and C) and Calu-3 cells (D and E) were infected with SARS-CoV-2 (0.1 MOI) and then treated with PBS or 2 μM of cell-penetrating peptides (R-Spike CD-SARS-CoV-2 or R-CP-1) at 6 h after virus infection (n = 3) in DMEM medium containing 2% FBS. The cells were cultured for 48 h and then analyzed by confocal microscopy after staining with anti-SARS-CoV-2 S Ab (B and D) or anti-SARS-CoV-2 N mAb (C and E) and then, Alexa Fluor 488-conjugated secondary antibody. Scale bar, 20 μm. These results are representative of two independent experiments.

    Techniques Used: Infection, Immunoprecipitation, Western Blot, Cell Culture, Confocal Microscopy, Staining

    12) Product Images from "Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion"

    Article Title: Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion

    Journal: bioRxiv

    doi: 10.1101/2020.06.08.141077

    CH25H and 25HC block SARS-CoV-2 S mediated membrane fusion (A) Wild-type (WT) HEK293-hACE2 cells or those stably expressing TMPRSS2 or TMPRSS4 were transfected with mock, IFITM2, IFITM3, or CH25H for 24 hr and infected with VSV-SARS-CoV-2 (MOI=1). At 24 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression. (B) HEK293-hACE2-TMPRSS2 cells with or without CH25H expression were infected with wild-type SARS-CoV-2 (MOI=0.5). At 24 hpi, the mRNA level of SARS-CoV-2 N was measured by RT-qPCR and normalized to GAPDH expression. (C) HEK293-hACE2-TMPRSS2 cells were co-transfected with GFP, either SARS-CoV S or SARS-CoV-2 S, and IFITM2, IFITM3, or CH25H for 24 hr. The red arrows highlight the syncytia formation. Enlarged images of mock condition are highlighted by red boxes and included as insets. Scale bar: 200 µm. (D) HEK293 cells were co-transfected with GFP, Western equine encephalomyelitis virus (WEEV) E1 and E2, VSV G, or reovirus FAST p10, with or without CH25H for 24 hr. The red arrows highlight the syncytia formation. Enlarged images of mock condition are highlighted by red boxes and included as insets. Scale bar: 200 µm. (E) HEK293-hACE2 cells stably expressing TMPRSS2 or TMPRSS4 were co-transfected with SARS-CoV-2 S and GFP with or without 25HC (10 µM) for 24 hr. The red arrows highlight the syncytia formation. Scale bar: 200 µm. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: CH25H and 25HC block SARS-CoV-2 S mediated membrane fusion (A) Wild-type (WT) HEK293-hACE2 cells or those stably expressing TMPRSS2 or TMPRSS4 were transfected with mock, IFITM2, IFITM3, or CH25H for 24 hr and infected with VSV-SARS-CoV-2 (MOI=1). At 24 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression. (B) HEK293-hACE2-TMPRSS2 cells with or without CH25H expression were infected with wild-type SARS-CoV-2 (MOI=0.5). At 24 hpi, the mRNA level of SARS-CoV-2 N was measured by RT-qPCR and normalized to GAPDH expression. (C) HEK293-hACE2-TMPRSS2 cells were co-transfected with GFP, either SARS-CoV S or SARS-CoV-2 S, and IFITM2, IFITM3, or CH25H for 24 hr. The red arrows highlight the syncytia formation. Enlarged images of mock condition are highlighted by red boxes and included as insets. Scale bar: 200 µm. (D) HEK293 cells were co-transfected with GFP, Western equine encephalomyelitis virus (WEEV) E1 and E2, VSV G, or reovirus FAST p10, with or without CH25H for 24 hr. The red arrows highlight the syncytia formation. Enlarged images of mock condition are highlighted by red boxes and included as insets. Scale bar: 200 µm. (E) HEK293-hACE2 cells stably expressing TMPRSS2 or TMPRSS4 were co-transfected with SARS-CoV-2 S and GFP with or without 25HC (10 µM) for 24 hr. The red arrows highlight the syncytia formation. Scale bar: 200 µm. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Blocking Assay, Stable Transfection, Expressing, Transfection, Infection, Quantitative RT-PCR, Western Blot

    CH25H and 25HC do not affect S cleavage or lipid raft organization. (A) VSV-SARS-CoV-2 was incubated with 25HC (10 µM) for 30 min. HEK293-hACE2 cells were treated with 25HC (10 µM) for 1 hr. At 6 hpi, cells were harvested and measured for GFP percentage and intensity by flow cytometry. (B) MA104 cells were treated with 25HC (10 µM) based on the scheme (right panel) and infected with VSV-SARS-CoV-2 (MOI=1). At 24 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression (left panel). (C) HEK293-hACE2 cells were transfected with SARS-CoV-2 for 24 hr. Some cells were also transfected with TMPRSS2 or treated with trypsin (0.5 µg/ml) or 25HC (10 µM). Cells were harvested for western blot and probed for SARS-CoV-2 S1, S2, and GAPDH protein levels. (D) HEK293-hACE2 cells stably expressing indicated ISGs were stained for lipid rafts (cholera toxin B, green) and nucleus (DAPI, blue). Scale bar: 30 µm. (E) HEK293 cells were treated with C4-TopFluor-25HC (10, 1, or 0.1 µM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=0.5) for 24 hr. Scale bar: 500 µm. (F) HEK293-hACE2 cells were transfected GFP-tagged wild-type (WT) or dominant negative (DN) mutants of Rab5 or Rab7 for 24 hr. Cells were harvested for western blot and probed for GFP and GAPDH protein levels. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: CH25H and 25HC do not affect S cleavage or lipid raft organization. (A) VSV-SARS-CoV-2 was incubated with 25HC (10 µM) for 30 min. HEK293-hACE2 cells were treated with 25HC (10 µM) for 1 hr. At 6 hpi, cells were harvested and measured for GFP percentage and intensity by flow cytometry. (B) MA104 cells were treated with 25HC (10 µM) based on the scheme (right panel) and infected with VSV-SARS-CoV-2 (MOI=1). At 24 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression (left panel). (C) HEK293-hACE2 cells were transfected with SARS-CoV-2 for 24 hr. Some cells were also transfected with TMPRSS2 or treated with trypsin (0.5 µg/ml) or 25HC (10 µM). Cells were harvested for western blot and probed for SARS-CoV-2 S1, S2, and GAPDH protein levels. (D) HEK293-hACE2 cells stably expressing indicated ISGs were stained for lipid rafts (cholera toxin B, green) and nucleus (DAPI, blue). Scale bar: 30 µm. (E) HEK293 cells were treated with C4-TopFluor-25HC (10, 1, or 0.1 µM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=0.5) for 24 hr. Scale bar: 500 µm. (F) HEK293-hACE2 cells were transfected GFP-tagged wild-type (WT) or dominant negative (DN) mutants of Rab5 or Rab7 for 24 hr. Cells were harvested for western blot and probed for GFP and GAPDH protein levels. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Incubation, Flow Cytometry, Infection, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Stable Transfection, Staining, Dominant Negative Mutation

    25HC inhibits endosomal cholesterol export to block SARS-CoV-2 fusion (A) HEK293-hACE2-TMPRSS2 cells were treated with or without C4 TopFluor-25HC (F-25HC, 3 µM) and co-cultured at 1:1 ratio with HEK293 cells transfected with SARS-CoV-2 and TdTomato for 24 hr. Note that the fused cells (red) stop at the boundary of 25HC treated cells (green). Scale bar: 200 µm. (B) HEK293 cells were incubated with C4 TopFluor-25HC (F-25HC, 2 µM) for 1 hr, fixed, and stained for early/recycling endosome (Rab4), late endosome (LBPA), lysosome (LAMP1), and nucleus (blue, DAPI). Scale bar: 30 µm. (C) HEK293-hACE2 cells were transfected with wild-type (WT) or dominant negative (DN) mutants of Rab5 or Rab7 for 24 hr and infected with VSV-SARS-CoV-2 (MOI=1) with or without 25HC (10 µM). At 24 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression. (D) HEK293 cells were treated with TopFluor-cholesterol (F-cholesterol, 2 µM) with or without 25HC (20 µM) for 1 hr. Scale bar: 30 µm. (E) MA104 cells were treated with 25HC at indicated concentrations in either complete or serum-free media (SFM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 24 hr. Cells were fixed and scanned with Typhoon. Green signals were quantified by ImageJ. (F) MA104 cells were treated with itraconazole (ICZ) or furin inhibitor (FI) decanoyl-RVKR-CMK at indicated concentrations in either complete or serum-free media for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 24 hr. Cells were fixed and scanned with Typhoon for green signals. (G) HEK293-hACE2-TMPRSS2 cells were treated with 25HC (10 µM) or ICZ (3 µM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 20 hr. Scale bar: 500 µm. (H) HEK293-ACE2-TMPRSS2 cells were transfected with SARS-CoV-2 S and TdTomato plasmids for 24 hr in the presence of chloroquine (10 µM), camostat (10 µM), methyl-β-cyclodextrin (MCBD, 1 mM), ICZ (3 µM), or 25HC (20 µM). Scale bar: 200 µm. (I) Vero-E6 cells were treated with ICZ or 25HC at indicated concentrations for 1 hr and infected with SARS-CoV-2-mNeonGreen (MOI=0.5) for 24 hr. Cells were fixed and green signals were scanned with Typhoon and quantified by ImageJ. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: 25HC inhibits endosomal cholesterol export to block SARS-CoV-2 fusion (A) HEK293-hACE2-TMPRSS2 cells were treated with or without C4 TopFluor-25HC (F-25HC, 3 µM) and co-cultured at 1:1 ratio with HEK293 cells transfected with SARS-CoV-2 and TdTomato for 24 hr. Note that the fused cells (red) stop at the boundary of 25HC treated cells (green). Scale bar: 200 µm. (B) HEK293 cells were incubated with C4 TopFluor-25HC (F-25HC, 2 µM) for 1 hr, fixed, and stained for early/recycling endosome (Rab4), late endosome (LBPA), lysosome (LAMP1), and nucleus (blue, DAPI). Scale bar: 30 µm. (C) HEK293-hACE2 cells were transfected with wild-type (WT) or dominant negative (DN) mutants of Rab5 or Rab7 for 24 hr and infected with VSV-SARS-CoV-2 (MOI=1) with or without 25HC (10 µM). At 24 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression. (D) HEK293 cells were treated with TopFluor-cholesterol (F-cholesterol, 2 µM) with or without 25HC (20 µM) for 1 hr. Scale bar: 30 µm. (E) MA104 cells were treated with 25HC at indicated concentrations in either complete or serum-free media (SFM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 24 hr. Cells were fixed and scanned with Typhoon. Green signals were quantified by ImageJ. (F) MA104 cells were treated with itraconazole (ICZ) or furin inhibitor (FI) decanoyl-RVKR-CMK at indicated concentrations in either complete or serum-free media for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 24 hr. Cells were fixed and scanned with Typhoon for green signals. (G) HEK293-hACE2-TMPRSS2 cells were treated with 25HC (10 µM) or ICZ (3 µM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 20 hr. Scale bar: 500 µm. (H) HEK293-ACE2-TMPRSS2 cells were transfected with SARS-CoV-2 S and TdTomato plasmids for 24 hr in the presence of chloroquine (10 µM), camostat (10 µM), methyl-β-cyclodextrin (MCBD, 1 mM), ICZ (3 µM), or 25HC (20 µM). Scale bar: 200 µm. (I) Vero-E6 cells were treated with ICZ or 25HC at indicated concentrations for 1 hr and infected with SARS-CoV-2-mNeonGreen (MOI=0.5) for 24 hr. Cells were fixed and green signals were scanned with Typhoon and quantified by ImageJ. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Blocking Assay, Cell Culture, Transfection, Incubation, Staining, Dominant Negative Mutation, Infection, Quantitative RT-PCR, Expressing

