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Addgene inc sars cov 2 nsp3 protein
Sars Cov 2 Nsp3 Protein, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sars+cov+2+nsp3+protein/pDONR207+SARS-CoV-2+NSP3+(Plasmid+%23141257)/pmc12219648__41467_2025_60949_MOESM1_ESM-80-4-29
Average 93 stars, based on 16 article reviews

sars cov 2 nsp3 protein - by Bioz Stars, 2026-09

93/100 stars

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Article Title: Human protein interaction networks of ancestral and variant SARS-CoV-2 in organ-specific cells and bodily fluids
Article Snippet: .. Using this method, the SARS-CoV-2 NSP3 protein was engineered with a V5 epitope tag by combining a pDONR207 SARS-CoV-2 NSP3 Gateway entry plasmid with a pLEX- 307 destination vector (Addgene 41392) containing a V5 peptide. ..

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Figure 1. Transmembrane SARS-CoV-2 Nsp3 localizes to the ER and delays degradation of INSIG-1. A, schematic representation of forms of FLAG- tagged Nsp3 used in this study. Amino acid numbers shown at the top correspond to the C termini of the constructs and the boundaries of PLpro. Transmembrane domains are indicated in gray, PLpro in blue. Mutation in the active site Cys (aa 111 of PLpro; aa 856 of Nsp3) is indicated by “x.” B, HEK293T cells were transfected with the indicated N terminally FLAG-tagged forms of Nsp3. Samples were evaluated for colocalization of Nsp3 with endogenous Calnexin by wide ﬁeld microscopy. The scale bar in lower left panel corresponds to 10 μm; and the scale bar in lower left panel of magniﬁed images corresponds to 5 μm. Assessment of colocalization was carried out as described in Experimental procedures and plotted as a function of distance from the beginning of the arrow, intensity is in arbitrary units. C, HEK293T cells transfected with either full-length Nsp3 or Nsp3TM were assessed for colocalization with Calnexin by SIM. The scale bar (left panels) corresponds to 10 μm, assessment of colocalization as in (B). D, HEK293T cells were transfected with full- length Nsp3, which includes a triple FLAG tag, or with vector control (Vector) along with INSIG-1 and assessed by cycloheximide (CHX) chase. E, HEK293T cells were transfected with Nsp3TM or soluble PLpro (Nsp3PLpro), both of which include a single N-terminal FLAG tag, and assessed as in (D). F, HEK293T cells were transfected with Nsp3TM, catalytically inactive NspTMC111A, or vector control and assessed as in (D). G, HEK293T cells were transfected with the indicated forms of Nsp3 and the Hong Kong Null variant of alpha-1 anti-trypsin (NHK). Degradation was assessed as in (D–F). H, HEK293T cells were transfected with INSIG-1, HA-tagged ubiquitin, and either Nsp3TM, Nsp3TMC111A, or vector control. Cells were treated overnight with MG132 to inhibit proteasomal degradation of ubiquitinated proteins prior to lysis. Samples were immunoprecipitated with anti-MYC to assess INSIG-1 ubiquitination, equal amounts of each lysate were also assessed for total cellular ubiquitination, arrow indicates migration of INSIG-1. In (D–G), GFP serves as a transfection efﬁciency control. ER, endoplasmic reticulum; NHK, Null Hong Kong; Nsp, nonstructural protein; PLpro, papain-like protease ; SARS-CoV, severe acute respiratory syndrome coronavirus; SIM, structured illumination microscopy; TM, transmembrane.

Journal: The Journal of biological chemistry

Article Title: SARS-CoV-2 papain-like protease plays multiple roles in regulating cellular proteins in the endoplasmic reticulum.

