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sars cov 1 2 spike protein  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc sars cov 1 2 spike protein
    PAD expression in vivo and in vitro after <t>SARS-CoV-2</t> infection (A) Volcano plot relative to the reanalysis of transcriptomic data from ten studies of healthy lungs and four studies of patients with COVID-19 (Delorey et al.). (B and C) Calu3 cells are infected with 2020B.1 (MOI 0.01), Delta (MOI 0.01), or Omicron (MOI 0.01) SARS-CoV-2 variants, and PAD4 (B) and PAD2 (C) mRNA are assessed by RT-qPCR. Data are normalized to the housekeeping gene PGK1 and expressed as mean fold change ±SEM over mock-infected cells of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test. (D) Protein lysates from Calu3 infected with SARS-CoV-2 (2020B.1 variant, MOI 0.1 PFU/cell) or uninfected cells (mock) at different hours post-infection (hpi) are analyzed for citrullinated proteins using an Rh-PG citrulline-specific probe (left panel) or an anti-CCP antibody (right panel). Samples are subjected to gel electrophoresis, and SARS-CoV-2 infection is confirmed using an anti-SARS-CoV-2 Spike (S SARS-CoV-2) antibody. β-actin is used as a loading control. One representative blot of three independent experiments is shown. (E) Volcano plot depicts host (gray dots) and viral (red dots) citrullinated proteins of SARS-infected vs. mock-infected cells at 48 hpi (2020B.1 variant, MOI 0.1 PFU/cell). The x axis represents the ratio of citrullination between mock and infected cells at the indicated time points, while the y axis indicates the statistical significance. Both variables are plotted on a logarithmic scale ( n = 3). (F) PANTHER classification of over-citrullinated cellular proteins based on protein classes.
    Sars Cov 1 2 Spike Protein, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/sars+cov+1+2+spike+protein/SARS-CoV-1%2F2+Spike+Protein+Mouse+mAb/pmc12966709-549-10-15
    Average 94 stars, based on 10 article reviews
    sars cov 1 2 spike protein - by Bioz Stars, 2026-09
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    Images

    1) Product Images from "Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response"

    Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

    Journal: iScience

    doi: 10.1016/j.isci.2026.115038

    PAD expression in vivo and in vitro after SARS-CoV-2 infection (A) Volcano plot relative to the reanalysis of transcriptomic data from ten studies of healthy lungs and four studies of patients with COVID-19 (Delorey et al.). (B and C) Calu3 cells are infected with 2020B.1 (MOI 0.01), Delta (MOI 0.01), or Omicron (MOI 0.01) SARS-CoV-2 variants, and PAD4 (B) and PAD2 (C) mRNA are assessed by RT-qPCR. Data are normalized to the housekeeping gene PGK1 and expressed as mean fold change ±SEM over mock-infected cells of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test. (D) Protein lysates from Calu3 infected with SARS-CoV-2 (2020B.1 variant, MOI 0.1 PFU/cell) or uninfected cells (mock) at different hours post-infection (hpi) are analyzed for citrullinated proteins using an Rh-PG citrulline-specific probe (left panel) or an anti-CCP antibody (right panel). Samples are subjected to gel electrophoresis, and SARS-CoV-2 infection is confirmed using an anti-SARS-CoV-2 Spike (S SARS-CoV-2) antibody. β-actin is used as a loading control. One representative blot of three independent experiments is shown. (E) Volcano plot depicts host (gray dots) and viral (red dots) citrullinated proteins of SARS-infected vs. mock-infected cells at 48 hpi (2020B.1 variant, MOI 0.1 PFU/cell). The x axis represents the ratio of citrullination between mock and infected cells at the indicated time points, while the y axis indicates the statistical significance. Both variables are plotted on a logarithmic scale ( n = 3). (F) PANTHER classification of over-citrullinated cellular proteins based on protein classes.
    Figure Legend Snippet: PAD expression in vivo and in vitro after SARS-CoV-2 infection (A) Volcano plot relative to the reanalysis of transcriptomic data from ten studies of healthy lungs and four studies of patients with COVID-19 (Delorey et al.). (B and C) Calu3 cells are infected with 2020B.1 (MOI 0.01), Delta (MOI 0.01), or Omicron (MOI 0.01) SARS-CoV-2 variants, and PAD4 (B) and PAD2 (C) mRNA are assessed by RT-qPCR. Data are normalized to the housekeeping gene PGK1 and expressed as mean fold change ±SEM over mock-infected cells of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test. (D) Protein lysates from Calu3 infected with SARS-CoV-2 (2020B.1 variant, MOI 0.1 PFU/cell) or uninfected cells (mock) at different hours post-infection (hpi) are analyzed for citrullinated proteins using an Rh-PG citrulline-specific probe (left panel) or an anti-CCP antibody (right panel). Samples are subjected to gel electrophoresis, and SARS-CoV-2 infection is confirmed using an anti-SARS-CoV-2 Spike (S SARS-CoV-2) antibody. β-actin is used as a loading control. One representative blot of three independent experiments is shown. (E) Volcano plot depicts host (gray dots) and viral (red dots) citrullinated proteins of SARS-infected vs. mock-infected cells at 48 hpi (2020B.1 variant, MOI 0.1 PFU/cell). The x axis represents the ratio of citrullination between mock and infected cells at the indicated time points, while the y axis indicates the statistical significance. Both variables are plotted on a logarithmic scale ( n = 3). (F) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

    Techniques Used: Expressing, In Vivo, In Vitro, Infection, Quantitative RT-PCR, Variant Assay, Nucleic Acid Electrophoresis, Control

    SARS-CoV-2 infection affects protein citrullination by modulating PAD expression in vivo (A and B) PAD4 and PAD2 mRNA expression in mouse lungs (A) and brains (B) assessed by RT-qPCR. Each dot represents an individual mouse sample ( n = 6). Data are normalized to the housekeeping gene β-actin and presented relative to one randomly selected non-infected sample. Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (C) Western blot analysis of protein lysates from pooled uninfected (mock) and SARS-CoV-2-infected (6 dpi, Delta variant) mouse lungs and brains. The analysis is performed using antibodies against PAD2, PAD4, and β-actin, the latter to ensure equal loading. A representative blot from three independent experiments is shown. (D) Multiplex immunofluorescence staining for the detection of SARS-CoV-2-targeted cells in mouse brains and lung sections at 6 dpi and mock control. S SARS-CoV-2 protein, PAD2, and PAD4 positive signals are represented in green, pink, and yellow, respectively. Cell nuclei are visualized by DAPI (blue). Original magnification 20×. (E) Quantification of PAD2-and PAD4-positive cells is performed using the inForm Image Analysis software (Akoya Biosciences). For each mouse ( n = 6), one representative section is analyzed to determine cell density (cells/mm 2 ). Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (F) Volcano plot shows citrullinated proteins in pooled protein lysates from SARS-CoV-2-infected vs. mock-infected mouse lungs at 6 dpi. Lysates are incubated with biotin-PG to isolate citrullinated proteins on streptavidin agarose, followed by on-bead tryptic digestion and LC-MS/MS analysis. Each dot represents an identified citrullinated protein. The x axis shows the citrullination ratio between mock and infected samples, and the y axis indicates statistical significance, both on a logarithmic scale ( n = 3). (G) PANTHER classification of over-citrullinated cellular proteins based on protein classes.
    Figure Legend Snippet: SARS-CoV-2 infection affects protein citrullination by modulating PAD expression in vivo (A and B) PAD4 and PAD2 mRNA expression in mouse lungs (A) and brains (B) assessed by RT-qPCR. Each dot represents an individual mouse sample ( n = 6). Data are normalized to the housekeeping gene β-actin and presented relative to one randomly selected non-infected sample. Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (C) Western blot analysis of protein lysates from pooled uninfected (mock) and SARS-CoV-2-infected (6 dpi, Delta variant) mouse lungs and brains. The analysis is performed using antibodies against PAD2, PAD4, and β-actin, the latter to ensure equal loading. A representative blot from three independent experiments is shown. (D) Multiplex immunofluorescence staining for the detection of SARS-CoV-2-targeted cells in mouse brains and lung sections at 6 dpi and mock control. S SARS-CoV-2 protein, PAD2, and PAD4 positive signals are represented in green, pink, and yellow, respectively. Cell nuclei are visualized by DAPI (blue). Original magnification 20×. (E) Quantification of PAD2-and PAD4-positive cells is performed using the inForm Image Analysis software (Akoya Biosciences). For each mouse ( n = 6), one representative section is analyzed to determine cell density (cells/mm 2 ). Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (F) Volcano plot shows citrullinated proteins in pooled protein lysates from SARS-CoV-2-infected vs. mock-infected mouse lungs at 6 dpi. Lysates are incubated with biotin-PG to isolate citrullinated proteins on streptavidin agarose, followed by on-bead tryptic digestion and LC-MS/MS analysis. Each dot represents an identified citrullinated protein. The x axis shows the citrullination ratio between mock and infected samples, and the y axis indicates statistical significance, both on a logarithmic scale ( n = 3). (G) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

    Techniques Used: Infection, Expressing, In Vivo, Quantitative RT-PCR, Western Blot, Variant Assay, Multiplex Assay, Immunofluorescence, Staining, Control, Software, Incubation, Liquid Chromatography with Mass Spectroscopy

    Antiviral effects of PAD inhibitors against SARS-CoV-2 in vitro (A and B) Quantification of SARS-CoV-2 2020B.1 (MOI 0.1) viral particle production in Calu-3 and Huh7.5 cells (at 24 hpi and 48 hpi, respectively) treated with the increasing concentrations of BB-Cl (A) or GSK199 (B), determined by plaque assay. Results are expressed as a percentage relative to vehicle-treated cells (DMSO, marked as 0 on the graph). Data are presented as means ± SEM from four independent experiments and are analyzed by one-way ANOVA followed by Bonferroni’s post-test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (C and D) Cell viability of uninfected Calu3 and Huh7.5 cells treated with the indicated concentrations of BB-Cl (C) and GSK199 (D) for 72 h, determined for each concentration by MTT assay. Values are expressed as means ± SEM of three independent experiments. (E) Viral titers of different SARS-CoV-2 variants are measured in Calu-3 cells at 24 hpi (MOI = 0.1) following treatment with BB-Cl (5 μM) or GSK199 (20 μM). Viral particle production is determined by plaque assay. Data are expressed as mean ± SEM from four independent experiments and analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (F) Viral titers of different SARS-CoV-2 variants (as indicated in the figure) are measured in Huh7.5 cells at 48 hpi (MOI = 0.1) following treatment with BB-Cl (5 μM) or GSK199 (20 μM). Viral particle production is determined by plaque assay. Data are expressed as mean ± SEM from four independent experiments and analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.
    Figure Legend Snippet: Antiviral effects of PAD inhibitors against SARS-CoV-2 in vitro (A and B) Quantification of SARS-CoV-2 2020B.1 (MOI 0.1) viral particle production in Calu-3 and Huh7.5 cells (at 24 hpi and 48 hpi, respectively) treated with the increasing concentrations of BB-Cl (A) or GSK199 (B), determined by plaque assay. Results are expressed as a percentage relative to vehicle-treated cells (DMSO, marked as 0 on the graph). Data are presented as means ± SEM from four independent experiments and are analyzed by one-way ANOVA followed by Bonferroni’s post-test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (C and D) Cell viability of uninfected Calu3 and Huh7.5 cells treated with the indicated concentrations of BB-Cl (C) and GSK199 (D) for 72 h, determined for each concentration by MTT assay. Values are expressed as means ± SEM of three independent experiments. (E) Viral titers of different SARS-CoV-2 variants are measured in Calu-3 cells at 24 hpi (MOI = 0.1) following treatment with BB-Cl (5 μM) or GSK199 (20 μM). Viral particle production is determined by plaque assay. Data are expressed as mean ± SEM from four independent experiments and analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (F) Viral titers of different SARS-CoV-2 variants (as indicated in the figure) are measured in Huh7.5 cells at 48 hpi (MOI = 0.1) following treatment with BB-Cl (5 μM) or GSK199 (20 μM). Viral particle production is determined by plaque assay. Data are expressed as mean ± SEM from four independent experiments and analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