    CH25H suppresses VSV-SARS-CoV-2 replication in HEK293-hACE2 cells. (A) HEK293-hACE2-mCherry cells were transfected with plasma membrane (PM)-localized GFP and stained for cell surface (green), ACE2 (red), nucleus (DAPI, blue), and actin (white). Scale bar: 30 µm. (B) Wild-type (WT) HEK293 or HEK293-hACE2-mCherry cells were infected with VSV-SARS-CoV-2 (MOI=1) for 8 hr. Scale bar: 200 µm. (C) Same as (B) except that infection was 24 hr and RNA was harvested for RT-qPCR measuring the mRNA level of VSV N compared to GAPDH expression. (D) Same as (B) except that infection was 24 hr and cell lysates were harvested for plaque assays. (E) HEK293-hACE2 cells stably expressing indicated ISGs were harvested for western blot and probed for V5-tagged ISG and GAPDH protein levels. (F) HEK293-hACE2 cells stably expressing indicated ISGs were infected with VSV-SARS-CoV-2 (MOI=1) for 24 hr. Scale bar: 200 µm. (G) HEK293 cells were transfected with mock, IFIH1, or CH25H plasmids for 24 hr or treated with 25HC (10 µM) for 1 hr. RNA was harvested and the mRNA levels of IFN-β (IFNB) and IFN-λ (IFNL3) were measured by RT-qPCR and normalized to GAPDH expression. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: CH25H suppresses VSV-SARS-CoV-2 replication in HEK293-hACE2 cells. (A) HEK293-hACE2-mCherry cells were transfected with plasma membrane (PM)-localized GFP and stained for cell surface (green), ACE2 (red), nucleus (DAPI, blue), and actin (white). Scale bar: 30 µm. (B) Wild-type (WT) HEK293 or HEK293-hACE2-mCherry cells were infected with VSV-SARS-CoV-2 (MOI=1) for 8 hr. Scale bar: 200 µm. (C) Same as (B) except that infection was 24 hr and RNA was harvested for RT-qPCR measuring the mRNA level of VSV N compared to GAPDH expression. (D) Same as (B) except that infection was 24 hr and cell lysates were harvested for plaque assays. (E) HEK293-hACE2 cells stably expressing indicated ISGs were harvested for western blot and probed for V5-tagged ISG and GAPDH protein levels. (F) HEK293-hACE2 cells stably expressing indicated ISGs were infected with VSV-SARS-CoV-2 (MOI=1) for 24 hr. Scale bar: 200 µm. (G) HEK293 cells were transfected with mock, IFIH1, or CH25H plasmids for 24 hr or treated with 25HC (10 µM) for 1 hr. RNA was harvested and the mRNA levels of IFN-β (IFNB) and IFN-λ (IFNL3) were measured by RT-qPCR and normalized to GAPDH expression. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Transfection, Staining, Infection, Quantitative RT-PCR, Expressing, Stable Transfection, Western Blot

    25HC restricts VSV-SARS-CoV-2 replication in MA104 cells. (A) MA104 cells were infected with serially diluted VSV-SARS-CoV-2 (10 5 shown here) with or without 25HC (10 µM). At 3 dpi, GFP signals were scanned with Typhoon. (B) Quantification of plaque sizes in (A). For all figures, experiments were repeated at least three times with similar results. Individual data point is indicated (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: 25HC restricts VSV-SARS-CoV-2 replication in MA104 cells. (A) MA104 cells were infected with serially diluted VSV-SARS-CoV-2 (10 5 shown here) with or without 25HC (10 µM). At 3 dpi, GFP signals were scanned with Typhoon. (B) Quantification of plaque sizes in (A). For all figures, experiments were repeated at least three times with similar results. Individual data point is indicated (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Infection

    25HC inhibits SARS-CoV-2 replication (A) HEK293-hACE2 cells were treated with 7-α, 25-OHC or 25HC at 0.1, 1, or 10 µM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=5). GFP signals were detected at 24 hpi. Scale bar: 200 µm. (B) MA104 cells were treated with 25HC at indicated concentrations for 1 hr and infected with VSV-SARS-CoV-2 (MOI=0.1) for 24 hr. GFP signals were quantified by ImageJ and plotted as percentage of inhibition. (C) HEK293-hACE2 cells were treated with 7-α, 25-OHC or 25HC at 0.1 or 10 µM for 1 hr and infected with SARS-CoV-2 (MOI=0.5). At 24 hpi, the mRNA level of SARS-CoV-2 N was measured by RT-qPCR and normalized to GAPDH expression. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments.
    Figure Legend Snippet: 25HC inhibits SARS-CoV-2 replication (A) HEK293-hACE2 cells were treated with 7-α, 25-OHC or 25HC at 0.1, 1, or 10 µM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=5). GFP signals were detected at 24 hpi. Scale bar: 200 µm. (B) MA104 cells were treated with 25HC at indicated concentrations for 1 hr and infected with VSV-SARS-CoV-2 (MOI=0.1) for 24 hr. GFP signals were quantified by ImageJ and plotted as percentage of inhibition. (C) HEK293-hACE2 cells were treated with 7-α, 25-OHC or 25HC at 0.1 or 10 µM for 1 hr and infected with SARS-CoV-2 (MOI=0.5). At 24 hpi, the mRNA level of SARS-CoV-2 N was measured by RT-qPCR and normalized to GAPDH expression. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments.

    Techniques Used: Infection, Inhibition, Quantitative RT-PCR, Expressing

    ISG screen identifies CH25H as an antiviral host factor that restricts SARS-CoV-2 infection (A) HEK293-hACE2-mCherry cells were transduced with lentiviral vectors encoding individual ISGs for 72 hr and infected with VSV-SARS-CoV or VSV-SARS-CoV-2 (MOI=1) for 24 hr. The percentage of GFP + cells were quantified and plotted. (B) Wild-type (WT) HEK293-hACE2 cells or HEK293-hACE2 cells stably expressing indicated ISGs were infected with VSV-SARS-CoV-2 (MOI=1). At 18 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression. (C) HEK293-hACE2 cells with or without CH25H expression were infected with wild-type VSV, VSV-SARS-CoV or VSV-SARS-CoV-2 (MOI=10) for 6 hr. Cells were harvested and measured for GFP percentage and intensity by flow cytometry. (D) HEK293-hACE2 cells with or without CH25H expression were infected with VSV-SARS-CoV, VSV-SARS-CoV-2, rotavirus RRV strain, or adenovirus serotype 5 (MOI=3) for 24 hr. Viral RNA levels were measured by RT-qPCR and normalized to GAPDH expression. (E) HEK293-hACE2 cells with or without CH25H expression were infected with wild-type SARS-CoV-2 (MOI=0.5). At 24 hpi, the mRNA level of SARS-CoV-2 N was measured by RT-qPCR and normalized to GAPDH expression. For all figures except A, experiments were repeated at least three times with similar results. Fig. 1A was performed twice with average numbers indicated on the graph. Raw data is listed in Dataset S1. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: ISG screen identifies CH25H as an antiviral host factor that restricts SARS-CoV-2 infection (A) HEK293-hACE2-mCherry cells were transduced with lentiviral vectors encoding individual ISGs for 72 hr and infected with VSV-SARS-CoV or VSV-SARS-CoV-2 (MOI=1) for 24 hr. The percentage of GFP + cells were quantified and plotted. (B) Wild-type (WT) HEK293-hACE2 cells or HEK293-hACE2 cells stably expressing indicated ISGs were infected with VSV-SARS-CoV-2 (MOI=1). At 18 hpi, the mRNA level of VSV N was measured by RT-qPCR and normalized to GAPDH expression. (C) HEK293-hACE2 cells with or without CH25H expression were infected with wild-type VSV, VSV-SARS-CoV or VSV-SARS-CoV-2 (MOI=10) for 6 hr. Cells were harvested and measured for GFP percentage and intensity by flow cytometry. (D) HEK293-hACE2 cells with or without CH25H expression were infected with VSV-SARS-CoV, VSV-SARS-CoV-2, rotavirus RRV strain, or adenovirus serotype 5 (MOI=3) for 24 hr. Viral RNA levels were measured by RT-qPCR and normalized to GAPDH expression. (E) HEK293-hACE2 cells with or without CH25H expression were infected with wild-type SARS-CoV-2 (MOI=0.5). At 24 hpi, the mRNA level of SARS-CoV-2 N was measured by RT-qPCR and normalized to GAPDH expression. For all figures except A, experiments were repeated at least three times with similar results. Fig. 1A was performed twice with average numbers indicated on the graph. Raw data is listed in Dataset S1. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Infection, Transduction, Stable Transfection, Expressing, Quantitative RT-PCR, Flow Cytometry

    CH25H and 25HC block SARS-CoV-2 S mediated fusion. (A) HEK293-hACE2-TMPRSS2 cells were infected with wild-type VSV, VSV-SARS-CoV or VSV-SARS-CoV-2 (MOI=10) for 6 hr. Cells were harvested and measured for GFP percentage and intensity by flow cytometry. (B) HEK293-hACE2-TMPRSS2 cells expressing GFP and indicated ISGs or treated with 25HC (10 µM) were mixed at 1:1 ratio and co-cultured with HEK293 cells expressing SARS-CoV-2 S and TdTomato for 24 hr. Note the formation of cell-cell fusion (yellow), highlighted by black arrows. Scale bar: 200 µm. (C) HEK293 cells were co-transfected with GFP, VSV G, or reovirus FAST p10, with or without 25HC (10 µM) for 24 hr. The red arrows highlight the syncytia formation. Scale bar: 200 µm. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM.
    Figure Legend Snippet: CH25H and 25HC block SARS-CoV-2 S mediated fusion. (A) HEK293-hACE2-TMPRSS2 cells were infected with wild-type VSV, VSV-SARS-CoV or VSV-SARS-CoV-2 (MOI=10) for 6 hr. Cells were harvested and measured for GFP percentage and intensity by flow cytometry. (B) HEK293-hACE2-TMPRSS2 cells expressing GFP and indicated ISGs or treated with 25HC (10 µM) were mixed at 1:1 ratio and co-cultured with HEK293 cells expressing SARS-CoV-2 S and TdTomato for 24 hr. Note the formation of cell-cell fusion (yellow), highlighted by black arrows. Scale bar: 200 µm. (C) HEK293 cells were co-transfected with GFP, VSV G, or reovirus FAST p10, with or without 25HC (10 µM) for 24 hr. The red arrows highlight the syncytia formation. Scale bar: 200 µm. For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM.

    Techniques Used: Blocking Assay, Infection, Flow Cytometry, Expressing, Cell Culture, Transfection

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.06.15.153064

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

    Techniques Used: Titration, Concentration Assay, Standard Deviation

    14) Product Images from "Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mice and non-human primates"

    Article Title: Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mice and non-human primates

    Journal: bioRxiv

    doi: 10.1101/2021.01.05.422952

    Quantitative assessment of humoral responses in two mouse strains. (A-B) Monthly monitoring of SARS-CoV-2 RBD-binding IgG titers in 6-10 week-old BALB/c (A) and C57BL/6 (B) mice injected IM with two doses (10 10 gc and 10 11 gc) of AC1 or AC3, n=20 (10 females and 10 males). Mean geometric titers (MGT) shown above each group. (C-D) Pseudovirus neutralizing titers of a subset of BALB/c (C) and C57BL/6 (D) animals (6 females and 6 males per group) from the studies described in A and B. TheGMT are shown above each group. (E-F) Correlation of pseudovirus neutralizing titers and RBD-binding IgG titers in BALB/c (E) and C57BL/6 (F). (G) Live SARS-CoV-2 neutralizing titers measured on a PRNT assay on week 4 samples harvested from BALB/c animals (n≥8, both genders). The GMT is shown above each group. (H) Correlation of SARS-CoV-2 neutralizing and pseudovirus neutralizing titers. (I) Titer of binding antibodies against SARS-CoV-2 RBD (SARS2 RBD), SARS-CoV-2 Spike ectodomain (SARS2 Ecto) and SARS-CoV RBD (SARS RBD) in female BALB/c sera 28 days after AC1 or AC3 injection. (J) RBD-binding antibody titers in BALB/c male animals (n=5) vaccinated with 10 11 gc of AC1 or AAV1-S (same genomic sequence packaged in different capsids), which were naïve (0 mg IVIG) or passively pre-immunized with 15 mg of human IVIG 24h and 2h prior to the vaccination. Ctr: unvaccinated control. (A-J) Data are represented as mean ± SD. For (A-D and G) groups were compared by one-way ANOVA and Tukey’s post-test. * p
    Figure Legend Snippet: Quantitative assessment of humoral responses in two mouse strains. (A-B) Monthly monitoring of SARS-CoV-2 RBD-binding IgG titers in 6-10 week-old BALB/c (A) and C57BL/6 (B) mice injected IM with two doses (10 10 gc and 10 11 gc) of AC1 or AC3, n=20 (10 females and 10 males). Mean geometric titers (MGT) shown above each group. (C-D) Pseudovirus neutralizing titers of a subset of BALB/c (C) and C57BL/6 (D) animals (6 females and 6 males per group) from the studies described in A and B. TheGMT are shown above each group. (E-F) Correlation of pseudovirus neutralizing titers and RBD-binding IgG titers in BALB/c (E) and C57BL/6 (F). (G) Live SARS-CoV-2 neutralizing titers measured on a PRNT assay on week 4 samples harvested from BALB/c animals (n≥8, both genders). The GMT is shown above each group. (H) Correlation of SARS-CoV-2 neutralizing and pseudovirus neutralizing titers. (I) Titer of binding antibodies against SARS-CoV-2 RBD (SARS2 RBD), SARS-CoV-2 Spike ectodomain (SARS2 Ecto) and SARS-CoV RBD (SARS RBD) in female BALB/c sera 28 days after AC1 or AC3 injection. (J) RBD-binding antibody titers in BALB/c male animals (n=5) vaccinated with 10 11 gc of AC1 or AAV1-S (same genomic sequence packaged in different capsids), which were naïve (0 mg IVIG) or passively pre-immunized with 15 mg of human IVIG 24h and 2h prior to the vaccination. Ctr: unvaccinated control. (A-J) Data are represented as mean ± SD. For (A-D and G) groups were compared by one-way ANOVA and Tukey’s post-test. * p

