rabbit anti sars cov 2 membrane glycoprotein polyclonal antibodies Search Results


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  • 93
    Rockland Immunochemicals rabbit polyclonal anti sars cov nucleocapsid antibody
    a Genome maps of recombinant VSV vectors. The original VSV contains five transcription units encoding the N, P, M, G, and L genes. The VSV vector was modified by replacing the G gene with a <t>modified</t> <t>SARS-CoV-2</t> spike gene and by inserting an additional transcription unit encoding GFP. M q denotes a quadruple mutant M gene encoding an M protein that lacks host shut-off activity. G RBD denotes a synthetic membrane protein presenting the receptor-binding domain (RBD) of <t>the</t> <t>SARS-CoV-2</t> spike protein. b Virus yield on Vero E6 and BHK-G43 cells. Vero E6 cells, BHK-G43 cells expressing the VSV glycoprotein (+), and BHK-G43 cells lacking VSV glycoprotein expression (−) were infected with either VSV*M q ΔG-S Δ21 (S Δ21 ), VSV*M q ΔG-S Δ21 (M q -S Δ21 ), or VSV*ΔG-G RBD (G RBD ) using 0.1 ffu/cell. At the 24 hours pi, infectious virus released into the cell culture was titrated on Vero E6 cells. Mean values and standard deviations of 3 infection experiments are shown. c Western blot analysis of recombinant VSV vector particles. VSV*ΔG-S Δ21 (S Δ21 ) and VSV*M q ΔG-S Δ21 (M q -S Δ21 ) were propagated on both Vero E6 and BHK-G43 cells, while VSV*ΔG-G RBD (G RBD ) were propagated only on BHK-G43 helper cells. At 24 hours pi, the virus particles were concentrated from the cell culture supernatant by ultracentrifugation and dissolved in SDS sample buffer. The viral proteins were separated by SDS-PAGE under non-reducing conditions and blotted onto nitrocellulose membrane. Antigens were detected with a COVID-19 convalescent serum (α-S) and a rabbit <t>polyclonal</t> immune serum directed to the VSV G and M proteins (α-G/M). The blots were derived from the same experiment and were not further processed. d Inhibition of virus entry using neutralizing antibodies directed to either the VSV G protein or the SARS-CoV-2 spike protein. Vero E6 cells were inoculated in the absence or presence of the indicated neutralizing antibodies with VSV*M q ΔG-S Δ21 produced on either Vero E6 or BHK-G43 cells and with VSV*ΔG-G RBD grown on BHK-G43 cells. Infected cells were detected 24 h pi taking advantage of the GFP reporter. Bar = 100 µm.
    Rabbit Polyclonal Anti Sars Cov Nucleocapsid Antibody, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti sars cov nucleocapsid antibody/product/Rockland Immunochemicals
    Average 93 stars, based on 1 article reviews
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    86
    Abcam rabbit anti sars cov 2 spike glycoprotein antibody
    Immunohistochemical/immunofluorescence expression of <t>Sars-Cov-2</t> antigens in relation to histopathological changes in thyroid gland obtained from patient who deceased from COVID-19. Sars-Cov-2 spike (S) protein was found notably expressed throughout the thyroid tissue and was localized diffusely in the cytoplasm of follicular cells (A, B, C, D, E, F and J, K, L, M, N, O). Concomitantly, the thyroid gland harboured copious mononuclear infiltrate (A, B, C, D, E, F, G and H) forming granuloma-like structures (B, C, D, E and F) with giant multinuclear cells (arrows on B, D, E, F), epithelial desquamation, colloid depletion and extensive follicular disruption (A, B, C, D, E, F, G, H and I, corresponding to findings consistent with subacute thyroiditis. Spike protein positivity was also found in epithelioid cells within the granulomatous tissue (F). Sars-Cov-2 nucleocapsid protein immunopositivity was noticeably sparser, arranged in punctiform inclusions and localized predominantly in the perinuclear region of follicular cells (arrows on G and H; J, K, L, M, N, O). The majority of remaining follicular cells showed positivity for cleaved caspase-3 (I). Staining was performed using rabbit anti-SARS-CoV-2 spike glycoprotein antibody (Abcam, <t>ab272504;</t> dilution 1:4000), mouse anti-SARS-CoV-2 nucleocapsid protein antibody (Cell Signaling Technology, clone E8R1L; dilution 1:200) and rabbit anti-caspase-3 antibody (Abcam, ab13847; diluted 1:50). Immunoreactions were visualized by DAKO EnVision+System (DAKO Cytomation) for immunohistochemistry or by Alexa Fluor 488 donkey anti-rabbit IgG (Thermo Fisher Scientific; diluted 1:300) and Alexa Fluor 555 goat anti-mouse IgG (Thermo Fisher Scientific; diluted 1:500) secondary antibodies for immunofluorescence studies. Magnifications: A × 100; B, C, D, E, I × 400; F, J, K, L × 600; G, H, M, N, O × 1000.
    Rabbit Anti Sars Cov 2 Spike Glycoprotein Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    86
    Jackson Immuno rabbit anti sars cov 2 spike glycoprotein antibody
    (a) Each panel presents the protein expressed by the DNA (black outline) for the gp96-Ig and <t>SARS-CoV-2</t> protein S vaccine antigen. Gp96-Ig and SARS-CoV-2-S DNA were cloned into the mammalian expression vectors B45 and pcDNA 3.1, which are transfected into HEK-293 and AD100. Stable transfection vaccine cell clones (1A, 1A6, 1D6) were generated after selection with L-Histidinol and Neomycin; (b) One million 293-gp96-Ig-S and AD-100-gp96-Ig-S (1D6) cells were plated in 1 mL for 24 hours and gp96-Ig production in the supernatant was determined by ELISA using antihuman IgG antibody for detection with mouse IgG1 (0.5 ug/mL) as a standard; (c) Cell lysates were analyzed under reduced conditions by SDS-PAGE and Western blotting using anti protein S antibody and recombinant protein S1 as a positive control; (d) IF for protein S (in green) expressed in AD100-gp96-Ig-S cells using rabbit anti-SARS-CoV-2 S antibody and antirabbit Ig-AF488 as secondary antibody. AD100 was used as a negative control and β-actin for protein quantification. Original magnification 40× with DAPI nuclear staining shown in blue. DNA, deoxyribonucleic acid; ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; N, amino terminus; C, carboxy terminus; IF, immunofluorescence; TM, transmembrane domain; KDEL, retention signal; CH2 CH3 gamma 1, heavy chain of IgG1. See text for explanation.
    Rabbit Anti Sars Cov 2 Spike Glycoprotein Antibody, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    86
    Abcam rabbit polyclonal anti sars cov 2 spike glycoprotein antibody
    Cobicistat is a candidate inhibitor of <t>SARS-CoV-2</t> replication. (A and B) In silico docking analysis of the putative mode and energy of binding of cobicistat to SARS-CoV-2 3CL pro . (A) Docking pose showing the ligand interaction of cobicistat to the active site of 3CL pro and the formation of hydrogen bonds to Asn142, Gly143, and Gln189 of 3CL pro . (B) Overlay of crystal structures of SARS-Cov-2 3CL pro showing the amino acids important for the binding of cobicistat to the active site of the enzyme. Residues of the catalytic dyad (Cys145 and His41) of 3CL pro were among the highest contributors to noncovalent binding to cobicistat. The source and list of structures used are detailed in Materials and Methods. (C) Schematic representation of time course experiments evaluating in vitro inhibition of SARS-CoV-2 replication by cobicistat (created with BioRender). (D and E) Effect of various concentrations of cobicistat, added according to the scheme of panel C, on intracellular and supernatant SARS-CoV-2 RNA content in Calu-3 cells. Viral RNA content was measured by qPCR using the 2019-nCoV_N1 primer set (Centers for Disease Control and Prevention). Fold change values in intracellular RNA (D) were calculated by the delta-delta C T method , using the Tata-binding protein (TBP) gene as housekeeper control. Expression levels in supernatant (E) were quantified using an in vitro -transcribed standard curve generated as described in Materials and Methods. Data are expressed as mean with standard deviation (SD) and were analyzed by two-way ANOVA followed by Dunnett’s posttest ( n = 3 independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
    Rabbit Polyclonal Anti Sars Cov 2 Spike Glycoprotein Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti sars cov 2 spike glycoprotein antibody/product/Abcam
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    rabbit polyclonal anti sars cov 2 spike glycoprotein antibody - by Bioz Stars, 2024-02
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    86
    Abcam anti sars cov 2 spike glycoprotein rabbit polyclonal antibody
    ( A-E ) The tracks of transient speed of VLP particles during entry from extracellular into intracellular cytoplasm upon untreated (Ctrl) (A), NP-G2-044 (50 μM for 2 h) (B), SIMFH2 (15 μM for 1 h) (C), CK666 (100 μM for 2 h) (D), Blebbistatin (17 μM for 1 h) (E), respectively. The original point on the X axis represents the viral entry time point. The negative number on the X axis represents the time before the virus enters the cell. The positive number on the X axis represents the time after the virus enters the cell. ( F ) Quantification of the mean velocities of VLP prior entering the untreated and inhibitors-treated cells. ( G ) Quantification of the duration of VLP particles enter into the untreated and inhibitors-treated cells. ( H ) Schematic diagram of drug treatment and authentic <t>SARS-CoV-2</t> infection. ( I ) Viral entry efficiency of SARS-CoV-2 in BSL3. VeroE6-ACE2 cells were pretreated with 50 μM ML141 or 50 μM NP-G2-044 for 5 h, and challenged with SARS-CoV-2 (MOI=1) for 45 min. Viral RNA levels of internalized virus were measured by qRT-PCR. The data are normalized to the mean of DMSO-treated cells and represent as means ± SEM from three independent experiments. ns (no significant difference), P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001 (One-way ANOVA test).
    Anti Sars Cov 2 Spike Glycoprotein Rabbit Polyclonal Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sars cov 2 spike glycoprotein rabbit polyclonal antibody/product/Abcam
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti sars cov 2 spike glycoprotein rabbit polyclonal antibody - by Bioz Stars, 2024-02
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    a Genome maps of recombinant VSV vectors. The original VSV contains five transcription units encoding the N, P, M, G, and L genes. The VSV vector was modified by replacing the G gene with a modified SARS-CoV-2 spike gene and by inserting an additional transcription unit encoding GFP. M q denotes a quadruple mutant M gene encoding an M protein that lacks host shut-off activity. G RBD denotes a synthetic membrane protein presenting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. b Virus yield on Vero E6 and BHK-G43 cells. Vero E6 cells, BHK-G43 cells expressing the VSV glycoprotein (+), and BHK-G43 cells lacking VSV glycoprotein expression (−) were infected with either VSV*M q ΔG-S Δ21 (S Δ21 ), VSV*M q ΔG-S Δ21 (M q -S Δ21 ), or VSV*ΔG-G RBD (G RBD ) using 0.1 ffu/cell. At the 24 hours pi, infectious virus released into the cell culture was titrated on Vero E6 cells. Mean values and standard deviations of 3 infection experiments are shown. c Western blot analysis of recombinant VSV vector particles. VSV*ΔG-S Δ21 (S Δ21 ) and VSV*M q ΔG-S Δ21 (M q -S Δ21 ) were propagated on both Vero E6 and BHK-G43 cells, while VSV*ΔG-G RBD (G RBD ) were propagated only on BHK-G43 helper cells. At 24 hours pi, the virus particles were concentrated from the cell culture supernatant by ultracentrifugation and dissolved in SDS sample buffer. The viral proteins were separated by SDS-PAGE under non-reducing conditions and blotted onto nitrocellulose membrane. Antigens were detected with a COVID-19 convalescent serum (α-S) and a rabbit polyclonal immune serum directed to the VSV G and M proteins (α-G/M). The blots were derived from the same experiment and were not further processed. d Inhibition of virus entry using neutralizing antibodies directed to either the VSV G protein or the SARS-CoV-2 spike protein. Vero E6 cells were inoculated in the absence or presence of the indicated neutralizing antibodies with VSV*M q ΔG-S Δ21 produced on either Vero E6 or BHK-G43 cells and with VSV*ΔG-G RBD grown on BHK-G43 cells. Infected cells were detected 24 h pi taking advantage of the GFP reporter. Bar = 100 µm.