doi: 10.1016/j.jbc.2023.105346

Figure Lengend Snippet: Figure 1. Transmembrane SARS-CoV-2 Nsp3 localizes to the ER and delays degradation of INSIG-1. A, schematic representation of forms of FLAG- tagged Nsp3 used in this study. Amino acid numbers shown at the top correspond to the C termini of the constructs and the boundaries of PLpro. Transmembrane domains are indicated in gray, PLpro in blue. Mutation in the active site Cys (aa 111 of PLpro; aa 856 of Nsp3) is indicated by “x.” B, HEK293T cells were transfected with the indicated N terminally FLAG-tagged forms of Nsp3. Samples were evaluated for colocalization of Nsp3 with endogenous Calnexin by wide ﬁeld microscopy. The scale bar in lower left panel corresponds to 10 μm; and the scale bar in lower left panel of magniﬁed images corresponds to 5 μm. Assessment of colocalization was carried out as described in Experimental procedures and plotted as a function of distance from the beginning of the arrow, intensity is in arbitrary units. C, HEK293T cells transfected with either full-length Nsp3 or Nsp3TM were assessed for colocalization with Calnexin by SIM. The scale bar (left panels) corresponds to 10 μm, assessment of colocalization as in (B). D, HEK293T cells were transfected with full- length Nsp3, which includes a triple FLAG tag, or with vector control (Vector) along with INSIG-1 and assessed by cycloheximide (CHX) chase. E, HEK293T cells were transfected with Nsp3TM or soluble PLpro (Nsp3PLpro), both of which include a single N-terminal FLAG tag, and assessed as in (D). F, HEK293T cells were transfected with Nsp3TM, catalytically inactive NspTMC111A, or vector control and assessed as in (D). G, HEK293T cells were transfected with the indicated forms of Nsp3 and the Hong Kong Null variant of alpha-1 anti-trypsin (NHK). Degradation was assessed as in (D–F). H, HEK293T cells were transfected with INSIG-1, HA-tagged ubiquitin, and either Nsp3TM, Nsp3TMC111A, or vector control. Cells were treated overnight with MG132 to inhibit proteasomal degradation of ubiquitinated proteins prior to lysis. Samples were immunoprecipitated with anti-MYC to assess INSIG-1 ubiquitination, equal amounts of each lysate were also assessed for total cellular ubiquitination, arrow indicates migration of INSIG-1. In (D–G), GFP serves as a transfection efﬁciency control. ER, endoplasmic reticulum; NHK, Null Hong Kong; Nsp, nonstructural protein; PLpro, papain-like protease ; SARS-CoV, severe acute respiratory syndrome coronavirus; SIM, structured illumination microscopy; TM, transmembrane.

Article Snippet: Complementary DNA encoding the full-length Nsp3 protein from the SARS-CoV-2 isolate Wuhan-Hu-1 (https://www. addgene.org/141257/), obtained from Addgene, was a gift from Dr Fritz Roth.

Techniques: Construct, Mutagenesis, Transfection, Microscopy, FLAG-tag, Plasmid Preparation, Control, Variant Assay, Ubiquitin Proteomics, Lysis, Immunoprecipitation, Migration

Figure 2. Differential effects of Nsp3 on SREBP-1 and SREBP-2. A, HEK293T cells were cotransfected with plasmid encoding T7 epitope-tagged SREBP-1 (isoform 1) or SREBP-2 and Nsp3TM and imaged by SIM. The scale bar (left panels) corresponds to 10 μm, assessment of colocalization as in Figure 1B. B and C, HEK293T cells were transfected with SREBP-1 and either vector control (−) or the indicated forms of Nsp3 and assessed by immunoblotting for SREBP-1 levels and degradation after 2 h of CHX. In (B), bracket indicates migration of N-terminal fragments of SREBP-1 that are inconsistently observed regardless of Nsp3 coexpression, arrow denotes migration of full-length SREBP-1, arrowhead indicates Nsp3-dependent species. D, HEK293T cells were transfected with the N-terminal domain (NTD) of SREBP-1, which includes the active transcription factor form of the protein (aa 1–520) and with the indicated forms of Nsp3 and analyzed as in (C). E and F, HEK293T cells were transfected with SREBP-2 and indicated forms of Nsp3 and analyzed as in (B and C). In (B–F), cells were split after transfection, β-actin serves as an equal loading control. CHX, cycloheximide; Nsp, nonstructural protein; SREBP, sterol regulatory element–binding protein; SIM, structured illumination microscopy; TM, transmembrane.

Journal: The Journal of biological chemistry

Article Title: SARS-CoV-2 papain-like protease plays multiple roles in regulating cellular proteins in the endoplasmic reticulum.

doi: 10.1016/j.jbc.2023.105346

Figure Lengend Snippet: Figure 2. Differential effects of Nsp3 on SREBP-1 and SREBP-2. A, HEK293T cells were cotransfected with plasmid encoding T7 epitope-tagged SREBP-1 (isoform 1) or SREBP-2 and Nsp3TM and imaged by SIM. The scale bar (left panels) corresponds to 10 μm, assessment of colocalization as in Figure 1B. B and C, HEK293T cells were transfected with SREBP-1 and either vector control (−) or the indicated forms of Nsp3 and assessed by immunoblotting for SREBP-1 levels and degradation after 2 h of CHX. In (B), bracket indicates migration of N-terminal fragments of SREBP-1 that are inconsistently observed regardless of Nsp3 coexpression, arrow denotes migration of full-length SREBP-1, arrowhead indicates Nsp3-dependent species. D, HEK293T cells were transfected with the N-terminal domain (NTD) of SREBP-1, which includes the active transcription factor form of the protein (aa 1–520) and with the indicated forms of Nsp3 and analyzed as in (C). E and F, HEK293T cells were transfected with SREBP-2 and indicated forms of Nsp3 and analyzed as in (B and C). In (B–F), cells were split after transfection, β-actin serves as an equal loading control. CHX, cycloheximide; Nsp, nonstructural protein; SREBP, sterol regulatory element–binding protein; SIM, structured illumination microscopy; TM, transmembrane.

