Journal: eLife

Article Title: Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

doi: 10.7554/eLife.90316

Figure Lengend Snippet: ( A ) Immunoblot analysis of lysates prepared from MRC-5-ACE2 human cells that were mock-infected or infected with SARS-CoV-2 at multiplicity of infection (MOI) of 5 for 24 or 48 hr. The immunoblot was probed with anti-TRMT1, actin, or SARS-CoV-2 nucleocapsid (N) antibodies. Circle represents endogenous full-length TRMT1. Asterisk (*) denotes a non-specific band. Size markers are noted in kiloDalton. ( B ) Quantification of TRMT1 signal intensity normalized to actin in the mock or SARS-CoV-2-infected cell lines. TRMT1 protein levels are expressed relative to mock-infected samples for each time point. ( C ) m2,2G levels in small RNAs isolated from MRC5 cells that were either mock-infected or infected with SARS-CoV-2 at MOI of 5 for 24 or 48 hr. m2,2G levels were normalized to A, C, G, and U. Samples were measured in biological replicates. Statistical significance for ( B ) and ( C ) was determined by two-way ANOVA with multiple comparisons test. ***p<0.001; ****p<0.0001; ns, non-significant. ( D ) Levels of the indicated RNA modifications in small RNAs isolated from MRC5 cells that were either mock-infected or infected with SARS-CoV-2 for 24 or 48 hr. RNA modification levels were normalized to A, C, G, and U. Y-axis represents the log2 fold change in the levels of the indicated tRNA modification between SARS-CoV-2 infected versus mock-infected MRC5 cells. The experiment in ( C ) was repeated as an independent biological replicate in . Figure 1—source data 1. Raw uncropped immunoblots for . Figure 1—source data 2. LC-MS measurements of RNA modifications.

Article Snippet: Membrane was blocked by Odyssey blocking buffer for 1 hrr at room temperature followed by immunoblotting with the following antibodies: mouse monoclonal anti-TRMT1 aa 201–229 (G3, sc-373687, Santa Cruz Biotechnologies), rabbit polyclonal anti-TRMT1 aa 609–659 (Bethyl, A304-205A); anti-Strep-tag II (NC9261069, Thermo Fisher), anti-FLAG epitope tag (L00018; F3165), Rabbit polyclonal anti-SARS-CoV-2 nucleoprotein N protein (40068-RP01; Sino Biological), and anti-actin (L00003; EMD Millipore).

Techniques: Western Blot, Infection, Isolation, RNA modification, Modification, Liquid Chromatography with Mass Spectroscopy

Journal: eLife

Article Title: Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

doi: 10.7554/eLife.90316

Figure Lengend Snippet: ( A ) Schematic of human TRMT1 primary structure with predicted Nsp5 cleavage site. Mitochondrial targeting signal (MTS), methyltransferase (MT) domain, and zinc finger motif are denoted. ( B ) Consensus sequence logo of cleavage sites in SARS-CoV-2 polyproteins. ( C ) Alpha-fold predicted structure of human TRMT1 with putative Nsp5 cleavage site denoted in magenta and arrowhead. ( D ) Immunoblot of input and strep-tactin purifications from human cells expressing empty vector, wild-type (WT) Nsp5, or Nsp5-C145A fused to the Strep-tag without or with co-expression with TRMT1-FLAG. The immunoblot was probed with anti-Strep, FLAG, and actin antibodies. Square represents TRMT1-FLAG, circle represents endogenous TRMT1. Size markers are noted to the left in kiloDalton. The experiment in ( D ) was repeated as an independent biological replicate in . Figure 2—source data 1. Raw uncropped immunoblots for .