    Journal: NPJ Vaccines

    Article Title: Optimized intramuscular immunization with VSV-vectored spike protein triggers a superior immune response to SARS-CoV-2

    doi: 10.1038/s41541-022-00508-7

    Figure Lengend Snippet: a Genome maps of recombinant VSV vectors. The original VSV contains five transcription units encoding the N, P, M, G, and L genes. The VSV vector was modified by replacing the G gene with a modified SARS-CoV-2 spike gene and by inserting an additional transcription unit encoding GFP. M q denotes a quadruple mutant M gene encoding an M protein that lacks host shut-off activity. G RBD denotes a synthetic membrane protein presenting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. b Virus yield on Vero E6 and BHK-G43 cells. Vero E6 cells, BHK-G43 cells expressing the VSV glycoprotein (+), and BHK-G43 cells lacking VSV glycoprotein expression (−) were infected with either VSV*M q ΔG-S Δ21 (S Δ21 ), VSV*M q ΔG-S Δ21 (M q -S Δ21 ), or VSV*ΔG-G RBD (G RBD ) using 0.1 ffu/cell. At the 24 hours pi, infectious virus released into the cell culture was titrated on Vero E6 cells. Mean values and standard deviations of 3 infection experiments are shown. c Western blot analysis of recombinant VSV vector particles. VSV*ΔG-S Δ21 (S Δ21 ) and VSV*M q ΔG-S Δ21 (M q -S Δ21 ) were propagated on both Vero E6 and BHK-G43 cells, while VSV*ΔG-G RBD (G RBD ) were propagated only on BHK-G43 helper cells. At 24 hours pi, the virus particles were concentrated from the cell culture supernatant by ultracentrifugation and dissolved in SDS sample buffer. The viral proteins were separated by SDS-PAGE under non-reducing conditions and blotted onto nitrocellulose membrane. Antigens were detected with a COVID-19 convalescent serum (α-S) and a rabbit polyclonal immune serum directed to the VSV G and M proteins (α-G/M). The blots were derived from the same experiment and were not further processed. d Inhibition of virus entry using neutralizing antibodies directed to either the VSV G protein or the SARS-CoV-2 spike protein. Vero E6 cells were inoculated in the absence or presence of the indicated neutralizing antibodies with VSV*M q ΔG-S Δ21 produced on either Vero E6 or BHK-G43 cells and with VSV*ΔG-G RBD grown on BHK-G43 cells. Infected cells were detected 24 h pi taking advantage of the GFP reporter. Bar = 100 µm.

    Article Snippet: A 1:3000 dilution of a rabbit polyclonal anti-SARS-CoV nucleocapsid antibody (Rockland, Cat. no. 200-401-A50) was used for SARS-CoV-2 immunohistochemical (IHC) analysis of the lung and brain.

    Techniques: Recombinant, Plasmid Preparation, Modification, Mutagenesis, Activity Assay, Binding Assay, Expressing, Infection, Cell Culture, Western Blot, SDS Page, Derivative Assay, Inhibition, Produced

    K18-hACE2 mice were immunized (i.m.) with either 10 4 or 10 5 focus-forming units (ffu) of the VSV*ΔG-S Δ21 vector vaccine which was produced on either Vero E6 or BHK-G43 cells. a Detection of spike-specific serum antibodies by ELISA 3 weeks after the first (prime) and 3 weeks after the second (boost) immunization. b Determination of the virus neutralization dose 50% (ND 50 ) in serum of immunized mice. c Determination of body weight of immunized mice following nasal infection with 10 5 pfu of SARS-CoV-2-S D614G . d , e Determination of virus load by RT-qPCR ( d ) in oropharyngeal swab samples collected at days 2 and 5 pi and ( e ) in the indicated organs prepared from the euthanized animals at day 5 pi. Mean values and standard deviations for n = 5 mice per group are shown. Statistical analysis was performed using the two-way ANOVA with either Tukey’s or Sidak’s multiple comparison test (* p < 0.05; ** p < 0.005; *** p < 0.0005; **** p < 0.0001).

    Journal: NPJ Vaccines

    Article Title: Optimized intramuscular immunization with VSV-vectored spike protein triggers a superior immune response to SARS-CoV-2

    doi: 10.1038/s41541-022-00508-7

    Figure Lengend Snippet: K18-hACE2 mice were immunized (i.m.) with either 10 4 or 10 5 focus-forming units (ffu) of the VSV*ΔG-S Δ21 vector vaccine which was produced on either Vero E6 or BHK-G43 cells. a Detection of spike-specific serum antibodies by ELISA 3 weeks after the first (prime) and 3 weeks after the second (boost) immunization. b Determination of the virus neutralization dose 50% (ND 50 ) in serum of immunized mice. c Determination of body weight of immunized mice following nasal infection with 10 5 pfu of SARS-CoV-2-S D614G . d , e Determination of virus load by RT-qPCR ( d ) in oropharyngeal swab samples collected at days 2 and 5 pi and ( e ) in the indicated organs prepared from the euthanized animals at day 5 pi. Mean values and standard deviations for n = 5 mice per group are shown. Statistical analysis was performed using the two-way ANOVA with either Tukey’s or Sidak’s multiple comparison test (* p < 0.05; ** p < 0.005; *** p < 0.0005; **** p < 0.0001).

    Article Snippet: A 1:3000 dilution of a rabbit polyclonal anti-SARS-CoV nucleocapsid antibody (Rockland, Cat. no. 200-401-A50) was used for SARS-CoV-2 immunohistochemical (IHC) analysis of the lung and brain.

