Journal: mBio

Article Title: A new mechanism of respiratory syncytial virus entry inhibition by small-molecule to overcome K394R-associated resistance

doi: 10.1128/mbio.01385-24

Figure Lengend Snippet: In vitro anti-RSV activity of CL-A3-7. (A) A schematic of the analysis of anti-RSV activity. (B) 3D structural view of CL-A3-7 bound to residues of prefusion RSV F (PDB 7UJA) with the K394R mutation. (C) Effects of CL-A3-7 on cell viability and viral infection in HEp-2 cells were determined by CCK-8 and cytopathic effect (CPE) inhibition assays. Values are normalized to those obtained for DMSO-treated cells and presented as mean ± SD, n = 3 biological replicates. (D) Viral NS1 mRNA level in HEp-2 cells in the presence of CL-A3-7 (10 µM) or DMSO. Growth kinetics of WT (E) and K394R variant (F) in HEp-2 cells treated with CL-A3-7 (40 µM) or DMSO. Data are mean ± SD, n = 3 biological replicates. Statistically significant differences in the comparison between DMSO-treated and WT-treated cells at each time point are indicated by asterisks. (G, H) Representative images of immunostaining of the viral F protein in WT- and K394R-infected cells at 48 hpi. Relative immunofluorescent intensity (RFI) in each group of cells was analyzed using ImageJ software (G and H, right). Bar, 100 µm. (I) HEp-2 cells were inoculated with RSV A2 in the presence of increasing concentrations of the inhibitors. The starting concentrations of CL-A3-7, BMS-433771, and JNJ-53718678 were 2 µM, 20 nM, and 2 nM, respectively. Supernatants from cell cultures exhibiting cytopathic effects were harvested for the next passage of infection. For the combination treatments, CL-A3-7 was used at 8 µM after the third passage. Resistance fold was calculated based on the IC 50 changes. (D–H) Data are mean ± SD, n = 3 biological replicates. The two-tailed Student’s t test was used to measure the statistical difference between groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To determine the binding of IGF1R to RSV F, Maxisorp (Nunc) ELISA plates were coated with IGF1R protein (SinoBiological, 10164-H49H1-B) for 12 h at 4°C.

Techniques: In Vitro, Activity Assay, Mutagenesis, Infection, CCK-8 Assay, Inhibition, Variant Assay, Comparison, Immunostaining, Software, Two Tailed Test

Journal: mBio

Article Title: A new mechanism of respiratory syncytial virus entry inhibition by small-molecule to overcome K394R-associated resistance

doi: 10.1128/mbio.01385-24

Figure Lengend Snippet: Action mechanism of CL-A3-7 against RSV. (A) HEp-2 cells were inoculated with octadecyl rhodamine B (R18)-labeled virions in the presence of CL-A3-7 (40 µM), ribavirin (40 µM), or heparin (4 µM) for 2 h at 37°C. Virus-cell fusion was measured by flow cytometry (FCM). Representative histograms of the FCM data (top panel) and the mean fluorescence intensity (MFI; bottom panel) are indicated. (B) HEK293T cells were transfected with F-WT or F-K394R plasmids and then treated with CL-A3-7 (20 or 40 µM) or DMSO. At 36 h after transfection, the cell–cell fusion activity was determined using a dual-luciferase system. RFA, relative fusion activity. (C) C636 mosquito cells were transfected with plasmids expressing IGF1R and treated with CL-A3-7 or DMSO, and then inoculated with the WT or K394R variant. Representative images of cells with DAPI (blue) and anti-RSV F (green) staining are shown. Scale bar, 20 µm. (D) Western blot and immunoprecipitation analysis of the binding of IGF1R with F-WT or F-K394R. The IGF1R band intensity after immunoblotting was calculated. (E) Binding of IGF1Rs on the cell surface in F-WT- or F-K394R- transfected cells. CL-A3-7 (20 µM). Representative FCM histograms (left panel) and the MFI correlated with the IGF1Rs on the cell surface were analyzed using FlowJo v.10.0 (right panel). (F, G) Left panel, the representative images of colocalization between IGF1R and F-WT (top) or between IGF1R and F-K394R (bottom). Right panel, viral F- and IGF1R-associated relative fluorescence intensity (RFI) are indicated. HEK293T cells were transfected with F-WT or F-K394R plasmids and then treated with IGF1R (2 µg) in the presence of CL-A3-7 (40 µM) or DMSO, followed by incubation with anti-IGF1R and anti-RSV F antibodies, and then stained with Alexa Fluor 488 and 594-conjugated antibodies, respectively, and subsequently detected with laser scanning microscope. Scale bar, 10 µm. (H) The interactions of IGF1R with F (ecto) - or with F (ecto) -K394R were determined by MST assay. The RED-NHS dye-labeled F (ecto) - and F (ecto) -K394R were premixed with CL-A3-7 (40 µM) and then treated with IGF1R protein. (I) Binding of IGF1R with F (ecto) - or F (ecto) -K394R in the presence of CL-A3-7 or DMSO was determined by ELISA. (A, B, E–G, I) Data are mean ± SD, n = 3 or 4 biological replicates. The two-tailed Student’s t test was used to measure the statistical difference between groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., no significant difference.