    Techniques Used: In Vitro, Plaque Assay, Concentration Assay, MTT Assay

    PAD4 inhibition blocks SARS-CoV-2 protein and genome synthesis (A) Relative viral RNA quantification in cell extracts from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO, determined by comparative RT-qPCR. Values are expressed relative to vehicle-treated samples and normalized to the housekeeping gene PGK1. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (B) Western blot analysis of viral protein expression in Huh7.5 and Calu3 cells, either mock-infected or infected with SARS-CoV-2 (MOI 0.1) and treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO. One representative blot from three independent experiments is shown. (C) Schematic representation of infection and treatment protocols used for the viral entry inhibition assay. (D) Viral entry assay on VERO-E6 (left) and TMPRSS2-rexpressing VERO-E6 (right) infected with VSV-Spike GFP (MOI 1). GFP-positive infected cells are microscopically counted, and the results are expressed as a percentage relative to vehicle-treated cells, and are analyzed by t test. Data represent mean ± SEM. (E) Absolute quantification of viral RNA released from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with DMSO (vehicle) or GSK199 (20 μM), either using standard treatment (total) or added at 2 hpi (Post), determined by qRT-PCR. Supernatants are collected at 24 hpi for Calu3 and 48 hpi for Huh7.5. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05 and ∗∗ p < 0.01.
    Figure Legend Snippet: PAD4 inhibition blocks SARS-CoV-2 protein and genome synthesis (A) Relative viral RNA quantification in cell extracts from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO, determined by comparative RT-qPCR. Values are expressed relative to vehicle-treated samples and normalized to the housekeeping gene PGK1. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (B) Western blot analysis of viral protein expression in Huh7.5 and Calu3 cells, either mock-infected or infected with SARS-CoV-2 (MOI 0.1) and treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO. One representative blot from three independent experiments is shown. (C) Schematic representation of infection and treatment protocols used for the viral entry inhibition assay. (D) Viral entry assay on VERO-E6 (left) and TMPRSS2-rexpressing VERO-E6 (right) infected with VSV-Spike GFP (MOI 1). GFP-positive infected cells are microscopically counted, and the results are expressed as a percentage relative to vehicle-treated cells, and are analyzed by t test. Data represent mean ± SEM. (E) Absolute quantification of viral RNA released from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with DMSO (vehicle) or GSK199 (20 μM), either using standard treatment (total) or added at 2 hpi (Post), determined by qRT-PCR. Supernatants are collected at 24 hpi for Calu3 and 48 hpi for Huh7.5. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05 and ∗∗ p < 0.01.

    Techniques Used: Inhibition, Infection, Quantitative RT-PCR, Western Blot, Expressing, Quantitative Proteomics

    In vivo antiviral activity of PAD inhibitors against SARS-CoV-2 (A) Schematic representation of the treatment and infection protocols in K18-hACE2 transgenic mice. (B) Quantification of viral genome copies in mouse lungs—treated and infected as described in A— by ddPCR. Each dot represents an individual mouse sample ( n = 6). (C) Plaque assay to determine the number of infectious viral particles in mouse nasal swabs. (D) Graphical representation of cumulative scores from for immunohistochemical staining of mouse lungs using an anti-SARS-CoV-2 nucleocapsid antibody ( n = 4). Y axis represents the cumulative score calculated according to the criteria described in . (E) Graphical representation of cumulative scores from for H&E staining of mouse lungs ( n = 4), showing histopathological alterations classified by anatomical site and summarized as a percentage of tissue involvement. (F) Quantification of viral genome copies in mouse brains—treated and infected, as represented in A— by ddPCR. Each dot represents an individual mouse sample ( n = 6). (G) Distribution of brains from vehicle- or PAD-inhibitor-treated infected mice based on viral load (low: < below 10 3 ; high: > below 10 3 ). (H) Graphical representation of cumulative scores from for immunohistochemical staining of mouse brains using anti-SARS-CoV-2 nucleocapsid antibody ( n = 4). Y axis represents the cumulative score calculated according to the criteria described in . (I) Graphical representation of cumulative scores from for H&E staining of mouse brains ( n = 4), showing histopathological alterations classified by anatomical site and summarized as a percentage of tissue involvement. Data from all experiments, which represent mean ± SEM, are analyzed using an unpaired two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.
    Figure Legend Snippet: In vivo antiviral activity of PAD inhibitors against SARS-CoV-2 (A) Schematic representation of the treatment and infection protocols in K18-hACE2 transgenic mice. (B) Quantification of viral genome copies in mouse lungs—treated and infected as described in A— by ddPCR. Each dot represents an individual mouse sample ( n = 6). (C) Plaque assay to determine the number of infectious viral particles in mouse nasal swabs. (D) Graphical representation of cumulative scores from for immunohistochemical staining of mouse lungs using an anti-SARS-CoV-2 nucleocapsid antibody ( n = 4). Y axis represents the cumulative score calculated according to the criteria described in . (E) Graphical representation of cumulative scores from for H&E staining of mouse lungs ( n = 4), showing histopathological alterations classified by anatomical site and summarized as a percentage of tissue involvement. (F) Quantification of viral genome copies in mouse brains—treated and infected, as represented in A— by ddPCR. Each dot represents an individual mouse sample ( n = 6). (G) Distribution of brains from vehicle- or PAD-inhibitor-treated infected mice based on viral load (low: < below 10 3 ; high: > below 10 3 ). (H) Graphical representation of cumulative scores from for immunohistochemical staining of mouse brains using anti-SARS-CoV-2 nucleocapsid antibody ( n = 4). Y axis represents the cumulative score calculated according to the criteria described in . (I) Graphical representation of cumulative scores from for H&E staining of mouse brains ( n = 4), showing histopathological alterations classified by anatomical site and summarized as a percentage of tissue involvement. Data from all experiments, which represent mean ± SEM, are analyzed using an unpaired two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

    Techniques Used: In Vivo, Activity Assay, Infection, Transgenic Assay, Plaque Assay, Immunohistochemical staining, Staining, Two Tailed Test

    PAD inhibition modulates SARS-CoV-2-induced inflammation (A–D) Relative mRNA levels of the indicated genes are quantified by comparative RT-qPCR from total RNA of Calu-3 cells infected with the SARS-CoV-2 Delta strain (gray bars; MOI 0.1, 24 hpi) and treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO (vehicle). Data from three independent experiments were normalized to the housekeeping gene PGK1 and plotted as mean fold change ±SEM over mock-infected cells (white bars). (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test). Red asterisks indicate comparisons with mock control, while black asterisks refer to comparisons among treatments. (E–J) Relative mRNA levels of the indicated cytokines are quantified by RT-qPCR from lung tissues of mice infected with SARS-CoV-2 Delta variant (10 5 PFU, i.n.) for 4 days and treated with BB-Cl (1 mg/kg), GSK199 (30 mg/kg), or DMSO (vehicle). Each dot represents one individual mouse. Data are normalized to the housekeeping gene β-actin and are presented relative to the mean ΔCt value of vehicle-treated mice. Statistical significance is assessed using an unpaired two-tailed t test (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).
    Figure Legend Snippet: PAD inhibition modulates SARS-CoV-2-induced inflammation (A–D) Relative mRNA levels of the indicated genes are quantified by comparative RT-qPCR from total RNA of Calu-3 cells infected with the SARS-CoV-2 Delta strain (gray bars; MOI 0.1, 24 hpi) and treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO (vehicle). Data from three independent experiments were normalized to the housekeeping gene PGK1 and plotted as mean fold change ±SEM over mock-infected cells (white bars). (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test). Red asterisks indicate comparisons with mock control, while black asterisks refer to comparisons among treatments. (E–J) Relative mRNA levels of the indicated cytokines are quantified by RT-qPCR from lung tissues of mice infected with SARS-CoV-2 Delta variant (10 5 PFU, i.n.) for 4 days and treated with BB-Cl (1 mg/kg), GSK199 (30 mg/kg), or DMSO (vehicle). Each dot represents one individual mouse. Data are normalized to the housekeeping gene β-actin and are presented relative to the mean ΔCt value of vehicle-treated mice. Statistical significance is assessed using an unpaired two-tailed t test (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

    Techniques Used: Inhibition, Quantitative RT-PCR, Infection, Control, Variant Assay, Two Tailed Test

    PAD inhibition protects against SARS-CoV-2-induced pathological changes (A–C) Volcano plots show citrullinated proteins in pooled protein lysates from SARS-CoV-2- vs. mock-infected mouse lungs at 4 dpi. Lysates are incubated with biotin-PG to isolate citrullinated proteins on streptavidin agarose, followed by on-bead tryptic digestion and LC-MS/MS analysis. Each dot represents an identified citrullinated protein. The x axis shows the citrullination ratio between mock and infected samples (A) and between infected mice treated with vehicle (DMSO) or with PAD inhibitors BB-Cl-amidine (B) or GSK199 (C), while the y axis indicates statistical significance, both on a logarithmic scale ( n = 3). (D) Heatmap shows the fold changes of differentially citrullinated proteins identified in lung tissues across the indicated pairwise comparisons: uninfected vs. infected, and vehicle-treated infected vs. BB-Cl–treated or GSK199–treated infected mice. The color scale represents relative citrullination levels expressed as log 2 fold change.
    Figure Legend Snippet: PAD inhibition protects against SARS-CoV-2-induced pathological changes (A–C) Volcano plots show citrullinated proteins in pooled protein lysates from SARS-CoV-2- vs. mock-infected mouse lungs at 4 dpi. Lysates are incubated with biotin-PG to isolate citrullinated proteins on streptavidin agarose, followed by on-bead tryptic digestion and LC-MS/MS analysis. Each dot represents an identified citrullinated protein. The x axis shows the citrullination ratio between mock and infected samples (A) and between infected mice treated with vehicle (DMSO) or with PAD inhibitors BB-Cl-amidine (B) or GSK199 (C), while the y axis indicates statistical significance, both on a logarithmic scale ( n = 3). (D) Heatmap shows the fold changes of differentially citrullinated proteins identified in lung tissues across the indicated pairwise comparisons: uninfected vs. infected, and vehicle-treated infected vs. BB-Cl–treated or GSK199–treated infected mice. The color scale represents relative citrullination levels expressed as log 2 fold change.

    Techniques Used: Inhibition, Infection, Incubation, Liquid Chromatography with Mass Spectroscopy

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    Article Snippet: Arbidol (ARB, Umifenovir), a broad-spectrum antiviral from Russia, lacks Food and Drug Administration (FDA) approval due to insufficient clinical data and undocumented toxicity concerns.. Its indole scaffold, with six unique substitutions, enables optimization for improved efficacy.. This study optimized ARB’s antiviral potency and safety by modifying the N1, C2, C3, and C4 positions.

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    Polymer:

    Article Title: Cellular tropism of SARS-CoV-2 in the respiratory tract of Syrian hamsters and B6.Cg-Tg(K18-ACE2)2Prlmn/J transgenic mice
    Article Snippet: For formalin-fixed and paraffin-embedded tissues monoplex DAB immunohistochemistry was conducted using a Chromomap DAB IHC kit (Roche) with CC1 antigen retrieval at 95 °C for 32 minutes. .. Primary antibody incubation was conducted with a mouse mAB specific to SARS-CoV-1/2 Spike Protein (Cell Signaling Technology, 2B3E5), followed with a rabbit anti-mouse linking antibody IgG1+IgG2a+IgG3 antibody (Abcam, ab133469) at 37 °C for 20 minutes (1:1,000), and finally with a pre-dilute ImmPRESS polymer goat anti-rabbit HRP conjugated secondary (Vector Labs MP-7451-50) for 20 min at 37 °C. .. Immunoreactivity was developed via DAB followed by nuclear counterstaining with hematoxylin and bluing reagents (Roche).