    Techniques Used: Binding Assay, Mouse Assay, Injection, Plaque Reduction Neutralization Test, Sequencing

    Characterization of humoral immune responses in NHP. (A) SARS-CoV-2 RBD-binding IgG titers 20-week follow up in Rhesus macaques (n=2, 1 female and 1 male) treated IM with 10 12 gc of AC1 or AC3. (B) Pseudovirus neutralizing antibody titers in NHPs for 20 weeks (left) and 60 convalescent human plasma samples of patients with different disease severity and NIBSC 20/130 (red dot) reference plasma (right). The Geometric Mean Titer (GMT) is shown for each cohort of convalescent plasma. (C) Live SARS-CoV-2 neutralizing titers. (D) Identification of RBD-binding B cells with a memory phenotype (CD27+ or CD27-IgD-) in peripheral blood of a representative macaque at multiple dates post-vaccination. (E) Frequency of RBD-binding B cells in memory B cell compartment. (F) Frequency of RBD-binding memory B cells with isotype-switched (IgD-IgM-) phenotype. (G) Quantification of S1 subunit concentration (pg/mL) in sera of animals treated with AC3 during the first month after vaccination. (H) Titration of binding antibodies against SARS-CoV-2 RBD (SARS2 RBD), SARS-CoV-2 Spike ectodomain (SARS2 Ecto) and SARS-CoV RBD (SARS RBD) 9 weeks after vaccination. (I) Ratio between RBD-binding IgG1 and IgG4 isotypes 8 weeks post-vaccination.
    Figure Legend Snippet: Characterization of humoral immune responses in NHP. (A) SARS-CoV-2 RBD-binding IgG titers 20-week follow up in Rhesus macaques (n=2, 1 female and 1 male) treated IM with 10 12 gc of AC1 or AC3. (B) Pseudovirus neutralizing antibody titers in NHPs for 20 weeks (left) and 60 convalescent human plasma samples of patients with different disease severity and NIBSC 20/130 (red dot) reference plasma (right). The Geometric Mean Titer (GMT) is shown for each cohort of convalescent plasma. (C) Live SARS-CoV-2 neutralizing titers. (D) Identification of RBD-binding B cells with a memory phenotype (CD27+ or CD27-IgD-) in peripheral blood of a representative macaque at multiple dates post-vaccination. (E) Frequency of RBD-binding B cells in memory B cell compartment. (F) Frequency of RBD-binding memory B cells with isotype-switched (IgD-IgM-) phenotype. (G) Quantification of S1 subunit concentration (pg/mL) in sera of animals treated with AC3 during the first month after vaccination. (H) Titration of binding antibodies against SARS-CoV-2 RBD (SARS2 RBD), SARS-CoV-2 Spike ectodomain (SARS2 Ecto) and SARS-CoV RBD (SARS RBD) 9 weeks after vaccination. (I) Ratio between RBD-binding IgG1 and IgG4 isotypes 8 weeks post-vaccination.

    Techniques Used: Binding Assay, Concentration Assay, Titration

    Quality of the host response to AAVCOVID. (A) Several RBD-binding antibody isotype titers (IgG, IgG1, IgG2a, IgG2b, IgG3, IgA and IgM) measured weekly in 6-10 week-old BALB/c (n=10, 5 females and 5 males) treated IM with two doses of AC1 and AC3. (B) Ratio of RBD-binding IgG2a and IgG1 antibody titers in serum samples harvested 28 days after vaccination of BALB/c mice as described in A. The Geometric Mean Titer (GMT) is shown above each group. (C and F) Cytokine concentration (pg/mL) in supernatants harvested from splenocytes stimulated for 48h with peptides spanning SARS-CoV-2 Spike protein. Splenocytes were extracted from BALB/c (C) and C57BL/6 (F) animals 4 and 6 weeks, respectively, after vaccination with 10 11 gc of AC1 or AC3. (D-E) Spot forming units (SFU) detected by IFN-γ (D) or IL-4 (E) ELISpot in splenocytes extracted from BALB/c animals 4 weeks after vaccination with 10 11 gc of AC1 or AC3 and stimulated with peptides spanning SARS-CoV-2 Spike protein for 48h. (G-H) Spot forming units (SFU) detected by IFN-γ (G) or IL-4 (H) ELISpot in splenocytes extracted from C57BL/6 animals 6 weeks after vaccination with 10 10 gc of AC1 or AC3 and stimulated with peptides spanning SARS-CoV-2 Spike protein for 48h. For (B-H) data are represented as mean ± SD and groups were compared by Kruskal Wallis and Dunn’s post-test.
    Figure Legend Snippet: Quality of the host response to AAVCOVID. (A) Several RBD-binding antibody isotype titers (IgG, IgG1, IgG2a, IgG2b, IgG3, IgA and IgM) measured weekly in 6-10 week-old BALB/c (n=10, 5 females and 5 males) treated IM with two doses of AC1 and AC3. (B) Ratio of RBD-binding IgG2a and IgG1 antibody titers in serum samples harvested 28 days after vaccination of BALB/c mice as described in A. The Geometric Mean Titer (GMT) is shown above each group. (C and F) Cytokine concentration (pg/mL) in supernatants harvested from splenocytes stimulated for 48h with peptides spanning SARS-CoV-2 Spike protein. Splenocytes were extracted from BALB/c (C) and C57BL/6 (F) animals 4 and 6 weeks, respectively, after vaccination with 10 11 gc of AC1 or AC3. (D-E) Spot forming units (SFU) detected by IFN-γ (D) or IL-4 (E) ELISpot in splenocytes extracted from BALB/c animals 4 weeks after vaccination with 10 11 gc of AC1 or AC3 and stimulated with peptides spanning SARS-CoV-2 Spike protein for 48h. (G-H) Spot forming units (SFU) detected by IFN-γ (G) or IL-4 (H) ELISpot in splenocytes extracted from C57BL/6 animals 6 weeks after vaccination with 10 10 gc of AC1 or AC3 and stimulated with peptides spanning SARS-CoV-2 Spike protein for 48h. For (B-H) data are represented as mean ± SD and groups were compared by Kruskal Wallis and Dunn’s post-test.

    Techniques Used: Binding Assay, Mouse Assay, Concentration Assay, Enzyme-linked Immunospot

    Composition and characterization of AAVCOVID vaccine candidates. (A) Schematic representation of the recombinant genome of AAVCOVID19-1 (AC1) and AAVCOVID19-3 (AC3) vaccine candidates. SV40: Simian virus 40 promoter. RBD: receptor binding domain. S1: SARS-CoV-2 Spike subunit 1. S2: SARS-CoV-2 Spike subunit 2. CMV: cytomegalovirus promoter. tPA-SP: tissue plasminogen activator signal peptide. WPRE: woodchuck hepatitis virus posttranscriptional regulatory element. bGH: bovine growth hormone. ITR: inverted terminal repeat. (B) Phylogenetic tree of several AAV clades and percentage of sequence identity with AAVrh32.33. (C) Percentage of seropositivity of neutralizing antibodies and titer range against AAV2, AAV8 and AAVrh32.33 among 50 donor plasma samples. (D) Productivity of several AC1 and AC3 (vector genome copies produced per producer cell or Gc/cell) compared to various AAV serotypes carrying a CMV-EGFP-WPRE transgene in small scale production and purification. Data are represented as mean ± SD. One-way ANOVA and Tukey’s tests were used to compare groups between them. * p
    Figure Legend Snippet: Composition and characterization of AAVCOVID vaccine candidates. (A) Schematic representation of the recombinant genome of AAVCOVID19-1 (AC1) and AAVCOVID19-3 (AC3) vaccine candidates. SV40: Simian virus 40 promoter. RBD: receptor binding domain. S1: SARS-CoV-2 Spike subunit 1. S2: SARS-CoV-2 Spike subunit 2. CMV: cytomegalovirus promoter. tPA-SP: tissue plasminogen activator signal peptide. WPRE: woodchuck hepatitis virus posttranscriptional regulatory element. bGH: bovine growth hormone. ITR: inverted terminal repeat. (B) Phylogenetic tree of several AAV clades and percentage of sequence identity with AAVrh32.33. (C) Percentage of seropositivity of neutralizing antibodies and titer range against AAV2, AAV8 and AAVrh32.33 among 50 donor plasma samples. (D) Productivity of several AC1 and AC3 (vector genome copies produced per producer cell or Gc/cell) compared to various AAV serotypes carrying a CMV-EGFP-WPRE transgene in small scale production and purification. Data are represented as mean ± SD. One-way ANOVA and Tukey’s tests were used to compare groups between them. * p

    Techniques Used: Recombinant, Binding Assay, Sequencing, Plasmid Preparation, Produced, Purification

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.10.15.339838

    Mannosidase-I blockade using kifunensine inhibits SARS-CoV-2 pseudovirus entry into 293T/ACE2 cells. A . VSVG, Spike-WT and Spike-mutant pseudovirus were produced in the presence of 15µM kifunensine or vehicle control. The 6 viruses were added to 293T/ACE2 at equal titer. B-D . Microscopy (panel B) and cytometry (panel C, D) show ∼90% loss of viral infection in the case of Spike-WT and Spike-mutant virus upon kifunensine treatment (* P
    Figure Legend Snippet: Mannosidase-I blockade using kifunensine inhibits SARS-CoV-2 pseudovirus entry into 293T/ACE2 cells. A . VSVG, Spike-WT and Spike-mutant pseudovirus were produced in the presence of 15µM kifunensine or vehicle control. The 6 viruses were added to 293T/ACE2 at equal titer. B-D . Microscopy (panel B) and cytometry (panel C, D) show ∼90% loss of viral infection in the case of Spike-WT and Spike-mutant virus upon kifunensine treatment (* P

    Techniques Used: Mutagenesis, Produced, Microscopy, Cytometry, Infection

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

    Techniques Used: Binding Assay, Expressing, Generated

    N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. A . Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] − and [N] − 293T cells. All 9 viruses were applied at equal titer to stable 293T/ACE2. B - C . O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. D . Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. E . Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] − 293T products due to truncation of glycan biosynthesis. F . Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] − 293Ts is almost fully proteolyzed during viral production (red arrowhead). * P
    Figure Legend Snippet: N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. A . Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] − and [N] − 293T cells. All 9 viruses were applied at equal titer to stable 293T/ACE2. B - C . O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. D . Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. E . Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] − 293T products due to truncation of glycan biosynthesis. F . Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] − 293Ts is almost fully proteolyzed during viral production (red arrowhead). * P

    Techniques Used: Modification, Expressing, Mutagenesis, Produced, Generated, Titration, Infection, Western Blot

    Glycan coverage of Spike-ACE2 co-complex: SARS-CoV-2 spike protein trimer (pink) bound to ACE2 (green). A . without glycans. B . with N-glycans (red) identified using LC-MS on Spike and ACE2. C . Molecular dynamics simulation analyzed the range of movement of each glycan. The space sampled by glycans is represented by a gray cloud. Glycans cover the Spike-ACE2 interface. They also surround the putative proteolysis site of furin (“S1-S2”, yellow) and S2’ (blue).
    Figure Legend Snippet: Glycan coverage of Spike-ACE2 co-complex: SARS-CoV-2 spike protein trimer (pink) bound to ACE2 (green). A . without glycans. B . with N-glycans (red) identified using LC-MS on Spike and ACE2. C . Molecular dynamics simulation analyzed the range of movement of each glycan. The space sampled by glycans is represented by a gray cloud. Glycans cover the Spike-ACE2 interface. They also surround the putative proteolysis site of furin (“S1-S2”, yellow) and S2’ (blue).