Article Snippet: Membrane was blocked by Odyssey blocking buffer for 1 hrr at room temperature followed by immunoblotting with the following antibodies: mouse monoclonal anti-TRMT1 aa 201–229 (G3, sc-373687, Santa Cruz Biotechnologies), rabbit polyclonal anti-TRMT1 aa 609–659 (Bethyl, A304-205A); anti-Strep-tag II (NC9261069, Thermo Fisher), anti-FLAG epitope tag (L00018; F3165), Rabbit polyclonal anti-SARS-CoV-2 nucleoprotein N protein (40068-RP01; Sino Biological), and anti-actin (L00003; EMD Millipore).

Techniques: Sequencing, Western Blot, Expressing, Plasmid Preparation, Strep-tag

Journal: eLife

Article Title: Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

doi: 10.7554/eLife.90316

Figure Lengend Snippet: ( A ) Immunoblot analysis of lysates prepared from the indicated 293T cell lines expressing empty vector or ACE2. The immunoblot was probed with anti-ACE2 and actin. ( B ) Immunoblot analysis of lysates prepared from 293T-ACE2 cell lines that were mock-infected (multiplicity of infection, MOI 0) or infected with SARS-CoV-2 at the indicated MOI. The blot was probed against the SARS-CoV-2 nucleocapsid ( N ) and actin. Figure 6—figure supplement 1—source data 1. Raw uncropped immunoblots for .

Article Snippet: Membrane was blocked by Odyssey blocking buffer for 1 hrr at room temperature followed by immunoblotting with the following antibodies: mouse monoclonal anti-TRMT1 aa 201–229 (G3, sc-373687, Santa Cruz Biotechnologies), rabbit polyclonal anti-TRMT1 aa 609–659 (Bethyl, A304-205A); anti-Strep-tag II (NC9261069, Thermo Fisher), anti-FLAG epitope tag (L00018; F3165), Rabbit polyclonal anti-SARS-CoV-2 nucleoprotein N protein (40068-RP01; Sino Biological), and anti-actin (L00003; EMD Millipore).

Techniques: Western Blot, Expressing, Plasmid Preparation, Infection

Journal: eLife

Article Title: Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

doi: 10.7554/eLife.90316

Figure Lengend Snippet: ( A ) Immunoblot of lysates prepared from 293T control-wild-type (WT) or TRMT1-knockout (KO) cell lines that were mock-infected (multiplicity of infection, MOI of 0) or infected with SARS-CoV-2 at MOI of 0.2 or 0.4 for 24 hr. The immunoblot was probed with antibodies against TRMT1 or actin. Circle represents endogenous full-length TRMT1. Asterisk (*) denotes a non-specific band. Size markers to the right in kiloDalton. ( B ) Normalized TRMT1 signal intensity relative to mock-infected cells (MOI of 0). Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test. ( C ) Immunoblot of lysates prepared from 293T control-wild-type (WT) or TRMT1-KO cell lines that were mock-infected (MOI of 0) or infected with SARS-CoV-2 at MOI of 0.1 or 5.0 for 24 hr. The immunoblot was probed with antibodies as in ( A ). ( D ) Normalized TRMT1 signal intensity relative to mock-infected cells (MOI of 0). ( E, F ) SARS-CoV-2 RNA copy number in control-WT or TRMT1-KO human 293T cell lines after infection at the indicated MOI for 24 hr. Viral copy number was measured by QRT-PCR and normalized to GAPDH. Samples were measured in triplicate. Statistical significance was determined by two-way ANOVA with Šídák’s multiple comparisons test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, non-significant. Figure 6—source data 1. Raw uncropped immunoblots for . Figure 6—source data 2. QRT-PCR measurements of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA.

Article Snippet: Membrane was blocked by Odyssey blocking buffer for 1 hrr at room temperature followed by immunoblotting with the following antibodies: mouse monoclonal anti-TRMT1 aa 201–229 (G3, sc-373687, Santa Cruz Biotechnologies), rabbit polyclonal anti-TRMT1 aa 609–659 (Bethyl, A304-205A); anti-Strep-tag II (NC9261069, Thermo Fisher), anti-FLAG epitope tag (L00018; F3165), Rabbit polyclonal anti-SARS-CoV-2 nucleoprotein N protein (40068-RP01; Sino Biological), and anti-actin (L00003; EMD Millipore).

