Journal: Journal of Virology

Article Title: Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

doi: 10.1128/jvi.01805-07

Figure Lengend Snippet: FIG. 6. Surface expression and soluble receptor binding of wild-type and mutant SARS-CoV S 19 proteins. (A) Cell surface expression of the wild-type or mutant SARS-CoV S 19 protein was measured by flow cytometry. (B and C) Binding of the soluble hACE2-Fc receptor protein by cells expressing wild-type or mutant SARS-CoV S 19 detached with trypsin-EDTA (B) or EDTA alone (C). (D to F) Representative flow cytometry overlays shown to the right of corresponding bar graphs. Mock-transfected cells are shown in dark gray, and SARS-CoV S 19 is shown by a black line; representative SARS-CoV S 19 mutant proteins are shown by color (SARS-CoV S 19 Y1188A, green; S 19 W1194A, red; S 19 Y1197A, orange; S 19 Y1188A/Y1191A, cyan; and S 19 W1194/W1199A, yellow, respectively). Error bars represent standard errors of percent wild-type levels between at least three independent experiments.

Article Snippet: HEK-293 cells stably expressing hACE2 were made by transfection of HEK-293 cells with pcDNA3.1 hACE2, selection with G418 (Invitrogen), and sorting for hACE2 expression with goat anti-human hACE2 AF933 (R&D Systems, Minneapolis, MN) and phycoerythrin (PE)-conjugated anti-goat immunoglobulin G (IgG) (Jackson Immunoresearch, West Grove, PA) antibodies.

Techniques: Expressing, Binding Assay, Mutagenesis, Cytometry, Transfection

Journal: Journal of Virology

Article Title: Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

doi: 10.1128/jvi.01805-07

Figure Lengend Snippet: FIG. 8. Surface expression of hACE2. HEK-293 cells stably ex- pressing high levels of hACE2 were selected by flow cytometry.

Article Snippet: HEK-293 cells stably expressing hACE2 were made by transfection of HEK-293 cells with pcDNA3.1 hACE2, selection with G418 (Invitrogen), and sorting for hACE2 expression with goat anti-human hACE2 AF933 (R&D Systems, Minneapolis, MN) and phycoerythrin (PE)-conjugated anti-goat immunoglobulin G (IgG) (Jackson Immunoresearch, West Grove, PA) antibodies.

Techniques: Expressing, Stable Transfection, Cytometry

Journal: Journal of Virology

Article Title: Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

doi: 10.1128/jvi.01805-07

Figure Lengend Snippet: FIG. 9. Alanine substitutions in the JMD of the SARS-CoV S 19 protein reduce receptor-dependent entry of pseudotyped viruses. Rel- ative transduction activity of pseudotyped viruses with wild-type or mutant SARS-CoV S 19 proteins is shown. Pseudotypes in filtered medium were used to transduce HEK-293/hACE2 cells. Beta-galacto- sidase expression was analyzed 2 days posttransduction. Transduction units per ml were calculated {TU/ml [(% cells expressing reporter/ 100) total number of cells per well]/ml inoculum}. Data are repre- sented as percent transduction activity of wild-type SARS-CoV S 19 proteins and are representative of three to six experiments. Error bars represent standard errors. Student’s t test was used to evaluate the significance of differences between the transducing activities of pseudotyped viruses with mutant or wild-type proteins. (*, P 0.01 to 0.05; **, P 0.01 to 0.001; ***, P 0.0001).

Article Snippet: HEK-293 cells stably expressing hACE2 were made by transfection of HEK-293 cells with pcDNA3.1 hACE2, selection with G418 (Invitrogen), and sorting for hACE2 expression with goat anti-human hACE2 AF933 (R&D Systems, Minneapolis, MN) and phycoerythrin (PE)-conjugated anti-goat immunoglobulin G (IgG) (Jackson Immunoresearch, West Grove, PA) antibodies.