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    Functional activity of PVI-2 antibodies compared to PVI-1 antibodies from the same clonal family. ( A ) Percent somatic hypermutation of IgG heavy chain and light chain among clonal lineage members. ( B ) Comparison of binding profiles between PVI-1 mAbs and clonal lineage members in PVI-2. The heatmap shows OD450 nm binding activity by ELISA, with the antigens tested shown to the left. The antibodies tested are indicated at the top with mAbs from PVI-1 in orange text and PVI-2 in black text. mAbs were tested in duplicate at a fixed concentration of 1,000 ng/mL. Green indicates binding with increasing shades of green indicating increasing OD450 values, as indicated in the key to the right. The cases with the darkest green (>3.0 OD450) are qualitative and not quantitative results, as the assay would be saturated for the most potent binding mAbs. Gray indicates no detectable binding. ( C ) Heatmap of neutralization IC50s (µg/mL) of the same antibodies as in panel B. Viruses tested are shown to the <t>left,</t> <t>with</t> <t>SARS-CoV-2</t> variants in the top half and more diverse sarbecoviruses in the bottom half. The color gradient represents neutralization activity as shown in the key to the right, with darker shades of blue correlating with IC50 potency. Gray indicates no detectable neutralization (IC50), and white indicates the mAbs were not tested because they did not bind SARS-CoV-1 spike trimer. IC50 values were averaged from 2 to 6 independent experiments performed in technical duplicate. IC50 values were calculated with GraphPad Prism, with a four-parameter non-linear regression model. ( D ) Geometric mean of the IC50 values (µg/mL) and 95% confidence intervals (CI) of the panel of viruses tested in panel C . IC50 >20 µg/mL was set to 20 µg/mL in this calculation. The number of viruses neutralized is displayed as a fraction of viruses that the antibody neutralizes with an IC50 <20 µg/mL, over the total number of viruses the antibody was tested against.
    Sars Cov 2 Delta Trimer, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    sars cov 2 wh 1 s1  (Sino Biological)
    96
    Sino Biological sars cov 2 wh 1 s1
    Functional activity of PVI-2 antibodies compared to PVI-1 antibodies from the same clonal family. ( A ) Percent somatic hypermutation of IgG heavy chain and light chain among clonal lineage members. ( B ) Comparison of binding profiles between PVI-1 mAbs and clonal lineage members in PVI-2. The heatmap shows OD450 nm binding activity by ELISA, with the antigens tested shown to the left. The antibodies tested are indicated at the top with mAbs from PVI-1 in orange text and PVI-2 in black text. mAbs were tested in duplicate at a fixed concentration of 1,000 ng/mL. Green indicates binding with increasing shades of green indicating increasing OD450 values, as indicated in the key to the right. The cases with the darkest green (>3.0 OD450) are qualitative and not quantitative results, as the assay would be saturated for the most potent binding mAbs. Gray indicates no detectable binding. ( C ) Heatmap of neutralization IC50s (µg/mL) of the same antibodies as in panel B. Viruses tested are shown to the left, with <t>SARS-CoV-2</t> variants in the top half and more diverse sarbecoviruses in the bottom half. The color gradient represents neutralization activity as shown in the key to the right, with darker shades of blue correlating with IC50 potency. Gray indicates no detectable neutralization (IC50), and white indicates the mAbs were not tested because they did not bind SARS-CoV-1 spike trimer. IC50 values were averaged from 2 to 6 independent experiments performed in technical duplicate. IC50 values were calculated with GraphPad Prism, with a four-parameter non-linear regression model. ( D ) Geometric mean of the IC50 values (µg/mL) and 95% confidence intervals (CI) of the panel of viruses tested in panel C . IC50 >20 µg/mL was set to 20 µg/mL in this calculation. The number of viruses neutralized is displayed as a fraction of viruses that the antibody neutralizes with an IC50 <20 µg/mL, over the total number of viruses the antibody was tested against.
    Sars Cov 2 Wh 1 S1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/sars+cov+1+2+spike+protein/SARS-CoV-2+(2019-nCoV)+Spike+RBD-His+Recombinant+Protein/pmc13059721-72-15-18
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    sars cov 2 b q 1 1 trimer  (Sino Biological)
    94
    Sino Biological sars cov 2 b q 1 1 trimer
    Functional activity of PVI-2 antibodies compared to PVI-1 antibodies from the same clonal family. ( A ) Percent somatic hypermutation of IgG heavy chain and light chain among clonal lineage members. ( B ) Comparison of binding profiles between PVI-1 mAbs and clonal lineage members in PVI-2. The heatmap shows OD450 nm binding activity by ELISA, with the antigens tested shown to the left. The antibodies tested are indicated at the top with mAbs from PVI-1 in orange text and PVI-2 in black text. mAbs were tested in duplicate at a fixed concentration of 1,000 ng/mL. Green indicates binding with increasing shades of green indicating increasing OD450 values, as indicated in the key to the right. The cases with the darkest green (>3.0 OD450) are qualitative and not quantitative results, as the assay would be saturated for the most potent binding mAbs. Gray indicates no detectable binding. ( C ) Heatmap of neutralization IC50s (µg/mL) of the same antibodies as in panel B. Viruses tested are shown to the <t>left,</t> <t>with</t> <t>SARS-CoV-2</t> variants in the top half and more diverse sarbecoviruses in the bottom half. The color gradient represents neutralization activity as shown in the key to the right, with darker shades of blue correlating with IC50 potency. Gray indicates no detectable neutralization (IC50), and white indicates the mAbs were not tested because they did not bind SARS-CoV-1 spike trimer. IC50 values were averaged from 2 to 6 independent experiments performed in technical duplicate. IC50 values were calculated with GraphPad Prism, with a four-parameter non-linear regression model. ( D ) Geometric mean of the IC50 values (µg/mL) and 95% confidence intervals (CI) of the panel of viruses tested in panel C . IC50 >20 µg/mL was set to 20 µg/mL in this calculation. The number of viruses neutralized is displayed as a fraction of viruses that the antibody neutralizes with an IC50 <20 µg/mL, over the total number of viruses the antibody was tested against.
    Sars Cov 2 B Q 1 1 Trimer, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    omicron  (Sino Biological)
    94
    Sino Biological omicron
    Functional activity of PVI-2 antibodies compared to PVI-1 antibodies from the same clonal family. ( A ) Percent somatic hypermutation of IgG heavy chain and light chain among clonal lineage members. ( B ) Comparison of binding profiles between PVI-1 mAbs and clonal lineage members in PVI-2. The heatmap shows OD450 nm binding activity by ELISA, with the antigens tested shown to the left. The antibodies tested are indicated at the top with mAbs from PVI-1 in orange text and PVI-2 in black text. mAbs were tested in duplicate at a fixed concentration of 1,000 ng/mL. Green indicates binding with increasing shades of green indicating increasing OD450 values, as indicated in the key to the right. The cases with the darkest green (>3.0 OD450) are qualitative and not quantitative results, as the assay would be saturated for the most potent binding mAbs. Gray indicates no detectable binding. ( C ) Heatmap of neutralization IC50s (µg/mL) of the same antibodies as in panel B. Viruses tested are shown to the <t>left,</t> <t>with</t> <t>SARS-CoV-2</t> variants in the top half and more diverse sarbecoviruses in the bottom half. The color gradient represents neutralization activity as shown in the key to the right, with darker shades of blue correlating with IC50 potency. Gray indicates no detectable neutralization (IC50), and white indicates the mAbs were not tested because they did not bind SARS-CoV-1 spike trimer. IC50 values were averaged from 2 to 6 independent experiments performed in technical duplicate. IC50 values were calculated with GraphPad Prism, with a four-parameter non-linear regression model. ( D ) Geometric mean of the IC50 values (µg/mL) and 95% confidence intervals (CI) of the panel of viruses tested in panel C . IC50 >20 µg/mL was set to 20 µg/mL in this calculation. The number of viruses neutralized is displayed as a fraction of viruses that the antibody neutralizes with an IC50 <20 µg/mL, over the total number of viruses the antibody was tested against.
    Omicron, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/sars+cov+1+2+spike+protein/SARS-CoV-2+BQ%2E1%2E1+(Omicron)Spike+RBD+Protein/pmc13119851-70-6-23
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    polyhistidine tag  (Sino Biological)
    94
    Sino Biological polyhistidine tag
    Functional activity of PVI-2 antibodies compared to PVI-1 antibodies from the same clonal family. ( A ) Percent somatic hypermutation of IgG heavy chain and light chain among clonal lineage members. ( B ) Comparison of binding profiles between PVI-1 mAbs and clonal lineage members in PVI-2. The heatmap shows OD450 nm binding activity by ELISA, with the antigens tested shown to the left. The antibodies tested are indicated at the top with mAbs from PVI-1 in orange text and PVI-2 in black text. mAbs were tested in duplicate at a fixed concentration of 1,000 ng/mL. Green indicates binding with increasing shades of green indicating increasing OD450 values, as indicated in the key to the right. The cases with the darkest green (>3.0 OD450) are qualitative and not quantitative results, as the assay would be saturated for the most potent binding mAbs. Gray indicates no detectable binding. ( C ) Heatmap of neutralization IC50s (µg/mL) of the same antibodies as in panel B. Viruses tested are shown to the <t>left,</t> <t>with</t> <t>SARS-CoV-2</t> variants in the top half and more diverse sarbecoviruses in the bottom half. The color gradient represents neutralization activity as shown in the key to the right, with darker shades of blue correlating with IC50 potency. Gray indicates no detectable neutralization (IC50), and white indicates the mAbs were not tested because they did not bind SARS-CoV-1 spike trimer. IC50 values were averaged from 2 to 6 independent experiments performed in technical duplicate. IC50 values were calculated with GraphPad Prism, with a four-parameter non-linear regression model. ( D ) Geometric mean of the IC50 values (µg/mL) and 95% confidence intervals (CI) of the panel of viruses tested in panel C . IC50 >20 µg/mL was set to 20 µg/mL in this calculation. The number of viruses neutralized is displayed as a fraction of viruses that the antibody neutralizes with an IC50 <20 µg/mL, over the total number of viruses the antibody was tested against.
    Polyhistidine Tag, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/sars+cov+1+2+spike+protein/SARS-CoV-2+BQ%2E1%2E1+(Omicron)Spike+RBD+Protein/pmc13119851-70-12-23
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    spike protein  (Sino Biological)
    94
    Sino Biological spike protein
    Functional activity of PVI-2 antibodies compared to PVI-1 antibodies from the same clonal family. ( A ) Percent somatic hypermutation of IgG heavy chain and light chain among clonal lineage members. ( B ) Comparison of binding profiles between PVI-1 mAbs and clonal lineage members in PVI-2. The heatmap shows OD450 nm binding activity by ELISA, with the antigens tested shown to the left. The antibodies tested are indicated at the top with mAbs from PVI-1 in orange text and PVI-2 in black text. mAbs were tested in duplicate at a fixed concentration of 1,000 ng/mL. Green indicates binding with increasing shades of green indicating increasing OD450 values, as indicated in the key to the right. The cases with the darkest green (>3.0 OD450) are qualitative and not quantitative results, as the assay would be saturated for the most potent binding mAbs. Gray indicates no detectable binding. ( C ) Heatmap of neutralization IC50s (µg/mL) of the same antibodies as in panel B. Viruses tested are shown to the <t>left,</t> <t>with</t> <t>SARS-CoV-2</t> variants in the top half and more diverse sarbecoviruses in the bottom half. The color gradient represents neutralization activity as shown in the key to the right, with darker shades of blue correlating with IC50 potency. Gray indicates no detectable neutralization (IC50), and white indicates the mAbs were not tested because they did not bind SARS-CoV-1 spike trimer. IC50 values were averaged from 2 to 6 independent experiments performed in technical duplicate. IC50 values were calculated with GraphPad Prism, with a four-parameter non-linear regression model. ( D ) Geometric mean of the IC50 values (µg/mL) and 95% confidence intervals (CI) of the panel of viruses tested in panel C . IC50 >20 µg/mL was set to 20 µg/mL in this calculation. The number of viruses neutralized is displayed as a fraction of viruses that the antibody neutralizes with an IC50 <20 µg/mL, over the total number of viruses the antibody was tested against.
    Spike Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    sars cov 1 2 spike protein  (Cell Signaling Technology Inc)
    94
    Cell Signaling Technology Inc sars cov 1 2 spike protein
    PAD expression in vivo and in vitro after <t>SARS-CoV-2</t> infection (A) Volcano plot relative to the reanalysis of transcriptomic data from ten studies of healthy lungs and four studies of patients with COVID-19 (Delorey et al.). (B and C) Calu3 cells are infected with 2020B.1 (MOI 0.01), Delta (MOI 0.01), or Omicron (MOI 0.01) SARS-CoV-2 variants, and PAD4 (B) and PAD2 (C) mRNA are assessed by RT-qPCR. Data are normalized to the housekeeping gene PGK1 and expressed as mean fold change ±SEM over mock-infected cells of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test. (D) Protein lysates from Calu3 infected with SARS-CoV-2 (2020B.1 variant, MOI 0.1 PFU/cell) or uninfected cells (mock) at different hours post-infection (hpi) are analyzed for citrullinated proteins using an Rh-PG citrulline-specific probe (left panel) or an anti-CCP antibody (right panel). Samples are subjected to gel electrophoresis, and SARS-CoV-2 infection is confirmed using an anti-SARS-CoV-2 Spike (S SARS-CoV-2) antibody. β-actin is used as a loading control. One representative blot of three independent experiments is shown. (E) Volcano plot depicts host (gray dots) and viral (red dots) citrullinated proteins of SARS-infected vs. mock-infected cells at 48 hpi (2020B.1 variant, MOI 0.1 PFU/cell). The x axis represents the ratio of citrullination between mock and infected cells at the indicated time points, while the y axis indicates the statistical significance. Both variables are plotted on a logarithmic scale ( n = 3). (F) PANTHER classification of over-citrullinated cellular proteins based on protein classes.
    Sars Cov 1 2 Spike Protein, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Functional activity of PVI-2 antibodies compared to PVI-1 antibodies from the same clonal family. ( A ) Percent somatic hypermutation of IgG heavy chain and light chain among clonal lineage members. ( B ) Comparison of binding profiles between PVI-1 mAbs and clonal lineage members in PVI-2. The heatmap shows OD450 nm binding activity by ELISA, with the antigens tested shown to the left. The antibodies tested are indicated at the top with mAbs from PVI-1 in orange text and PVI-2 in black text. mAbs were tested in duplicate at a fixed concentration of 1,000 ng/mL. Green indicates binding with increasing shades of green indicating increasing OD450 values, as indicated in the key to the right. The cases with the darkest green (>3.0 OD450) are qualitative and not quantitative results, as the assay would be saturated for the most potent binding mAbs. Gray indicates no detectable binding. ( C ) Heatmap of neutralization IC50s (µg/mL) of the same antibodies as in panel B. Viruses tested are shown to the left, with SARS-CoV-2 variants in the top half and more diverse sarbecoviruses in the bottom half. The color gradient represents neutralization activity as shown in the key to the right, with darker shades of blue correlating with IC50 potency. Gray indicates no detectable neutralization (IC50), and white indicates the mAbs were not tested because they did not bind SARS-CoV-1 spike trimer. IC50 values were averaged from 2 to 6 independent experiments performed in technical duplicate. IC50 values were calculated with GraphPad Prism, with a four-parameter non-linear regression model. ( D ) Geometric mean of the IC50 values (µg/mL) and 95% confidence intervals (CI) of the panel of viruses tested in panel C . IC50 >20 µg/mL was set to 20 µg/mL in this calculation. The number of viruses neutralized is displayed as a fraction of viruses that the antibody neutralizes with an IC50 <20 µg/mL, over the total number of viruses the antibody was tested against.