    Techniques Used: Liquid Chromatography with Mass Spectroscopy

    16) Product Images from "Native-like SARS-CoV-2 spike glycoprotein expressed by ChAdOx1 nCoV-19/AZD1222 vaccine"

    Article Title: Native-like SARS-CoV-2 spike glycoprotein expressed by ChAdOx1 nCoV-19/AZD1222 vaccine

    Journal: bioRxiv

    doi: 10.1101/2021.01.15.426463

    Site-specific glycan processing of SARS-CoV-2 S upon infection with ChAdOx1 nCoV-19. (A) Western blot analysis of SARS-CoV-2 spike proteins, using anti-S1 and anti-S1+S2 antibodies. Lane 1= Protein pellet from 293F cell lysates infected with ChAdOx1 nCoV-19. Lane 2= Reduced protein pellet from 293F infected with ChAdOx1 nCoV-19. Lane 3=2P-stablilsed SARS-CoV-2 S protein. The white boxes correspond to gel bands that were excised for mass spectrometric analysis. (B) Site-specific N-linked glycosylation of SARS-CoV-2 S0 and S1/S2 glycoproteins. The bar graphs represent the relative quantities of digested glycopeptides possessing the identifiers of oligomannose/hybrid-type glycans (green), complex-type glycans (pink), unoccupied PNGs (grey), or not determined (N.D.) at each N-linked glycan sequon on the S protein, listed from N to C terminus. (C) Glycosylated model of the cleaved (S1/S2) SARS-CoV-2 spike. The pie charts summarise the mass spectrometric analysis of the oligomannose/hybrid (green), complex (pink), or unoccupied (grey) N-linked glycan populations. Representative glycans are modelled onto the prefusion structure of trimeric SARS-CoV-2 S glycoprotein (PDB ID: 6VSB) ( 3 ), with one RBD in the “up” conformation. The modelled glycans are coloured according to oligomannose/hybrid-glycan content with glycan sites labelled in green (80-100%), orange (30-79%), pink (0-29%) or grey (not detected).
    Figure Legend Snippet: Site-specific glycan processing of SARS-CoV-2 S upon infection with ChAdOx1 nCoV-19. (A) Western blot analysis of SARS-CoV-2 spike proteins, using anti-S1 and anti-S1+S2 antibodies. Lane 1= Protein pellet from 293F cell lysates infected with ChAdOx1 nCoV-19. Lane 2= Reduced protein pellet from 293F infected with ChAdOx1 nCoV-19. Lane 3=2P-stablilsed SARS-CoV-2 S protein. The white boxes correspond to gel bands that were excised for mass spectrometric analysis. (B) Site-specific N-linked glycosylation of SARS-CoV-2 S0 and S1/S2 glycoproteins. The bar graphs represent the relative quantities of digested glycopeptides possessing the identifiers of oligomannose/hybrid-type glycans (green), complex-type glycans (pink), unoccupied PNGs (grey), or not determined (N.D.) at each N-linked glycan sequon on the S protein, listed from N to C terminus. (C) Glycosylated model of the cleaved (S1/S2) SARS-CoV-2 spike. The pie charts summarise the mass spectrometric analysis of the oligomannose/hybrid (green), complex (pink), or unoccupied (grey) N-linked glycan populations. Representative glycans are modelled onto the prefusion structure of trimeric SARS-CoV-2 S glycoprotein (PDB ID: 6VSB) ( 3 ), with one RBD in the “up” conformation. The modelled glycans are coloured according to oligomannose/hybrid-glycan content with glycan sites labelled in green (80-100%), orange (30-79%), pink (0-29%) or grey (not detected).

    Techniques Used: Infection, Western Blot

    ChAdOx1 nCoV-19 produces membrane associated SARS-CoV-2 S glycoprotein in native conformations able to bind its host receptor, ACE2. (A) Schematic representation of the vaccine encoded SARS-CoV-2 S protein, showing the position of N-linked glycosylation amino-acid sequons (NXS/T, where X≠P) as branches. Protein domains are illustrated: N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), and transmembrane domain (TM), with the additional tPA secretion signal at the N-terminus. (B) HeLa S3 cells were infected with ChAdOx1 nCoV-19 and incubated with either recombinant ACE2, anti-ChAdOx1 nCoV-19 (derived from vaccinated mice) or a panel of human mAbs (Ab44, Ab45, Ab71 and Ab111, which recognise, S2, RBD, trimeric S and NTD respectively) and compared to non-infected controls, analysed by flow cytometry. (Left). Relative frequency of cells and AlexaFluor 488 fluorescence associated with anti-spike detection is plotted. Left, (blue) anti-ChAdOx1 nCoV-19, middle (red), ACE2 and right (shades of green) human mAbs. In dark grey cells infected with an irrelevant ChAdOx1 vaccine and in light grey non-infected cells are shown as a control. Experimental replicates were performed two times and representative data shown.
    Figure Legend Snippet: ChAdOx1 nCoV-19 produces membrane associated SARS-CoV-2 S glycoprotein in native conformations able to bind its host receptor, ACE2. (A) Schematic representation of the vaccine encoded SARS-CoV-2 S protein, showing the position of N-linked glycosylation amino-acid sequons (NXS/T, where X≠P) as branches. Protein domains are illustrated: N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), and transmembrane domain (TM), with the additional tPA secretion signal at the N-terminus. (B) HeLa S3 cells were infected with ChAdOx1 nCoV-19 and incubated with either recombinant ACE2, anti-ChAdOx1 nCoV-19 (derived from vaccinated mice) or a panel of human mAbs (Ab44, Ab45, Ab71 and Ab111, which recognise, S2, RBD, trimeric S and NTD respectively) and compared to non-infected controls, analysed by flow cytometry. (Left). Relative frequency of cells and AlexaFluor 488 fluorescence associated with anti-spike detection is plotted. Left, (blue) anti-ChAdOx1 nCoV-19, middle (red), ACE2 and right (shades of green) human mAbs. In dark grey cells infected with an irrelevant ChAdOx1 vaccine and in light grey non-infected cells are shown as a control. Experimental replicates were performed two times and representative data shown.

    Techniques Used: Binding Assay, Infection, Incubation, Recombinant, Derivative Assay, Mouse Assay, Flow Cytometry, Fluorescence

    Cryo-ET and subtomogram average of ChAdOx1 nCoV-19 derived spike. (A) Tomographic slice of U2OS cell transduced with ChAdOx1 nCoV-19. The slice is 6.4 Å thick; PM = plasma membrane, scale bar = 100 nm (B) Detailed view of the boxed area marked in (A) . White arrowheads indicate spike proteins on the cell surface; scale bar = 50 nm. (C-E) Subtomogram average of ChAdOx1 nCoV-19 spikes at 11.6 Å resolution as indicated by Fourier-Shell correlation at 0.5 cut-off (C) , shown from side view (D) , and top view (E). SARS-CoV-2 atomic model (PDB 6ZB5) ( 29 ) is fitted for reference.
    Figure Legend Snippet: Cryo-ET and subtomogram average of ChAdOx1 nCoV-19 derived spike. (A) Tomographic slice of U2OS cell transduced with ChAdOx1 nCoV-19. The slice is 6.4 Å thick; PM = plasma membrane, scale bar = 100 nm (B) Detailed view of the boxed area marked in (A) . White arrowheads indicate spike proteins on the cell surface; scale bar = 50 nm. (C-E) Subtomogram average of ChAdOx1 nCoV-19 spikes at 11.6 Å resolution as indicated by Fourier-Shell correlation at 0.5 cut-off (C) , shown from side view (D) , and top view (E). SARS-CoV-2 atomic model (PDB 6ZB5) ( 29 ) is fitted for reference.

    Techniques Used: Derivative Assay, Transduction

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

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

    Journal: bioRxiv

    doi: 10.1101/2021.01.29.428890

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

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

    18) Product Images from "Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response"

    Article Title: Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response

    Journal: bioRxiv

    doi: 10.1101/2020.06.27.175166

    Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.
    Figure Legend Snippet: Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.

    Techniques Used: Mouse Assay, Staining, Immunofluorescence, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Binding Assay

    Neutralizing activity against SARS-CoV-2. (A) Percent neutralization of SARS-CoV-2 virus expressing GFP. Serum collected from the naïve animals used as negative controls. (B) Neutralization titer against SARS-CoV-2 virus expressing GFP. (C, D) Correlations between neutralization titer and ELISA binding titer.
    Figure Legend Snippet: Neutralizing activity against SARS-CoV-2. (A) Percent neutralization of SARS-CoV-2 virus expressing GFP. Serum collected from the naïve animals used as negative controls. (B) Neutralization titer against SARS-CoV-2 virus expressing GFP. (C, D) Correlations between neutralization titer and ELISA binding titer.

    Techniques Used: Activity Assay, Neutralization, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay

    Analyzing SARS-CoV-2 RBD and S1 proteins affinities to human ACE2 (hACE2) proteins using biolayer interferometry (BLI). (A) Bio-Layer Interferometry sensograms of the binding of SARS-CoV-2 S1 and RBD proteins to immobilized Fc-human ACE2, after incubation of the analytes at 25°C for 0and 60 minutes. The traces represent BLI response curves for SARS-CoV-2 proteins serially diluted from 800nM to 12.5nM, as indicated. Dotted lines show raw response values, while bold solid lines show the fitted trace. Association and dissociation phases were monitored for 300s and 600s, respectively. The data was globally fit using a 1:1 binding model to estimate binding affinity. (B) Binding affinity specifications of S1 and RBD proteins against hu-ACE2.
    Figure Legend Snippet: Analyzing SARS-CoV-2 RBD and S1 proteins affinities to human ACE2 (hACE2) proteins using biolayer interferometry (BLI). (A) Bio-Layer Interferometry sensograms of the binding of SARS-CoV-2 S1 and RBD proteins to immobilized Fc-human ACE2, after incubation of the analytes at 25°C for 0and 60 minutes. The traces represent BLI response curves for SARS-CoV-2 proteins serially diluted from 800nM to 12.5nM, as indicated. Dotted lines show raw response values, while bold solid lines show the fitted trace. Association and dissociation phases were monitored for 300s and 600s, respectively. The data was globally fit using a 1:1 binding model to estimate binding affinity. (B) Binding affinity specifications of S1 and RBD proteins against hu-ACE2.

    Techniques Used: Binding Assay, Incubation

    Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.
    Figure Legend Snippet: Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.

    Techniques Used: Mouse Assay, Recombinant, Expressing, Binding Assay, Enzyme-linked Immunosorbent Assay, Luminex, Fluorescence

    19) Product Images from "An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2"

    Article Title: An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2

    Journal: bioRxiv

    doi: 10.1101/2020.08.19.258244

    The immunogenicity, efficacy and safety of C-Vac for SARS-CoV-2 infection. (A) Flow cytometry-based detection of MHC(HLA-A2)-peptide complex binding affinity in T2 cells. (B) The expression of antigens in 293T-based C-Vac is confirmed by Western Blot. N, 293T cells transfected with the plasmid expressing a full length N gene. (C D) Pseudovirus neutralization titers of hamster serum at day 7 after the first immunization and day 21 (boosted at day 14) after vaccination with 293T-based C-Vac, MiT C-Vac: Mitomycin C-treated C-Vac, Lys C-Vac: Lysed C-Vac. (E) Histological characteristics of hamster lung at day 7 after the first vaccination. Original magnification 200× (F) Allograft volume of transformed fibroblasts expressing RBD-truncated N protein in the Syrian hamsters immunized with different regime. 5×10 6 BHK21 cells expressing C-Vac antigen (RBD-Ntap) were subcutaneously injected into immunized hamsters for challenge at day 45 after boost, and the volume of allografts were measured at 14 days after inoculation of the BHK21 cells expressing RBD-Ntap into the immunized hamsters.
    Figure Legend Snippet: The immunogenicity, efficacy and safety of C-Vac for SARS-CoV-2 infection. (A) Flow cytometry-based detection of MHC(HLA-A2)-peptide complex binding affinity in T2 cells. (B) The expression of antigens in 293T-based C-Vac is confirmed by Western Blot. N, 293T cells transfected with the plasmid expressing a full length N gene. (C D) Pseudovirus neutralization titers of hamster serum at day 7 after the first immunization and day 21 (boosted at day 14) after vaccination with 293T-based C-Vac, MiT C-Vac: Mitomycin C-treated C-Vac, Lys C-Vac: Lysed C-Vac. (E) Histological characteristics of hamster lung at day 7 after the first vaccination. Original magnification 200× (F) Allograft volume of transformed fibroblasts expressing RBD-truncated N protein in the Syrian hamsters immunized with different regime. 5×10 6 BHK21 cells expressing C-Vac antigen (RBD-Ntap) were subcutaneously injected into immunized hamsters for challenge at day 45 after boost, and the volume of allografts were measured at 14 days after inoculation of the BHK21 cells expressing RBD-Ntap into the immunized hamsters.