    Techniques: Plasmid Preparation, Produced, Enzyme-linked Immunosorbent Assay, Neutralization, Infection, Quantitative RT-PCR

    K18-hACE2 mice were immunized twice (i.m.) with one of the following vaccines: VSV*ΔG-S Δ21 ( n = 10), VSV*M q ΔG-S Δ21 (n = 10), VSV*ΔG-G RBD ( n = 10), and the VSV*ΔG control vaccine ( n = 5). Five animals of each vaccine group were euthanized at day 5 pi, while the others were maintained until day 14 pi. a Determination of neutralizing serum antibody titers (ND 50 ) 21 days after the primary immunization (prime) and 18 days after the secondary immunization (boost). b , c Weight loss ( b ) and survival ( c ) of K18-hACE2 mice (d14 group) following nasal infection with 10 5 pfu of SARS-CoV-2-S D614G . d Quantification (RT-qPCR) of SARS-CoV-2 RNA copies in oropharyngeal swab samples collected at 2 and 4 days pi. e Determination of neutralizing serum antibody titers (ND 50 ) 18 days after the boost and 14 days postchallenge infection. f , g RT-qPCR determination of viral genome copy numbers in lung ( f ) or brain ( g ) homogenates prepared from animals that were euthanized at either day 5 or day 14 pi. Mean values and standard deviations (SD) are shown. Statistically significant differences as computed by either the two-way ANOVA test ( a , d , e ) or the one-way ANOVA test ( f , g ) are indicated (* p < 0.05; ** p < 0.005; *** p < 0.0005; **** p < 0.0001).

    Journal: NPJ Vaccines

    Article Title: Optimized intramuscular immunization with VSV-vectored spike protein triggers a superior immune response to SARS-CoV-2

    doi: 10.1038/s41541-022-00508-7

    Figure Lengend Snippet: K18-hACE2 mice were immunized twice (i.m.) with one of the following vaccines: VSV*ΔG-S Δ21 ( n = 10), VSV*M q ΔG-S Δ21 (n = 10), VSV*ΔG-G RBD ( n = 10), and the VSV*ΔG control vaccine ( n = 5). Five animals of each vaccine group were euthanized at day 5 pi, while the others were maintained until day 14 pi. a Determination of neutralizing serum antibody titers (ND 50 ) 21 days after the primary immunization (prime) and 18 days after the secondary immunization (boost). b , c Weight loss ( b ) and survival ( c ) of K18-hACE2 mice (d14 group) following nasal infection with 10 5 pfu of SARS-CoV-2-S D614G . d Quantification (RT-qPCR) of SARS-CoV-2 RNA copies in oropharyngeal swab samples collected at 2 and 4 days pi. e Determination of neutralizing serum antibody titers (ND 50 ) 18 days after the boost and 14 days postchallenge infection. f , g RT-qPCR determination of viral genome copy numbers in lung ( f ) or brain ( g ) homogenates prepared from animals that were euthanized at either day 5 or day 14 pi. Mean values and standard deviations (SD) are shown. Statistically significant differences as computed by either the two-way ANOVA test ( a , d , e ) or the one-way ANOVA test ( f , g ) are indicated (* p < 0.05; ** p < 0.005; *** p < 0.0005; **** p < 0.0001).

    Article Snippet: A 1:3000 dilution of a rabbit polyclonal anti-SARS-CoV nucleocapsid antibody (Rockland, Cat. no. 200-401-A50) was used for SARS-CoV-2 immunohistochemical (IHC) analysis of the lung and brain.

    Techniques: Infection, Quantitative RT-PCR

    K18-hACE2 mice (group size n = 10) were immunized with the indicated vaccines and challenged with 10 5 pfu of SARS-CoV-2-S D614G via the nasal route. Five animal groups of each vaccine group were euthanized at day 5 pi, while the remaining animals of each group were sacrificed at day 14 pi. a Lung and brain tissue sections were analyzed by immunohistochemistry (IHC) using a rabbit polyclonal antibody directed to the SARS-CoV nucleoprotein antigen. Lung and brain sections prepared from mock-infected non-vaccinated animals served as control. Arrows indicate lung and brain areas where the viral nucleoprotein antigen was detected. In parallel, lung and brain tissue sections were stained by hematoxylin-eosin (HE). Arrow heads indicate perivascular/peribronchiolar infiltration. Stars mark tissue consolidation. Large size and small size bars indicate 100 and 500 µm, respectively. b , c Histopathological scoring of lung tissue sections obtained on days 5 ( b ) and 14 ( c ) pi. Mean values and SD are indicated. Statistically significant differences as computed by the one-way ANOVA test are shown (* p < 0.05; ** p < 0.01).

    Journal: NPJ Vaccines

    Article Title: Optimized intramuscular immunization with VSV-vectored spike protein triggers a superior immune response to SARS-CoV-2

    doi: 10.1038/s41541-022-00508-7

    Figure Lengend Snippet: K18-hACE2 mice (group size n = 10) were immunized with the indicated vaccines and challenged with 10 5 pfu of SARS-CoV-2-S D614G via the nasal route. Five animal groups of each vaccine group were euthanized at day 5 pi, while the remaining animals of each group were sacrificed at day 14 pi. a Lung and brain tissue sections were analyzed by immunohistochemistry (IHC) using a rabbit polyclonal antibody directed to the SARS-CoV nucleoprotein antigen. Lung and brain sections prepared from mock-infected non-vaccinated animals served as control. Arrows indicate lung and brain areas where the viral nucleoprotein antigen was detected. In parallel, lung and brain tissue sections were stained by hematoxylin-eosin (HE). Arrow heads indicate perivascular/peribronchiolar infiltration. Stars mark tissue consolidation. Large size and small size bars indicate 100 and 500 µm, respectively. b , c Histopathological scoring of lung tissue sections obtained on days 5 ( b ) and 14 ( c ) pi. Mean values and SD are indicated. Statistically significant differences as computed by the one-way ANOVA test are shown (* p < 0.05; ** p < 0.01).

    Article Snippet: A 1:3000 dilution of a rabbit polyclonal anti-SARS-CoV nucleocapsid antibody (Rockland, Cat. no. 200-401-A50) was used for SARS-CoV-2 immunohistochemical (IHC) analysis of the lung and brain.

    Techniques: Immunohistochemistry, Infection, Staining

    Six-week-old offspring of naïve (red) or of immunized and subsequently infected with SARS-CoV-2-S D614G (green) K18-hACE2 mice were challenged intranasally with 3 × 10 4 TCID 50 SARS-CoV-2-S D614G . a Determination of virus neutralization titers (ND 50 ) in serum of offspring 1 week before and 5 days after challenge infection. b Body weight at the indicated times pi. c Determination of infectious SARS-CoV-2 titers in lung and brain tissue homogenates 5 days pi. Geometric means and geometric SD are indicated. Statistically significant differences were determined by the two-way ANOVA test ( a ) or by the two-tailed unpaired Student’s t test ( c ) (* p < 0.05; ** p < 0.005, **** p < 0.0001).

    Journal: NPJ Vaccines

    Article Title: Optimized intramuscular immunization with VSV-vectored spike protein triggers a superior immune response to SARS-CoV-2

    doi: 10.1038/s41541-022-00508-7

    Figure Lengend Snippet: Six-week-old offspring of naïve (red) or of immunized and subsequently infected with SARS-CoV-2-S D614G (green) K18-hACE2 mice were challenged intranasally with 3 × 10 4 TCID 50 SARS-CoV-2-S D614G . a Determination of virus neutralization titers (ND 50 ) in serum of offspring 1 week before and 5 days after challenge infection. b Body weight at the indicated times pi. c Determination of infectious SARS-CoV-2 titers in lung and brain tissue homogenates 5 days pi. Geometric means and geometric SD are indicated. Statistically significant differences were determined by the two-way ANOVA test ( a ) or by the two-tailed unpaired Student’s t test ( c ) (* p < 0.05; ** p < 0.005, **** p < 0.0001).

    Article Snippet: A 1:3000 dilution of a rabbit polyclonal anti-SARS-CoV nucleocapsid antibody (Rockland, Cat. no. 200-401-A50) was used for SARS-CoV-2 immunohistochemical (IHC) analysis of the lung and brain.

    Techniques: Infection, Neutralization, Two Tailed Test

    Immune sera were prepared from K18-hACE2 mice 4 weeks after the second immunization with either ( a ) VSV*ΔG-S Δ21 ( n = 10), ( b ) VSV*M q ΔG-S Δ21 ( n = 10), and ( c ) VSV*ΔG-S RBD ( n = 5). Virus neutralization tests were performed using the VSV*ΔG(FLuc) vector pseudotyped with the S protein of the indicated SARS-CoV-2 VOC. At 20 h pi of Vero E6 cells, the reciprocal serum dilution causing 50% inhibition of the pseudotype virus-encoded firefly luciferase reporter expression was determined and expressed as pseudotype virus neutralization dose 50% (PVND 50 ). Lines connect the corresponding PVND 50 titers of individual immune sera. The bars represent the mean neutralization titers. Statistically significant differences as determined by the two-tailed paired Student’s t test are indicated.