Techniques: Plasmid Preparation, Transfection, Control, Western Blot, Migration, Binding Assay, Microscopy

Figure 3. SREBP-1 is cleaved at multiple sites by Nsp3. A, top, linear representation of untagged SREBP-1 (isoform 1). Transmembrane domains (TMD) are indicated in gray, 520 is the S2P site of cleavage that results in the N-terminal domain (NTD) active transcription factor. Regions corresponding to aa 313 to 349 and 1166 to 1177 (C terminus of SREBP-1) are shown below with mutations and deletions in this study denoted by the single letter of the substituted amino acid or by “x” in the case of deletions. Shorthand terminology for mutations and deletions in each region of the protein are to the right (point mutations are underlined). Nomenclature for amino acids in proposed cleavage sites (P1-P5) is from Schechter and Berger (6). B, HEK293T cells were transfected with truncated forms of SREBP-1 alone (numbers indicate terminal amino acid included in sequence) or with full-length SREBP-1 with or without cotransfection of Nsp3TM. After treatment with phosphatase (P’tase) samples were resolved by SDS-PAGE and immunoblotted. C, HEK293T cells were transfected with or without Nsp3TM and either WT SREBP-1 (lanes 1 and 2) or the indicated mutations or deletions (lanes 3–5) and immunoblotted. D, HEK293T cells were transfected as in (C). Lanes 5 to 10 include the SREBP-1 A317-318 mutation with either the WT site 2 (lane 5) or single amino acid substitutions in the region of site 2 from 337 to 341 (lane 6–10). See schematic (A) “site 2 and mutations.”Nsp, nonstructural protein; SREBP, sterol regulatory element–binding protein; TM, transmembrane.

Journal: The Journal of biological chemistry

Article Title: SARS-CoV-2 papain-like protease plays multiple roles in regulating cellular proteins in the endoplasmic reticulum.

doi: 10.1016/j.jbc.2023.105346

Figure Lengend Snippet: Figure 3. SREBP-1 is cleaved at multiple sites by Nsp3. A, top, linear representation of untagged SREBP-1 (isoform 1). Transmembrane domains (TMD) are indicated in gray, 520 is the S2P site of cleavage that results in the N-terminal domain (NTD) active transcription factor. Regions corresponding to aa 313 to 349 and 1166 to 1177 (C terminus of SREBP-1) are shown below with mutations and deletions in this study denoted by the single letter of the substituted amino acid or by “x” in the case of deletions. Shorthand terminology for mutations and deletions in each region of the protein are to the right (point mutations are underlined). Nomenclature for amino acids in proposed cleavage sites (P1-P5) is from Schechter and Berger (6). B, HEK293T cells were transfected with truncated forms of SREBP-1 alone (numbers indicate terminal amino acid included in sequence) or with full-length SREBP-1 with or without cotransfection of Nsp3TM. After treatment with phosphatase (P’tase) samples were resolved by SDS-PAGE and immunoblotted. C, HEK293T cells were transfected with or without Nsp3TM and either WT SREBP-1 (lanes 1 and 2) or the indicated mutations or deletions (lanes 3–5) and immunoblotted. D, HEK293T cells were transfected as in (C). Lanes 5 to 10 include the SREBP-1 A317-318 mutation with either the WT site 2 (lane 5) or single amino acid substitutions in the region of site 2 from 337 to 341 (lane 6–10). See schematic (A) “site 2 and mutations.”Nsp, nonstructural protein; SREBP, sterol regulatory element–binding protein; TM, transmembrane.

Techniques: Transfection, Sequencing, Cotransfection, SDS Page, Mutagenesis, Binding Assay

Figure 4. Assessment of sites 1 and 3 of SREBP-1. A, HEK293T cells were transfected with either WT SREBP-1 in lanes 1 to 2 or mutants that abrogate cutting in site 1 (S1) and site 2 (S2) (A317–318 and RLAAA) in lanes 3 to 8, with either the native sequence (lane 3) or the indicated point mutations (underlined) in the C-terminal cleavage site (site 3) encompassing aa 1168 to 1172. Lanes 2 to 8 were cotransfected with Nsp3TM. B, HEK293T cells were transfected with either Nsp3TM or vector control (−) and C-terminal GFP fusions of SREBP-1. SREBP-1-GFP used in lanes 3 to 8 all include the indicated mutations in the ﬁrst and second sites as in (A) and either the native sequence (lanes 3–4) or the indicated point mutations in site 3 (lanes 5–8). C, HEK293T cells were transfected with SREBP-1-GFP having WT site 3 (RLGGG) and mutated in site 1 and site 2 as in (B), and the indicated forms of Nsp3 or vector control (−), and immunoblotted as indicated. D, HEK293T cells were transfected with plasmid encoding WT SREBP-1 (lane 1) or forms that are cleavage resistant in site 2 (S2) and site 3 (S3) (lanes 2–8) and having the indicated sequences in site 1, and cotransfected with Nsp3TM as indicated. Middle panel is a long exposure of the relevant area of the T7 blot showing residual cleavage of the LVSGG to LASGG mutation. E, HEK293T cells were transfected with WT SREBP-1 (lanes 1–4) or SREBP-1 that is cleavage resistant in sites 1 and 2 (lane 5–8) and either Nsp3TM or catalytically inactive Nsp3TMC111A and assessed for loss of the full-length protein by CHX chase. In (E), cells were split after transfection, β-actin serves as an equal loading control. CHX, cycloheximide; Nsp, nonstructural protein; SREBP, sterol regulatory element–binding protein; TM, transmembrane.