Techniques: Western Blot, Knock-Out, Infection, Quantitative RT-PCR

Journal: eLife

Article Title: Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

doi: 10.7554/eLife.90316

Figure Lengend Snippet: ( A ) Immunoblot of lysates prepared from the indicated 293T TRMT1-knockout (KO) cell lines that were mock-infected (multiplicity of infection, MOI of 0) or infected with SARS-CoV-2 for 24 hr. The immunoblot was probed with antibodies against TRMT1 or actin. Square represents full-length TRMT1-FLAG. Asterisk (*) denotes a non-specific band. Size markers to the left in kiloDalton. ( B ) Normalized TRMT1-WT signal intensity relative to mock-infected cells (MOI of 0). ( C ) Normalized TRMT1-Q530N signal intensity relative to mock-infected cells (MOI of 0). Statistical significance was determined in ( B ) and ( C ) by one-way ANOVA with Dunnett’s multiple comparisons test. ( D ) SARS-CoV-2 RNA copy number in control-wild-type (WT) or TRMT1-KO human 293T cell lines after infection at the indicated MOI. Viral copy number was measured by QRT-PCR and normalized to GAPDH. Statistical significance was determined by two-way ANOVA with Dunnett’s multiple comparisons test. *p<0.05; **p<0.01; ****p<0.0001; ns, non-significant. Figure 7—source data 1. Raw uncropped immunoblots for . Figure 7—source data 2. QRT-PCR measurements of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA for .

Article Snippet: Membrane was blocked by Odyssey blocking buffer for 1 hrr at room temperature followed by immunoblotting with the following antibodies: mouse monoclonal anti-TRMT1 aa 201–229 (G3, sc-373687, Santa Cruz Biotechnologies), rabbit polyclonal anti-TRMT1 aa 609–659 (Bethyl, A304-205A); anti-Strep-tag II (NC9261069, Thermo Fisher), anti-FLAG epitope tag (L00018; F3165), Rabbit polyclonal anti-SARS-CoV-2 nucleoprotein N protein (40068-RP01; Sino Biological), and anti-actin (L00003; EMD Millipore).

Techniques: Western Blot, Knock-Out, Infection, Quantitative RT-PCR

Journal: eLife

Article Title: Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

doi: 10.7554/eLife.90316

Figure Lengend Snippet: ( A ) Viral titer of supernatants collected from the indicated cell lines infected with SARS-CoV-2. Infectious titer was determined by TCID 50 endpoint dilution assay in VeroE6 cells and expressed in focus forming units per mL of supernatant (FFU/mL). ( B ) Infectivity of SARS-CoV-2 particles generated from cell lines in ( A ). Infectivity of viral particles was calculated with the formula [(FFU/mL)/(viral genomic RNA copies/mL)], and expressed in FFU per 100 genomic copies. Figure 8—source data 1. Infectious titers and QRT-PCR results for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections.

Article Snippet: Membrane was blocked by Odyssey blocking buffer for 1 hrr at room temperature followed by immunoblotting with the following antibodies: mouse monoclonal anti-TRMT1 aa 201–229 (G3, sc-373687, Santa Cruz Biotechnologies), rabbit polyclonal anti-TRMT1 aa 609–659 (Bethyl, A304-205A); anti-Strep-tag II (NC9261069, Thermo Fisher), anti-FLAG epitope tag (L00018; F3165), Rabbit polyclonal anti-SARS-CoV-2 nucleoprotein N protein (40068-RP01; Sino Biological), and anti-actin (L00003; EMD Millipore).

Techniques: Infection, Endpoint Dilution Assay, Generated, Quantitative RT-PCR

Journal: eLife

Article Title: Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

doi: 10.7554/eLife.90316

Figure Lengend Snippet:

Article Snippet: Membrane was blocked by Odyssey blocking buffer for 1 hrr at room temperature followed by immunoblotting with the following antibodies: mouse monoclonal anti-TRMT1 aa 201–229 (G3, sc-373687, Santa Cruz Biotechnologies), rabbit polyclonal anti-TRMT1 aa 609–659 (Bethyl, A304-205A); anti-Strep-tag II (NC9261069, Thermo Fisher), anti-FLAG epitope tag (L00018; F3165), Rabbit polyclonal anti-SARS-CoV-2 nucleoprotein N protein (40068-RP01; Sino Biological), and anti-actin (L00003; EMD Millipore).

Techniques: Virus, Software, Recombinant, Plasmid Preparation, Expressing, Variant Assay, Suspension, Protein Purification, Western Blot, Imaging, Sequencing

Journal: Vaccine

Article Title: Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models

doi: 10.1016/j.vaccine.2023.04.020

Figure Lengend Snippet: Humoral responses to single treatment with Ad5-RBD administered intranasally (i.n.) or intramuscularly (I.m.) in HLA-DQ6 vs. Balb/c mice. Groups of adult female mice (n = 3) were inoculated with Ad5-RBD at doses ranging from 10 5 -10 10 viral particles/mouse. Anti-spike IgG serum antibodies (a; area under the curve (AUC)), SARS-CoV-2 pseudovirus neutralization (b; calculated serum dilution producing 50% inhibition (ID 50 )), and anti-spike IgG and IgA bronchoalveolar lavage fluid antibodies (c, d; AUC) were measured after 25 days. For statistical comparisons, one-way ANOVA and Tukey’s test for multiple comparisons (a, c, d) or Kruskal-Wallis test and Dunn’s test for multiple comparisons (b) were used. Adjusted p-values are displayed as * <0.05, ** <0.01 and *** <0.001. To improve readability, adjusted p-values calculated in comparisons involving groups treated with doses 10 5 or 10 6 are not shown. Lines and bars represent geometric means and standard deviations. Dotted line represents the limit of detection of the assay.

Article Snippet: IHC was performed in an autostainer (Agilent) using a rabbit polyclonal anti-SARS-CoV nucleoprotein antibody (Rockland, 200–402-A50) and the horseradish peroxidase (HRP) method.

Techniques: Neutralization, Inhibition

Journal: Vaccine

Article Title: Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models

doi: 10.1016/j.vaccine.2023.04.020

Figure Lengend Snippet: Humoral responses to one vs. two treatments with Ad5-RBD administered intranasally (i.n.) or intramuscularly (i.m.) in HLA-DQ6 mice, and lack of effect of pre-treatment with Ad5 vector on priming with Ad5-RBD. Groups of adult male and female mice (n = 3) were inoculated either once or twice (14 days apart) with Ad5-RBD at doses 10 7 or 10 9 viral particles/mouse (a-e). Anti-spike IgG or IgA serum antibodies (a, b; area under the curve (AUC)), SARS-CoV-2 pseudovirus neutralization (c; calculated serum dilution producing 50% inhibition (ID 50 )), and anti-spike IgG and IgA bronchoalveolar lavage fluid antibodies (d, e; AUC) were measured after 28 days. In a different experiment, one group of HLA-DQ6 mice was pre-treated i.n. with Ad5-vector encoding β-galactosidase (Ad5-LacZ; 10 9 vp/mouse), while a second group remained untreated (n = 3; 3f). After 21 days, a priming dose of Ad5-RBD i.n. was administered to both groups (10 9 vp/mouse). Serum was collected after 42 days, and anti-spike IgG or IgA serum antibodies were measured (f; AUC). For statistical comparisons, one-way ANOVA and Tukey’s test for multiple comparisons (a, b, d, e, f) or Kruskal-Wallis test and Dunn’s test for multiple comparisons (c) were used. Adjusted p-values are displayed as * <0.05, ** <0.01 and *** <0.001. Lines and bars represent geometric means and standard deviations. Dotted line represents the limit of detection of the assay.

Article Snippet: IHC was performed in an autostainer (Agilent) using a rabbit polyclonal anti-SARS-CoV nucleoprotein antibody (Rockland, 200–402-A50) and the horseradish peroxidase (HRP) method.

Techniques: Plasmid Preparation, Neutralization, Inhibition

Journal: Vaccine

Article Title: Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models

doi: 10.1016/j.vaccine.2023.04.020

Figure Lengend Snippet: Cellular responses to treatment with Ad5-RBD administered intranasally (i.n.) or intramuscularly (i.m.) in HLA-DQ6 mice. Groups of female HLA-DQ6 mice (n = 3) were inoculated once with Ad5-RBD at doses ranging from 10 5 -10 10 viral particles/mouse (a, b). Groups of male or female HLA-DQ6 mice were inoculated once or twice (14 days apart) with Ad5-RBD at doses 10 7 or 10 9 viral particles/mouse (c). After 25–28 days, spleen cells were restimulated in vitro with recombinant SARS-CoV-2 spike protein receptor binding domain (RBD). Supernatants were harvested after 3 days, and the effector proteins IFNg and granzyme B were measured by ELISA. Results expressed as concentrations (pg/ml). Statistical comparisons were performed using Kruskal-Wallis test and Dunn’s test for multiple comparisons. Group differences were non-significant (adjusted p-values > 0.05).

Article Snippet: IHC was performed in an autostainer (Agilent) using a rabbit polyclonal anti-SARS-CoV nucleoprotein antibody (Rockland, 200–402-A50) and the horseradish peroxidase (HRP) method.

Techniques: In Vitro, Recombinant, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Vaccine

Article Title: Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models

doi: 10.1016/j.vaccine.2023.04.020

Figure Lengend Snippet: Transcriptomic profiles of RBD-stimulated spleen cells from mice treated with Ad5-RBD administered intranasally (i.n.) or intramuscularly (i.m.). Groups of female HLA-DQ6 mice (n = 3) were inoculated once with Ad5-RBD at doses ranging from 10 5 -10 10 viral particles/mouse. After 25 days, spleen cells were restimulated in vitro with recombinant SARS-CoV-2 spike protein receptor binding domain (RBD). Cells were harvested after 3 days of culture, and bulk RNA was isolated. Heatmaps depicting the up- (a; red color, 86 genes) or down-regulation (b; blue color, 62 genes) of altogether 148 genes differentially expressed after treatment with Ad5-RBD in at least one of two separate comparisons, and are ordered to show the genes with the highest log2 fold changes from the top [Ad5-RBD at doses 10 5 /10 6 viral particles i.n. (ineffective treatment) vs. doses 10 7 /10 8 viral particles i.m. or doses 10 9 /10 10 viral particles i.n. (n = 6; adjusted P value, P ≤ 0.001; RNA sequencing)]. Statistical analyses were performed using edgeR. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: IHC was performed in an autostainer (Agilent) using a rabbit polyclonal anti-SARS-CoV nucleoprotein antibody (Rockland, 200–402-A50) and the horseradish peroxidase (HRP) method.

Techniques: In Vitro, Recombinant, Binding Assay, Isolation, RNA Sequencing

Journal: Vaccine

Article Title: Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models

doi: 10.1016/j.vaccine.2023.04.020

Figure Lengend Snippet: Upregulation of selected immune genes, differentially expressed in RBD-stimulated spleen cells from mice treated with Ad5-RBD administered intranasally (i.n.) or intramuscularly (i.m.). Groups of female HLA-DQ6 mice (n = 3) were inoculated once with Ad5-RBD at doses ranging from 10 5 -10 10 viral particles/mouse. After 25 days, spleen cells were restimulated in vitro with recombinant SARS-CoV-2 spike protein receptor binding domain (RBD). Cells were harvested after 3 days of culture, and bulk RNA was isolated for transcriptomic analyses (RNA sequencing; compare ). Panels depict the results for 9 selected immune genes [T helper 1/cytotoxic T cell markers: Interferon gamma (Ifng), perforin 1 (Prf1), granzyme B (Gzmb); Interferon gamma-related genes: Interferon regulatory factor 1 (Irf1), signal transducer and activator of transcription 1 (Stat1), chemokine (C-X-C motif) ligand 9 (CXCL9); Lymphocyte activation-related genes: Interleukin 2-receptor alpha (Il2ra), chemokine (C motif) ligand (XCL1), thymocyte differentiation antigen 1 (Thy1)]. Results expressed as read counts.

Article Snippet: IHC was performed in an autostainer (Agilent) using a rabbit polyclonal anti-SARS-CoV nucleoprotein antibody (Rockland, 200–402-A50) and the horseradish peroxidase (HRP) method.

Techniques: In Vitro, Recombinant, Binding Assay, Isolation, RNA Sequencing, Activation Assay

Journal: Vaccine

Article Title: Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models

doi: 10.1016/j.vaccine.2023.04.020

Figure Lengend Snippet: Humoral responses to a single treatment with either Ad5-RBD, Ad5-S or ChAdOx1 nCoV-19 (AstraZeneca, “AZ”) administered intranasally in C57BL/6 mice. Groups of adult male or female mice (n = 3) were inoculated with 3 different vaccines, at doses ranging from 10 7 -10 9 viral particles/mouse, as indicated. Anti-spike IgG serum antibodies (a; area under the curve (AUC)), anti-spike IgA serum antibodies (b; AUC) and SARS-CoV-2 pseudovirus neutralization (c; calculated serum dilution producing 50% inhibition (ID 50 )) were measured after 25 days. For statistical comparisons, one-way ANOVA and Tukey’s test for multiple comparisons (a, b) or Kruskal-Wallis test and Dunn’s test for multiple comparisons (c) were used. Adjusted p-values are displayed as * <0.05, ** <0.01 and *** <0.001. Lines and bars represent geometric means and standard deviations. Dotted line represents the limit of detection of the assay.

Article Snippet: IHC was performed in an autostainer (Agilent) using a rabbit polyclonal anti-SARS-CoV nucleoprotein antibody (Rockland, 200–402-A50) and the horseradish peroxidase (HRP) method.

Techniques: Vaccines, Neutralization, Inhibition

Journal: Vaccine

Article Title: Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models

doi: 10.1016/j.vaccine.2023.04.020

Figure Lengend Snippet: Humoral responses to single treatment with Ad5-RBD or Ad5-S administered intranasally in HLA-DQ6 vs. HLA-DQ8 mice, and responses to Ad5-S administered intranasally after priming with mRNA vaccine Comirnaty (Pfizer-BioNTech, “COM”) intramuscularly. Groups of adult female HLA-DQ6 or -DQ8 mice (n = 3) were inoculated with 10 7 viral particles (vp)/mouse of either Ad5-RBD or Ad5-S (a, b), as indicated. Anti-spike IgG serum antibodies (a; AUC) and SARS-CoV-2 pseudovirus neutralization (b; pooled sera, dots representing the values of triplicates; calculated serum dilution producing 50% inhibition (ID 50 )) were measured after three weeks. Groups of adult male and female HLA-DQ8 mice (n = 4) were inoculated either with Comirnaty (2 µg/dose, i.m.), Ad5-S (10 9 vp/dose, i.n.) or were not pre-treated (c-g). Three weeks later, they received either Comirnaty i.m. or Ad5-S i.n. at the same doses, as indicated. The Ad5-S vector used in this experiment encoded for the beta variant strain of SARS-CoV-2, containing three major receptor-binding domain mutations. Anti-spike IgG and IgA serum or bronchoalveolar lavage (BAL) antibodies (c, d, f, g; AUC) and pseudovirus neutralization (e; individual sera; ID 50 ) were measured after six weeks. For statistical comparisons, one-way ANOVA and Tukey’s test for multiple comparisons (a, c, d, f, g) or Kruskal-Wallis test and Dunn’s test for multiple comparisons (b, e) were used. Adjusted p-values are displayed as * <0.05, ** <0.01 and *** <0.001. Lines and bars represent geometric means and standard deviations. Dotted line represents the limit of detection of the assay.

Article Snippet: IHC was performed in an autostainer (Agilent) using a rabbit polyclonal anti-SARS-CoV nucleoprotein antibody (Rockland, 200–402-A50) and the horseradish peroxidase (HRP) method.

Techniques: Neutralization, Inhibition, Plasmid Preparation, Variant Assay, Binding Assay

Journal: Vaccine

Article Title: Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models

doi: 10.1016/j.vaccine.2023.04.020

Figure Lengend Snippet: Infectious challenge of Balb/c mice with SARS-CoV-2 after single treatment with Ad5-RBD or Ad5-S administered intranasally (I). Groups of adult female mice (n = 3) were inoculated with 10 9 viral particles/mouse of either Ad5-RBD or Ad5-S, or received no pre-treatment (n = 6). Anti-spike IgG or IgA serum antibodies (a; AUC) and SARS-CoV-2 pseudovirus neutralization (b; pooled sera, dots representing the values of triplicates; calculated serum dilution producing 50% inhibition (ID 50 )) were measured after three weeks. Lines and bars represent geometric means and standard deviations. After 5 weeks, all mice were inoculated intranasally with 2 × 10 5 plaque-forming units of the beta variant of SARS-CoV-2. Three days later, viral RNA transcripts for (genomic) RNA-dependent RNA polymerase or (subgenomic) E were quantified in tissue of the right lung ( c ; RNA copies per ng of total lung RNA, based on SARS-CoV-2 genomic RNA standard included in the RT-qPCR run).

Article Snippet: IHC was performed in an autostainer (Agilent) using a rabbit polyclonal anti-SARS-CoV nucleoprotein antibody (Rockland, 200–402-A50) and the horseradish peroxidase (HRP) method.

Techniques: Neutralization, Inhibition, Variant Assay, Quantitative RT-PCR

Journal: Vaccine

Article Title: Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models

doi: 10.1016/j.vaccine.2023.04.020

Figure Lengend Snippet: Infectious challenge of Balb/c mice with SARS-CoV-2 after single treatment with Ad5-RBD or Ad5-S administered intranasally (II). The left lung was subjected to histological (H/E; a-c ) and immunohistochemical (SARS-CoV-2 nucleoprotein, hematoxylin counterstain; d-f ) examination on day 3 post intranasal infection with 2 × 10 5 plaque-forming units of the beta variant of SARS-CoV-2. Unvaccinated animal no. 3 ( a, d ): There is extensive SARS-CoV-2 nucleoprotein expression in bronchial and bronchiolar epithelial cells and, focally (arrow heads) in the parenchyma. Ad5-RBD vaccinated mouse no. 1 ( b, e ): The lung is widely unaltered and there is no evidence of viral antigen expression. Ad5-S vaccinated animal no. 1 ( c, f ): The lung is widely unaltered and there is no evidence of viral antigen expression. Bars = 1 mm.

Article Snippet: IHC was performed in an autostainer (Agilent) using a rabbit polyclonal anti-SARS-CoV nucleoprotein antibody (Rockland, 200–402-A50) and the horseradish peroxidase (HRP) method.

Techniques: Immunohistochemical staining, Infection, Variant Assay, Expressing