    Journal: mBio

    Article Title: Re-infection with SARS-CoV-2 is associated with increased antibody breadth and potency against diverse sarbecovirus strains

    doi: 10.1128/mbio.03612-25

    Figure Lengend Snippet: Functional activity of PVI-2 antibodies compared to PVI-1 antibodies from the same clonal family. ( A ) Percent somatic hypermutation of IgG heavy chain and light chain among clonal lineage members. ( B ) Comparison of binding profiles between PVI-1 mAbs and clonal lineage members in PVI-2. The heatmap shows OD450 nm binding activity by ELISA, with the antigens tested shown to the left. The antibodies tested are indicated at the top with mAbs from PVI-1 in orange text and PVI-2 in black text. mAbs were tested in duplicate at a fixed concentration of 1,000 ng/mL. Green indicates binding with increasing shades of green indicating increasing OD450 values, as indicated in the key to the right. The cases with the darkest green (>3.0 OD450) are qualitative and not quantitative results, as the assay would be saturated for the most potent binding mAbs. Gray indicates no detectable binding. ( C ) Heatmap of neutralization IC50s (µg/mL) of the same antibodies as in panel B. Viruses tested are shown to the left, with SARS-CoV-2 variants in the top half and more diverse sarbecoviruses in the bottom half. The color gradient represents neutralization activity as shown in the key to the right, with darker shades of blue correlating with IC50 potency. Gray indicates no detectable neutralization (IC50), and white indicates the mAbs were not tested because they did not bind SARS-CoV-1 spike trimer. IC50 values were averaged from 2 to 6 independent experiments performed in technical duplicate. IC50 values were calculated with GraphPad Prism, with a four-parameter non-linear regression model. ( D ) Geometric mean of the IC50 values (µg/mL) and 95% confidence intervals (CI) of the panel of viruses tested in panel C . IC50 >20 µg/mL was set to 20 µg/mL in this calculation. The number of viruses neutralized is displayed as a fraction of viruses that the antibody neutralizes with an IC50 <20 µg/mL, over the total number of viruses the antibody was tested against.

    Article Snippet: In addition, a subset was also tested for binding to SARS-CoV-2 Delta trimer (Sino Biological, cat. 40589-V08H10) and SARS-CoV-2 B.Q.1.1 trimer (Sino Biological, cat. 40589-V08H41).

    Techniques: Functional Assay, Activity Assay, Comparison, Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Neutralization

    Binding and neutralization of antibodies representing new clonal lineages specific to PVI-2. ( A ) Heatmap shows binding (green) and neutralization IC50s (blue, last two columns). Viruses tested are shown at the top, and mAbs are indicated to the left, after the clonal family designation. IC50 values were averaged from two independent experiments performed in technical duplicate. Figure details are as described in . ( B ) Comparison of IC50s for SARS-CoV-2 XBB1.5 and SARS-CoV-1 for antibodies from PVI-2 specific clonal lineages. The P value is calculated from the Wilcoxon matched-paired signed rank test. Antibodies with no neutralization activity (IC50 > 20 µg/mL) were set to 20 µg/mL for this comparison.

    Journal: mBio

    Article Title: Re-infection with SARS-CoV-2 is associated with increased antibody breadth and potency against diverse sarbecovirus strains

    doi: 10.1128/mbio.03612-25

    Figure Lengend Snippet: Binding and neutralization of antibodies representing new clonal lineages specific to PVI-2. ( A ) Heatmap shows binding (green) and neutralization IC50s (blue, last two columns). Viruses tested are shown at the top, and mAbs are indicated to the left, after the clonal family designation. IC50 values were averaged from two independent experiments performed in technical duplicate. Figure details are as described in . ( B ) Comparison of IC50s for SARS-CoV-2 XBB1.5 and SARS-CoV-1 for antibodies from PVI-2 specific clonal lineages. The P value is calculated from the Wilcoxon matched-paired signed rank test. Antibodies with no neutralization activity (IC50 > 20 µg/mL) were set to 20 µg/mL for this comparison.

    Article Snippet: In addition, a subset was also tested for binding to SARS-CoV-2 Delta trimer (Sino Biological, cat. 40589-V08H10) and SARS-CoV-2 B.Q.1.1 trimer (Sino Biological, cat. 40589-V08H41).

    Techniques: Binding Assay, Neutralization, Comparison, Activity Assay

    Functional characterization of a pan-sarbecovirus neutralizing antibody C68.490. ( A ) Binding breadth of C68.490 and previously characterized antibodies with known pan-sarbecovirus activity against a library of yeast-display sarbecovirus RBDs. Antibodies tested are shown to the left with the specific RBD indicated at the bottom. RBDs are classified by clades as shown above and color-coded. Data for VIR-7229, SA55, and S2X259 were previously published by reference and shown for comparison. ( B ) Comparison of C68.490 binding with other pan-sarbecovirus antibodies by sarbecovirus clade. Statistically significant differences in mean EC50 values between C68.490 and other antibodies were assessed using the Friedman test with Dunn’s multiple comparisons test. Sarbecovirus RBDs were assigned to clades based on existing clade definitions . ( C ) Neutralization of SARS-CoV-2 and sarbecovirus variants by C68.490. Antibodies tested are described at the top and include two control mAbs for comparison. The viruses tested are shown to the left, with a line separating SARS-CoV-2 variants from the more diverse sarbecoviruses. Neutralization data are represented as IC50s (µg/mL) and color-coded as shown in the table below. IC50 values were averaged from 2 to 4 independent experiments performed in technical duplicate, with the exception of neutralization of S2X259 against SARS-CoV-1, which was only tested once. Data for C68.61, which was tested in parallel to the C68.490, was also reported in reference . Other details are as in .

    Journal: mBio

    Article Title: Re-infection with SARS-CoV-2 is associated with increased antibody breadth and potency against diverse sarbecovirus strains

    doi: 10.1128/mbio.03612-25

    Figure Lengend Snippet: Functional characterization of a pan-sarbecovirus neutralizing antibody C68.490. ( A ) Binding breadth of C68.490 and previously characterized antibodies with known pan-sarbecovirus activity against a library of yeast-display sarbecovirus RBDs. Antibodies tested are shown to the left with the specific RBD indicated at the bottom. RBDs are classified by clades as shown above and color-coded. Data for VIR-7229, SA55, and S2X259 were previously published by reference and shown for comparison. ( B ) Comparison of C68.490 binding with other pan-sarbecovirus antibodies by sarbecovirus clade. Statistically significant differences in mean EC50 values between C68.490 and other antibodies were assessed using the Friedman test with Dunn’s multiple comparisons test. Sarbecovirus RBDs were assigned to clades based on existing clade definitions . ( C ) Neutralization of SARS-CoV-2 and sarbecovirus variants by C68.490. Antibodies tested are described at the top and include two control mAbs for comparison. The viruses tested are shown to the left, with a line separating SARS-CoV-2 variants from the more diverse sarbecoviruses. Neutralization data are represented as IC50s (µg/mL) and color-coded as shown in the table below. IC50 values were averaged from 2 to 4 independent experiments performed in technical duplicate, with the exception of neutralization of S2X259 against SARS-CoV-1, which was only tested once. Data for C68.61, which was tested in parallel to the C68.490, was also reported in reference . Other details are as in .

    Article Snippet: In addition, a subset was also tested for binding to SARS-CoV-2 Delta trimer (Sino Biological, cat. 40589-V08H10) and SARS-CoV-2 B.Q.1.1 trimer (Sino Biological, cat. 40589-V08H41).

    Techniques: Functional Assay, Binding Assay, Activity Assay, Comparison, Neutralization, Control

    Characterization of C68.490 escape mutations. ( A ) Top, sites of binding escape and residues conferring escape from C68.490 in different viral backgrounds (WH1 = SARS-CoV-2 Wuhan-Hu-1, BA.2 = SARS-CoV-2 Omicron BA.2, SARS1 = SARS-CoV-1 Urbani) by deep mutational scanning of the RBD. Amino acid numbering based on the SARS-CoV-2 WH1 sequence. Bottom, sites of escape mapped onto surface representation of the RBD (key interacting motifs of ACE2 shown as the gray ribbon). Color gradient represents the escape fraction, with darker red encompassing degree of escape. ( B ) Multi-clade sarbecovirus sequence alignments. Conserved sites (in blue) and variable residues (in white) across sarbecoviruses around the epitope of C68.490 defined in panel A are depicted. C68.490 sites of escape are indicated with yellow arrows. ( C ) Genotype at SARS-CoV-2 spike amino acid sites 378 and 384 from 2,967 GenBank sequences. Adapted from Nextstrain.org (retrieved 16 November 2025).

    Journal: mBio

    Article Title: Re-infection with SARS-CoV-2 is associated with increased antibody breadth and potency against diverse sarbecovirus strains

    doi: 10.1128/mbio.03612-25

    Figure Lengend Snippet: Characterization of C68.490 escape mutations. ( A ) Top, sites of binding escape and residues conferring escape from C68.490 in different viral backgrounds (WH1 = SARS-CoV-2 Wuhan-Hu-1, BA.2 = SARS-CoV-2 Omicron BA.2, SARS1 = SARS-CoV-1 Urbani) by deep mutational scanning of the RBD. Amino acid numbering based on the SARS-CoV-2 WH1 sequence. Bottom, sites of escape mapped onto surface representation of the RBD (key interacting motifs of ACE2 shown as the gray ribbon). Color gradient represents the escape fraction, with darker red encompassing degree of escape. ( B ) Multi-clade sarbecovirus sequence alignments. Conserved sites (in blue) and variable residues (in white) across sarbecoviruses around the epitope of C68.490 defined in panel A are depicted. C68.490 sites of escape are indicated with yellow arrows. ( C ) Genotype at SARS-CoV-2 spike amino acid sites 378 and 384 from 2,967 GenBank sequences. Adapted from Nextstrain.org (retrieved 16 November 2025).

    Article Snippet: In addition, a subset was also tested for binding to SARS-CoV-2 Delta trimer (Sino Biological, cat. 40589-V08H10) and SARS-CoV-2 B.Q.1.1 trimer (Sino Biological, cat. 40589-V08H41).

    Techniques: Binding Assay, Sequencing

    Functional activity of PVI-2 antibodies compared to PVI-1 antibodies from the same clonal family. ( A ) Percent somatic hypermutation of IgG heavy chain and light chain among clonal lineage members. ( B ) Comparison of binding profiles between PVI-1 mAbs and clonal lineage members in PVI-2. The heatmap shows OD450 nm binding activity by ELISA, with the antigens tested shown to the left. The antibodies tested are indicated at the top with mAbs from PVI-1 in orange text and PVI-2 in black text. mAbs were tested in duplicate at a fixed concentration of 1,000 ng/mL. Green indicates binding with increasing shades of green indicating increasing OD450 values, as indicated in the key to the right. The cases with the darkest green (>3.0 OD450) are qualitative and not quantitative results, as the assay would be saturated for the most potent binding mAbs. Gray indicates no detectable binding. ( C ) Heatmap of neutralization IC50s (µg/mL) of the same antibodies as in panel B. Viruses tested are shown to the left, with SARS-CoV-2 variants in the top half and more diverse sarbecoviruses in the bottom half. The color gradient represents neutralization activity as shown in the key to the right, with darker shades of blue correlating with IC50 potency. Gray indicates no detectable neutralization (IC50), and white indicates the mAbs were not tested because they did not bind SARS-CoV-1 spike trimer. IC50 values were averaged from 2 to 6 independent experiments performed in technical duplicate. IC50 values were calculated with GraphPad Prism, with a four-parameter non-linear regression model. ( D ) Geometric mean of the IC50 values (µg/mL) and 95% confidence intervals (CI) of the panel of viruses tested in panel C . IC50 >20 µg/mL was set to 20 µg/mL in this calculation. The number of viruses neutralized is displayed as a fraction of viruses that the antibody neutralizes with an IC50 <20 µg/mL, over the total number of viruses the antibody was tested against.

    Journal: mBio

    Article Title: Re-infection with SARS-CoV-2 is associated with increased antibody breadth and potency against diverse sarbecovirus strains

    doi: 10.1128/mbio.03612-25

    Figure Lengend Snippet: Functional activity of PVI-2 antibodies compared to PVI-1 antibodies from the same clonal family. ( A ) Percent somatic hypermutation of IgG heavy chain and light chain among clonal lineage members. ( B ) Comparison of binding profiles between PVI-1 mAbs and clonal lineage members in PVI-2. The heatmap shows OD450 nm binding activity by ELISA, with the antigens tested shown to the left. The antibodies tested are indicated at the top with mAbs from PVI-1 in orange text and PVI-2 in black text. mAbs were tested in duplicate at a fixed concentration of 1,000 ng/mL. Green indicates binding with increasing shades of green indicating increasing OD450 values, as indicated in the key to the right. The cases with the darkest green (>3.0 OD450) are qualitative and not quantitative results, as the assay would be saturated for the most potent binding mAbs. Gray indicates no detectable binding. ( C ) Heatmap of neutralization IC50s (µg/mL) of the same antibodies as in panel B. Viruses tested are shown to the left, with SARS-CoV-2 variants in the top half and more diverse sarbecoviruses in the bottom half. The color gradient represents neutralization activity as shown in the key to the right, with darker shades of blue correlating with IC50 potency. Gray indicates no detectable neutralization (IC50), and white indicates the mAbs were not tested because they did not bind SARS-CoV-1 spike trimer. IC50 values were averaged from 2 to 6 independent experiments performed in technical duplicate. IC50 values were calculated with GraphPad Prism, with a four-parameter non-linear regression model. ( D ) Geometric mean of the IC50 values (µg/mL) and 95% confidence intervals (CI) of the panel of viruses tested in panel C . IC50 >20 µg/mL was set to 20 µg/mL in this calculation. The number of viruses neutralized is displayed as a fraction of viruses that the antibody neutralizes with an IC50 <20 µg/mL, over the total number of viruses the antibody was tested against.

    Article Snippet: All described antibodies were tested for binding to SARS-CoV-2 WH-1 RBD (Sino Biological, cat. 40592-V08H), SARS-CoV-2 WH-1 S1 (Sino Biological, cat. 40591-V08H), SARS-CoV-2 XBB.1.5 trimer (40589-V08H45), and SARS-CoV-1 GD01 trimer (Acro Biosystems, cat. SPN-S52Ht).

    Techniques: Functional Assay, Activity Assay, Comparison, Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Neutralization

    Binding and neutralization of antibodies representing new clonal lineages specific to PVI-2. ( A ) Heatmap shows binding (green) and neutralization IC50s (blue, last two columns). Viruses tested are shown at the top, and mAbs are indicated to the left, after the clonal family designation. IC50 values were averaged from two independent experiments performed in technical duplicate. Figure details are as described in . ( B ) Comparison of IC50s for SARS-CoV-2 XBB1.5 and SARS-CoV-1 for antibodies from PVI-2 specific clonal lineages. The P value is calculated from the Wilcoxon matched-paired signed rank test. Antibodies with no neutralization activity (IC50 > 20 µg/mL) were set to 20 µg/mL for this comparison.

    Journal: mBio

    Article Title: Re-infection with SARS-CoV-2 is associated with increased antibody breadth and potency against diverse sarbecovirus strains

    doi: 10.1128/mbio.03612-25

    Figure Lengend Snippet: Binding and neutralization of antibodies representing new clonal lineages specific to PVI-2. ( A ) Heatmap shows binding (green) and neutralization IC50s (blue, last two columns). Viruses tested are shown at the top, and mAbs are indicated to the left, after the clonal family designation. IC50 values were averaged from two independent experiments performed in technical duplicate. Figure details are as described in . ( B ) Comparison of IC50s for SARS-CoV-2 XBB1.5 and SARS-CoV-1 for antibodies from PVI-2 specific clonal lineages. The P value is calculated from the Wilcoxon matched-paired signed rank test. Antibodies with no neutralization activity (IC50 > 20 µg/mL) were set to 20 µg/mL for this comparison.

    Article Snippet: All described antibodies were tested for binding to SARS-CoV-2 WH-1 RBD (Sino Biological, cat. 40592-V08H), SARS-CoV-2 WH-1 S1 (Sino Biological, cat. 40591-V08H), SARS-CoV-2 XBB.1.5 trimer (40589-V08H45), and SARS-CoV-1 GD01 trimer (Acro Biosystems, cat. SPN-S52Ht).

    Techniques: Binding Assay, Neutralization, Comparison, Activity Assay

    Functional characterization of a pan-sarbecovirus neutralizing antibody C68.490. ( A ) Binding breadth of C68.490 and previously characterized antibodies with known pan-sarbecovirus activity against a library of yeast-display sarbecovirus RBDs. Antibodies tested are shown to the left with the specific RBD indicated at the bottom. RBDs are classified by clades as shown above and color-coded. Data for VIR-7229, SA55, and S2X259 were previously published by reference and shown for comparison. ( B ) Comparison of C68.490 binding with other pan-sarbecovirus antibodies by sarbecovirus clade. Statistically significant differences in mean EC50 values between C68.490 and other antibodies were assessed using the Friedman test with Dunn’s multiple comparisons test. Sarbecovirus RBDs were assigned to clades based on existing clade definitions . ( C ) Neutralization of SARS-CoV-2 and sarbecovirus variants by C68.490. Antibodies tested are described at the top and include two control mAbs for comparison. The viruses tested are shown to the left, with a line separating SARS-CoV-2 variants from the more diverse sarbecoviruses. Neutralization data are represented as IC50s (µg/mL) and color-coded as shown in the table below. IC50 values were averaged from 2 to 4 independent experiments performed in technical duplicate, with the exception of neutralization of S2X259 against SARS-CoV-1, which was only tested once. Data for C68.61, which was tested in parallel to the C68.490, was also reported in reference . Other details are as in .

    Journal: mBio

    Article Title: Re-infection with SARS-CoV-2 is associated with increased antibody breadth and potency against diverse sarbecovirus strains

    doi: 10.1128/mbio.03612-25

    Figure Lengend Snippet: Functional characterization of a pan-sarbecovirus neutralizing antibody C68.490. ( A ) Binding breadth of C68.490 and previously characterized antibodies with known pan-sarbecovirus activity against a library of yeast-display sarbecovirus RBDs. Antibodies tested are shown to the left with the specific RBD indicated at the bottom. RBDs are classified by clades as shown above and color-coded. Data for VIR-7229, SA55, and S2X259 were previously published by reference and shown for comparison. ( B ) Comparison of C68.490 binding with other pan-sarbecovirus antibodies by sarbecovirus clade. Statistically significant differences in mean EC50 values between C68.490 and other antibodies were assessed using the Friedman test with Dunn’s multiple comparisons test. Sarbecovirus RBDs were assigned to clades based on existing clade definitions . ( C ) Neutralization of SARS-CoV-2 and sarbecovirus variants by C68.490. Antibodies tested are described at the top and include two control mAbs for comparison. The viruses tested are shown to the left, with a line separating SARS-CoV-2 variants from the more diverse sarbecoviruses. Neutralization data are represented as IC50s (µg/mL) and color-coded as shown in the table below. IC50 values were averaged from 2 to 4 independent experiments performed in technical duplicate, with the exception of neutralization of S2X259 against SARS-CoV-1, which was only tested once. Data for C68.61, which was tested in parallel to the C68.490, was also reported in reference . Other details are as in .

    Article Snippet: All described antibodies were tested for binding to SARS-CoV-2 WH-1 RBD (Sino Biological, cat. 40592-V08H), SARS-CoV-2 WH-1 S1 (Sino Biological, cat. 40591-V08H), SARS-CoV-2 XBB.1.5 trimer (40589-V08H45), and SARS-CoV-1 GD01 trimer (Acro Biosystems, cat. SPN-S52Ht).