    Techniques Used: Infection, Flow Cytometry, Binding Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Neutralization, Transformation Assay, Injection

    The RBD domain of Spike is crucial for the SARS-CoV2 Vaccine. (A) The functional domain of SARS-CoV-2 spike protein. (B) Potential B cell antigen of RBD domain from SARS-CoV2 is predicted by Discotope software based on their 3D structure. (C) Potential linear B cell epitopes of SARS-CoV-2 full S protein are analysed with the IEDB database. (D) The location of potential antigens in RBD domain (SARS-CoV-2:red, SARS-CoV: Purple) and interaction model between RBD and ACE2 receptor (interface is marked yellow) are marked with Discovery Studio. (E) The expression of Spike and nucleocapsid with wild-type sequence in 293T cells are detected by Western Blot assay. (F) The expression of Spike and its derivatives with codon optimization (opt).
    Figure Legend Snippet: The RBD domain of Spike is crucial for the SARS-CoV2 Vaccine. (A) The functional domain of SARS-CoV-2 spike protein. (B) Potential B cell antigen of RBD domain from SARS-CoV2 is predicted by Discotope software based on their 3D structure. (C) Potential linear B cell epitopes of SARS-CoV-2 full S protein are analysed with the IEDB database. (D) The location of potential antigens in RBD domain (SARS-CoV-2:red, SARS-CoV: Purple) and interaction model between RBD and ACE2 receptor (interface is marked yellow) are marked with Discovery Studio. (E) The expression of Spike and nucleocapsid with wild-type sequence in 293T cells are detected by Western Blot assay. (F) The expression of Spike and its derivatives with codon optimization (opt).

    Techniques Used: Functional Assay, Software, Expressing, Sequencing, Western Blot

    Construction of chimeric vaccine for SARS-CoV-2. (A) Potential B-cell epitopes of N protein is predicted by IEDB database. (B) Potential MHCI-binding peptides of N. (C) Functional domain of SARS-CoV N protein (Upper) and its antibody epitope map reported in previous study. (D) The skeleton of Chimeric Vaccine for SARS-CoV-2, RBD: spike RBD domain (306-541 aa), Ntap: T-cell-associated peptide of N (211-339 aa). (E) Characterization of SARS-CoV-2-derived protein and C-Vac antigen by SARS-CoV-2 antisera and commercial antibodies against SARS-CoV2 spike RBD or Nucleocapsid.
    Figure Legend Snippet: Construction of chimeric vaccine for SARS-CoV-2. (A) Potential B-cell epitopes of N protein is predicted by IEDB database. (B) Potential MHCI-binding peptides of N. (C) Functional domain of SARS-CoV N protein (Upper) and its antibody epitope map reported in previous study. (D) The skeleton of Chimeric Vaccine for SARS-CoV-2, RBD: spike RBD domain (306-541 aa), Ntap: T-cell-associated peptide of N (211-339 aa). (E) Characterization of SARS-CoV-2-derived protein and C-Vac antigen by SARS-CoV-2 antisera and commercial antibodies against SARS-CoV2 spike RBD or Nucleocapsid.

    Techniques Used: Binding Assay, Functional Assay, Derivative Assay

    20) Product Images from "Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model"

    Article Title: Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model

    Journal: bioRxiv

    doi: 10.1101/2020.09.24.312165

    JIB-04 exhibits distinct post-entry antiviral mechanisms (A) Drug combination dose-response matrix and VSV-SARS-CoV-2 replication. MA104 cells were treated with JIB-04 and chloroquine or JIB-04 and NTZ for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. (B) Time of compound addition and VSV-SARS-CoV-2 replication. MA104 cells were treated with NTZ or JIB-04 (10 μM) at indicated time points relative to VSV-SARS-CoV-2 infection (MOI=3, 0 hpi). GFP signals at 8 hpi were quantified to calculate the percentage of inhibition. (C) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. S RNA levels were measured by RT-qPCR. (D) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. FL: full-length. S2: cleaved S2 fragment. (* non-specific band) (E) Histone demethylase siRNA knockdown and RV replication. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01). Viral RNA copy numbers at 12 hpi were quantified by RT-qPCR. (F) Volcano plot of differentially expressed transcripts with JIB-04 treatment and RV infection. HEK293 cells were treated with DMSO or JIB-04 (10 μM) for 12 hr, and mock-infected (left panel) or infected with porcine RV (MOI=0.01, right panel) for another 12 hr. Red dots represent upregulated genes and green dots represent downregulated genes in JIB-04 treated cells. (G) Expression of three top genes in (F) with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 12 hr and mock-infected or infected porcine RV (MOI=0.01) for 12 hr. mRNA levels of CYP1A1, CYP1B1, and AHRR at 12 hpi were measured by RT-qPCR. (H) Dose-response analysis of VSV-SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. (I) Dose-response analysis of wild-type SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels at 24 hpi were quantified based on immunofluorescence. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. For all panels except A and I, experiments were repeated at least three times with similar results. Fig. 3A was performed twice. Inhibition assay in Fig. 3I was performed once and cytotoxicity assay was performed in triplicates. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: JIB-04 exhibits distinct post-entry antiviral mechanisms (A) Drug combination dose-response matrix and VSV-SARS-CoV-2 replication. MA104 cells were treated with JIB-04 and chloroquine or JIB-04 and NTZ for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. (B) Time of compound addition and VSV-SARS-CoV-2 replication. MA104 cells were treated with NTZ or JIB-04 (10 μM) at indicated time points relative to VSV-SARS-CoV-2 infection (MOI=3, 0 hpi). GFP signals at 8 hpi were quantified to calculate the percentage of inhibition. (C) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. S RNA levels were measured by RT-qPCR. (D) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. FL: full-length. S2: cleaved S2 fragment. (* non-specific band) (E) Histone demethylase siRNA knockdown and RV replication. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01). Viral RNA copy numbers at 12 hpi were quantified by RT-qPCR. (F) Volcano plot of differentially expressed transcripts with JIB-04 treatment and RV infection. HEK293 cells were treated with DMSO or JIB-04 (10 μM) for 12 hr, and mock-infected (left panel) or infected with porcine RV (MOI=0.01, right panel) for another 12 hr. Red dots represent upregulated genes and green dots represent downregulated genes in JIB-04 treated cells. (G) Expression of three top genes in (F) with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 12 hr and mock-infected or infected porcine RV (MOI=0.01) for 12 hr. mRNA levels of CYP1A1, CYP1B1, and AHRR at 12 hpi were measured by RT-qPCR. (H) Dose-response analysis of VSV-SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. (I) Dose-response analysis of wild-type SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels at 24 hpi were quantified based on immunofluorescence. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. For all panels except A and I, experiments were repeated at least three times with similar results. Fig. 3A was performed twice. Inhibition assay in Fig. 3I was performed once and cytotoxicity assay was performed in triplicates. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Infection, Inhibition, Quantitative RT-PCR, Western Blot, Transfection, Expressing, Immunofluorescence, Cytotoxicity Assay

    Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Chemical structures of NTZ and JIB-04 E-isomer from ChemSpider database. (B) Representative images of Vero E6 cells infected by SARS-CoV-2-mNeonGreen (MOI=0.5) at 24 hpi in Fig. 1A . Experiments were repeated at least three times with similar results.
    Figure Legend Snippet: Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Chemical structures of NTZ and JIB-04 E-isomer from ChemSpider database. (B) Representative images of Vero E6 cells infected by SARS-CoV-2-mNeonGreen (MOI=0.5) at 24 hpi in Fig. 1A . Experiments were repeated at least three times with similar results.

    Techniques Used: Infection

    Nitazoxanide and JIB-04 inhibit the replication of multiple viruses (A) Mean fluorescence intensity of GFP positive cells in Fig. 2B was quantified by flow cytometry. (B) Dose-response analysis of VSV-SARS-CoV-2 replication with NTZ or JIB-04 treatment. MA104 cells were treated with compounds at indicated concentrations for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). At 24 hpi, images of GFP positive infected cells were acquired by the ECHO fluorescence microscope. (C) Same as (B) except that cells were infected with an MOI of 0.1. (D) Dose-response analysis of intracellular viral RNA levels with NTZ, JIB-04, or chloroquine treatment. MA104 cells were treated with compounds at 0.1 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (E) Western blot analysis of RV antigen VP6 levels with JIB-04 treatment. HEK293 cells were treated with JIB-04 at 1, 5, or 10 μM for 6 hr and infected with porcine RV (MOI=0.01) for 12 hr. GAPDH was used as a loading control. All experiments were repeated at least three times with similar results. Data are represented as mean ± SEM.
    Figure Legend Snippet: Nitazoxanide and JIB-04 inhibit the replication of multiple viruses (A) Mean fluorescence intensity of GFP positive cells in Fig. 2B was quantified by flow cytometry. (B) Dose-response analysis of VSV-SARS-CoV-2 replication with NTZ or JIB-04 treatment. MA104 cells were treated with compounds at indicated concentrations for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). At 24 hpi, images of GFP positive infected cells were acquired by the ECHO fluorescence microscope. (C) Same as (B) except that cells were infected with an MOI of 0.1. (D) Dose-response analysis of intracellular viral RNA levels with NTZ, JIB-04, or chloroquine treatment. MA104 cells were treated with compounds at 0.1 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (E) Western blot analysis of RV antigen VP6 levels with JIB-04 treatment. HEK293 cells were treated with JIB-04 at 1, 5, or 10 μM for 6 hr and infected with porcine RV (MOI=0.01) for 12 hr. GAPDH was used as a loading control. All experiments were repeated at least three times with similar results. Data are represented as mean ± SEM.

    Techniques Used: Fluorescence, Flow Cytometry, Infection, Microscopy, Quantitative RT-PCR, Western Blot

    Nitazoxanide and JIB-04 broadly inhibit DNA and RNA viruses in different cell types (A) Dose-response analysis of VSV and VSV-SARS-CoV-2 replication with 15 compounds. MA104 cells were treated with indicated compounds at 0.01 to 30 μM for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. EC 50 values for VSV and VSV-SARS-CoV-2 are shown in each graph in red and blue, respectively. (B) Virus infectivity with NTZ or JIB-04 treatment. Vero E6-TMPRSS2 cells were treated with compounds (10 μM) for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). At 6 hpi, percentages of GFP positive cells were quantified by flow cytometry. (C) Dose-response analysis of VSV-SARS-CoV-2 replication and cytotoxicity with NTZ or JIB-04 treatment. For EC 50 measurement, MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3) for 24 hr. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 3 mM for 25 hr. SI: selectivity index. (D) Intracellular viral RNA levels with NTZ or JIB-04 treatment. MA104 cells were treated with compounds (10 μM) for 1 hr and infected with vaccinia virus (VACV), herpes simplex virus-1 (HSV-1), or rotavirus (RV, RRV and UK strains) (MOI=1). Viral RNA levels at 24 hpi were measured by RT-qPCR for VACV B10R, HSV-1 ICP-27, and RV NSP5, respectively. (E) Viral RNA copy numbers with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 6 hr and infected with porcine rotavirus (MOI=0.01) for 6 hr. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with transmissible gastroenteritis virus (TGEV) (MOI=0.01) for 12 hr. Viral RNA copy numbers were measured by RT-qPCR. (F) TGEV titers in the cell supernatant with JIB-04 treatment. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with TGEV (MOI=0.01). Virus titers at 6 and 12 hpi were measured by plaque assays. (G) Intracellular viral RNA levels with NTZ or JIB-04 treatment in different cell types. HEK293-hACE2, HEK293-hACE2-TMPRSS2, and Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1). VSV RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 2A was performed once. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: Nitazoxanide and JIB-04 broadly inhibit DNA and RNA viruses in different cell types (A) Dose-response analysis of VSV and VSV-SARS-CoV-2 replication with 15 compounds. MA104 cells were treated with indicated compounds at 0.01 to 30 μM for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. EC 50 values for VSV and VSV-SARS-CoV-2 are shown in each graph in red and blue, respectively. (B) Virus infectivity with NTZ or JIB-04 treatment. Vero E6-TMPRSS2 cells were treated with compounds (10 μM) for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). At 6 hpi, percentages of GFP positive cells were quantified by flow cytometry. (C) Dose-response analysis of VSV-SARS-CoV-2 replication and cytotoxicity with NTZ or JIB-04 treatment. For EC 50 measurement, MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3) for 24 hr. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 3 mM for 25 hr. SI: selectivity index. (D) Intracellular viral RNA levels with NTZ or JIB-04 treatment. MA104 cells were treated with compounds (10 μM) for 1 hr and infected with vaccinia virus (VACV), herpes simplex virus-1 (HSV-1), or rotavirus (RV, RRV and UK strains) (MOI=1). Viral RNA levels at 24 hpi were measured by RT-qPCR for VACV B10R, HSV-1 ICP-27, and RV NSP5, respectively. (E) Viral RNA copy numbers with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 6 hr and infected with porcine rotavirus (MOI=0.01) for 6 hr. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with transmissible gastroenteritis virus (TGEV) (MOI=0.01) for 12 hr. Viral RNA copy numbers were measured by RT-qPCR. (F) TGEV titers in the cell supernatant with JIB-04 treatment. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with TGEV (MOI=0.01). Virus titers at 6 and 12 hpi were measured by plaque assays. (G) Intracellular viral RNA levels with NTZ or JIB-04 treatment in different cell types. HEK293-hACE2, HEK293-hACE2-TMPRSS2, and Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1). VSV RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 2A was performed once. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Infection, Inhibition, Flow Cytometry, Quantitative RT-PCR

    Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Small molecule inhibitor screen. Vero E6 cells were treated with individual compounds (listed in Table S1) at 10 μM for 1 hour (hr) and infected with SARS-CoV-2-mNeonGreen (MOI=0.5). At 24 hr post infection (hpi), cells were fixed and nuclei were stained by Hoechst 33342. The intensities of mNeonGreen and Hoechst were quantified by the Typhoon biomolecular imager and Cytation plate reader, respectively. The ratio of mNeonGreen and Hoechst is plotted as percentage of inhibition. (B) Dose-response analysis of wild-type SARS-CoV-2 replication with NTZ or JIB-04 treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels were quantified at 24 hpi based on immunofluorescence. For CC 50 measurement, cells were treated with inhibitors at 0.3 μM to 1 mM for 25 hr. SI: selectivity index. (C) Dose-response analysis of intracellular viral RNA levels with compounds. Vero E6 cells were treated with NTZ (10 μM), JIB-04 (10 μM), chloroquine (10 μM), remdesivir (3 μM), or camostat (10 μM) for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). SARS-CoV-2 RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 1A was performed once with raw data included in Dataset S1. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05).
    Figure Legend Snippet: Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Small molecule inhibitor screen. Vero E6 cells were treated with individual compounds (listed in Table S1) at 10 μM for 1 hour (hr) and infected with SARS-CoV-2-mNeonGreen (MOI=0.5). At 24 hr post infection (hpi), cells were fixed and nuclei were stained by Hoechst 33342. The intensities of mNeonGreen and Hoechst were quantified by the Typhoon biomolecular imager and Cytation plate reader, respectively. The ratio of mNeonGreen and Hoechst is plotted as percentage of inhibition. (B) Dose-response analysis of wild-type SARS-CoV-2 replication with NTZ or JIB-04 treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels were quantified at 24 hpi based on immunofluorescence. For CC 50 measurement, cells were treated with inhibitors at 0.3 μM to 1 mM for 25 hr. SI: selectivity index. (C) Dose-response analysis of intracellular viral RNA levels with compounds. Vero E6 cells were treated with NTZ (10 μM), JIB-04 (10 μM), chloroquine (10 μM), remdesivir (3 μM), or camostat (10 μM) for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). SARS-CoV-2 RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 1A was performed once with raw data included in Dataset S1. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05).

    Techniques Used: Infection, Staining, Inhibition, Immunofluorescence, Quantitative RT-PCR

    Inhibition or knockdown of specific KDM histone demethylases inhibits virus replication (A) Expression of IFN and IFN-stimulated genes with NTZ or JIB-04 treatment. HEK293 cells were treated with NTZ (10 μM), JIB-04 (3 μM), or transfected with low-molecular-weight poly(I:C) (100 ng/ml) for 24 hr. mRNA levels of IFNL3 and CXCL10 were measured by RT-qPCR. (B) Autophagy formation with compound treatment. HEK293 cells were transfected with EGFP-LC3 plasmid for 24 hr and treated with rapamycin (100 nM), NTZ (10 μM), or JIB-04 (3 μM) for another 18 hr. GFP positive punctate structures indicate autophagy activation. Scale bar, 20 μm. (C) Intracellular viral RNA levels with JIB-04 and camostat treatment. Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (D) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. S RNA levels were measured by RT-qPCR. (E) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. FL: full-length. S2: cleaved S2 fragment. (F) siRNA-mediated knockdown of JIB-04 target histone demethylases. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr. mRNA levels of indicated histone demethylases were measured by RT-qPCR. (G) Western blot analysis of RV antigen VP6 levels in cells with histone demethylase siRNA knockdown. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01) for 12 hr. (H) Pathway enrichment analysis of gene expression regulated by JIB-04 treatment. Downregulated genes in Fig. 3F with p values
    Figure Legend Snippet: Inhibition or knockdown of specific KDM histone demethylases inhibits virus replication (A) Expression of IFN and IFN-stimulated genes with NTZ or JIB-04 treatment. HEK293 cells were treated with NTZ (10 μM), JIB-04 (3 μM), or transfected with low-molecular-weight poly(I:C) (100 ng/ml) for 24 hr. mRNA levels of IFNL3 and CXCL10 were measured by RT-qPCR. (B) Autophagy formation with compound treatment. HEK293 cells were transfected with EGFP-LC3 plasmid for 24 hr and treated with rapamycin (100 nM), NTZ (10 μM), or JIB-04 (3 μM) for another 18 hr. GFP positive punctate structures indicate autophagy activation. Scale bar, 20 μm. (C) Intracellular viral RNA levels with JIB-04 and camostat treatment. Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (D) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. S RNA levels were measured by RT-qPCR. (E) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. FL: full-length. S2: cleaved S2 fragment. (F) siRNA-mediated knockdown of JIB-04 target histone demethylases. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr. mRNA levels of indicated histone demethylases were measured by RT-qPCR. (G) Western blot analysis of RV antigen VP6 levels in cells with histone demethylase siRNA knockdown. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01) for 12 hr. (H) Pathway enrichment analysis of gene expression regulated by JIB-04 treatment. Downregulated genes in Fig. 3F with p values

    Techniques Used: Inhibition, Expressing, Transfection, Molecular Weight, Quantitative RT-PCR, Plasmid Preparation, Activation Assay, Infection, Western Blot

    21) Product Images from "Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response"

    Article Title: Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response

    Journal: bioRxiv

    doi: 10.1101/2020.06.27.175166

    Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.
    Figure Legend Snippet: Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.

    Techniques Used: Mouse Assay, Staining, Immunofluorescence, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Binding Assay

    Neutralizing activity against SARS-CoV-2. (A) Percent neutralization of SARS-CoV-2 virus expressing GFP. Serum collected from the naïve animals used as negative controls. (B) Neutralization titer against SARS-CoV-2 virus expressing GFP. (C, D) Correlations between neutralization titer and ELISA binding titer.
    Figure Legend Snippet: Neutralizing activity against SARS-CoV-2. (A) Percent neutralization of SARS-CoV-2 virus expressing GFP. Serum collected from the naïve animals used as negative controls. (B) Neutralization titer against SARS-CoV-2 virus expressing GFP. (C, D) Correlations between neutralization titer and ELISA binding titer.

    Techniques Used: Activity Assay, Neutralization, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay

    Analyzing SARS-CoV-2 RBD and S1 proteins affinities to human ACE2 (hACE2) proteins using biolayer interferometry (BLI). (A) Bio-Layer Interferometry sensograms of the binding of SARS-CoV-2 S1 and RBD proteins to immobilized Fc-human ACE2, after incubation of the analytes at 25°C for 0and 60 minutes. The traces represent BLI response curves for SARS-CoV-2 proteins serially diluted from 800nM to 12.5nM, as indicated. Dotted lines show raw response values, while bold solid lines show the fitted trace. Association and dissociation phases were monitored for 300s and 600s, respectively. The data was globally fit using a 1:1 binding model to estimate binding affinity. (B) Binding affinity specifications of S1 and RBD proteins against hu-ACE2.
    Figure Legend Snippet: Analyzing SARS-CoV-2 RBD and S1 proteins affinities to human ACE2 (hACE2) proteins using biolayer interferometry (BLI). (A) Bio-Layer Interferometry sensograms of the binding of SARS-CoV-2 S1 and RBD proteins to immobilized Fc-human ACE2, after incubation of the analytes at 25°C for 0and 60 minutes. The traces represent BLI response curves for SARS-CoV-2 proteins serially diluted from 800nM to 12.5nM, as indicated. Dotted lines show raw response values, while bold solid lines show the fitted trace. Association and dissociation phases were monitored for 300s and 600s, respectively. The data was globally fit using a 1:1 binding model to estimate binding affinity. (B) Binding affinity specifications of S1 and RBD proteins against hu-ACE2.

    Techniques Used: Binding Assay, Incubation

    Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.
    Figure Legend Snippet: Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.

    Techniques Used: Mouse Assay, Recombinant, Expressing, Binding Assay, Enzyme-linked Immunosorbent Assay, Luminex, Fluorescence

    22) Product Images from "mRNA vaccine CVnCoV protects non-human primates from SARS-CoV-2 challenge infection"

    Article Title: mRNA vaccine CVnCoV protects non-human primates from SARS-CoV-2 challenge infection

    Journal: bioRxiv

    doi: 10.1101/2020.12.23.424138

    CVnCoV induces humoral response in non-human primates. (A) Schematic drawing of study setup. Rhesus macaques (n=6; 3 male, 3 female/group) were vaccinated IM on day 0 and day 28 with 0.5 μg or 8 μg of CVnCoV or remained unvaccinated. All animals were challenge with 5.0 x 106 PFU of SARS-CoV-2 on d56. Two animals of each group were terminated on d62, d63 and d64, respectively (B) Trimeric Spike protein or (C) RBD specific binding IgG antibodies, displayed as endpoint titres at different time points as indicated (C) Virus neutralising antibodies determined via focus reduction neutralisation test at different time points as indicated. All values are displayed as median with range. Square symbols represent male, round symbols female animals. Dotted lines represent vaccinations and challenge infection, respectively. RBD receptor binding domain; VNT virus neutralising titre
    Figure Legend Snippet: CVnCoV induces humoral response in non-human primates. (A) Schematic drawing of study setup. Rhesus macaques (n=6; 3 male, 3 female/group) were vaccinated IM on day 0 and day 28 with 0.5 μg or 8 μg of CVnCoV or remained unvaccinated. All animals were challenge with 5.0 x 106 PFU of SARS-CoV-2 on d56. Two animals of each group were terminated on d62, d63 and d64, respectively (B) Trimeric Spike protein or (C) RBD specific binding IgG antibodies, displayed as endpoint titres at different time points as indicated (C) Virus neutralising antibodies determined via focus reduction neutralisation test at different time points as indicated. All values are displayed as median with range. Square symbols represent male, round symbols female animals. Dotted lines represent vaccinations and challenge infection, respectively. RBD receptor binding domain; VNT virus neutralising titre

    Techniques Used: Binding Assay, Infection

    Exemplary sections showing histopathology (H E) and SARS-CoV-2 in situ hybridisation (ISH). A. Alveolar necrosis and inflammatory exudates (*) in the alveolar spaces and type II pneumocyte hyperplasia (arrows). B) Mild perivascular cuffing (arrow). C) Inflammatory cell infiltration in the alveolar spaces and the interalveolar septa (*) and type II pneumocyte hyperplasia (arrows). D) SARS-CoV-2 ISH staining in abundant cell within inflammatory foci (arrows). E. SARS-CoV-2 ISH staining in a single cell within an interalveolar septum (arrow). F. Abundant foci of SARS-CoV-2 ISH stained cells within the alveolar lining and the interalveolar septa (arrows). Bar = 100μm. ISH in situ hybridisation
    Figure Legend Snippet: Exemplary sections showing histopathology (H E) and SARS-CoV-2 in situ hybridisation (ISH). A. Alveolar necrosis and inflammatory exudates (*) in the alveolar spaces and type II pneumocyte hyperplasia (arrows). B) Mild perivascular cuffing (arrow). C) Inflammatory cell infiltration in the alveolar spaces and the interalveolar septa (*) and type II pneumocyte hyperplasia (arrows). D) SARS-CoV-2 ISH staining in abundant cell within inflammatory foci (arrows). E. SARS-CoV-2 ISH staining in a single cell within an interalveolar septum (arrow). F. Abundant foci of SARS-CoV-2 ISH stained cells within the alveolar lining and the interalveolar septa (arrows). Bar = 100μm. ISH in situ hybridisation

    Techniques Used: Histopathology, In Situ, Hybridization, In Situ Hybridization, Staining

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.06.15.153064

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

    Techniques Used: Polymerase Chain Reaction

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

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

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

    Techniques Used: Enzyme-linked Immunosorbent Assay

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

    Techniques Used: Incubation

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

    Techniques Used: Titration, Concentration Assay, Standard Deviation

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

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

    Journal: eLife

    doi: 10.7554/eLife.61552

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

    Techniques Used: Binding Assay, Expressing, Generated

    N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. ( A ) Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] - and [N] - 293 T cells. All nine viruses were applied at equal titer to stable 293T/ACE2. ( B–C ) O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. ( D ) Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. ( E ) Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] - 293T products due to truncation of glycan biosynthesis. ( F ) Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] - 293Ts is almost fully proteolyzed during viral production (red arrowhead). *p
    Figure Legend Snippet: N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. ( A ) Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] - and [N] - 293 T cells. All nine viruses were applied at equal titer to stable 293T/ACE2. ( B–C ) O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. ( D ) Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. ( E ) Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] - 293T products due to truncation of glycan biosynthesis. ( F ) Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] - 293Ts is almost fully proteolyzed during viral production (red arrowhead). *p

    Techniques Used: Modification, Expressing, Mutagenesis, Produced, Generated, Titration, Infection, Western Blot

    Glycan coverage of Spike-ACE2 co-complex. SARS-CoV-2 Spike protein trimer (pink) bound to ACE2 (green). ( A ) Without glycans. ( B ) With N-glycans (red) identified using LC-MS on Spike and ACE2. ( C ) Molecular dynamics simulation analyzed the range of movement of each glycan. The space sampled by glycans is represented by a gray cloud. Glycans cover the Spike-ACE2 interface. They also surround the putative proteolysis site of furin (‘S1-S2’, yellow) and S2’ (blue).
    Figure Legend Snippet: Glycan coverage of Spike-ACE2 co-complex. SARS-CoV-2 Spike protein trimer (pink) bound to ACE2 (green). ( A ) Without glycans. ( B ) With N-glycans (red) identified using LC-MS on Spike and ACE2. ( C ) Molecular dynamics simulation analyzed the range of movement of each glycan. The space sampled by glycans is represented by a gray cloud. Glycans cover the Spike-ACE2 interface. They also surround the putative proteolysis site of furin (‘S1-S2’, yellow) and S2’ (blue).