Journal: The Journal of biological chemistry

Article Title: SARS-CoV-2 papain-like protease plays multiple roles in regulating cellular proteins in the endoplasmic reticulum.

doi: 10.1016/j.jbc.2023.105346

Figure Lengend Snippet: Figure 4. Assessment of sites 1 and 3 of SREBP-1. A, HEK293T cells were transfected with either WT SREBP-1 in lanes 1 to 2 or mutants that abrogate cutting in site 1 (S1) and site 2 (S2) (A317–318 and RLAAA) in lanes 3 to 8, with either the native sequence (lane 3) or the indicated point mutations (underlined) in the C-terminal cleavage site (site 3) encompassing aa 1168 to 1172. Lanes 2 to 8 were cotransfected with Nsp3TM. B, HEK293T cells were transfected with either Nsp3TM or vector control (−) and C-terminal GFP fusions of SREBP-1. SREBP-1-GFP used in lanes 3 to 8 all include the indicated mutations in the ﬁrst and second sites as in (A) and either the native sequence (lanes 3–4) or the indicated point mutations in site 3 (lanes 5–8). C, HEK293T cells were transfected with SREBP-1-GFP having WT site 3 (RLGGG) and mutated in site 1 and site 2 as in (B), and the indicated forms of Nsp3 or vector control (−), and immunoblotted as indicated. D, HEK293T cells were transfected with plasmid encoding WT SREBP-1 (lane 1) or forms that are cleavage resistant in site 2 (S2) and site 3 (S3) (lanes 2–8) and having the indicated sequences in site 1, and cotransfected with Nsp3TM as indicated. Middle panel is a long exposure of the relevant area of the T7 blot showing residual cleavage of the LVSGG to LASGG mutation. E, HEK293T cells were transfected with WT SREBP-1 (lanes 1–4) or SREBP-1 that is cleavage resistant in sites 1 and 2 (lane 5–8) and either Nsp3TM or catalytically inactive Nsp3TMC111A and assessed for loss of the full-length protein by CHX chase. In (E), cells were split after transfection, β-actin serves as an equal loading control. CHX, cycloheximide; Nsp, nonstructural protein; SREBP, sterol regulatory element–binding protein; TM, transmembrane.

Techniques: Transfection, Sequencing, Plasmid Preparation, Control, Mutagenesis, Binding Assay

Figure 5. Nsp3 increases SREBP-2 stability. A, HEK293T cells transfected with either WT SREBP-2 (KLGGG), a double mutation of Gly 1134 and 1135 to Ala (KLGAA), or a truncation after Gly 1135 (STOP) were assessed by CHX chase. B, C-terminal GFP fusions of WT SREBP-2 or the double Ala mutant were cotransfected with the indicated forms of Nsp3 in HEK293T cells and immunoblotted as indicated. C, GFP fusion of WT SREBP-2 was cotransfected with the indicated forms of Nsp3 and assessed as in (B). D, SREBP-2 plasmids described in (A) were cotransfected with WT or mutant Nsp3TM in HEK293T cells and levels assessed over a 3 h CHX chase. Samples on the left and right are identical exposures of material run on two gels that were transferred and evaluated simultaneously (see Fig. S6D for KLGAA samples run side-by side). Cells were split after transfection, β-actin serves as an equal loading control. E, HEK293T were transfected with the indicated forms of SREBP-2 and of Nsp3TM together with HA-ubiquitin. T7 immunoprecipitates were assessed for ubiquitin and SREBP-2 after treatment with MG132 for 3 h to allow for accumulation of ubiquitinated protein. Equal amounts of cell lysate were evaluated to ensure that changes in ubiquitination in immunoprecipitates did not reﬂect altered total cellular ubiquitination. F, cells transfected with C-terminal MYC epitope-tagged SCAP and the indicated forms of Nsp3 or vector control were treated with 20 μM MG132 for 4 h prior to lysis. Immunoblotting was with anti-MYC or with a SCAP antibody directed against a region N terminal to the LGGG sequence. Cotransfected GFP serves as transfection efﬁciency control. CHX, cycloheximide; Nsp, nonstructural protein; SREBP, sterol regulatory element–binding protein; SCAP, SREBP cleavage–activating protein; TM, transmembrane.