Techniques: Transduction, Activity Assay, Mutagenesis, Expressing

Journal: Journal of Virology

Article Title: Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

doi: 10.1128/jvi.01805-07

Figure Lengend Snippet: FIG. 10. Trypsin enhances receptor-dependent cell-cell fusion induced by wild-type SARS-CoV S 19 protein. The time course of syncytium formation was determined after 30 min, 1 h, 2 h, or 3 h. HEK-293 cells transfected with cDNA encoding the wild-type SARS-CoV S 19 glycoprotein (green) were stained with Celltracker Green (Molecular Probes) and were detached from the monolayer with EDTA alone or trypsin-EDTA and added to a 50% confluent monolayer of HEK-293/hACE2 cells (blue) stained with Hoechst 33342 (Sigma) at a ratio of one S-expressing cell to three hACE2-expressing cells. Cells were fixed with paraformaldehyde 2 h postmixing. Pictures were taken with a 20 objective. Pictures are representative of two independent experiments..

Article Snippet: HEK-293 cells stably expressing hACE2 were made by transfection of HEK-293 cells with pcDNA3.1 hACE2, selection with G418 (Invitrogen), and sorting for hACE2 expression with goat anti-human hACE2 AF933 (R&D Systems, Minneapolis, MN) and phycoerythrin (PE)-conjugated anti-goat immunoglobulin G (IgG) (Jackson Immunoresearch, West Grove, PA) antibodies.

Techniques: Transfection, Staining, Expressing

Journal: Journal of Virology

Article Title: Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

doi: 10.1128/jvi.01805-07

Figure Lengend Snippet: FIG. 11. JMD mutations reduce receptor-dependent cell-cell fu- sion induced by SARS S 19 proteins. (A) Fusion of HEK-293 cells expressing wild-type or mutant SARS-CoV S 19 proteins or empty vector with HEK-293/hACE2 cells. Fusion was scored as the percent- age of nuclei present in syncytia of any size at 2 h after mixing cells expressing spike and receptor. Background levels of fusion up to 15% were seen in HEK-293 cells. Data are expressed as a percentage of wild-type SARS-CoV S 19 fusion with background levels of fusion subtracted. Data are representative of three independent experiments. P values are 0.02 for all mutant proteins relative to results for wild- type SARS-CoV S 19. (B and C) Analysis of syncytium size induced by tyrosine-to-alanine (B) or tryptophan-to-alanine (C) mutants of the SARS-CoV S 19 protein. Each individual syncytium was scored for size (number of nuclei). Results are plotted as the cumulative percent- age of cells containing a given number of nuclei or less.

Article Snippet: HEK-293 cells stably expressing hACE2 were made by transfection of HEK-293 cells with pcDNA3.1 hACE2, selection with G418 (Invitrogen), and sorting for hACE2 expression with goat anti-human hACE2 AF933 (R&D Systems, Minneapolis, MN) and phycoerythrin (PE)-conjugated anti-goat immunoglobulin G (IgG) (Jackson Immunoresearch, West Grove, PA) antibodies.

Techniques: Expressing, Mutagenesis, Plasmid Preparation

Journal: JCI insight

Article Title: Highly susceptible SARS-CoV-2 model in CAG promoter-driven hACE2-transgenic mice.

doi: 10.1172/jci.insight.152529

Figure Lengend Snippet: Figure 1. The pathogenesis of SARS-CoV-2 infection is exacerbated in a viral dose-dependent manner. (A) Western blot of human ACE2 protein using various organs in WT and CAG-hACE2 mice. The top and bottom rows show hACE2 and GAPDH, respectively. (B) Schematic diagram of experimental schedule. Male and female C57BL/6 and CAG-hACE2 mice were infected via respiratory tract with SARS-CoV-2 (2 × 102 TCID50, n = 5; 2 × 103 TCID50, n = 6; 2 × 104 TCID50, n = 5) and were administrated with an equal volume of PBS for mock infection controls (WT, n = 3; CAG-hACE2 mice, n = 3). Body weight and survival were recorded daily for up to 14 days. (C and D) Percentage of initial body weight (C) and survival rate (D). Numbers in C represent the number of mice measured for body weight at each time. White and black circles indicate WT with mock infection and WT infected with 2 × 104 TCID50, respectively. Triangles denote CAG-hACE2 mice. White, orange, blue, and red triangles represent PBS, 2 × 102 TCID50, 2 × 103 TCID50, and 2 × 104 TCID50, respectively.

Article Snippet: Then, the membranes were incubated with HRP-conjugated goat anti-mouse polyclonal IgG for hACE2 (1:2000, catalog 172-1011, Bio-Rad) or HRP-conjugated goat anti-rabbit polyclonal IgG for GAPDH (1:2000, catalog 172-1019, Bio-Rad) for 1 hour at room temperature, and the protein signals were detected using a chemiluminescence detection reagent (Chemi-Lumi One L; Nacalai Tesque Inc.) and visualized using the ImageQuant LAS 4000 system (Cytiva).

Techniques: Infection, Western Blot

Journal: JCI insight

Article Title: Highly susceptible SARS-CoV-2 model in CAG promoter-driven hACE2-transgenic mice.

doi: 10.1172/jci.insight.152529

Figure Lengend Snippet: Figure 7. Characterization of CAG-promoter hACE2-transgenic mice. (A) Chromosome map of annotated hACE2-containing reads. The pink horizontal bars indicate the number of annotated ACE2-containing reads. Annotated site of integrated hACE2 is indicated as a blue box. (B) Detail of annotated site in chromosome 1. The horizontal red and pink bars indicate the ACE2-containing reads mapped to the plus and minus strand of the mouse reference genome (GRCh38/mm10), respectively. The blue box represents the duplicated region. (C) Schematic diagrams and sequences are shown as the hACE2- integrated site inside the first intron region of the Colgalt2 gene locus. The blue boxes denote duplicated regions. Each yellow arrow indicates the entire hACE2 sequence. Consensus sequences of reads at 5′- and 3′-junction sites are shown.

Article Snippet: Then, the membranes were incubated with HRP-conjugated goat anti-mouse polyclonal IgG for hACE2 (1:2000, catalog 172-1011, Bio-Rad) or HRP-conjugated goat anti-rabbit polyclonal IgG for GAPDH (1:2000, catalog 172-1019, Bio-Rad) for 1 hour at room temperature, and the protein signals were detected using a chemiluminescence detection reagent (Chemi-Lumi One L; Nacalai Tesque Inc.) and visualized using the ImageQuant LAS 4000 system (Cytiva).

Techniques: Transgenic Assay, Sequencing

Journal: JCI insight

Article Title: Highly susceptible SARS-CoV-2 model in CAG promoter-driven hACE2-transgenic mice.

doi: 10.1172/jci.insight.152529

Figure Lengend Snippet: Figure 8. Protection against SARS-CoV-2 by immunization of SARS-CoV-2 RBD protein. (A) Schematic showing the experimental schedule. Male and female CAG-hACE2 mice were immunized with RBD-mFc/AddaVax twice before infection and were infected via respiratory tract with SARS-CoV-2 (2 × 103 TCID50, n = 4; 1 × 104 TCID50, n = 4). PBS/AddaVax–immunized CAG-hACE2 mice were used as a control (2 × 103 TCID50, n = 6; 1 × 104 TCID50, n = 6). (B) Expression of RBD-sIgG in plasma from PBS/AddaVax or RBD-mFc/AddaVax–immunized CAG-hACE2 mice at 2 days before infection. Blue and red indicate 2 × 103 TCID50 and 1 × 104 TCID50, respectively. (C and D) Percentage of initial body weight (C) and survival rate (D). Numbers in C represent the number of mice measured for body weight at each time. White and colored circles indicate mice injected with PBS/AddaVax or RBD-mFc/AddaVax, respectively. Blue and red denote infection doses of 2 × 103 TCID50 and 1 × 104 TCID50, respectively. Data are presented as the mean (B) and the mean ± SEM (C). Statistical analyses were performed using 2-tailed unpaired t test for body weight loss (B and C) and log-rank (mantel-cox) test for survival rate (D). P < 0.05 for comparison with RBD-mFc/AddaVax (2 × 103 TCID50) or PBS/AddaVax–immunized mice, respectively.

Article Snippet: Then, the membranes were incubated with HRP-conjugated goat anti-mouse polyclonal IgG for hACE2 (1:2000, catalog 172-1011, Bio-Rad) or HRP-conjugated goat anti-rabbit polyclonal IgG for GAPDH (1:2000, catalog 172-1019, Bio-Rad) for 1 hour at room temperature, and the protein signals were detected using a chemiluminescence detection reagent (Chemi-Lumi One L; Nacalai Tesque Inc.) and visualized using the ImageQuant LAS 4000 system (Cytiva).

Techniques: Infection, Control, Expressing, Clinical Proteomics, Injection, Comparison

Journal: JCI insight

Article Title: Highly susceptible SARS-CoV-2 model in CAG promoter-driven hACE2-transgenic mice.

doi: 10.1172/jci.insight.152529

Figure Lengend Snippet: Figure 9. Protection against SARS-CoV-2 infection by the injection of RBD-Fc–immunized mouse plasma. (A) Schematic showing the experimental schedule. Male and female CAG-hACE2 mice were intravenously injected with pooled plasma from RBD-mFc/AddaVax–immunized mice at doses of 15 μg/shot (n = 6), 50 μg/shot (n = 5), and 150 μg/shot (n = 6) 1 day before SARS-CoV-2 infection and were infected via respiratory tract with SARS-CoV-2 at a dose of 1 × 104 TCID50. CAG-hACE2 mice that were injected with PBS-treated pooled plasma were used as a control (n = 6). (B) The measurement of SARS-CoV-2–neutralizing antibodies in pooled plasma. Black circles and white, violet, and gray diamonds indicate the standard, pooled plasma from PBS/ AddaVax–treated mice, pooled plasma from RBD-mFc/AddaVax–immunized mice, and mouse neutralizing antibody, respectively. (C and D) Percentage of initial body weight (C) and survival rate (D). Numbers in C represent the number of mice measured for body weight at each time. Black circles and orange, blue, and red triangles indicate plasma from PBS/AddaVax–immunized mice and RBD-mFc/AddaVax–immunized mice at doses of 150 ng/mL (n = 6), 500 ng/mL (n = 5), and 1500 ng/mL (n = 6), respectively. Data were presented as the mean (B) and the mean ± SEM (C). Statistical analyses were performed using log-rank (mantel-cox) test for survival rate (D). P < 0.05 for the comparison with PBS-treated pool plasma.

Article Snippet: Then, the membranes were incubated with HRP-conjugated goat anti-mouse polyclonal IgG for hACE2 (1:2000, catalog 172-1011, Bio-Rad) or HRP-conjugated goat anti-rabbit polyclonal IgG for GAPDH (1:2000, catalog 172-1019, Bio-Rad) for 1 hour at room temperature, and the protein signals were detected using a chemiluminescence detection reagent (Chemi-Lumi One L; Nacalai Tesque Inc.) and visualized using the ImageQuant LAS 4000 system (Cytiva).

Techniques: Infection, Injection, Clinical Proteomics, Control, Comparison

Journal: Nature Communications

Article Title: Individual bat virome analysis reveals co-infection and spillover among bats and virus zoonotic potential

doi: 10.1038/s41467-023-39835-1

Figure Lengend Snippet: a Top, homology-modeling structure of the receptor-binding domain (RBD) of Bat SARS-like coronavirus CX1 in complex with human angiotensin-converting enzyme 2 (hACE2). Blue-colored residues on RBD indicate amino acid differences compared with SARS-CoV-2 Wuhan-Hu-1. Bottom, alignment of RBD sequences (residues T333 to G526 of spike protein) of Bat SARS-like coronavirus CX1, SARS-CoV-2 Wuhan-Hu-1 and two closely related bat coronavirus. Only polymorphic sites are shown. The five amino acid differences in the RBD of Bat SARS-like coronavirus CX1 compared to SARS-CoV-2 Wuhan-Hu-1 are marked with blue dots. b Molecular dynamics simulation results of binding energy (top) and binding stability (bottom) of Bat SARS-like coronavirus CX1 RBD-hACE2 complex. c The binding capability of SARS-CoV, SARS-CoV-2 and Bat SARS-like coronavirus CX1 RBD proteins to hACE2 protein was tested with various concentrations of the RBD proteins via ELISA. d The binding kinetics was determined by the biolayer interferometry (BLI) binding analysis. The purified hACE2 were coated on the sensor followed by the injection of various concentrations of SARS-CoV, SARS-CoV-2 and Bat SARS-like coronavirus CX1 RBD proteins. Source data are provided as a Source Data file.

Article Snippet: To detect the binding of hACE2 Horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody (polyclonal) diluted 1:2000 (Jackson ImmunoResearch, catalog number #109-035-098) was added and incubated for 1 h at 37 °C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Purification, Injection

Journal: Journal of Virology

Article Title: A Glycosylated RBD Protein Induces Enhanced Neutralizing Antibodies against Omicron and Other Variants with Improved Protection against SARS-CoV-2 Infection

doi: 10.1128/jvi.00118-22

Figure Lengend Snippet: Introduction of glycan probe and characterization of glycosylated mutant SARS-CoV-2 RBD protein. (A) Crystal structure of SARS-CoV-2 RBD (PDB access code: 6M0J ). The core structure is colored in cyan, and the receptor-binding motif (RBM) in magenta. Mutated residue (Asn519) is shown where an N-linked glycan probe was introduced. (B) Receptor-binding affinity of mutant receptor-binding domain (RBD) (MU-RBD) subunit vaccine. An enyzme-linked immunosorbent assay (ELISA) was carried out to assess the binding of MU-RBD protein to soluble human angiotensin-converting enzyme 2 (hACE2) protein. Prototypic wild-type (WT)-RBD protein was included as comparison. (C to G) Antibody-binding affinity of MU-RBD subunit vaccine. ELISA was carried out to detect the binding of MU-RBD protein to SARS-CoV-2 RBD-specific neutralizing nanobodies Nanosota-1C-Fc (C) and Ty1 (D), neutralizing MAbs CV30 (E) and EY6A (F), and neutralizing human sera (G). WT-RBD was used as comparison. Data (panels B to G) are presented as mean ± standard error of the mean (SEM) of quadruple wells. The experiments were repeated twice, resulting in similar results.

Article Snippet: Briefly, the ELISA plates were coated with respective RBD protein (2 μg/mL) at 4°C overnight and blocked with 2% fat-free milk in PBS containing 0.05% Tween 20 (PBST) at 37°C for 2 h. The plates were then incubated with hACE2 protein (20 or 5 μg/mL, R&D Systems) at 37°C for 2 h. After washing 3 times using PBST, the binding was detected by goat anti-hACE2 IgG antibody (0.2 μg/mL, R&D Systems), followed by horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG antibody (1:5,000, R&D Systems).

Techniques: Glycoproteomics, Mutagenesis, Binding Assay, Residue, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Journal of Virology

Article Title: A Glycosylated RBD Protein Induces Enhanced Neutralizing Antibodies against Omicron and Other Variants with Improved Protection against SARS-CoV-2 Infection

doi: 10.1128/jvi.00118-22

Figure Lengend Snippet: Immunization and challenge schedules. C57BL/6 and hACE2-transgenic (Tg) mice were immunized with the prototypic WT-RBD, MU-RBD, or phosphate-buffered saline (PBS) control in the presence of adjuvants for three times at 3-week intervals. Sera were collected at 10 days after the second and third immunizations, and tested for IgG antibodies and neutralizing antibodies against pseudotyped and authentic SARS-CoV-2 original strain and variants. Immunized hACE2-Tg mice were challenged with SARS-CoV-2 original strain or Delta variant and observed for survival and weight changes for 14 days. Immunized C57BL/6 mice were challenged with a mouse-adapted SARS-CoV-2 variant and evaluated for viral titers in the lungs at day 2 after virus challenge.

Article Snippet: Briefly, the ELISA plates were coated with respective RBD protein (2 μg/mL) at 4°C overnight and blocked with 2% fat-free milk in PBS containing 0.05% Tween 20 (PBST) at 37°C for 2 h. The plates were then incubated with hACE2 protein (20 or 5 μg/mL, R&D Systems) at 37°C for 2 h. After washing 3 times using PBST, the binding was detected by goat anti-hACE2 IgG antibody (0.2 μg/mL, R&D Systems), followed by horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG antibody (1:5,000, R&D Systems).

Techniques: Transgenic Assay, Saline, Control, Variant Assay, Virus

Journal: Journal of Virology

Article Title: A Glycosylated RBD Protein Induces Enhanced Neutralizing Antibodies against Omicron and Other Variants with Improved Protection against SARS-CoV-2 Infection

doi: 10.1128/jvi.00118-22

Figure Lengend Snippet: Glycosylated mutant SARS-CoV-2 RBD protein elicited improved neutralizing antibodies against SARS-CoV-2 Alpha, Beta, Gamma, and Epsilon variants. hACE2-Tg mice were immunized with the prototypic WT-RBD or MU-RBD protein and boosted twice at 3 weeks. Mice injected with PBS were included as control. Mouse sera collected 10 days after the third immunization (A to E) and 10 days after the second immunization (F to J) were assessed for neutralizing activity against infection of pseudoviruses expressing S protein of the SARS-CoV-2 original strain and each variant harboring mutation(s) at the indicated amino acid(s), respectively. Alpha variant (B.1.1.7 lineage) contains all 10 amino acid mutations (69 to 70 deletion, 145 deletion, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H) in the S protein of SARS-CoV-2. Neutralizing activity was expressed as 50% neutralizing antibody titers (NT 50 ) against pseudovirus infection in 293T cells expressing hACE2 receptor (hACE2/293T). Data are presented as mean ± SEM of quadruple wells from pooled sera of five mice in each group. ***, ( P < 0.001) indicates significant differences between the MU-RBD and WT-RBD groups. The experiments were repeated twice, resulting in similar results. (K to L) Calculated neutralizing immunogenicity index (NII) values based on the neutralizing antibody titers (NT 50 ) and the following formula: (NT 50-WT – NT 50-MU )/NT 50-WT , where NT 50-WT and NT 50-MU represent NT 50 induced by the WT-RBD and MU-RBD, respectively.

Article Snippet: Briefly, the ELISA plates were coated with respective RBD protein (2 μg/mL) at 4°C overnight and blocked with 2% fat-free milk in PBS containing 0.05% Tween 20 (PBST) at 37°C for 2 h. The plates were then incubated with hACE2 protein (20 or 5 μg/mL, R&D Systems) at 37°C for 2 h. After washing 3 times using PBST, the binding was detected by goat anti-hACE2 IgG antibody (0.2 μg/mL, R&D Systems), followed by horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG antibody (1:5,000, R&D Systems).

Techniques: Mutagenesis, Injection, Control, Activity Assay, Infection, Expressing, Variant Assay, Immunopeptidomics

Journal: Journal of Virology

Article Title: A Glycosylated RBD Protein Induces Enhanced Neutralizing Antibodies against Omicron and Other Variants with Improved Protection against SARS-CoV-2 Infection

doi: 10.1128/jvi.00118-22

Figure Lengend Snippet: Antibody responses induced by glycosylated mutant SARS-CoV-2 RBD protein. C57BL/6 and hACE2-Tg mice were immunized with the prototypic WT-RBD or MU-RBD protein, or with PBS control as described above, and collected for sera 10 days after the second and third immunizations. ELISA for detection of IgG antibodies specific to the SARS-CoV-2 WT-RBD (A, E, I, and M), Delta-RBD (B, F, J, and N), Omicron BA.1-RBD (C, G, K, and O), and Omicron BA.2-RBD (D, H, L, and P) from sera of C57BL/6 mice (A to H) and hACE2-Tg (I to P) mice after the second (A to D, I to L) and third (E to H, M to P) immunizations. Data are presented as mean ± SEM of quadruple wells from pooled sera of five mice in each group. The experiments were repeated twice, resulting in similar results.

Article Snippet: Briefly, the ELISA plates were coated with respective RBD protein (2 μg/mL) at 4°C overnight and blocked with 2% fat-free milk in PBS containing 0.05% Tween 20 (PBST) at 37°C for 2 h. The plates were then incubated with hACE2 protein (20 or 5 μg/mL, R&D Systems) at 37°C for 2 h. After washing 3 times using PBST, the binding was detected by goat anti-hACE2 IgG antibody (0.2 μg/mL, R&D Systems), followed by horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG antibody (1:5,000, R&D Systems).

Techniques: Mutagenesis, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: A Glycosylated RBD Protein Induces Enhanced Neutralizing Antibodies against Omicron and Other Variants with Improved Protection against SARS-CoV-2 Infection

doi: 10.1128/jvi.00118-22

Figure Lengend Snippet: Glycosylated mutant SARS-CoV-2 RBD protein elicited improved neutralizing antibodies against SARS-CoV-2 Delta and Omicron variants. The same sera described in were tested for neutralizing antibodies against infection of pseudoviruses expressing S protein of the SARS-CoV-2 original strain (A, D, G, and J), Delta variant (harboring L452R-T478K-P681R mutations in the S1 region) (B, E, H, and K), and Omicron variant (harboring 38 amino acid mutations in the S protein) (C, F, I, and L), respectively. These sera were collected from C57BL/6 (A to F) and hACE2-Tg (G to L) mice 10 days after the second (A to C, G to I) and third (D to F, J to L) immunizations. Sera from C57BL/6 (M to O) and hACE2-Tg (P to R) mice after the third immunization were also tested for neutralizing antibodies against infection of authentic SARS-CoV-2 original strain (M and P), Delta (N and Q), and Omicron (O and R) variants. Neutralizing activity was calculated as NT 50 against infection of each pseudotyped or authentic SARS-CoV-2. **, ( P < 0.01) and ***, ( P < 0.001) indicate significant differences between the MU-RBD and WT-RBD groups. Data (panels A to R) are shown as mean ± SEM of quadruple wells from pooled sera of five mice in each group. The experiments were repeated twice, resulting in similar results. (S to X) Calculated neutralizing NII values based on the neutralizing antibody titers (NT 50 ) and the formula described in .

Article Snippet: Briefly, the ELISA plates were coated with respective RBD protein (2 μg/mL) at 4°C overnight and blocked with 2% fat-free milk in PBS containing 0.05% Tween 20 (PBST) at 37°C for 2 h. The plates were then incubated with hACE2 protein (20 or 5 μg/mL, R&D Systems) at 37°C for 2 h. After washing 3 times using PBST, the binding was detected by goat anti-hACE2 IgG antibody (0.2 μg/mL, R&D Systems), followed by horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG antibody (1:5,000, R&D Systems).

Techniques: Mutagenesis, Infection, Expressing, Variant Assay, Activity Assay

Journal: Journal of Virology

Article Title: A Glycosylated RBD Protein Induces Enhanced Neutralizing Antibodies against Omicron and Other Variants with Improved Protection against SARS-CoV-2 Infection

doi: 10.1128/jvi.00118-22

Figure Lengend Snippet: Glycosylated mutant SARS-CoV-2 RBD protein induced enhanced protective efficacy against infection of the SARS-CoV-2 original strain and Delta variant. At 25 days after the third immunization, hACE2-Tg mice (5 mice/group) were challenged with prototype SARS-CoV-2 human strain (2019n-CoV/USA-WA1/2009, 5,000 PFU/mouse), and observed for survival (A) and body weight changes (B) for 14 days post-challenge. In addition, 50 days after the third immunization, hACE2-Tg mice (5 mice/group) were challenged with SARS-CoV-2 Delta variant (10,000 PFU/mouse) and observed for survival (C) and body weight changes (D) for 14 days post-challenge. At 50 days after the third immunization, C57BL/6 mice were challenged with mouse-adapted strain of SARS-CoV-2 (SARS2-N501Y MA30 , 5,000 PFU/mouse), and measured for lung viral titers on day 2 post-challenge. Data (in panels B, D, and E) are shown as mean ± SEM of 5 mice in each group. *, ( P < 0.05); **, ( P < 0.01); and ***, ( P < 0.001) indicate significant differences between the WT-RBD and PBS (cyan), MU-RBD and PBS (red), or WT-RBD and MU-RBD (black) groups.

Article Snippet: Briefly, the ELISA plates were coated with respective RBD protein (2 μg/mL) at 4°C overnight and blocked with 2% fat-free milk in PBS containing 0.05% Tween 20 (PBST) at 37°C for 2 h. The plates were then incubated with hACE2 protein (20 or 5 μg/mL, R&D Systems) at 37°C for 2 h. After washing 3 times using PBST, the binding was detected by goat anti-hACE2 IgG antibody (0.2 μg/mL, R&D Systems), followed by horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG antibody (1:5,000, R&D Systems).

Techniques: Mutagenesis, Infection, Variant Assay