    Techniques Used: Liquid Chromatography with Mass Spectroscopy

    25) Product Images from "Single-dose intranasal administration of AdCOVID elicits systemic and mucosal immunity against SARS-CoV-2 in mice"

    Article Title: Single-dose intranasal administration of AdCOVID elicits systemic and mucosal immunity against SARS-CoV-2 in mice

    Journal: bioRxiv

    doi: 10.1101/2020.10.10.331348

    SARS-CoV-2 neutralizing antibody responses in serum following single intranasal administration of the S1 and RBD vectors. (A) Neutralizing antibody response by C57BL/6J or CD-1 mice vaccinated 28 days earlier with the mid or high dose of the S1 or RBD vector as indicated. Results are expressed as the reciprocal of the dilution of serum samples required to achieve 50% neutralization (FRNT 50 ) of wild-type SARS-CoV-2 infection of permissive Vero E6 cells. Line represent the group median value. (B-C) Correlation between neutralizing antibody response and Spike-specific IgG response in serum of vaccinated animals. Correlation analysis was performed with a two-tailed Spearman test.
    Figure Legend Snippet: SARS-CoV-2 neutralizing antibody responses in serum following single intranasal administration of the S1 and RBD vectors. (A) Neutralizing antibody response by C57BL/6J or CD-1 mice vaccinated 28 days earlier with the mid or high dose of the S1 or RBD vector as indicated. Results are expressed as the reciprocal of the dilution of serum samples required to achieve 50% neutralization (FRNT 50 ) of wild-type SARS-CoV-2 infection of permissive Vero E6 cells. Line represent the group median value. (B-C) Correlation between neutralizing antibody response and Spike-specific IgG response in serum of vaccinated animals. Correlation analysis was performed with a two-tailed Spearman test.

    Techniques Used: Mouse Assay, Plasmid Preparation, Neutralization, Infection, Two Tailed Test

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    Article Title: Development and pre-clinical evaluation of Newcastle disease virus-vectored SARS-CoV-2 intranasal vaccine candidate
    Article Snippet: .. Then cells were washed three times with DPBS, and incubated the monolayer with the rabbit polyclonal antibody specific to SARS-CoV-2 RBD protein (1:200) (cat. n° 40592-T62, Sino Biological, Beijing, China), and a chicken antiserum specific to Newcastle disease virus (1:200) (10100482, Charles River Avian Vaccine Services, Norwich, CT, USA) for 1.5h at RT. .. Afterwards, the monolayer was incubated with Donkey Anti-Rabbit IgG H & L-Alexa Fluor® 594 (1:250) (cat. n° ab150072, Abcam, Cambridge, MA, USA) and Goat Anti-Chicken IgY H & L-Alexa Fluor® 488 (1:1000) (cat. n° ab150169, Abcam, Cambridge, MA, USA) for 60 min at RT.

    Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity
    Article Snippet: To reduce proteins, DTT (final concentration 62.5 mM) was added to the 4× Laemmli sample buffer (Bio-Rad). .. The membranes were blocked with 5% w/v milk in TBS/Tween 0.1% (TBS/T) and incubated with rabbit polyclonal anti-His (catalog 2365S, Cell Signaling Technology, 1:1000), rabbit polyclonal anti–SARS-CoV-2 Spike RBD (40592-T62, Sino Biological, 1:1000), or rabbit polyclonal anti–SARS-CoV2-Nucleocapsid protein (40588-T62, Sino Biological, 1:2000) antibodies in 2% BSA TBS/T (see ). .. Secondary antibodies used were donkey anti-rabbit HRP (ab16284, Abcam, 1:2000) in 2% BSA TBS/T.

    Article Title: Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response
    Article Snippet: Lysates were kept on ice for 10 min, centrifuged, and resolved by SDS PAGE using precast 4–15% SDS polyacrylamide gels (BioRad). .. Proteins were transferred to a nitrocellulose membrane, blocked with 1% casein blocker overnight (Cat#1610782 Biorad), and incubated for 1 h at room temperature with anti-SARS-CoV-2 spike mouse mAb (Cat # GTX632604, GeneTex) for MVA/S and rabbit SARS-CoV-2 RBD polyclonal antibody (Cat# 40592-T62, Sino Biological) for MVA/S1 diluted 1:2500 in blocking buffer, respectively. .. The membrane was washed in PBS containing Tween-20 (0.05%) and was incubated for 1 h with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (Southern Biotech) diluted 1:20,000 accordingly.

    SDS Page:

    Article Title: Mice immunized with the vaccine candidate HexaPro spike produce neutralizing antibodies against SARS-CoV-2
    Article Snippet: The protein was further purified with Sephacryl S-300 HR (GE Healthcare) with PBS. .. Fractions which contain HexaPro protein were pooled and analyzed with SDS-PAGE and western blot against the SARS-CoV-2 RBD protein (Sino Biological, Cat#40592-T62) or pooled convalescent sera. .. Immunofluorescence stainingHeLa cells were transiently transfected with the plasmid encoding HexaPro using lipofectamine 3000 (Invitrogen, Cat#L3000008).

    Western Blot:

    Article Title: Mice immunized with the vaccine candidate HexaPro spike produce neutralizing antibodies against SARS-CoV-2
    Article Snippet: The protein was further purified with Sephacryl S-300 HR (GE Healthcare) with PBS. .. Fractions which contain HexaPro protein were pooled and analyzed with SDS-PAGE and western blot against the SARS-CoV-2 RBD protein (Sino Biological, Cat#40592-T62) or pooled convalescent sera. .. Immunofluorescence stainingHeLa cells were transiently transfected with the plasmid encoding HexaPro using lipofectamine 3000 (Invitrogen, Cat#L3000008).

    Blocking Assay:

    Article Title: Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response
    Article Snippet: Lysates were kept on ice for 10 min, centrifuged, and resolved by SDS PAGE using precast 4–15% SDS polyacrylamide gels (BioRad). .. Proteins were transferred to a nitrocellulose membrane, blocked with 1% casein blocker overnight (Cat#1610782 Biorad), and incubated for 1 h at room temperature with anti-SARS-CoV-2 spike mouse mAb (Cat # GTX632604, GeneTex) for MVA/S and rabbit SARS-CoV-2 RBD polyclonal antibody (Cat# 40592-T62, Sino Biological) for MVA/S1 diluted 1:2500 in blocking buffer, respectively. .. The membrane was washed in PBS containing Tween-20 (0.05%) and was incubated for 1 h with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (Southern Biotech) diluted 1:20,000 accordingly.

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    Sino Biological sars cov 2 2019 ncov spike rbd antibody rabbit pab
    ELISA ( x -axis) vs. LFRET ( y -axis) results by disease severity. ( a ) Anti-NP IgA ELISA vs. anti-NP LFRET (N = 81, R = 0.25). ( b ) anti-NP IgG ELISA vs. anti-NP LFRET (N = 129, R = 0.62). ( c ) anti-NP IgM ELISA vs. anti-NP LFRET (N = 81, R = 0.13). ( d ) anti-SP IgA ELISA vs. anti-SP LFRET (N = 129, R = 0.53). ( e ) anti-SP IgG ELISA vs. anti-SP LFRET (N = 129, R = 0.62). ( f ) anti-SP IgM ELISA vs. anti-SP LFRET (N = 81, R = 0.56). Color of the dot indicates <t>SARS-CoV-2</t> PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal and vertical black lines indicate LFRET and ELISA cutoffs. On the x -axis, ELISA absorbance on a logarithmic scale and on the y -axis, LFRET signal on a logarithmic scale. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. R = Pearson’s correlation coefficient.
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    ELISA ( x -axis) vs. LFRET ( y -axis) results by disease severity. ( a ) Anti-NP IgA ELISA vs. anti-NP LFRET (N = 81, R = 0.25). ( b ) anti-NP IgG ELISA vs. anti-NP LFRET (N = 129, R = 0.62). ( c ) anti-NP IgM ELISA vs. anti-NP LFRET (N = 81, R = 0.13). ( d ) anti-SP IgA ELISA vs. anti-SP LFRET (N = 129, R = 0.53). ( e ) anti-SP IgG ELISA vs. anti-SP LFRET (N = 129, R = 0.62). ( f ) anti-SP IgM ELISA vs. anti-SP LFRET (N = 81, R = 0.56). Color of the dot indicates SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal and vertical black lines indicate LFRET and ELISA cutoffs. On the x -axis, ELISA absorbance on a logarithmic scale and on the y -axis, LFRET signal on a logarithmic scale. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. R = Pearson’s correlation coefficient.

    Journal: Viruses

    Article Title: A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

    doi: 10.3390/v13020143

    Figure Lengend Snippet: ELISA ( x -axis) vs. LFRET ( y -axis) results by disease severity. ( a ) Anti-NP IgA ELISA vs. anti-NP LFRET (N = 81, R = 0.25). ( b ) anti-NP IgG ELISA vs. anti-NP LFRET (N = 129, R = 0.62). ( c ) anti-NP IgM ELISA vs. anti-NP LFRET (N = 81, R = 0.13). ( d ) anti-SP IgA ELISA vs. anti-SP LFRET (N = 129, R = 0.53). ( e ) anti-SP IgG ELISA vs. anti-SP LFRET (N = 129, R = 0.62). ( f ) anti-SP IgM ELISA vs. anti-SP LFRET (N = 81, R = 0.56). Color of the dot indicates SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal and vertical black lines indicate LFRET and ELISA cutoffs. On the x -axis, ELISA absorbance on a logarithmic scale and on the y -axis, LFRET signal on a logarithmic scale. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. R = Pearson’s correlation coefficient.

    Article Snippet: At 48 h, the medium was analyzed for the presence of SARS-CoV-2 SP by dot blotting; briefly via drying 2.5 µL of the supernatant onto a nitrocellulose membrane, which then was blocked (3% skim milk in Tris-buffered saline with 0.05% Tween-20), washed, probed with rabbit anti-RBD (40592-T62, Sino Biological, Beijing, China), washed, probed with anti-rabbit IRDye800 (LI-COR Biosciences, Lincoln, NE, USA), washed, and read using Odyssey Infrared Imaging System (LI-COR Biosciences).

    Techniques: Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Förster Resonance Energy Transfer

    Microneutralization vs. LFRET and ELISA. Microneutralization titers are on the x -axis and LFRET signal or ELISA absorbance on the y -axis. Logarithmic scale is used on both axes. ( a ) Microneutralization titer vs. anti-SP LFRET signal (N = 107, ρ = 0.87). ( b – d ) Microneutralization titer vs. anti-SP IgG, IgA and IgM ELISA (N = 107, 107 and 67, ρ = 0.68, 0.86 and 0.81). ( e ) Microneutralization titer vs. anti-NP LFRET signal (N = 107, ρ = 0.83). ( f – h ) Microneutralization titer vs. anti-NP IgG, IgA and IgM ELISA (N = 107, 67 and 67, ρ = 0.81, 0.69 and 0.61). Color of the dots indicate SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal black lines indicate LFRET/ELISA cutoffs. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. ρ = Spearman’s rank correlation coefficient.

    Journal: Viruses

    Article Title: A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

    doi: 10.3390/v13020143

    Figure Lengend Snippet: Microneutralization vs. LFRET and ELISA. Microneutralization titers are on the x -axis and LFRET signal or ELISA absorbance on the y -axis. Logarithmic scale is used on both axes. ( a ) Microneutralization titer vs. anti-SP LFRET signal (N = 107, ρ = 0.87). ( b – d ) Microneutralization titer vs. anti-SP IgG, IgA and IgM ELISA (N = 107, 107 and 67, ρ = 0.68, 0.86 and 0.81). ( e ) Microneutralization titer vs. anti-NP LFRET signal (N = 107, ρ = 0.83). ( f – h ) Microneutralization titer vs. anti-NP IgG, IgA and IgM ELISA (N = 107, 67 and 67, ρ = 0.81, 0.69 and 0.61). Color of the dots indicate SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal black lines indicate LFRET/ELISA cutoffs. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. ρ = Spearman’s rank correlation coefficient.