Journal: The Journal of biological chemistry

Article Title: SARS-CoV-2 papain-like protease plays multiple roles in regulating cellular proteins in the endoplasmic reticulum.

doi: 10.1016/j.jbc.2023.105346

Figure Lengend Snippet: Figure 5. Nsp3 increases SREBP-2 stability. A, HEK293T cells transfected with either WT SREBP-2 (KLGGG), a double mutation of Gly 1134 and 1135 to Ala (KLGAA), or a truncation after Gly 1135 (STOP) were assessed by CHX chase. B, C-terminal GFP fusions of WT SREBP-2 or the double Ala mutant were cotransfected with the indicated forms of Nsp3 in HEK293T cells and immunoblotted as indicated. C, GFP fusion of WT SREBP-2 was cotransfected with the indicated forms of Nsp3 and assessed as in (B). D, SREBP-2 plasmids described in (A) were cotransfected with WT or mutant Nsp3TM in HEK293T cells and levels assessed over a 3 h CHX chase. Samples on the left and right are identical exposures of material run on two gels that were transferred and evaluated simultaneously (see Fig. S6D for KLGAA samples run side-by side). Cells were split after transfection, β-actin serves as an equal loading control. E, HEK293T were transfected with the indicated forms of SREBP-2 and of Nsp3TM together with HA-ubiquitin. T7 immunoprecipitates were assessed for ubiquitin and SREBP-2 after treatment with MG132 for 3 h to allow for accumulation of ubiquitinated protein. Equal amounts of cell lysate were evaluated to ensure that changes in ubiquitination in immunoprecipitates did not reﬂect altered total cellular ubiquitination. F, cells transfected with C-terminal MYC epitope-tagged SCAP and the indicated forms of Nsp3 or vector control were treated with 20 μM MG132 for 4 h prior to lysis. Immunoblotting was with anti-MYC or with a SCAP antibody directed against a region N terminal to the LGGG sequence. Cotransfected GFP serves as transfection efﬁciency control. CHX, cycloheximide; Nsp, nonstructural protein; SREBP, sterol regulatory element–binding protein; SCAP, SREBP cleavage–activating protein; TM, transmembrane.

Techniques: Transfection, Mutagenesis, Control, Ubiquitin Proteomics, Plasmid Preparation, Lysis, Western Blot, Sequencing, Binding Assay

Sequence homology between pathogenic bacteria and SARS-CoV-2. Amino acid sequence alignment of pathogenic bacteria (top) and SARS-CoV-2 (bottom) used in this study. KP- Klebsiella pneumoniae , SE- Salmonella Enteritidis, SA- Staphylococcus aureus , CD- Clostridioides difficile , CL- Clostridium spp., NSP3- non-structural protein 3 of SARS-CoV-2, S- spike glycoprotein of SARS-CoV-2. Red amino acid- identity. Yellow amino acid- similarity. Grey amino acid- no identity or similarity.

Journal: Frontiers in Immunology

Article Title: Heterologous Immunity Between SARS-CoV-2 and Pathogenic Bacteria

doi: 10.3389/fimmu.2022.821595

Figure Lengend Snippet: Sequence homology between pathogenic bacteria and SARS-CoV-2. Amino acid sequence alignment of pathogenic bacteria (top) and SARS-CoV-2 (bottom) used in this study. KP- Klebsiella pneumoniae , SE- Salmonella Enteritidis, SA- Staphylococcus aureus , CD- Clostridioides difficile , CL- Clostridium spp., NSP3- non-structural protein 3 of SARS-CoV-2, S- spike glycoprotein of SARS-CoV-2. Red amino acid- identity. Yellow amino acid- similarity. Grey amino acid- no identity or similarity.

Article Snippet: SARS-CoV-2 recombinant proteins NSP3 and spike glycoprotein were purchased from MyBioSource.

Techniques: Sequencing

Bacterial peptide priming enhances SARS-CoV-2 T cell cytokine expression in responders. Pathogenic bacterial peptide (bac) primed T cells restimulated with SARS-CoV-2-peptide measured by flow cytometry with background control values subtracted. Unshaded bars- Control primed (irrelevant peptide, PVSKMRMATPLLMQA), then restimulated with SARS-CoV-2 homologous peptide. Shaded bars– bac primed (KP1, KP2, SE1, SA1, CD1 or CL1) then SARS-CoV-2-peptide homologue restimulated. NoP – no peptide negative control. (A) CD8+ IFN-γ+ responses ( n =7-12), (B) CD4 + IFN-γ + responses ( n =5-10), (C) CD8 + TNF + responses ( n =5-9), (D) CD4 + TNF + responses ( n =4-15), (E) Representative TNF (y-axis) and IFN-γ (x-axis) dot plots of a responder donor with their corresponding SARS-CoV-2 primary response control. * P < 0.05, ** P < 0.01, *** P < 0.001 by Wilcoxon-matched-pairs-signed-rank test, comparing magnitude of response to SARS-CoV-2 peptides with or without bacterial peptide priming.