    Journal: NPJ Vaccines

    Article Title: Optimized intramuscular immunization with VSV-vectored spike protein triggers a superior immune response to SARS-CoV-2

    doi: 10.1038/s41541-022-00508-7

    Figure Lengend Snippet: Immune sera were prepared from K18-hACE2 mice 4 weeks after the second immunization with either ( a ) VSV*ΔG-S Δ21 ( n = 10), ( b ) VSV*M q ΔG-S Δ21 ( n = 10), and ( c ) VSV*ΔG-S RBD ( n = 5). Virus neutralization tests were performed using the VSV*ΔG(FLuc) vector pseudotyped with the S protein of the indicated SARS-CoV-2 VOC. At 20 h pi of Vero E6 cells, the reciprocal serum dilution causing 50% inhibition of the pseudotype virus-encoded firefly luciferase reporter expression was determined and expressed as pseudotype virus neutralization dose 50% (PVND 50 ). Lines connect the corresponding PVND 50 titers of individual immune sera. The bars represent the mean neutralization titers. Statistically significant differences as determined by the two-tailed paired Student’s t test are indicated.

    Article Snippet: A 1:3000 dilution of a rabbit polyclonal anti-SARS-CoV nucleocapsid antibody (Rockland, Cat. no. 200-401-A50) was used for SARS-CoV-2 immunohistochemical (IHC) analysis of the lung and brain.

    Techniques: Neutralization, Plasmid Preparation, Inhibition, Luciferase, Expressing, Two Tailed Test

    K18-hACE2 mice ( n = 10) were immunized (i.m.) twice with 10 6 ffu of VSV*ΔG-S Δ21. a Determination of VSV*ΔG-S Δ21 -neutralizing antibodies in serum collected 3 weeks after the first (prime) and 3 weeks after the second immunization (boost). b Determination of the pseudotype virus neutralization dose 50 (PVND 50 ) using the VSV*ΔG(FLuc) reporter virus pseudotyped with the S D614G or S Delta protein. c – h Four weeks after the second immunization, animals were infected with 8×10 4 pfu of SARS-CoV-2 Delta VOC via the nasal route. A group of naïve K18-hACE2 mice ( n = 5) served as control. c Relative body weight of mice in the course of infection up to day 5 pi. Some Individual curves were labeled with an ID number to identify the animals in the histological analysis (Supplementary Fig. ). d , e Quantification of SARS-CoV-2 RNA copies by RT-qPCR in oropharyngeal swab samples collected at days 2 and 5 pi ( d ), and in the indicated organs prepared at day 5 and 14 pi ( e ). f Determination of infectious SARS-CoV-2 Delta titers in homogenates of lung and brain at day 5 pi. g Relative body weight of vaccinated mice in the course of infection with SARS-CoV-2 Delta up to day 14 pi. h Determination of neutralizing serum antibody titers (ND50) 18 days after the boost and 14 days post challenge infection. Mean values and SD are shown. Statistical significance was calculated by the unpaired Student’s t test ( a , e , f , h ), by the paired Student’s t test ( b ), and by the two-way ANOVA test ( d ) (* p < 0.05, ** p < 0.01; *** p < 0.005; **** p < 0.0001).

    Journal: NPJ Vaccines

    Article Title: Optimized intramuscular immunization with VSV-vectored spike protein triggers a superior immune response to SARS-CoV-2

    doi: 10.1038/s41541-022-00508-7

    Figure Lengend Snippet: K18-hACE2 mice ( n = 10) were immunized (i.m.) twice with 10 6 ffu of VSV*ΔG-S Δ21. a Determination of VSV*ΔG-S Δ21 -neutralizing antibodies in serum collected 3 weeks after the first (prime) and 3 weeks after the second immunization (boost). b Determination of the pseudotype virus neutralization dose 50 (PVND 50 ) using the VSV*ΔG(FLuc) reporter virus pseudotyped with the S D614G or S Delta protein. c – h Four weeks after the second immunization, animals were infected with 8×10 4 pfu of SARS-CoV-2 Delta VOC via the nasal route. A group of naïve K18-hACE2 mice ( n = 5) served as control. c Relative body weight of mice in the course of infection up to day 5 pi. Some Individual curves were labeled with an ID number to identify the animals in the histological analysis (Supplementary Fig. ). d , e Quantification of SARS-CoV-2 RNA copies by RT-qPCR in oropharyngeal swab samples collected at days 2 and 5 pi ( d ), and in the indicated organs prepared at day 5 and 14 pi ( e ). f Determination of infectious SARS-CoV-2 Delta titers in homogenates of lung and brain at day 5 pi. g Relative body weight of vaccinated mice in the course of infection with SARS-CoV-2 Delta up to day 14 pi. h Determination of neutralizing serum antibody titers (ND50) 18 days after the boost and 14 days post challenge infection. Mean values and SD are shown. Statistical significance was calculated by the unpaired Student’s t test ( a , e , f , h ), by the paired Student’s t test ( b ), and by the two-way ANOVA test ( d ) (* p < 0.05, ** p < 0.01; *** p < 0.005; **** p < 0.0001).

    Article Snippet: A 1:3000 dilution of a rabbit polyclonal anti-SARS-CoV nucleocapsid antibody (Rockland, Cat. no. 200-401-A50) was used for SARS-CoV-2 immunohistochemical (IHC) analysis of the lung and brain.

    Techniques: Neutralization, Infection, Labeling, Quantitative RT-PCR

    Immunohistochemical/immunofluorescence expression of Sars-Cov-2 antigens in relation to histopathological changes in thyroid gland obtained from patient who deceased from COVID-19. Sars-Cov-2 spike (S) protein was found notably expressed throughout the thyroid tissue and was localized diffusely in the cytoplasm of follicular cells (A, B, C, D, E, F and J, K, L, M, N, O). Concomitantly, the thyroid gland harboured copious mononuclear infiltrate (A, B, C, D, E, F, G and H) forming granuloma-like structures (B, C, D, E and F) with giant multinuclear cells (arrows on B, D, E, F), epithelial desquamation, colloid depletion and extensive follicular disruption (A, B, C, D, E, F, G, H and I, corresponding to findings consistent with subacute thyroiditis. Spike protein positivity was also found in epithelioid cells within the granulomatous tissue (F). Sars-Cov-2 nucleocapsid protein immunopositivity was noticeably sparser, arranged in punctiform inclusions and localized predominantly in the perinuclear region of follicular cells (arrows on G and H; J, K, L, M, N, O). The majority of remaining follicular cells showed positivity for cleaved caspase-3 (I). Staining was performed using rabbit anti-SARS-CoV-2 spike glycoprotein antibody (Abcam, ab272504; dilution 1:4000), mouse anti-SARS-CoV-2 nucleocapsid protein antibody (Cell Signaling Technology, clone E8R1L; dilution 1:200) and rabbit anti-caspase-3 antibody (Abcam, ab13847; diluted 1:50). Immunoreactions were visualized by DAKO EnVision+System (DAKO Cytomation) for immunohistochemistry or by Alexa Fluor 488 donkey anti-rabbit IgG (Thermo Fisher Scientific; diluted 1:300) and Alexa Fluor 555 goat anti-mouse IgG (Thermo Fisher Scientific; diluted 1:500) secondary antibodies for immunofluorescence studies. Magnifications: A × 100; B, C, D, E, I × 400; F, J, K, L × 600; G, H, M, N, O × 1000.

    Journal: European Thyroid Journal

    Article Title: Detection of Sars-Cov-2 antigens in thyroid gland showing histopathological features of subacute thyroiditis

    doi: 10.1530/ETJ-22-0005

    Figure Lengend Snippet: Immunohistochemical/immunofluorescence expression of Sars-Cov-2 antigens in relation to histopathological changes in thyroid gland obtained from patient who deceased from COVID-19. Sars-Cov-2 spike (S) protein was found notably expressed throughout the thyroid tissue and was localized diffusely in the cytoplasm of follicular cells (A, B, C, D, E, F and J, K, L, M, N, O). Concomitantly, the thyroid gland harboured copious mononuclear infiltrate (A, B, C, D, E, F, G and H) forming granuloma-like structures (B, C, D, E and F) with giant multinuclear cells (arrows on B, D, E, F), epithelial desquamation, colloid depletion and extensive follicular disruption (A, B, C, D, E, F, G, H and I, corresponding to findings consistent with subacute thyroiditis. Spike protein positivity was also found in epithelioid cells within the granulomatous tissue (F). Sars-Cov-2 nucleocapsid protein immunopositivity was noticeably sparser, arranged in punctiform inclusions and localized predominantly in the perinuclear region of follicular cells (arrows on G and H; J, K, L, M, N, O). The majority of remaining follicular cells showed positivity for cleaved caspase-3 (I). Staining was performed using rabbit anti-SARS-CoV-2 spike glycoprotein antibody (Abcam, ab272504; dilution 1:4000), mouse anti-SARS-CoV-2 nucleocapsid protein antibody (Cell Signaling Technology, clone E8R1L; dilution 1:200) and rabbit anti-caspase-3 antibody (Abcam, ab13847; diluted 1:50). Immunoreactions were visualized by DAKO EnVision+System (DAKO Cytomation) for immunohistochemistry or by Alexa Fluor 488 donkey anti-rabbit IgG (Thermo Fisher Scientific; diluted 1:300) and Alexa Fluor 555 goat anti-mouse IgG (Thermo Fisher Scientific; diluted 1:500) secondary antibodies for immunofluorescence studies. Magnifications: A × 100; B, C, D, E, I × 400; F, J, K, L × 600; G, H, M, N, O × 1000.