    Techniques: Functional Assay, Binding Assay, Activity Assay, Comparison, Neutralization, Control

    Characterization of C68.490 escape mutations. ( A ) Top, sites of binding escape and residues conferring escape from C68.490 in different viral backgrounds (WH1 = SARS-CoV-2 Wuhan-Hu-1, BA.2 = SARS-CoV-2 Omicron BA.2, SARS1 = SARS-CoV-1 Urbani) by deep mutational scanning of the RBD. Amino acid numbering based on the SARS-CoV-2 WH1 sequence. Bottom, sites of escape mapped onto surface representation of the RBD (key interacting motifs of ACE2 shown as the gray ribbon). Color gradient represents the escape fraction, with darker red encompassing degree of escape. ( B ) Multi-clade sarbecovirus sequence alignments. Conserved sites (in blue) and variable residues (in white) across sarbecoviruses around the epitope of C68.490 defined in panel A are depicted. C68.490 sites of escape are indicated with yellow arrows. ( C ) Genotype at SARS-CoV-2 spike amino acid sites 378 and 384 from 2,967 GenBank sequences. Adapted from Nextstrain.org (retrieved 16 November 2025).

    Journal: mBio

    Article Title: Re-infection with SARS-CoV-2 is associated with increased antibody breadth and potency against diverse sarbecovirus strains

    doi: 10.1128/mbio.03612-25

    Figure Lengend Snippet: Characterization of C68.490 escape mutations. ( A ) Top, sites of binding escape and residues conferring escape from C68.490 in different viral backgrounds (WH1 = SARS-CoV-2 Wuhan-Hu-1, BA.2 = SARS-CoV-2 Omicron BA.2, SARS1 = SARS-CoV-1 Urbani) by deep mutational scanning of the RBD. Amino acid numbering based on the SARS-CoV-2 WH1 sequence. Bottom, sites of escape mapped onto surface representation of the RBD (key interacting motifs of ACE2 shown as the gray ribbon). Color gradient represents the escape fraction, with darker red encompassing degree of escape. ( B ) Multi-clade sarbecovirus sequence alignments. Conserved sites (in blue) and variable residues (in white) across sarbecoviruses around the epitope of C68.490 defined in panel A are depicted. C68.490 sites of escape are indicated with yellow arrows. ( C ) Genotype at SARS-CoV-2 spike amino acid sites 378 and 384 from 2,967 GenBank sequences. Adapted from Nextstrain.org (retrieved 16 November 2025).

    Article Snippet: All described antibodies were tested for binding to SARS-CoV-2 WH-1 RBD (Sino Biological, cat. 40592-V08H), SARS-CoV-2 WH-1 S1 (Sino Biological, cat. 40591-V08H), SARS-CoV-2 XBB.1.5 trimer (40589-V08H45), and SARS-CoV-1 GD01 trimer (Acro Biosystems, cat. SPN-S52Ht).

    Techniques: Binding Assay, Sequencing

    Functional activity of PVI-2 antibodies compared to PVI-1 antibodies from the same clonal family. ( A ) Percent somatic hypermutation of IgG heavy chain and light chain among clonal lineage members. ( B ) Comparison of binding profiles between PVI-1 mAbs and clonal lineage members in PVI-2. The heatmap shows OD450 nm binding activity by ELISA, with the antigens tested shown to the left. The antibodies tested are indicated at the top with mAbs from PVI-1 in orange text and PVI-2 in black text. mAbs were tested in duplicate at a fixed concentration of 1,000 ng/mL. Green indicates binding with increasing shades of green indicating increasing OD450 values, as indicated in the key to the right. The cases with the darkest green (>3.0 OD450) are qualitative and not quantitative results, as the assay would be saturated for the most potent binding mAbs. Gray indicates no detectable binding. ( C ) Heatmap of neutralization IC50s (µg/mL) of the same antibodies as in panel B. Viruses tested are shown to the left, with SARS-CoV-2 variants in the top half and more diverse sarbecoviruses in the bottom half. The color gradient represents neutralization activity as shown in the key to the right, with darker shades of blue correlating with IC50 potency. Gray indicates no detectable neutralization (IC50), and white indicates the mAbs were not tested because they did not bind SARS-CoV-1 spike trimer. IC50 values were averaged from 2 to 6 independent experiments performed in technical duplicate. IC50 values were calculated with GraphPad Prism, with a four-parameter non-linear regression model. ( D ) Geometric mean of the IC50 values (µg/mL) and 95% confidence intervals (CI) of the panel of viruses tested in panel C . IC50 >20 µg/mL was set to 20 µg/mL in this calculation. The number of viruses neutralized is displayed as a fraction of viruses that the antibody neutralizes with an IC50 <20 µg/mL, over the total number of viruses the antibody was tested against.

    Journal: mBio

    Article Title: Re-infection with SARS-CoV-2 is associated with increased antibody breadth and potency against diverse sarbecovirus strains

    doi: 10.1128/mbio.03612-25

    Figure Lengend Snippet: Functional activity of PVI-2 antibodies compared to PVI-1 antibodies from the same clonal family. ( A ) Percent somatic hypermutation of IgG heavy chain and light chain among clonal lineage members. ( B ) Comparison of binding profiles between PVI-1 mAbs and clonal lineage members in PVI-2. The heatmap shows OD450 nm binding activity by ELISA, with the antigens tested shown to the left. The antibodies tested are indicated at the top with mAbs from PVI-1 in orange text and PVI-2 in black text. mAbs were tested in duplicate at a fixed concentration of 1,000 ng/mL. Green indicates binding with increasing shades of green indicating increasing OD450 values, as indicated in the key to the right. The cases with the darkest green (>3.0 OD450) are qualitative and not quantitative results, as the assay would be saturated for the most potent binding mAbs. Gray indicates no detectable binding. ( C ) Heatmap of neutralization IC50s (µg/mL) of the same antibodies as in panel B. Viruses tested are shown to the left, with SARS-CoV-2 variants in the top half and more diverse sarbecoviruses in the bottom half. The color gradient represents neutralization activity as shown in the key to the right, with darker shades of blue correlating with IC50 potency. Gray indicates no detectable neutralization (IC50), and white indicates the mAbs were not tested because they did not bind SARS-CoV-1 spike trimer. IC50 values were averaged from 2 to 6 independent experiments performed in technical duplicate. IC50 values were calculated with GraphPad Prism, with a four-parameter non-linear regression model. ( D ) Geometric mean of the IC50 values (µg/mL) and 95% confidence intervals (CI) of the panel of viruses tested in panel C . IC50 >20 µg/mL was set to 20 µg/mL in this calculation. The number of viruses neutralized is displayed as a fraction of viruses that the antibody neutralizes with an IC50 <20 µg/mL, over the total number of viruses the antibody was tested against.

    Article Snippet: In addition, a subset was also tested for binding to SARS-CoV-2 Delta trimer (Sino Biological, cat. 40589-V08H10) and SARS-CoV-2 B.Q.1.1 trimer (Sino Biological, cat. 40589-V08H41).

    Techniques: Functional Assay, Activity Assay, Comparison, Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Neutralization

    Binding and neutralization of antibodies representing new clonal lineages specific to PVI-2. ( A ) Heatmap shows binding (green) and neutralization IC50s (blue, last two columns). Viruses tested are shown at the top, and mAbs are indicated to the left, after the clonal family designation. IC50 values were averaged from two independent experiments performed in technical duplicate. Figure details are as described in . ( B ) Comparison of IC50s for SARS-CoV-2 XBB1.5 and SARS-CoV-1 for antibodies from PVI-2 specific clonal lineages. The P value is calculated from the Wilcoxon matched-paired signed rank test. Antibodies with no neutralization activity (IC50 > 20 µg/mL) were set to 20 µg/mL for this comparison.

    Journal: mBio

    Article Title: Re-infection with SARS-CoV-2 is associated with increased antibody breadth and potency against diverse sarbecovirus strains

    doi: 10.1128/mbio.03612-25

    Figure Lengend Snippet: Binding and neutralization of antibodies representing new clonal lineages specific to PVI-2. ( A ) Heatmap shows binding (green) and neutralization IC50s (blue, last two columns). Viruses tested are shown at the top, and mAbs are indicated to the left, after the clonal family designation. IC50 values were averaged from two independent experiments performed in technical duplicate. Figure details are as described in . ( B ) Comparison of IC50s for SARS-CoV-2 XBB1.5 and SARS-CoV-1 for antibodies from PVI-2 specific clonal lineages. The P value is calculated from the Wilcoxon matched-paired signed rank test. Antibodies with no neutralization activity (IC50 > 20 µg/mL) were set to 20 µg/mL for this comparison.

    Article Snippet: In addition, a subset was also tested for binding to SARS-CoV-2 Delta trimer (Sino Biological, cat. 40589-V08H10) and SARS-CoV-2 B.Q.1.1 trimer (Sino Biological, cat. 40589-V08H41).

    Techniques: Binding Assay, Neutralization, Comparison, Activity Assay

    Functional characterization of a pan-sarbecovirus neutralizing antibody C68.490. ( A ) Binding breadth of C68.490 and previously characterized antibodies with known pan-sarbecovirus activity against a library of yeast-display sarbecovirus RBDs. Antibodies tested are shown to the left with the specific RBD indicated at the bottom. RBDs are classified by clades as shown above and color-coded. Data for VIR-7229, SA55, and S2X259 were previously published by reference and shown for comparison. ( B ) Comparison of C68.490 binding with other pan-sarbecovirus antibodies by sarbecovirus clade. Statistically significant differences in mean EC50 values between C68.490 and other antibodies were assessed using the Friedman test with Dunn’s multiple comparisons test. Sarbecovirus RBDs were assigned to clades based on existing clade definitions . ( C ) Neutralization of SARS-CoV-2 and sarbecovirus variants by C68.490. Antibodies tested are described at the top and include two control mAbs for comparison. The viruses tested are shown to the left, with a line separating SARS-CoV-2 variants from the more diverse sarbecoviruses. Neutralization data are represented as IC50s (µg/mL) and color-coded as shown in the table below. IC50 values were averaged from 2 to 4 independent experiments performed in technical duplicate, with the exception of neutralization of S2X259 against SARS-CoV-1, which was only tested once. Data for C68.61, which was tested in parallel to the C68.490, was also reported in reference . Other details are as in .

    Journal: mBio

    Article Title: Re-infection with SARS-CoV-2 is associated with increased antibody breadth and potency against diverse sarbecovirus strains

    doi: 10.1128/mbio.03612-25

    Figure Lengend Snippet: Functional characterization of a pan-sarbecovirus neutralizing antibody C68.490. ( A ) Binding breadth of C68.490 and previously characterized antibodies with known pan-sarbecovirus activity against a library of yeast-display sarbecovirus RBDs. Antibodies tested are shown to the left with the specific RBD indicated at the bottom. RBDs are classified by clades as shown above and color-coded. Data for VIR-7229, SA55, and S2X259 were previously published by reference and shown for comparison. ( B ) Comparison of C68.490 binding with other pan-sarbecovirus antibodies by sarbecovirus clade. Statistically significant differences in mean EC50 values between C68.490 and other antibodies were assessed using the Friedman test with Dunn’s multiple comparisons test. Sarbecovirus RBDs were assigned to clades based on existing clade definitions . ( C ) Neutralization of SARS-CoV-2 and sarbecovirus variants by C68.490. Antibodies tested are described at the top and include two control mAbs for comparison. The viruses tested are shown to the left, with a line separating SARS-CoV-2 variants from the more diverse sarbecoviruses. Neutralization data are represented as IC50s (µg/mL) and color-coded as shown in the table below. IC50 values were averaged from 2 to 4 independent experiments performed in technical duplicate, with the exception of neutralization of S2X259 against SARS-CoV-1, which was only tested once. Data for C68.61, which was tested in parallel to the C68.490, was also reported in reference . Other details are as in .

    Article Snippet: In addition, a subset was also tested for binding to SARS-CoV-2 Delta trimer (Sino Biological, cat. 40589-V08H10) and SARS-CoV-2 B.Q.1.1 trimer (Sino Biological, cat. 40589-V08H41).

    Techniques: Functional Assay, Binding Assay, Activity Assay, Comparison, Neutralization, Control

    Characterization of C68.490 escape mutations. ( A ) Top, sites of binding escape and residues conferring escape from C68.490 in different viral backgrounds (WH1 = SARS-CoV-2 Wuhan-Hu-1, BA.2 = SARS-CoV-2 Omicron BA.2, SARS1 = SARS-CoV-1 Urbani) by deep mutational scanning of the RBD. Amino acid numbering based on the SARS-CoV-2 WH1 sequence. Bottom, sites of escape mapped onto surface representation of the RBD (key interacting motifs of ACE2 shown as the gray ribbon). Color gradient represents the escape fraction, with darker red encompassing degree of escape. ( B ) Multi-clade sarbecovirus sequence alignments. Conserved sites (in blue) and variable residues (in white) across sarbecoviruses around the epitope of C68.490 defined in panel A are depicted. C68.490 sites of escape are indicated with yellow arrows. ( C ) Genotype at SARS-CoV-2 spike amino acid sites 378 and 384 from 2,967 GenBank sequences. Adapted from Nextstrain.org (retrieved 16 November 2025).

    Journal: mBio

    Article Title: Re-infection with SARS-CoV-2 is associated with increased antibody breadth and potency against diverse sarbecovirus strains

    doi: 10.1128/mbio.03612-25

    Figure Lengend Snippet: Characterization of C68.490 escape mutations. ( A ) Top, sites of binding escape and residues conferring escape from C68.490 in different viral backgrounds (WH1 = SARS-CoV-2 Wuhan-Hu-1, BA.2 = SARS-CoV-2 Omicron BA.2, SARS1 = SARS-CoV-1 Urbani) by deep mutational scanning of the RBD. Amino acid numbering based on the SARS-CoV-2 WH1 sequence. Bottom, sites of escape mapped onto surface representation of the RBD (key interacting motifs of ACE2 shown as the gray ribbon). Color gradient represents the escape fraction, with darker red encompassing degree of escape. ( B ) Multi-clade sarbecovirus sequence alignments. Conserved sites (in blue) and variable residues (in white) across sarbecoviruses around the epitope of C68.490 defined in panel A are depicted. C68.490 sites of escape are indicated with yellow arrows. ( C ) Genotype at SARS-CoV-2 spike amino acid sites 378 and 384 from 2,967 GenBank sequences. Adapted from Nextstrain.org (retrieved 16 November 2025).

    Article Snippet: In addition, a subset was also tested for binding to SARS-CoV-2 Delta trimer (Sino Biological, cat. 40589-V08H10) and SARS-CoV-2 B.Q.1.1 trimer (Sino Biological, cat. 40589-V08H41).

    Techniques: Binding Assay, Sequencing

    PAD expression in vivo and in vitro after SARS-CoV-2 infection (A) Volcano plot relative to the reanalysis of transcriptomic data from ten studies of healthy lungs and four studies of patients with COVID-19 (Delorey et al.). (B and C) Calu3 cells are infected with 2020B.1 (MOI 0.01), Delta (MOI 0.01), or Omicron (MOI 0.01) SARS-CoV-2 variants, and PAD4 (B) and PAD2 (C) mRNA are assessed by RT-qPCR. Data are normalized to the housekeeping gene PGK1 and expressed as mean fold change ±SEM over mock-infected cells of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test. (D) Protein lysates from Calu3 infected with SARS-CoV-2 (2020B.1 variant, MOI 0.1 PFU/cell) or uninfected cells (mock) at different hours post-infection (hpi) are analyzed for citrullinated proteins using an Rh-PG citrulline-specific probe (left panel) or an anti-CCP antibody (right panel). Samples are subjected to gel electrophoresis, and SARS-CoV-2 infection is confirmed using an anti-SARS-CoV-2 Spike (S SARS-CoV-2) antibody. β-actin is used as a loading control. One representative blot of three independent experiments is shown. (E) Volcano plot depicts host (gray dots) and viral (red dots) citrullinated proteins of SARS-infected vs. mock-infected cells at 48 hpi (2020B.1 variant, MOI 0.1 PFU/cell). The x axis represents the ratio of citrullination between mock and infected cells at the indicated time points, while the y axis indicates the statistical significance. Both variables are plotted on a logarithmic scale ( n = 3). (F) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

    Journal: iScience

    Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

    doi: 10.1016/j.isci.2026.115038

    Figure Lengend Snippet: PAD expression in vivo and in vitro after SARS-CoV-2 infection (A) Volcano plot relative to the reanalysis of transcriptomic data from ten studies of healthy lungs and four studies of patients with COVID-19 (Delorey et al.). (B and C) Calu3 cells are infected with 2020B.1 (MOI 0.01), Delta (MOI 0.01), or Omicron (MOI 0.01) SARS-CoV-2 variants, and PAD4 (B) and PAD2 (C) mRNA are assessed by RT-qPCR. Data are normalized to the housekeeping gene PGK1 and expressed as mean fold change ±SEM over mock-infected cells of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test. (D) Protein lysates from Calu3 infected with SARS-CoV-2 (2020B.1 variant, MOI 0.1 PFU/cell) or uninfected cells (mock) at different hours post-infection (hpi) are analyzed for citrullinated proteins using an Rh-PG citrulline-specific probe (left panel) or an anti-CCP antibody (right panel). Samples are subjected to gel electrophoresis, and SARS-CoV-2 infection is confirmed using an anti-SARS-CoV-2 Spike (S SARS-CoV-2) antibody. β-actin is used as a loading control. One representative blot of three independent experiments is shown. (E) Volcano plot depicts host (gray dots) and viral (red dots) citrullinated proteins of SARS-infected vs. mock-infected cells at 48 hpi (2020B.1 variant, MOI 0.1 PFU/cell). The x axis represents the ratio of citrullination between mock and infected cells at the indicated time points, while the y axis indicates the statistical significance. Both variables are plotted on a logarithmic scale ( n = 3). (F) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

    Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

    Techniques: Expressing, In Vivo, In Vitro, Infection, Quantitative RT-PCR, Variant Assay, Nucleic Acid Electrophoresis, Control

    SARS-CoV-2 infection affects protein citrullination by modulating PAD expression in vivo (A and B) PAD4 and PAD2 mRNA expression in mouse lungs (A) and brains (B) assessed by RT-qPCR. Each dot represents an individual mouse sample ( n = 6). Data are normalized to the housekeeping gene β-actin and presented relative to one randomly selected non-infected sample. Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (C) Western blot analysis of protein lysates from pooled uninfected (mock) and SARS-CoV-2-infected (6 dpi, Delta variant) mouse lungs and brains. The analysis is performed using antibodies against PAD2, PAD4, and β-actin, the latter to ensure equal loading. A representative blot from three independent experiments is shown. (D) Multiplex immunofluorescence staining for the detection of SARS-CoV-2-targeted cells in mouse brains and lung sections at 6 dpi and mock control. S SARS-CoV-2 protein, PAD2, and PAD4 positive signals are represented in green, pink, and yellow, respectively. Cell nuclei are visualized by DAPI (blue). Original magnification 20×. (E) Quantification of PAD2-and PAD4-positive cells is performed using the inForm Image Analysis software (Akoya Biosciences). For each mouse ( n = 6), one representative section is analyzed to determine cell density (cells/mm 2 ). Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (F) Volcano plot shows citrullinated proteins in pooled protein lysates from SARS-CoV-2-infected vs. mock-infected mouse lungs at 6 dpi. Lysates are incubated with biotin-PG to isolate citrullinated proteins on streptavidin agarose, followed by on-bead tryptic digestion and LC-MS/MS analysis. Each dot represents an identified citrullinated protein. The x axis shows the citrullination ratio between mock and infected samples, and the y axis indicates statistical significance, both on a logarithmic scale ( n = 3). (G) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

    Journal: iScience

    Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

    doi: 10.1016/j.isci.2026.115038

    Figure Lengend Snippet: SARS-CoV-2 infection affects protein citrullination by modulating PAD expression in vivo (A and B) PAD4 and PAD2 mRNA expression in mouse lungs (A) and brains (B) assessed by RT-qPCR. Each dot represents an individual mouse sample ( n = 6). Data are normalized to the housekeeping gene β-actin and presented relative to one randomly selected non-infected sample. Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (C) Western blot analysis of protein lysates from pooled uninfected (mock) and SARS-CoV-2-infected (6 dpi, Delta variant) mouse lungs and brains. The analysis is performed using antibodies against PAD2, PAD4, and β-actin, the latter to ensure equal loading. A representative blot from three independent experiments is shown. (D) Multiplex immunofluorescence staining for the detection of SARS-CoV-2-targeted cells in mouse brains and lung sections at 6 dpi and mock control. S SARS-CoV-2 protein, PAD2, and PAD4 positive signals are represented in green, pink, and yellow, respectively. Cell nuclei are visualized by DAPI (blue). Original magnification 20×. (E) Quantification of PAD2-and PAD4-positive cells is performed using the inForm Image Analysis software (Akoya Biosciences). For each mouse ( n = 6), one representative section is analyzed to determine cell density (cells/mm 2 ). Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (F) Volcano plot shows citrullinated proteins in pooled protein lysates from SARS-CoV-2-infected vs. mock-infected mouse lungs at 6 dpi. Lysates are incubated with biotin-PG to isolate citrullinated proteins on streptavidin agarose, followed by on-bead tryptic digestion and LC-MS/MS analysis. Each dot represents an identified citrullinated protein. The x axis shows the citrullination ratio between mock and infected samples, and the y axis indicates statistical significance, both on a logarithmic scale ( n = 3). (G) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

    Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

    Techniques: Infection, Expressing, In Vivo, Quantitative RT-PCR, Western Blot, Variant Assay, Multiplex Assay, Immunofluorescence, Staining, Control, Software, Incubation, Liquid Chromatography with Mass Spectroscopy

    Antiviral effects of PAD inhibitors against SARS-CoV-2 in vitro (A and B) Quantification of SARS-CoV-2 2020B.1 (MOI 0.1) viral particle production in Calu-3 and Huh7.5 cells (at 24 hpi and 48 hpi, respectively) treated with the increasing concentrations of BB-Cl (A) or GSK199 (B), determined by plaque assay. Results are expressed as a percentage relative to vehicle-treated cells (DMSO, marked as 0 on the graph). Data are presented as means ± SEM from four independent experiments and are analyzed by one-way ANOVA followed by Bonferroni’s post-test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (C and D) Cell viability of uninfected Calu3 and Huh7.5 cells treated with the indicated concentrations of BB-Cl (C) and GSK199 (D) for 72 h, determined for each concentration by MTT assay. Values are expressed as means ± SEM of three independent experiments. (E) Viral titers of different SARS-CoV-2 variants are measured in Calu-3 cells at 24 hpi (MOI = 0.1) following treatment with BB-Cl (5 μM) or GSK199 (20 μM). Viral particle production is determined by plaque assay. Data are expressed as mean ± SEM from four independent experiments and analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (F) Viral titers of different SARS-CoV-2 variants (as indicated in the figure) are measured in Huh7.5 cells at 48 hpi (MOI = 0.1) following treatment with BB-Cl (5 μM) or GSK199 (20 μM). Viral particle production is determined by plaque assay. Data are expressed as mean ± SEM from four independent experiments and analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

    Journal: iScience

    Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

    doi: 10.1016/j.isci.2026.115038

    Figure Lengend Snippet: Antiviral effects of PAD inhibitors against SARS-CoV-2 in vitro (A and B) Quantification of SARS-CoV-2 2020B.1 (MOI 0.1) viral particle production in Calu-3 and Huh7.5 cells (at 24 hpi and 48 hpi, respectively) treated with the increasing concentrations of BB-Cl (A) or GSK199 (B), determined by plaque assay. Results are expressed as a percentage relative to vehicle-treated cells (DMSO, marked as 0 on the graph). Data are presented as means ± SEM from four independent experiments and are analyzed by one-way ANOVA followed by Bonferroni’s post-test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (C and D) Cell viability of uninfected Calu3 and Huh7.5 cells treated with the indicated concentrations of BB-Cl (C) and GSK199 (D) for 72 h, determined for each concentration by MTT assay. Values are expressed as means ± SEM of three independent experiments. (E) Viral titers of different SARS-CoV-2 variants are measured in Calu-3 cells at 24 hpi (MOI = 0.1) following treatment with BB-Cl (5 μM) or GSK199 (20 μM). Viral particle production is determined by plaque assay. Data are expressed as mean ± SEM from four independent experiments and analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (F) Viral titers of different SARS-CoV-2 variants (as indicated in the figure) are measured in Huh7.5 cells at 48 hpi (MOI = 0.1) following treatment with BB-Cl (5 μM) or GSK199 (20 μM). Viral particle production is determined by plaque assay. Data are expressed as mean ± SEM from four independent experiments and analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

    Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

    Techniques: In Vitro, Plaque Assay, Concentration Assay, MTT Assay

    PAD4 inhibition blocks SARS-CoV-2 protein and genome synthesis (A) Relative viral RNA quantification in cell extracts from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO, determined by comparative RT-qPCR. Values are expressed relative to vehicle-treated samples and normalized to the housekeeping gene PGK1. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (B) Western blot analysis of viral protein expression in Huh7.5 and Calu3 cells, either mock-infected or infected with SARS-CoV-2 (MOI 0.1) and treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO. One representative blot from three independent experiments is shown. (C) Schematic representation of infection and treatment protocols used for the viral entry inhibition assay. (D) Viral entry assay on VERO-E6 (left) and TMPRSS2-rexpressing VERO-E6 (right) infected with VSV-Spike GFP (MOI 1). GFP-positive infected cells are microscopically counted, and the results are expressed as a percentage relative to vehicle-treated cells, and are analyzed by t test. Data represent mean ± SEM. (E) Absolute quantification of viral RNA released from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with DMSO (vehicle) or GSK199 (20 μM), either using standard treatment (total) or added at 2 hpi (Post), determined by qRT-PCR. Supernatants are collected at 24 hpi for Calu3 and 48 hpi for Huh7.5. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05 and ∗∗ p < 0.01.

    Journal: iScience

    Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

    doi: 10.1016/j.isci.2026.115038

    Figure Lengend Snippet: PAD4 inhibition blocks SARS-CoV-2 protein and genome synthesis (A) Relative viral RNA quantification in cell extracts from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO, determined by comparative RT-qPCR. Values are expressed relative to vehicle-treated samples and normalized to the housekeeping gene PGK1. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (B) Western blot analysis of viral protein expression in Huh7.5 and Calu3 cells, either mock-infected or infected with SARS-CoV-2 (MOI 0.1) and treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO. One representative blot from three independent experiments is shown. (C) Schematic representation of infection and treatment protocols used for the viral entry inhibition assay. (D) Viral entry assay on VERO-E6 (left) and TMPRSS2-rexpressing VERO-E6 (right) infected with VSV-Spike GFP (MOI 1). GFP-positive infected cells are microscopically counted, and the results are expressed as a percentage relative to vehicle-treated cells, and are analyzed by t test. Data represent mean ± SEM. (E) Absolute quantification of viral RNA released from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with DMSO (vehicle) or GSK199 (20 μM), either using standard treatment (total) or added at 2 hpi (Post), determined by qRT-PCR. Supernatants are collected at 24 hpi for Calu3 and 48 hpi for Huh7.5. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05 and ∗∗ p < 0.01.

    Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

    Techniques: Inhibition, Infection, Quantitative RT-PCR, Western Blot, Expressing, Quantitative Proteomics

    In vivo antiviral activity of PAD inhibitors against SARS-CoV-2 (A) Schematic representation of the treatment and infection protocols in K18-hACE2 transgenic mice. (B) Quantification of viral genome copies in mouse lungs—treated and infected as described in A— by ddPCR. Each dot represents an individual mouse sample ( n = 6). (C) Plaque assay to determine the number of infectious viral particles in mouse nasal swabs. (D) Graphical representation of cumulative scores from for immunohistochemical staining of mouse lungs using an anti-SARS-CoV-2 nucleocapsid antibody ( n = 4). Y axis represents the cumulative score calculated according to the criteria described in . (E) Graphical representation of cumulative scores from for H&E staining of mouse lungs ( n = 4), showing histopathological alterations classified by anatomical site and summarized as a percentage of tissue involvement. (F) Quantification of viral genome copies in mouse brains—treated and infected, as represented in A— by ddPCR. Each dot represents an individual mouse sample ( n = 6). (G) Distribution of brains from vehicle- or PAD-inhibitor-treated infected mice based on viral load (low: < below 10 3 ; high: > below 10 3 ). (H) Graphical representation of cumulative scores from for immunohistochemical staining of mouse brains using anti-SARS-CoV-2 nucleocapsid antibody ( n = 4). Y axis represents the cumulative score calculated according to the criteria described in . (I) Graphical representation of cumulative scores from for H&E staining of mouse brains ( n = 4), showing histopathological alterations classified by anatomical site and summarized as a percentage of tissue involvement. Data from all experiments, which represent mean ± SEM, are analyzed using an unpaired two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

    Journal: iScience

    Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

    doi: 10.1016/j.isci.2026.115038

    Figure Lengend Snippet: In vivo antiviral activity of PAD inhibitors against SARS-CoV-2 (A) Schematic representation of the treatment and infection protocols in K18-hACE2 transgenic mice. (B) Quantification of viral genome copies in mouse lungs—treated and infected as described in A— by ddPCR. Each dot represents an individual mouse sample ( n = 6). (C) Plaque assay to determine the number of infectious viral particles in mouse nasal swabs. (D) Graphical representation of cumulative scores from for immunohistochemical staining of mouse lungs using an anti-SARS-CoV-2 nucleocapsid antibody ( n = 4). Y axis represents the cumulative score calculated according to the criteria described in . (E) Graphical representation of cumulative scores from for H&E staining of mouse lungs ( n = 4), showing histopathological alterations classified by anatomical site and summarized as a percentage of tissue involvement. (F) Quantification of viral genome copies in mouse brains—treated and infected, as represented in A— by ddPCR. Each dot represents an individual mouse sample ( n = 6). (G) Distribution of brains from vehicle- or PAD-inhibitor-treated infected mice based on viral load (low: < below 10 3 ; high: > below 10 3 ). (H) Graphical representation of cumulative scores from for immunohistochemical staining of mouse brains using anti-SARS-CoV-2 nucleocapsid antibody ( n = 4). Y axis represents the cumulative score calculated according to the criteria described in . (I) Graphical representation of cumulative scores from for H&E staining of mouse brains ( n = 4), showing histopathological alterations classified by anatomical site and summarized as a percentage of tissue involvement. Data from all experiments, which represent mean ± SEM, are analyzed using an unpaired two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

    Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

    Techniques: In Vivo, Activity Assay, Infection, Transgenic Assay, Plaque Assay, Immunohistochemical staining, Staining, Two Tailed Test

    PAD inhibition modulates SARS-CoV-2-induced inflammation (A–D) Relative mRNA levels of the indicated genes are quantified by comparative RT-qPCR from total RNA of Calu-3 cells infected with the SARS-CoV-2 Delta strain (gray bars; MOI 0.1, 24 hpi) and treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO (vehicle). Data from three independent experiments were normalized to the housekeeping gene PGK1 and plotted as mean fold change ±SEM over mock-infected cells (white bars). (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test). Red asterisks indicate comparisons with mock control, while black asterisks refer to comparisons among treatments. (E–J) Relative mRNA levels of the indicated cytokines are quantified by RT-qPCR from lung tissues of mice infected with SARS-CoV-2 Delta variant (10 5 PFU, i.n.) for 4 days and treated with BB-Cl (1 mg/kg), GSK199 (30 mg/kg), or DMSO (vehicle). Each dot represents one individual mouse. Data are normalized to the housekeeping gene β-actin and are presented relative to the mean ΔCt value of vehicle-treated mice. Statistical significance is assessed using an unpaired two-tailed t test (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

    Journal: iScience

    Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

    doi: 10.1016/j.isci.2026.115038

    Figure Lengend Snippet: PAD inhibition modulates SARS-CoV-2-induced inflammation (A–D) Relative mRNA levels of the indicated genes are quantified by comparative RT-qPCR from total RNA of Calu-3 cells infected with the SARS-CoV-2 Delta strain (gray bars; MOI 0.1, 24 hpi) and treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO (vehicle). Data from three independent experiments were normalized to the housekeeping gene PGK1 and plotted as mean fold change ±SEM over mock-infected cells (white bars). (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test). Red asterisks indicate comparisons with mock control, while black asterisks refer to comparisons among treatments. (E–J) Relative mRNA levels of the indicated cytokines are quantified by RT-qPCR from lung tissues of mice infected with SARS-CoV-2 Delta variant (10 5 PFU, i.n.) for 4 days and treated with BB-Cl (1 mg/kg), GSK199 (30 mg/kg), or DMSO (vehicle). Each dot represents one individual mouse. Data are normalized to the housekeeping gene β-actin and are presented relative to the mean ΔCt value of vehicle-treated mice. Statistical significance is assessed using an unpaired two-tailed t test (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

    Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

    Techniques: Inhibition, Quantitative RT-PCR, Infection, Control, Variant Assay, Two Tailed Test

    PAD inhibition protects against SARS-CoV-2-induced pathological changes (A–C) Volcano plots show citrullinated proteins in pooled protein lysates from SARS-CoV-2- vs. mock-infected mouse lungs at 4 dpi. Lysates are incubated with biotin-PG to isolate citrullinated proteins on streptavidin agarose, followed by on-bead tryptic digestion and LC-MS/MS analysis. Each dot represents an identified citrullinated protein. The x axis shows the citrullination ratio between mock and infected samples (A) and between infected mice treated with vehicle (DMSO) or with PAD inhibitors BB-Cl-amidine (B) or GSK199 (C), while the y axis indicates statistical significance, both on a logarithmic scale ( n = 3). (D) Heatmap shows the fold changes of differentially citrullinated proteins identified in lung tissues across the indicated pairwise comparisons: uninfected vs. infected, and vehicle-treated infected vs. BB-Cl–treated or GSK199–treated infected mice. The color scale represents relative citrullination levels expressed as log 2 fold change.

    Journal: iScience

    Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

    doi: 10.1016/j.isci.2026.115038

    Figure Lengend Snippet: PAD inhibition protects against SARS-CoV-2-induced pathological changes (A–C) Volcano plots show citrullinated proteins in pooled protein lysates from SARS-CoV-2- vs. mock-infected mouse lungs at 4 dpi. Lysates are incubated with biotin-PG to isolate citrullinated proteins on streptavidin agarose, followed by on-bead tryptic digestion and LC-MS/MS analysis. Each dot represents an identified citrullinated protein. The x axis shows the citrullination ratio between mock and infected samples (A) and between infected mice treated with vehicle (DMSO) or with PAD inhibitors BB-Cl-amidine (B) or GSK199 (C), while the y axis indicates statistical significance, both on a logarithmic scale ( n = 3). (D) Heatmap shows the fold changes of differentially citrullinated proteins identified in lung tissues across the indicated pairwise comparisons: uninfected vs. infected, and vehicle-treated infected vs. BB-Cl–treated or GSK199–treated infected mice. The color scale represents relative citrullination levels expressed as log 2 fold change.

    Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

    Techniques: Inhibition, Infection, Incubation, Liquid Chromatography with Mass Spectroscopy