Journal: Frontiers in Immunology

Article Title: Heterologous Immunity Between SARS-CoV-2 and Pathogenic Bacteria

doi: 10.3389/fimmu.2022.821595

Figure Lengend Snippet: Bacterial peptide priming enhances SARS-CoV-2 T cell cytokine expression in responders. Pathogenic bacterial peptide (bac) primed T cells restimulated with SARS-CoV-2-peptide measured by flow cytometry with background control values subtracted. Unshaded bars- Control primed (irrelevant peptide, PVSKMRMATPLLMQA), then restimulated with SARS-CoV-2 homologous peptide. Shaded bars– bac primed (KP1, KP2, SE1, SA1, CD1 or CL1) then SARS-CoV-2-peptide homologue restimulated. NoP – no peptide negative control. (A) CD8+ IFN-γ+ responses ( n =7-12), (B) CD4 + IFN-γ + responses ( n =5-10), (C) CD8 + TNF + responses ( n =5-9), (D) CD4 + TNF + responses ( n =4-15), (E) Representative TNF (y-axis) and IFN-γ (x-axis) dot plots of a responder donor with their corresponding SARS-CoV-2 primary response control. * P < 0.05, ** P < 0.01, *** P < 0.001 by Wilcoxon-matched-pairs-signed-rank test, comparing magnitude of response to SARS-CoV-2 peptides with or without bacterial peptide priming.

Article Snippet: SARS-CoV-2 recombinant proteins NSP3 and spike glycoprotein were purchased from MyBioSource.

Techniques: Expressing, Flow Cytometry, Negative Control

| Bacterial peptide priming enhances IL-2, CD69 and Perforin T cell responses against SARS-CoV-2 in responders. Pathogenic bacterial-peptide (KP1, KP2, SE1, SA1, CD1 or CL1) primed (shaded bars) or control primed with irrelevant peptide (unshaded bars) CD3 + T cells were restimulated with SARS-CoV-2-peptide-homologue-pulsed DCs for 6 hours and analysed by flow cytometry with background control values subtracted. NoP – no peptide negative control. *P < 0.05, **P < 0.01, ***P < 0.001 by Wilcoxon matched-pairs signed rank test. (A) CD8 + IL-2 + responses (n=8-12). (B) CD4 + IL-2 + responses (n=6-14). (C) CD8 + CD69 + responses (n=5-9). (D) CD4 + CD69 + responses (n=5-12). (E) CD8 + Perforin + responses (n=4-15). (F) Representative donor T cell IL-2, CD69 and perforin dot plots of responder control-primed (top) and bacterial peptide (bac)-primed (bottom).

Journal: Frontiers in Immunology

Article Title: Heterologous Immunity Between SARS-CoV-2 and Pathogenic Bacteria

doi: 10.3389/fimmu.2022.821595

Figure Lengend Snippet: | Bacterial peptide priming enhances IL-2, CD69 and Perforin T cell responses against SARS-CoV-2 in responders. Pathogenic bacterial-peptide (KP1, KP2, SE1, SA1, CD1 or CL1) primed (shaded bars) or control primed with irrelevant peptide (unshaded bars) CD3 + T cells were restimulated with SARS-CoV-2-peptide-homologue-pulsed DCs for 6 hours and analysed by flow cytometry with background control values subtracted. NoP – no peptide negative control. *P < 0.05, **P < 0.01, ***P < 0.001 by Wilcoxon matched-pairs signed rank test. (A) CD8 + IL-2 + responses (n=8-12). (B) CD4 + IL-2 + responses (n=6-14). (C) CD8 + CD69 + responses (n=5-9). (D) CD4 + CD69 + responses (n=5-12). (E) CD8 + Perforin + responses (n=4-15). (F) Representative donor T cell IL-2, CD69 and perforin dot plots of responder control-primed (top) and bacterial peptide (bac)-primed (bottom).

Article Snippet: SARS-CoV-2 recombinant proteins NSP3 and spike glycoprotein were purchased from MyBioSource.