    Article Snippet: Staining was performed using rabbit anti-SARS-CoV-2 spike glycoprotein antibody (Abcam, ab272504; dilution 1:4000), mouse anti-SARS-CoV-2 nucleocapsid protein antibody (Cell Signaling Technology, clone E8R1L; dilution 1:200) and rabbit anti-caspase-3 antibody (Abcam, ab13847; diluted 1:50).

    Techniques: Immunohistochemical staining, Immunofluorescence, Expressing, Staining, Immunohistochemistry

    (a) Each panel presents the protein expressed by the DNA (black outline) for the gp96-Ig and SARS-CoV-2 protein S vaccine antigen. Gp96-Ig and SARS-CoV-2-S DNA were cloned into the mammalian expression vectors B45 and pcDNA 3.1, which are transfected into HEK-293 and AD100. Stable transfection vaccine cell clones (1A, 1A6, 1D6) were generated after selection with L-Histidinol and Neomycin; (b) One million 293-gp96-Ig-S and AD-100-gp96-Ig-S (1D6) cells were plated in 1 mL for 24 hours and gp96-Ig production in the supernatant was determined by ELISA using antihuman IgG antibody for detection with mouse IgG1 (0.5 ug/mL) as a standard; (c) Cell lysates were analyzed under reduced conditions by SDS-PAGE and Western blotting using anti protein S antibody and recombinant protein S1 as a positive control; (d) IF for protein S (in green) expressed in AD100-gp96-Ig-S cells using rabbit anti-SARS-CoV-2 S antibody and antirabbit Ig-AF488 as secondary antibody. AD100 was used as a negative control and β-actin for protein quantification. Original magnification 40× with DAPI nuclear staining shown in blue. DNA, deoxyribonucleic acid; ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; N, amino terminus; C, carboxy terminus; IF, immunofluorescence; TM, transmembrane domain; KDEL, retention signal; CH2 CH3 gamma 1, heavy chain of IgG1. See text for explanation.

    Journal: bioRxiv

    Article Title: Induction of SARS-CoV-2 protein S-specific CD8+ T cells in the lungs of gp96-Ig-S vaccinated mice

    doi: 10.1101/2020.08.24.265090

    Figure Lengend Snippet: (a) Each panel presents the protein expressed by the DNA (black outline) for the gp96-Ig and SARS-CoV-2 protein S vaccine antigen. Gp96-Ig and SARS-CoV-2-S DNA were cloned into the mammalian expression vectors B45 and pcDNA 3.1, which are transfected into HEK-293 and AD100. Stable transfection vaccine cell clones (1A, 1A6, 1D6) were generated after selection with L-Histidinol and Neomycin; (b) One million 293-gp96-Ig-S and AD-100-gp96-Ig-S (1D6) cells were plated in 1 mL for 24 hours and gp96-Ig production in the supernatant was determined by ELISA using antihuman IgG antibody for detection with mouse IgG1 (0.5 ug/mL) as a standard; (c) Cell lysates were analyzed under reduced conditions by SDS-PAGE and Western blotting using anti protein S antibody and recombinant protein S1 as a positive control; (d) IF for protein S (in green) expressed in AD100-gp96-Ig-S cells using rabbit anti-SARS-CoV-2 S antibody and antirabbit Ig-AF488 as secondary antibody. AD100 was used as a negative control and β-actin for protein quantification. Original magnification 40× with DAPI nuclear staining shown in blue. DNA, deoxyribonucleic acid; ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; N, amino terminus; C, carboxy terminus; IF, immunofluorescence; TM, transmembrane domain; KDEL, retention signal; CH2 CH3 gamma 1, heavy chain of IgG1. See text for explanation.

    Article Snippet: Protein expression was verified by SDS-page and Western blotting using rabbit anti-SARS-CoV-2 spike glycoprotein antibody (MBS 150780) at 1/1000 dilution and secondary antibody: Peroxidase AffiniPure F(ab’) 2 Fragment Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories) horseradish peroxidase conjugated anti-rabbit IgG (Jackson ImmunoResearch) at 1/10,000 dilution.

    Techniques: Clone Assay, Expressing, Transfection, Stable Transfection, Generated, Selection, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot, Recombinant, Positive Control, Negative Control, Staining, Immunofluorescence

    5 days after the vaccination of C57Bl6 mice, splenocytes and lung cells were isolated from vaccinated and control mice (PBS) and in vitro restimulated with S1 and S2 overlapping peptides from SARS-CoV-2 protein in the presence of protein transport inhibitor, brefeldin A for the last 5 hours of culture. After 20 hours of culture, ICS was preformed to quantify protein S-specific CD8+ and CD4+ T-cell responses. Cytokine expression in the presence of no peptides was considered background and it was subtracted from the responses measured from peptide pool stimulated samples for each individual mouse. (a–b) CD8+ T cells from spleen and lungs expressing IFNγ, TNFα and IL-2 in response to S1 and S2 peptide pool; (c–d) CD4+ T cells from spleen and lungs expressing IFNγ, TNFα and IL-2 in response to S1 and S2 peptide pool; (e) Proportion of antigen (protein S)-experienced CD8+ and CD4+ T cells isolated from spleen and lung tissue expressing IFNγ, TNFα, or IL-2 after o/n stimulation with S1 + S2 peptides. Pie charts corresponding to cytokine profiles of CD8+ and CD4+ T cells isolated from spleen and lung tissue; (f) Polyfunctional profiles of antigen experienced CD8+ and CD4+ T cells. Pie charts corresponding to polyfunctional profiles of CD8+ CD4+ T cells isolated from spleen and lung tissue after o/n stimulation with S1 + S2 peptides. Assessment of the mean proportion of cells making any combination of 1–3 cytokines (IFN-γ, TNFα, IL-2). Data represent at least 2 technical replicates with 3–6 independent biologic replicates per group. *p<0.05, **p<0.01, ***p<0.001. Kruskal-Wallis ANOVA with Dunn’s multiple comparisons tests were applied. Asterisks (*) above or inside the column denote significant differences between indicated T cells producing cytokines in vaccine versus control (PBS) at 0.05 alpha level. ANOVA, analysis of variance; ICS, intracellular cytokine staining; IFN, interferon; IL, interleukin; PBS, phosphate-buffered saline; TNF, tumor necrosis factor.

    Journal: bioRxiv

    Article Title: Induction of SARS-CoV-2 protein S-specific CD8+ T cells in the lungs of gp96-Ig-S vaccinated mice

    doi: 10.1101/2020.08.24.265090

    Figure Lengend Snippet: 5 days after the vaccination of C57Bl6 mice, splenocytes and lung cells were isolated from vaccinated and control mice (PBS) and in vitro restimulated with S1 and S2 overlapping peptides from SARS-CoV-2 protein in the presence of protein transport inhibitor, brefeldin A for the last 5 hours of culture. After 20 hours of culture, ICS was preformed to quantify protein S-specific CD8+ and CD4+ T-cell responses. Cytokine expression in the presence of no peptides was considered background and it was subtracted from the responses measured from peptide pool stimulated samples for each individual mouse. (a–b) CD8+ T cells from spleen and lungs expressing IFNγ, TNFα and IL-2 in response to S1 and S2 peptide pool; (c–d) CD4+ T cells from spleen and lungs expressing IFNγ, TNFα and IL-2 in response to S1 and S2 peptide pool; (e) Proportion of antigen (protein S)-experienced CD8+ and CD4+ T cells isolated from spleen and lung tissue expressing IFNγ, TNFα, or IL-2 after o/n stimulation with S1 + S2 peptides. Pie charts corresponding to cytokine profiles of CD8+ and CD4+ T cells isolated from spleen and lung tissue; (f) Polyfunctional profiles of antigen experienced CD8+ and CD4+ T cells. Pie charts corresponding to polyfunctional profiles of CD8+ CD4+ T cells isolated from spleen and lung tissue after o/n stimulation with S1 + S2 peptides. Assessment of the mean proportion of cells making any combination of 1–3 cytokines (IFN-γ, TNFα, IL-2). Data represent at least 2 technical replicates with 3–6 independent biologic replicates per group. *p<0.05, **p<0.01, ***p<0.001. Kruskal-Wallis ANOVA with Dunn’s multiple comparisons tests were applied. Asterisks (*) above or inside the column denote significant differences between indicated T cells producing cytokines in vaccine versus control (PBS) at 0.05 alpha level. ANOVA, analysis of variance; ICS, intracellular cytokine staining; IFN, interferon; IL, interleukin; PBS, phosphate-buffered saline; TNF, tumor necrosis factor.