Article Snippet: To determine the binding of IGF1R to RSV F, Maxisorp (Nunc) ELISA plates were coated with IGF1R protein (SinoBiological, 10164-H49H1-B) for 12 h at 4°C.

Techniques: Labeling, Virus, Flow Cytometry, Fluorescence, Transfection, Activity Assay, Luciferase, Expressing, Variant Assay, Staining, Western Blot, Immunoprecipitation, Binding Assay, Incubation, Laser-Scanning Microscopy, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Biotechnology Reports

Article Title: Immunogenicity of a recombinant plant-produced respiratory syncytial virus F subunit vaccine in mice

doi: 10.1016/j.btre.2023.e00826

Figure Lengend Snippet: Schematic representation of the T-DNA region of the pBYR2e RSV-F Fc fusion plant expression vector. The T-DNA region plays a crucial role in facilitating the transfer of the gene of interest into plant cells. It includes the left and right borders, known as RB and LB, which serve as the boundaries for gene transfer. The Pin II 3′ sequence derived from potato proteinase inhibitor II acts as a border element. The vector also incorporates several important components, such as the Tomato Bushy Stunt Virus (TBSV) RNA silencing suppressor, P19; the Cauliflower Mosaic Virus (CaMV) 35s promoter, P35s; the CaMV enhancer, P35s × 2; the tobacco extension gene region, Ext3′ FL, 3′; the tobacco RB7 promoter, Rb7 5′ del; the Bean Yellow Dwarf Virus (BeYDV) short intergenic region, SIR; the BeYDV long intergenic region, LIR; and the BeYDV replication initiation proteins, Rep and RepA, along with C2/C1.

Article Snippet: Human RSV fusion glycoprotein was detected with rabbit anti RSV-F mAb (Sino Biological, Cat#11049-R302, Beijing, China) diluted 1:3000 in 1x PBS containing 0.5 % BSA and 0.01 % Tween-20 added to each well and incubated for 1 h at 37 °C.

Techniques: Expressing, Plasmid Preparation, Sequencing, Derivative Assay, Virus

Journal: Biotechnology Reports

Article Title: Immunogenicity of a recombinant plant-produced respiratory syncytial virus F subunit vaccine in mice

doi: 10.1016/j.btre.2023.e00826

Figure Lengend Snippet: SDS-PAGE and western blot analysis of plant-produced RSV-F Fc fusion protein. SDS-PAGE stained with Instant Blue™ under non-reducing (a) and reducing condition (b) whereas, lane M, Protein ladder; lane 1, purified RSV F-Native Fc fusion protein; lane 2, purified SC-TM Fc fusion protein. Western blot analysis under non-reducing conditions (c) and reducing condition (d), the membrane was probed with anti-human IgG Fc HRP. The arrowhead indicates the major band.

Article Snippet: Human RSV fusion glycoprotein was detected with rabbit anti RSV-F mAb (Sino Biological, Cat#11049-R302, Beijing, China) diluted 1:3000 in 1x PBS containing 0.5 % BSA and 0.01 % Tween-20 added to each well and incubated for 1 h at 37 °C.

Techniques: SDS Page, Western Blot, Produced, Staining, Purification, Membrane

Journal: Biotechnology Reports

Article Title: Immunogenicity of a recombinant plant-produced respiratory syncytial virus F subunit vaccine in mice

doi: 10.1016/j.btre.2023.e00826

Figure Lengend Snippet: The binding activity of the plant-produced RSV-F Fc fusion protein to Motavizumab (anti-RSV mAb) was analyzed by ELISA. The plant-produced RBD-Fc was used as a negative control. Data are presented as mean ± SD of triplicates.

Article Snippet: Human RSV fusion glycoprotein was detected with rabbit anti RSV-F mAb (Sino Biological, Cat#11049-R302, Beijing, China) diluted 1:3000 in 1x PBS containing 0.5 % BSA and 0.01 % Tween-20 added to each well and incubated for 1 h at 37 °C.

Techniques: Binding Assay, Activity Assay, Produced, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Biotechnology Reports

Article Title: Immunogenicity of a recombinant plant-produced respiratory syncytial virus F subunit vaccine in mice

doi: 10.1016/j.btre.2023.e00826

Figure Lengend Snippet: Schematic illustration of immunization protocol and blood collection. Groups of mice (five mice per group) were intramuscularly immunized with 5 µg of RSV-F Fc fusion protein with 3M-SE or 3M-Alum adjuvant. A booster dose was administered 21 days after the first immunization. Mice sera were collected on day 0 (pre-immune sera) and day 35 post-immunization.

Article Snippet: Human RSV fusion glycoprotein was detected with rabbit anti RSV-F mAb (Sino Biological, Cat#11049-R302, Beijing, China) diluted 1:3000 in 1x PBS containing 0.5 % BSA and 0.01 % Tween-20 added to each well and incubated for 1 h at 37 °C.

Techniques: Adjuvant

Journal: Biotechnology Reports

Article Title: Immunogenicity of a recombinant plant-produced respiratory syncytial virus F subunit vaccine in mice

doi: 10.1016/j.btre.2023.e00826

Figure Lengend Snippet: Immunogenicity of RSV-F Fc fusion protein in mice. The mice sera was collected on day 35 and RSV specific titer was analyzed by ELISA using RSV-F his protein as the capture antigen. Data were presented as GMT ± 95 % CI of the endpoint titer ( n = 5). A nonparametric test was used to compare each group. (∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001).

Article Snippet: Human RSV fusion glycoprotein was detected with rabbit anti RSV-F mAb (Sino Biological, Cat#11049-R302, Beijing, China) diluted 1:3000 in 1x PBS containing 0.5 % BSA and 0.01 % Tween-20 added to each well and incubated for 1 h at 37 °C.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Mucosal immunology

Article Title: Respiratory syncytial virus activates epidermal growth factor receptor to suppress interferon regulatory factor 1-dependent interferon-lambda and antiviral defense in airway epithelium

doi: 10.1038/mi.2017.120

Figure Lengend Snippet: (A) BEAS-2b cells were treated with UV-inactivated RSV (UV RSV MOI 1; filled columns) (n=5 independent experiments, mean ± SEM; *** p<0.0001 vs. UV RSV). (B) NADPH activity was measured in BEAS-2b cells at 2 h. Cells were treated with serum-free medium alone (Control, empty columns), DPI (3 μM) alone, with UV RSV (MOI 1; filled columns) alone, or with the addition of DPI (n=3–6 independent experiments, mean ± SEM; ** p<0.005 vs. control; ### p<0.005 vs. UV RSV alone). (C) BEAS-2b cells were treated with serum-free medium alone (empty columns), Poly I:C alone (Poly I:C 100 μg/mL; filled colums), and Poly I:C plus RSV F protein (F protein 20 μg/mL; filled columns), and IRF-1 mRNA was analyzed by quantitative RT-PCR (n=6 independent experiments; *** p<0.0001 vs. serum-free medium; ### p<0.001 vs. RSV alone).

Article Snippet: RSV F protein was obtained from Sino Biological (Beijing, China).

Techniques: Activity Assay, Quantitative RT-PCR

Journal: Human Vaccines & Immunotherapeutics

Article Title: Neutralizing activity of anti-respiratory syncytial virus monoclonal antibody produced in Nicotiana benthamiana

doi: 10.1080/21645515.2024.2327142

Figure Lengend Snippet: Schematic representation of the T-DNA region of the pBYR2e RSV-F Fc fusion plant expression vector. The T-DNA region plays a crucial role in facilitating the transfer of the gene of interest into plant cells. It includes the left border (LB) and right border (RB) which serve as the boundaries for gene transfer. The Pin II 3’ sequence derived from potato proteinase inhibitor II acts as a border element helping to facilitate the insertion of the desired genes into the plant genome. The vector also incorporates several important components, such as the Tomato Bushy Stunt Virus (TBSV) RNA silencing suppressor, P19; the Cauliflower Mosaic Virus (CaMV) 35s promoter, P35s; the CaMV enhancer, P35s×2; the tobacco extension gene region, Ext3’ FL, 3‘; the tobacco RB7 promoter, Rb7 5’ del; the Bean Yellow Dwarf Virus (BeYDV) short intergenic region, SIR; the BeYDV long intergenic region, LIR; and the BeYDV replication initiation proteins, Rep and RepA, along with C2/C1. .

Article Snippet: Human RSV fusion glycoprotein was detected with rabbit anti-RSV-F mAb (Cat:11049-R302-H, Sino Biological, Beijing, China) diluted 1:3000 in PBS containing 0.5% BSA and 0.01% Tween-20 added to each well and incubated for 1 h at 37°C.

Techniques: Expressing, Plasmid Preparation, Sequencing, Derivative Assay, Virus

Journal: Human Vaccines & Immunotherapeutics

Article Title: Neutralizing activity of anti-respiratory syncytial virus monoclonal antibody produced in Nicotiana benthamiana

doi: 10.1080/21645515.2024.2327142

Figure Lengend Snippet: Binding characteristics of plant-produced anti-RSV mAbs. ELISA was used to quantify the specific binding to the RSV-F his-tagged monomer protein. The purified plant produced anti-RSV mAbs, and the plant produced nivolumab (anti PD-1, as a negative control). The binding activity of the antibody was detected with HRP-conjugated anti-human IgG antibody. The data are shown as the mean of triplicates and error bars represent standard deviation. The EC 50 was calculated by using GraphPad Prism software 9.3.

Article Snippet: Human RSV fusion glycoprotein was detected with rabbit anti-RSV-F mAb (Cat:11049-R302-H, Sino Biological, Beijing, China) diluted 1:3000 in PBS containing 0.5% BSA and 0.01% Tween-20 added to each well and incubated for 1 h at 37°C.

Techniques: Binding Assay, Produced, Enzyme-linked Immunosorbent Assay, Purification, Negative Control, Activity Assay, Standard Deviation, Software