Techniques: Flow Cytometry, Negative Control

Bacterial peptide priming increases T cell proliferation in responders. (A) CD8+ proliferation from restimulation is enhanced by pathogenic bacterial peptide priming ( n =1-10). (B) CD4+ proliferation from restimulation is enhanced by bacterial peptide priming ( n =5-10). Unshaded bars- Control-primed (irrelevant peptide, PVSKMRMATPLLMQA), then SARS-CoV-2-peptide restimulated. Shaded bars- bacterial peptide primed then SARS-CoV-2-peptide homologue restimulated. * P < 0.05, ** P < 0.01 by Wilcoxon-matched-pairs-signed rank test, comparing the magnitude of response to SARS-CoV-2 peptides with or without bacterial peptide priming after subtraction of the background control. (C) Representative CellTrace Yellow (CTY) vs. CellTrace Violet (CTV) dot plots of CD8+ and CD4+ cultured cells. Top right quadrant gate (CTY hi CTV hi cells) did not proliferate upon priming or restimulation. Top left quadrant gate (CTY lo CTV hi cells) proliferated upon priming but not with restimulation. Bottom left quadrant gate (CTY lo CTV lo cells) proliferated upon both priming and restimulation.

Journal: Frontiers in Immunology

Article Title: Heterologous Immunity Between SARS-CoV-2 and Pathogenic Bacteria

doi: 10.3389/fimmu.2022.821595

Figure Lengend Snippet: Bacterial peptide priming increases T cell proliferation in responders. (A) CD8+ proliferation from restimulation is enhanced by pathogenic bacterial peptide priming ( n =1-10). (B) CD4+ proliferation from restimulation is enhanced by bacterial peptide priming ( n =5-10). Unshaded bars- Control-primed (irrelevant peptide, PVSKMRMATPLLMQA), then SARS-CoV-2-peptide restimulated. Shaded bars- bacterial peptide primed then SARS-CoV-2-peptide homologue restimulated. * P < 0.05, ** P < 0.01 by Wilcoxon-matched-pairs-signed rank test, comparing the magnitude of response to SARS-CoV-2 peptides with or without bacterial peptide priming after subtraction of the background control. (C) Representative CellTrace Yellow (CTY) vs. CellTrace Violet (CTV) dot plots of CD8+ and CD4+ cultured cells. Top right quadrant gate (CTY hi CTV hi cells) did not proliferate upon priming or restimulation. Top left quadrant gate (CTY lo CTV hi cells) proliferated upon priming but not with restimulation. Bottom left quadrant gate (CTY lo CTV lo cells) proliferated upon both priming and restimulation.

Article Snippet: SARS-CoV-2 recombinant proteins NSP3 and spike glycoprotein were purchased from MyBioSource.

Techniques: Cell Culture

SARS-CoV-2 vaccination enhances T cell cytokine reactivity to pathogenic bacterial homologues. Direct ex-vivo T cell responses of donors (n=6) pre-SARS-CoV-2 vaccination (unshaded dots) and post-SARS-CoV-2 vaccination (shaded dots) to pathogenic bacterial peptides sharing homology with SARS-CoV-2 spike (SA1, SE1 and KP2) measured by intracellular cytokine staining. No Pep – no peptide negative control. *P < 0.05 by Wilcoxon matched-pairs signed rank test. (A) CD4 + IFN-γ + responses (n= 6). (B) CD4 + TNF + responses (n= 5-6). (C) CD4 + IL-2 + responses (n= 6). (D) CD8 + IFN-γ + responses (n= 6). (E) CD8 + TNF + responses (n= 6). (F) CD8 + IL-2 + responses (n= 6). (G) Representative donor T cell TNF (x-axis) and IFN-γ (y-axis) dot plots of pre-vaccinated CD4 + and CD8 + (top) and post-vaccinated CD4+ and CD8+ (bottom) responses to bacterial peptide SE1.

Journal: Frontiers in Immunology

Article Title: Heterologous Immunity Between SARS-CoV-2 and Pathogenic Bacteria

doi: 10.3389/fimmu.2022.821595

Figure Lengend Snippet: SARS-CoV-2 vaccination enhances T cell cytokine reactivity to pathogenic bacterial homologues. Direct ex-vivo T cell responses of donors (n=6) pre-SARS-CoV-2 vaccination (unshaded dots) and post-SARS-CoV-2 vaccination (shaded dots) to pathogenic bacterial peptides sharing homology with SARS-CoV-2 spike (SA1, SE1 and KP2) measured by intracellular cytokine staining. No Pep – no peptide negative control. *P < 0.05 by Wilcoxon matched-pairs signed rank test. (A) CD4 + IFN-γ + responses (n= 6). (B) CD4 + TNF + responses (n= 5-6). (C) CD4 + IL-2 + responses (n= 6). (D) CD8 + IFN-γ + responses (n= 6). (E) CD8 + TNF + responses (n= 6). (F) CD8 + IL-2 + responses (n= 6). (G) Representative donor T cell TNF (x-axis) and IFN-γ (y-axis) dot plots of pre-vaccinated CD4 + and CD8 + (top) and post-vaccinated CD4+ and CD8+ (bottom) responses to bacterial peptide SE1.

Article Snippet: SARS-CoV-2 recombinant proteins NSP3 and spike glycoprotein were purchased from MyBioSource.

Techniques: Ex Vivo, Staining, Negative Control

SARS-CoV-2 vaccination enhances T memory cell reactivity to pathogenic bacterial homologues by AIM and proliferation markers Direct ex-vivo T memory (Mem) cell responses of donors (n=6) pre-SARS-CoV-2 vaccination (unshaded dots) and post-SARS-CoV-2 vaccination (shaded dots) to pathogenic bacterial peptides sharing homology with SARS-CoV-2 spike (SA1, SE1 and KP2) measured by activation-induced markers (AIM) signifying antigen-specific responses (A–D) . 7-day proliferation assay of donors (n=6) pre-vaccination (unshaded bars) and post-vaccination (shaded bars) (E, F) . No Pep – no peptide negative control. MFI – mean fluorescence intensity. *P < 0.05 by Wilcoxon matched-pairs signed rank test. (A) AIM + CD4 + T memory cell responses measured by co-expression of CD134 and CD137 (n= 5-6). (B) AIM + CD4 + T memory responses measured by co-expression of CD154 and CD69 (n= 5-6). (C) AIM + CD8 + T memory cell responses measured by co-expression of CD69 and CD137 (n= 4-5). (D) AIM + CD8 + T memory cell responses measured by co-expression of CD154 and CD69 (n= 4-5). (E) CD4 + T cell proliferation in response to bacterial peptide homologues pre-and post SARS-CoV-2 vaccination (n= 6). (F) CD8 + T cell proliferation in response to bacterial peptide homologues pre-and post SARS-CoV-2 vaccination (n= 6).

Journal: Frontiers in Immunology

Article Title: Heterologous Immunity Between SARS-CoV-2 and Pathogenic Bacteria

doi: 10.3389/fimmu.2022.821595

Figure Lengend Snippet: SARS-CoV-2 vaccination enhances T memory cell reactivity to pathogenic bacterial homologues by AIM and proliferation markers Direct ex-vivo T memory (Mem) cell responses of donors (n=6) pre-SARS-CoV-2 vaccination (unshaded dots) and post-SARS-CoV-2 vaccination (shaded dots) to pathogenic bacterial peptides sharing homology with SARS-CoV-2 spike (SA1, SE1 and KP2) measured by activation-induced markers (AIM) signifying antigen-specific responses (A–D) . 7-day proliferation assay of donors (n=6) pre-vaccination (unshaded bars) and post-vaccination (shaded bars) (E, F) . No Pep – no peptide negative control. MFI – mean fluorescence intensity. *P < 0.05 by Wilcoxon matched-pairs signed rank test. (A) AIM + CD4 + T memory cell responses measured by co-expression of CD134 and CD137 (n= 5-6). (B) AIM + CD4 + T memory responses measured by co-expression of CD154 and CD69 (n= 5-6). (C) AIM + CD8 + T memory cell responses measured by co-expression of CD69 and CD137 (n= 4-5). (D) AIM + CD8 + T memory cell responses measured by co-expression of CD154 and CD69 (n= 4-5). (E) CD4 + T cell proliferation in response to bacterial peptide homologues pre-and post SARS-CoV-2 vaccination (n= 6). (F) CD8 + T cell proliferation in response to bacterial peptide homologues pre-and post SARS-CoV-2 vaccination (n= 6).

Article Snippet: SARS-CoV-2 recombinant proteins NSP3 and spike glycoprotein were purchased from MyBioSource.

Techniques: Ex Vivo, Activation Assay, Proliferation Assay, Negative Control, Fluorescence, Expressing

Journal: Frontiers in Immunology

Article Title: Heterologous Immunity Between SARS-CoV-2 and Pathogenic Bacteria

doi: 10.3389/fimmu.2022.821595

Article Snippet: SARS-CoV-2 recombinant proteins NSP3 and spike glycoprotein were purchased from MyBioSource.

Techniques: Sequencing, Bacteria

Properties of recombinant rRV/NSP3-2A-CoV2 strains.

Journal: Vaccines

Article Title: Rotavirus as an Expression Platform of Domains of the SARS-CoV-2 Spike Protein

doi: 10.3390/vaccines9050449

Figure Lengend Snippet: Properties of recombinant rRV/NSP3-2A-CoV2 strains.

Article Snippet: The RBD antibody recognized the fExRBD product of the rSA11/NSP3-fExRBD virus, but not the fRBD product of rSA11/NSP3-fRBD ( B), presumably because the latter product lacked the peptide sequence used in generating the ProSci RBD antibody.

Techniques: Recombinant

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