    Article Snippet: Protein expression was verified by SDS-page and Western blotting using rabbit anti-SARS-CoV-2 spike glycoprotein antibody (MBS 150780) at 1/1000 dilution and secondary antibody: Peroxidase AffiniPure F(ab’) 2 Fragment Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories) horseradish peroxidase conjugated anti-rabbit IgG (Jackson ImmunoResearch) at 1/10,000 dilution.

    Techniques: Isolation, In Vitro, Expressing, Staining

    Cobicistat is a candidate inhibitor of SARS-CoV-2 replication. (A and B) In silico docking analysis of the putative mode and energy of binding of cobicistat to SARS-CoV-2 3CL pro . (A) Docking pose showing the ligand interaction of cobicistat to the active site of 3CL pro and the formation of hydrogen bonds to Asn142, Gly143, and Gln189 of 3CL pro . (B) Overlay of crystal structures of SARS-Cov-2 3CL pro showing the amino acids important for the binding of cobicistat to the active site of the enzyme. Residues of the catalytic dyad (Cys145 and His41) of 3CL pro were among the highest contributors to noncovalent binding to cobicistat. The source and list of structures used are detailed in Materials and Methods. (C) Schematic representation of time course experiments evaluating in vitro inhibition of SARS-CoV-2 replication by cobicistat (created with BioRender). (D and E) Effect of various concentrations of cobicistat, added according to the scheme of panel C, on intracellular and supernatant SARS-CoV-2 RNA content in Calu-3 cells. Viral RNA content was measured by qPCR using the 2019-nCoV_N1 primer set (Centers for Disease Control and Prevention). Fold change values in intracellular RNA (D) were calculated by the delta-delta C T method , using the Tata-binding protein (TBP) gene as housekeeper control. Expression levels in supernatant (E) were quantified using an in vitro -transcribed standard curve generated as described in Materials and Methods. Data are expressed as mean with standard deviation (SD) and were analyzed by two-way ANOVA followed by Dunnett’s posttest ( n = 3 independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Journal: mBio

    Article Title: The FDA-Approved Drug Cobicistat Synergizes with Remdesivir To Inhibit SARS-CoV-2 Replication In Vitro and Decreases Viral Titers and Disease Progression in Syrian Hamsters

    doi: 10.1128/mbio.03705-21

    Figure Lengend Snippet: Cobicistat is a candidate inhibitor of SARS-CoV-2 replication. (A and B) In silico docking analysis of the putative mode and energy of binding of cobicistat to SARS-CoV-2 3CL pro . (A) Docking pose showing the ligand interaction of cobicistat to the active site of 3CL pro and the formation of hydrogen bonds to Asn142, Gly143, and Gln189 of 3CL pro . (B) Overlay of crystal structures of SARS-Cov-2 3CL pro showing the amino acids important for the binding of cobicistat to the active site of the enzyme. Residues of the catalytic dyad (Cys145 and His41) of 3CL pro were among the highest contributors to noncovalent binding to cobicistat. The source and list of structures used are detailed in Materials and Methods. (C) Schematic representation of time course experiments evaluating in vitro inhibition of SARS-CoV-2 replication by cobicistat (created with BioRender). (D and E) Effect of various concentrations of cobicistat, added according to the scheme of panel C, on intracellular and supernatant SARS-CoV-2 RNA content in Calu-3 cells. Viral RNA content was measured by qPCR using the 2019-nCoV_N1 primer set (Centers for Disease Control and Prevention). Fold change values in intracellular RNA (D) were calculated by the delta-delta C T method , using the Tata-binding protein (TBP) gene as housekeeper control. Expression levels in supernatant (E) were quantified using an in vitro -transcribed standard curve generated as described in Materials and Methods. Data are expressed as mean with standard deviation (SD) and were analyzed by two-way ANOVA followed by Dunnett’s posttest ( n = 3 independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Article Snippet: After washing, cells were stained with the primary rabbit polyclonal anti-SARS-CoV-2 spike glycoprotein antibody (1:1,000; Abcam) for 1 h at room temperature or overnight at 4°C.

    Techniques: In Silico, Binding Assay, In Vitro, Inhibition, Expressing, Generated, Standard Deviation

    Cobicistat decreases replication of SARS-CoV-2 and rescues viability of infected cells in multiple in vitro models. (A and B) Effect of serial dilutions of cobicistat on SARS-CoV-2 RNA concentration in supernatants (A) and on the viability of infected and uninfected cell lines of lung (Calu-3), gut (T84), and kidney (Vero E6) origin (A and B). Cells were infected with SARS-CoV-2 at two different MOIs (0.05 and 0.5) and left untreated or treated with cobicistat 2 h postinfection. Forty-eight hours postinfection, supernatants were collected and viral RNA was assayed by qPCR while cellular viability was measured by MTT assay (A) or by crystal violet staining (B). Inhibition of viral replication was calculated as described in Materials and Methods while viability data were normalized to the uninfected or to the untreated control. Half-maximal inhibitory concentration (IC 50 ) values were calculated by nonlinear regression. Each point in panel A represents a mean from 3 independent experiments. Pictures in panel B are derived from infections at MOI 0.5 (Calu-3 and T84 cells) or MOI 0.05 (Vero E6 cells). (C) Comparison between the IC 50 and CC 50 values of cobicistat determined in vitro and the peak plasma levels detectable in mice (Pharmacology Review of Cobicistat - application number: 203-094) and in humans ( , ) after administration of a single dose of the drug. Determination of in vitro CC 50 values is based on the data shown in <xref ref-type=Fig. S2 . " width="100%" height="100%">

    Journal: mBio

    Article Title: The FDA-Approved Drug Cobicistat Synergizes with Remdesivir To Inhibit SARS-CoV-2 Replication In Vitro and Decreases Viral Titers and Disease Progression in Syrian Hamsters

    doi: 10.1128/mbio.03705-21

    Figure Lengend Snippet: Cobicistat decreases replication of SARS-CoV-2 and rescues viability of infected cells in multiple in vitro models. (A and B) Effect of serial dilutions of cobicistat on SARS-CoV-2 RNA concentration in supernatants (A) and on the viability of infected and uninfected cell lines of lung (Calu-3), gut (T84), and kidney (Vero E6) origin (A and B). Cells were infected with SARS-CoV-2 at two different MOIs (0.05 and 0.5) and left untreated or treated with cobicistat 2 h postinfection. Forty-eight hours postinfection, supernatants were collected and viral RNA was assayed by qPCR while cellular viability was measured by MTT assay (A) or by crystal violet staining (B). Inhibition of viral replication was calculated as described in Materials and Methods while viability data were normalized to the uninfected or to the untreated control. Half-maximal inhibitory concentration (IC 50 ) values were calculated by nonlinear regression. Each point in panel A represents a mean from 3 independent experiments. Pictures in panel B are derived from infections at MOI 0.5 (Calu-3 and T84 cells) or MOI 0.05 (Vero E6 cells). (C) Comparison between the IC 50 and CC 50 values of cobicistat determined in vitro and the peak plasma levels detectable in mice (Pharmacology Review of Cobicistat - application number: 203-094) and in humans ( , ) after administration of a single dose of the drug. Determination of in vitro CC 50 values is based on the data shown in Fig. S2 .

    Article Snippet: After washing, cells were stained with the primary rabbit polyclonal anti-SARS-CoV-2 spike glycoprotein antibody (1:1,000; Abcam) for 1 h at room temperature or overnight at 4°C.

    Techniques: Infection, In Vitro, Concentration Assay, MTT Assay, Staining, Inhibition, Derivative Assay

    Cobicistat decreases SARS-CoV-2 S-protein content and fusion to target cells. (A and B) Effect of cobicistat on the expression of S- and N-proteins in SARS-CoV-2-infected Vero E6 cells. Cells were infected at 0.5 MOI and left untreated or treated, 2 h postinfection, with various concentrations of cobicistat, of the RdRp inhibitor remdesivir, or of the 3CL pro inhibitor GC376. Cells were harvested 24 h posttreatment and subjected to protein extraction and subsequent analysis by Western blotting. Expression of S- and N-proteins, and expression of the housekeeping protein actin-β, was detected using primary monoclonal antibodies followed by incubation with fluorescence-conjugated secondary antibodies and detection on a Li-Cor Odyssey CLx instrument (B). Relative protein levels were quantified using Fiji‐Image J and normalized to the untreated control. Data (mean ± range of three independent experiments) were analyzed by linear regression. n.s., not significant. (C and D) Effect of cobicistat on S-protein-mediated syncytium formation. Vero E6 cells were transfected with the SARS-CoV-2 S-protein and left untreated or treated with various concentrations of cobicistat or with sera isolated from convalescent SARS-CoV-2 patients (1:100 dilution). Syncytium formation was examined 24 h posttransfection by immunofluorescence (IF) staining for DAPI and S-protein (C) and quantified as the number of cells forming syncytia (D). (E) Effect of cobicistat treatment on S-glycoprotein-mediated fusion. TZM-bl cells stably expressing the S-glycoprotein were incubated with different concentrations of cobicistat for 1 h and mixed with cells stably expressing human ACE2 . Cell fusion was assessed by measuring firefly luciferase activity after 24 h. RLU, relative light units. Data in panels C and D were analyzed using the nonparametric Kruskal-Wallis test followed by Dunn’s posttest. Horizontal lines represent mean values. **, P < 0.01; ****, P < 0.0001. Scale bar = 50 μm.