    Article Snippet: At 48 h, the medium was analyzed for the presence of SARS-CoV-2 SP by dot blotting; briefly via drying 2.5 µL of the supernatant onto a nitrocellulose membrane, which then was blocked (3% skim milk in Tris-buffered saline with 0.05% Tween-20), washed, probed with rabbit anti-RBD (40592-T62, Sino Biological, Beijing, China), washed, probed with anti-rabbit IRDye800 (LI-COR Biosciences, Lincoln, NE, USA), washed, and read using Odyssey Infrared Imaging System (LI-COR Biosciences).

    Techniques: Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Förster Resonance Energy Transfer

    Simplified protocol for SARS-CoV-2 NP and SP LFRET assay. Eu-NP/-SP = Europium-labeled nucleoprotein/spike glycoprotein. AF-L = Alexa Fluor™ 647 -labeled protein L. TR-FRET = time-resolved Förster resonance energy transfer. RT = room temperature. TBS+BSA (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.2% BSA) was used for all dilutions. On-plate dilutions were 5 nM Eu-NP/500 nM AF-L/serum 1/25 for anti-NP and 5 nM Eu-SP/250 nM AF-L/serum 1/100 for anti-SP LFRET. For further details see the prior publication [ 5 ].

    Journal: Viruses

    Article Title: A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

    doi: 10.3390/v13020143

    Figure Lengend Snippet: Simplified protocol for SARS-CoV-2 NP and SP LFRET assay. Eu-NP/-SP = Europium-labeled nucleoprotein/spike glycoprotein. AF-L = Alexa Fluor™ 647 -labeled protein L. TR-FRET = time-resolved Förster resonance energy transfer. RT = room temperature. TBS+BSA (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.2% BSA) was used for all dilutions. On-plate dilutions were 5 nM Eu-NP/500 nM AF-L/serum 1/25 for anti-NP and 5 nM Eu-SP/250 nM AF-L/serum 1/100 for anti-SP LFRET. For further details see the prior publication [ 5 ].

    Article Snippet: At 48 h, the medium was analyzed for the presence of SARS-CoV-2 SP by dot blotting; briefly via drying 2.5 µL of the supernatant onto a nitrocellulose membrane, which then was blocked (3% skim milk in Tris-buffered saline with 0.05% Tween-20), washed, probed with rabbit anti-RBD (40592-T62, Sino Biological, Beijing, China), washed, probed with anti-rabbit IRDye800 (LI-COR Biosciences, Lincoln, NE, USA), washed, and read using Odyssey Infrared Imaging System (LI-COR Biosciences).

    Techniques: Labeling, Förster Resonance Energy Transfer

    Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.

    Journal: bioRxiv

    Article Title: Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response

    doi: 10.1101/2020.06.27.175166

    Figure Lengend Snippet: Inducible Bronchus Associated Lymphoid Tissues (iBALT) formation upon MVA/S and MVA/S1 vaccination. Frozen lung sections from vaccinated mice were either stained for H E to analyze tissue structure and formation of iBALT aggregates (A), or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route (right panel), and compared with unvaccinated control mice (left panel). Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups (C). The p value was calculated using non parametric mann-whitney test. (D) Lung immune responses in bronchoalveolar lavage (BAL) samples collected after euthanizations (three weeks post-boost) were measured using ELISA. SARS-CoV-2 S protein-specific binding IgG and IgA antibodies measured, and titters were presented in column graphs. The data represent mean responses in each group (n = 5) ± SEM.

    Article Snippet: Proteins were transferred to a nitrocellulose membrane, blocked with 1% casein blocker overnight (Cat#1610782 Biorad), and incubated for 1 h at room temperature with anti-SARS-CoV-2 spike mouse mAb (Cat # GTX632604, GeneTex) for MVA/S and rabbit SARS-CoV-2 RBD polyclonal antibody (Cat# 40592-T62, Sino Biological) for MVA/S1 diluted 1:2500 in blocking buffer, respectively.

    Techniques: Mouse Assay, Staining, Immunofluorescence, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Binding Assay

    Neutralizing activity against SARS-CoV-2. (A) Percent neutralization of SARS-CoV-2 virus expressing GFP. Serum collected from the naïve animals used as negative controls. (B) Neutralization titer against SARS-CoV-2 virus expressing GFP. (C, D) Correlations between neutralization titer and ELISA binding titer.

    Journal: bioRxiv

    Article Title: Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response

    doi: 10.1101/2020.06.27.175166

    Figure Lengend Snippet: Neutralizing activity against SARS-CoV-2. (A) Percent neutralization of SARS-CoV-2 virus expressing GFP. Serum collected from the naïve animals used as negative controls. (B) Neutralization titer against SARS-CoV-2 virus expressing GFP. (C, D) Correlations between neutralization titer and ELISA binding titer.

    Article Snippet: Proteins were transferred to a nitrocellulose membrane, blocked with 1% casein blocker overnight (Cat#1610782 Biorad), and incubated for 1 h at room temperature with anti-SARS-CoV-2 spike mouse mAb (Cat # GTX632604, GeneTex) for MVA/S and rabbit SARS-CoV-2 RBD polyclonal antibody (Cat# 40592-T62, Sino Biological) for MVA/S1 diluted 1:2500 in blocking buffer, respectively.

    Techniques: Activity Assay, Neutralization, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay

    Analyzing SARS-CoV-2 RBD and S1 proteins affinities to human ACE2 (hACE2) proteins using biolayer interferometry (BLI). (A) Bio-Layer Interferometry sensograms of the binding of SARS-CoV-2 S1 and RBD proteins to immobilized Fc-human ACE2, after incubation of the analytes at 25°C for 0and 60 minutes. The traces represent BLI response curves for SARS-CoV-2 proteins serially diluted from 800nM to 12.5nM, as indicated. Dotted lines show raw response values, while bold solid lines show the fitted trace. Association and dissociation phases were monitored for 300s and 600s, respectively. The data was globally fit using a 1:1 binding model to estimate binding affinity. (B) Binding affinity specifications of S1 and RBD proteins against hu-ACE2.

    Journal: bioRxiv

    Article Title: Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response

    doi: 10.1101/2020.06.27.175166

    Figure Lengend Snippet: Analyzing SARS-CoV-2 RBD and S1 proteins affinities to human ACE2 (hACE2) proteins using biolayer interferometry (BLI). (A) Bio-Layer Interferometry sensograms of the binding of SARS-CoV-2 S1 and RBD proteins to immobilized Fc-human ACE2, after incubation of the analytes at 25°C for 0and 60 minutes. The traces represent BLI response curves for SARS-CoV-2 proteins serially diluted from 800nM to 12.5nM, as indicated. Dotted lines show raw response values, while bold solid lines show the fitted trace. Association and dissociation phases were monitored for 300s and 600s, respectively. The data was globally fit using a 1:1 binding model to estimate binding affinity. (B) Binding affinity specifications of S1 and RBD proteins against hu-ACE2.

    Article Snippet: Proteins were transferred to a nitrocellulose membrane, blocked with 1% casein blocker overnight (Cat#1610782 Biorad), and incubated for 1 h at room temperature with anti-SARS-CoV-2 spike mouse mAb (Cat # GTX632604, GeneTex) for MVA/S and rabbit SARS-CoV-2 RBD polyclonal antibody (Cat# 40592-T62, Sino Biological) for MVA/S1 diluted 1:2500 in blocking buffer, respectively.

    Techniques: Binding Assay, Incubation

    Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.

    Journal: bioRxiv

    Article Title: Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response

    doi: 10.1101/2020.06.27.175166

    Figure Lengend Snippet: Antibody responses induced by MVA/S or MVA/S1 in mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVAs expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy. Unvaccinated (naïve) animals served as controls (n=5). (A) Binding IgG antibody response for individual proteins measured using ELISA at two weeks after boost. (B) Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. The data show mean response in each group (n = 5) ± SEM. (C) Binding antibody response determined using Luminex assay at 3 weeks post boost. The pie graphs show the relative proportions of binding to three proteins in each group. (D) IgG subclass and soluble Fc receptor binding analysis of RBD and S1 specific IgG measured using the Luminex assay. Raw values are presented as in mean fluorescence intensity (MFI) in bar graph. The data represent mean responses in each group (n = 5) ± SEM.

    Article Snippet: Proteins were transferred to a nitrocellulose membrane, blocked with 1% casein blocker overnight (Cat#1610782 Biorad), and incubated for 1 h at room temperature with anti-SARS-CoV-2 spike mouse mAb (Cat # GTX632604, GeneTex) for MVA/S and rabbit SARS-CoV-2 RBD polyclonal antibody (Cat# 40592-T62, Sino Biological) for MVA/S1 diluted 1:2500 in blocking buffer, respectively.

    Techniques: Mouse Assay, Recombinant, Expressing, Binding Assay, Enzyme-linked Immunosorbent Assay, Luminex, Fluorescence

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

    Journal: JCI Insight

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

    doi: 10.1172/jci.insight.142386

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

    Article Snippet: The membranes were blocked with 5% w/v milk in TBS/Tween 0.1% (TBS/T) and incubated with rabbit polyclonal anti-His (catalog 2365S, Cell Signaling Technology, 1:1000), rabbit polyclonal anti–SARS-CoV-2 Spike RBD (40592-T62, Sino Biological, 1:1000), or rabbit polyclonal anti–SARS-CoV2-Nucleocapsid protein (40588-T62, Sino Biological, 1:2000) antibodies in 2% BSA TBS/T (see ).

    Techniques: Enzyme-linked Immunosorbent Assay

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

    Journal: JCI Insight

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

    doi: 10.1172/jci.insight.142386

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

    Article Snippet: The membranes were blocked with 5% w/v milk in TBS/Tween 0.1% (TBS/T) and incubated with rabbit polyclonal anti-His (catalog 2365S, Cell Signaling Technology, 1:1000), rabbit polyclonal anti–SARS-CoV-2 Spike RBD (40592-T62, Sino Biological, 1:1000), or rabbit polyclonal anti–SARS-CoV2-Nucleocapsid protein (40588-T62, Sino Biological, 1:2000) antibodies in 2% BSA TBS/T (see ).

    Techniques: Binding Assay, Polymerase Chain Reaction, Negative Control

    Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.

    Journal: JCI Insight

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

    doi: 10.1172/jci.insight.142386

    Figure Lengend Snippet: Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.

    Article Snippet: The membranes were blocked with 5% w/v milk in TBS/Tween 0.1% (TBS/T) and incubated with rabbit polyclonal anti-His (catalog 2365S, Cell Signaling Technology, 1:1000), rabbit polyclonal anti–SARS-CoV-2 Spike RBD (40592-T62, Sino Biological, 1:1000), or rabbit polyclonal anti–SARS-CoV2-Nucleocapsid protein (40588-T62, Sino Biological, 1:2000) antibodies in 2% BSA TBS/T (see ).

    Techniques: Neutralization, Activity Assay, Binding Assay, Standard Deviation

    The recombinant SARS-CoV-2 HexaPro spike protein. (A) Schematic representation of the prefusion-stabilized SARS-CoV-2 HexaPro ectodomain showing the S1 and S2 subunits. Four additional proline substitutions from S-2P construct are indicated by the red arrows shown below the construct. (B) The HexaPro protein expressed in HEK293T cells was purified and characterized by SDS-PAGE (left), western blot using a commercial anti-RBD (middle), and western blot using pooled convalescence sera (right).

    Journal: bioRxiv

    Article Title: Mice immunized with the vaccine candidate HexaPro spike produce neutralizing antibodies against SARS-CoV-2

    doi: 10.1101/2021.02.27.433054

    Figure Lengend Snippet: The recombinant SARS-CoV-2 HexaPro spike protein. (A) Schematic representation of the prefusion-stabilized SARS-CoV-2 HexaPro ectodomain showing the S1 and S2 subunits. Four additional proline substitutions from S-2P construct are indicated by the red arrows shown below the construct. (B) The HexaPro protein expressed in HEK293T cells was purified and characterized by SDS-PAGE (left), western blot using a commercial anti-RBD (middle), and western blot using pooled convalescence sera (right).

    Article Snippet: Cells were fixed with 4% PFA and were incubated with either a polyclonal antibody against the SARS-CoV-2 RBD protein (Sino Biological, Cat#40592-T62) or a monoclonal antibody against the SARS-CoV-2 S1 protein (MyBioSource, Cat#MBS434277).

    Techniques: Recombinant, Construct, Purification, SDS Page, Western Blot