    Journal: mBio

    Article Title: The FDA-Approved Drug Cobicistat Synergizes with Remdesivir To Inhibit SARS-CoV-2 Replication In Vitro and Decreases Viral Titers and Disease Progression in Syrian Hamsters

    doi: 10.1128/mbio.03705-21

    Figure Lengend Snippet: Cobicistat decreases SARS-CoV-2 S-protein content and fusion to target cells. (A and B) Effect of cobicistat on the expression of S- and N-proteins in SARS-CoV-2-infected Vero E6 cells. Cells were infected at 0.5 MOI and left untreated or treated, 2 h postinfection, with various concentrations of cobicistat, of the RdRp inhibitor remdesivir, or of the 3CL pro inhibitor GC376. Cells were harvested 24 h posttreatment and subjected to protein extraction and subsequent analysis by Western blotting. Expression of S- and N-proteins, and expression of the housekeeping protein actin-β, was detected using primary monoclonal antibodies followed by incubation with fluorescence-conjugated secondary antibodies and detection on a Li-Cor Odyssey CLx instrument (B). Relative protein levels were quantified using Fiji‐Image J and normalized to the untreated control. Data (mean ± range of three independent experiments) were analyzed by linear regression. n.s., not significant. (C and D) Effect of cobicistat on S-protein-mediated syncytium formation. Vero E6 cells were transfected with the SARS-CoV-2 S-protein and left untreated or treated with various concentrations of cobicistat or with sera isolated from convalescent SARS-CoV-2 patients (1:100 dilution). Syncytium formation was examined 24 h posttransfection by immunofluorescence (IF) staining for DAPI and S-protein (C) and quantified as the number of cells forming syncytia (D). (E) Effect of cobicistat treatment on S-glycoprotein-mediated fusion. TZM-bl cells stably expressing the S-glycoprotein were incubated with different concentrations of cobicistat for 1 h and mixed with cells stably expressing human ACE2 . Cell fusion was assessed by measuring firefly luciferase activity after 24 h. RLU, relative light units. Data in panels C and D were analyzed using the nonparametric Kruskal-Wallis test followed by Dunn’s posttest. Horizontal lines represent mean values. **, P < 0.01; ****, P < 0.0001. Scale bar = 50 μm.

    Article Snippet: After washing, cells were stained with the primary rabbit polyclonal anti-SARS-CoV-2 spike glycoprotein antibody (1:1,000; Abcam) for 1 h at room temperature or overnight at 4°C.

    Techniques: Expressing, Infection, Protein Extraction, Western Blot, Incubation, Fluorescence, Transfection, Isolation, Immunofluorescence, Staining, Stable Transfection, Luciferase, Activity Assay

    Expression of the metabolic targets of cobicistat and its role in the antiviral activity of remdesivir. (A) Effect of the knockdown of CYP3A4 , CYP3A5 , and P-gp genes on the antiviral efficacy of remdesivir. Vero E6 cells were transfected with 40 nM siRNAs against either gene target or with nontargeting siRNAs. At 48 h posttransfection cells were infected at MOI 0.05, and 2 h postinfection, they were treated with 0.5 μM remdesivir. Intracellular SARS-CoV-2 RNA expression was analyzed by qPCR 24 h postinfection. (B) Relative expression of CYP3A4/5 and P-gp in SARS-CoV-2-infected or mock-infected cells. Infections were carried out at MOI 0.5 for 48 h, and gene expression was analyzed by qPCR. For both panels, raw data were used to calculate delta C T values by using the TBP gene as housekeeping control. Fold changes were calculated using the delta-delta C T method, as described in reference . Data are expressed as mean ± SD ( n = 2 for panel A and n = 3 for panel B).

    Journal: mBio

    Article Title: The FDA-Approved Drug Cobicistat Synergizes with Remdesivir To Inhibit SARS-CoV-2 Replication In Vitro and Decreases Viral Titers and Disease Progression in Syrian Hamsters

    doi: 10.1128/mbio.03705-21

    Figure Lengend Snippet: Expression of the metabolic targets of cobicistat and its role in the antiviral activity of remdesivir. (A) Effect of the knockdown of CYP3A4 , CYP3A5 , and P-gp genes on the antiviral efficacy of remdesivir. Vero E6 cells were transfected with 40 nM siRNAs against either gene target or with nontargeting siRNAs. At 48 h posttransfection cells were infected at MOI 0.05, and 2 h postinfection, they were treated with 0.5 μM remdesivir. Intracellular SARS-CoV-2 RNA expression was analyzed by qPCR 24 h postinfection. (B) Relative expression of CYP3A4/5 and P-gp in SARS-CoV-2-infected or mock-infected cells. Infections were carried out at MOI 0.5 for 48 h, and gene expression was analyzed by qPCR. For both panels, raw data were used to calculate delta C T values by using the TBP gene as housekeeping control. Fold changes were calculated using the delta-delta C T method, as described in reference . Data are expressed as mean ± SD ( n = 2 for panel A and n = 3 for panel B).

    Article Snippet: After washing, cells were stained with the primary rabbit polyclonal anti-SARS-CoV-2 spike glycoprotein antibody (1:1,000; Abcam) for 1 h at room temperature or overnight at 4°C.

    Techniques: Expressing, Activity Assay, Transfection, Infection, RNA Expression

    The combination of cobicistat and remdesivir synergistically inhibits SARS-CoV-2 activity. (A to F) Synergistic activity of cobicistat and remdesivir in inhibiting replication and cytopathic effects of SARS-CoV-2 in Vero E6 cells. Cells were infected at 0.5 MOI and left untreated or treated with the drugs at the indicated concentrations 2 h postinfection. Forty-eight hours posttreatment, cells were fixed for immunofluorescence (IF) staining (A and B), supernatants were collected for qPCR (C to E), or cellular viability was analyzed (F). For IF detection, cells were stained with sera of SARS-CoV-2 patients and with the J2 antibody, which binds to double-stranded RNA . The percentage of infected cells was determined by automatic acquisition of nine images per well (A), as described in Materials and Methods. Scale bar = 100 μm. Viral RNA in supernatants was detected by qPCR using an in vitro -transcribed standard curve for absolute quantification (C to E). Data, expressed as mean ± SD, were transformed as log 10 to restore normality and analyzed by one-way ANOVA, followed by the Holm-Sidak posttest (C). Cellular viability was measured by MTT assay (F). Isobologram analysis of synergism (D) was performed using the IC 90 values for SARS-CoV-2 replication of cobicistat, remdesivir, or their combination, calculated by nonlinear regression. Synergism analyses of the inhibition of viral replication (E) or cytopathic effects (F) were performed with the SynergyFinder web tool using the Zero Interaction Potency (ZIP) model based on inhibition values calculated as described in Materials and Methods. (G) Effect of the combination of cobicistat and remdesivir on SARS-CoV-2 RNA expression in supernatants of a primary human colon organoid. Treatment with cobicistat/remdesivir was performed 2 h postinfection, and supernatants were collected 48 h posttreatment. Viral RNA was quantified as described for panel C. For all panels, n equals 3 independent experiments, except for panel E ( n = 2 independent experiments) and panel G ( n = 2 replicates from one colon organoid donor). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Journal: mBio

    Article Title: The FDA-Approved Drug Cobicistat Synergizes with Remdesivir To Inhibit SARS-CoV-2 Replication In Vitro and Decreases Viral Titers and Disease Progression in Syrian Hamsters

    doi: 10.1128/mbio.03705-21

    Figure Lengend Snippet: The combination of cobicistat and remdesivir synergistically inhibits SARS-CoV-2 activity. (A to F) Synergistic activity of cobicistat and remdesivir in inhibiting replication and cytopathic effects of SARS-CoV-2 in Vero E6 cells. Cells were infected at 0.5 MOI and left untreated or treated with the drugs at the indicated concentrations 2 h postinfection. Forty-eight hours posttreatment, cells were fixed for immunofluorescence (IF) staining (A and B), supernatants were collected for qPCR (C to E), or cellular viability was analyzed (F). For IF detection, cells were stained with sera of SARS-CoV-2 patients and with the J2 antibody, which binds to double-stranded RNA . The percentage of infected cells was determined by automatic acquisition of nine images per well (A), as described in Materials and Methods. Scale bar = 100 μm. Viral RNA in supernatants was detected by qPCR using an in vitro -transcribed standard curve for absolute quantification (C to E). Data, expressed as mean ± SD, were transformed as log 10 to restore normality and analyzed by one-way ANOVA, followed by the Holm-Sidak posttest (C). Cellular viability was measured by MTT assay (F). Isobologram analysis of synergism (D) was performed using the IC 90 values for SARS-CoV-2 replication of cobicistat, remdesivir, or their combination, calculated by nonlinear regression. Synergism analyses of the inhibition of viral replication (E) or cytopathic effects (F) were performed with the SynergyFinder web tool using the Zero Interaction Potency (ZIP) model based on inhibition values calculated as described in Materials and Methods. (G) Effect of the combination of cobicistat and remdesivir on SARS-CoV-2 RNA expression in supernatants of a primary human colon organoid. Treatment with cobicistat/remdesivir was performed 2 h postinfection, and supernatants were collected 48 h posttreatment. Viral RNA was quantified as described for panel C. For all panels, n equals 3 independent experiments, except for panel E ( n = 2 independent experiments) and panel G ( n = 2 replicates from one colon organoid donor). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Article Snippet: After washing, cells were stained with the primary rabbit polyclonal anti-SARS-CoV-2 spike glycoprotein antibody (1:1,000; Abcam) for 1 h at room temperature or overnight at 4°C.

    Techniques: Activity Assay, Infection, Immunofluorescence, Staining, In Vitro, Transformation Assay, MTT Assay, Inhibition, RNA Expression

    The combination of cobicistat and remdesivir inhibits SARS-CoV-2 replication and disease progression in Syrian hamsters. (A) Schematic representation (created with BioRender) of the in vivo dosing and sample collection of Syrian hamsters infected with SARS-CoV-2 and treated with placebo, cobicistat, remdesivir, or a combination of cobicistat and remdesivir. (B) Weight loss progression over time in the placebo and each treatment group. Data are expressed as the mean ± SD of the percentage over the baseline (day 0 postinfection [p.i.]) weight of each animal ( n = 6 until day 3 p.i. and n = 3 at days 4 to 5 p.i.). Data were analyzed by linear regression for each experimental group, followed by the parametric F-test to assess differences among slopes. (C and D) Replication-competent viral titers as PFU on Vero E6 cells (C) and gRNA viral levels in the lung as measured at day 3 ( n = 3) and 5 ( n = 3) p.i. by plaque assay (C) and qPCR (D) quantification. Data were analyzed by two-way ANOVA followed by Tukey’s posttest, comparing the cumulative effects of treatments at both day 3 and day 5 p.i. Before the statistical analysis, an appropriate transformation was applied to make the results uniform (i.e., exponential transformation for PFU and standard log transposition for viral RNA copy numbers, due to the respective size-dependent restriction or amplification of the signal derived from the tests adopted). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

    Journal: mBio

    Article Title: The FDA-Approved Drug Cobicistat Synergizes with Remdesivir To Inhibit SARS-CoV-2 Replication In Vitro and Decreases Viral Titers and Disease Progression in Syrian Hamsters

    doi: 10.1128/mbio.03705-21

    Figure Lengend Snippet: The combination of cobicistat and remdesivir inhibits SARS-CoV-2 replication and disease progression in Syrian hamsters. (A) Schematic representation (created with BioRender) of the in vivo dosing and sample collection of Syrian hamsters infected with SARS-CoV-2 and treated with placebo, cobicistat, remdesivir, or a combination of cobicistat and remdesivir. (B) Weight loss progression over time in the placebo and each treatment group. Data are expressed as the mean ± SD of the percentage over the baseline (day 0 postinfection [p.i.]) weight of each animal ( n = 6 until day 3 p.i. and n = 3 at days 4 to 5 p.i.). Data were analyzed by linear regression for each experimental group, followed by the parametric F-test to assess differences among slopes. (C and D) Replication-competent viral titers as PFU on Vero E6 cells (C) and gRNA viral levels in the lung as measured at day 3 ( n = 3) and 5 ( n = 3) p.i. by plaque assay (C) and qPCR (D) quantification. Data were analyzed by two-way ANOVA followed by Tukey’s posttest, comparing the cumulative effects of treatments at both day 3 and day 5 p.i. Before the statistical analysis, an appropriate transformation was applied to make the results uniform (i.e., exponential transformation for PFU and standard log transposition for viral RNA copy numbers, due to the respective size-dependent restriction or amplification of the signal derived from the tests adopted). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

    Article Snippet: After washing, cells were stained with the primary rabbit polyclonal anti-SARS-CoV-2 spike glycoprotein antibody (1:1,000; Abcam) for 1 h at room temperature or overnight at 4°C.

    Techniques: In Vivo, Infection, Plaque Assay, Transformation Assay, Amplification, Derivative Assay

    ( A-E ) The tracks of transient speed of VLP particles during entry from extracellular into intracellular cytoplasm upon untreated (Ctrl) (A), NP-G2-044 (50 μM for 2 h) (B), SIMFH2 (15 μM for 1 h) (C), CK666 (100 μM for 2 h) (D), Blebbistatin (17 μM for 1 h) (E), respectively. The original point on the X axis represents the viral entry time point. The negative number on the X axis represents the time before the virus enters the cell. The positive number on the X axis represents the time after the virus enters the cell. ( F ) Quantification of the mean velocities of VLP prior entering the untreated and inhibitors-treated cells. ( G ) Quantification of the duration of VLP particles enter into the untreated and inhibitors-treated cells. ( H ) Schematic diagram of drug treatment and authentic SARS-CoV-2 infection. ( I ) Viral entry efficiency of SARS-CoV-2 in BSL3. VeroE6-ACE2 cells were pretreated with 50 μM ML141 or 50 μM NP-G2-044 for 5 h, and challenged with SARS-CoV-2 (MOI=1) for 45 min. Viral RNA levels of internalized virus were measured by qRT-PCR. The data are normalized to the mean of DMSO-treated cells and represent as means ± SEM from three independent experiments. ns (no significant difference), P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001 (One-way ANOVA test).

    Journal: bioRxiv

    Article Title: SARS-CoV-2 infected cells sprout actin-rich filopodia that facilitate viral invasion

    doi: 10.1101/2022.10.19.512957

    Figure Lengend Snippet: ( A-E ) The tracks of transient speed of VLP particles during entry from extracellular into intracellular cytoplasm upon untreated (Ctrl) (A), NP-G2-044 (50 μM for 2 h) (B), SIMFH2 (15 μM for 1 h) (C), CK666 (100 μM for 2 h) (D), Blebbistatin (17 μM for 1 h) (E), respectively. The original point on the X axis represents the viral entry time point. The negative number on the X axis represents the time before the virus enters the cell. The positive number on the X axis represents the time after the virus enters the cell. ( F ) Quantification of the mean velocities of VLP prior entering the untreated and inhibitors-treated cells. ( G ) Quantification of the duration of VLP particles enter into the untreated and inhibitors-treated cells. ( H ) Schematic diagram of drug treatment and authentic SARS-CoV-2 infection. ( I ) Viral entry efficiency of SARS-CoV-2 in BSL3. VeroE6-ACE2 cells were pretreated with 50 μM ML141 or 50 μM NP-G2-044 for 5 h, and challenged with SARS-CoV-2 (MOI=1) for 45 min. Viral RNA levels of internalized virus were measured by qRT-PCR. The data are normalized to the mean of DMSO-treated cells and represent as means ± SEM from three independent experiments. ns (no significant difference), P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001 (One-way ANOVA test).

    Article Snippet: The following antibodies were used in this study: anti-SARS-CoV-2 spike glycoprotein rabbit polyclonal antibody (dilution 1:1000; #ab272420, Abcam); anti-SARS-CoV-2 nucleocapsid protein rabbit polyclonal antibody (dilution 1:1000; #ab273167, Abcam); Alexa Fluor 488 goat anti-rabbit IgG (H+L) (dilution 1:1000; #A11008, Invitrogen); Alexa Fluor 555 phalloidin (dilution 1:500; #A34055, Invitrogen).

    Techniques: Infection, Quantitative RT-PCR

    Schematic diagram of SARS-CoV-2 induced filopodia formation in host cells to facilitate viral entry.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 infected cells sprout actin-rich filopodia that facilitate viral invasion

    doi: 10.1101/2022.10.19.512957

    Figure Lengend Snippet: Schematic diagram of SARS-CoV-2 induced filopodia formation in host cells to facilitate viral entry.

    Article Snippet: The following antibodies were used in this study: anti-SARS-CoV-2 spike glycoprotein rabbit polyclonal antibody (dilution 1:1000; #ab272420, Abcam); anti-SARS-CoV-2 nucleocapsid protein rabbit polyclonal antibody (dilution 1:1000; #ab273167, Abcam); Alexa Fluor 488 goat anti-rabbit IgG (H+L) (dilution 1:1000; #A11008, Invitrogen); Alexa Fluor 555 phalloidin (dilution 1:500; #A34055, Invitrogen).

    Techniques: