Journal: PLoS Pathogens

Article Title: Endocytosis of flavivirus NS1 is required for NS1-mediated endothelial hyperpermeability and is abolished by a single N-glycosylation site mutation

doi: 10.1371/journal.ppat.1007938

Figure Lengend Snippet: (A) Structural model of a DENV2 NS1 dimer showing site-specific mutations. Color-coding highlights two NS1 monomers (one in green, one in yellow) and amino acid changes in corresponding constructs (blue highlights changes in the green monomer; pink highlights changes in the yellow monomer). WT, N130Q, N207Q, and N130Q+N207Q are all on the DENV2 NS1 backbone. PDB ID: 4O6B . ( B) Western blot of SDS-PAGE of in-house purified and dialyzed DENV NS1-WT and NS1-N207Q mutant, showing monomeric form, using anti-NS1 mAb (7E11) for detection. (C) Silver stain of native PAGE showing oligomeric forms of both purified and dialyzed in-house DENV NS1-WT and NS1-N207Q mutant (MW: >250 kDa). (D) Silver stain of SDS-PAGE gels of purified and dialyzed in-house DENV NS1-WT and NS1-N207Q NS1 mutant, showing purity, using the Pierce Silver Stain Kit. Positive control, NS1 + , DENV NS1 from Native Antigen Company; Neg, dialyzed elution buffer. Molecular weight marker, 10-250-kDa protein ladder. (E-F) DENV NS1 triggers endothelial permeability of HPMEC ( E ) and HBMEC ( F ) monolayers evaluated by TEER (Ohms) at the indicated time-points. Relative TEER was calculated as previously described . Data are representative of 2–3 individual experiments. NS1+, NS1 from Native Antigen Company.

Article Snippet: Confluent HPMEC monolayers grown on gelatin-coated coverslips (0.2%, Sigma) were treated with 5 μg/ml of DENV2 NS1+ from the Native Antigen Company (Oxfordshire, UK) as a positive control, WT DENV NS1 produced in-house, or the DENV NS1-N207Q mutant and incubated for 1 hour at 37°C/4°C or for 6 hours at 37°C, depending on the experiment.

Techniques: Construct, Western Blot, SDS Page, Purification, Mutagenesis, Silver Staining, Clear Native PAGE, Positive Control, Molecular Weight, Marker, Permeability

Journal: PLoS Pathogens

Article Title: Endocytosis of flavivirus NS1 is required for NS1-mediated endothelial hyperpermeability and is abolished by a single N-glycosylation site mutation

doi: 10.1371/journal.ppat.1007938

Figure Lengend Snippet: (A) NS1 protein binding (green, top row) to HPMEC 6 hpt at 37°C was visualized via IFA. The integrity of the EGL was assessed by the presence of sialic acid (Sia) surface expression, stained with WGA-A647 (red, 2 nd row; merge of NS1 and Sia), as well as cathepsin L (CTSL) activity (red, 3 rd row) and heparan sulfate surface expression (green, bottom row), in HPMEC 6 hpt with NS1 at 37°C, as visualized via IFA. Nuclei were stained with Hoechst (blue). Images (20X; scale bars, 50 μm) are representative of 2 independent experiments. (B) Mean Fluorescence Intensity (MFI) was used to quantify the amount of NS1 binding to the cell surface, (C) sialic acid surface expression, (D) cathepsin L activity, and (E) heparan sulfate expression on HPMEC in Fig 2A . The means ± standard error of the mean (SEM) of two individual experiments run in duplicate are shown. ns = not significant; *, p<0.05; ***, p<0.001; ****, p<0.0001.

Article Snippet: Confluent HPMEC monolayers grown on gelatin-coated coverslips (0.2%, Sigma) were treated with 5 μg/ml of DENV2 NS1+ from the Native Antigen Company (Oxfordshire, UK) as a positive control, WT DENV NS1 produced in-house, or the DENV NS1-N207Q mutant and incubated for 1 hour at 37°C/4°C or for 6 hours at 37°C, depending on the experiment.

Techniques: Protein Binding, Expressing, Staining, Activity Assay, Fluorescence, Binding Assay

Journal: PLoS Pathogens

Article Title: Endocytosis of flavivirus NS1 is required for NS1-mediated endothelial hyperpermeability and is abolished by a single N-glycosylation site mutation

doi: 10.1371/journal.ppat.1007938

Figure Lengend Snippet: (A) NS1 protein binding (green) to HPMEC 1 hpt at 4°C (top row) or 37°C (bottom row) was visualized via IFA. Images (20X; scale bars, 50 μm) are representative of 2 independent experiments. (B) MFI was used to quantify the amount of NS1 binding to the cell surface 1 hpt at 4°C and 37°C in Fig 3A . The means ± SEM of two individual experiments run in duplicate are shown. ns = not significant; ****, p<0.0001. (C) Confluent HPMEC monolayers were exposed to 10 μg/ml of different NS1 proteins and incubated at 37°C for 1 hours. Trypsin was used to remove surface-bound NS1, and cell lysates were analyzed by Western blot. Western blot shows detection of the internalized DENV NS1 protein, GAPDH (loading control), and Rab5, an early endosome marker. ( D ) Quantitation of C. Each bar represents the mean ± standard error of the mean (SEM) of densitometry values normalized to the control protein α-tubulin from two independent experiments. (E) Co-localization of NS1 proteins (red), as indicated, with Rab5 (green) in HPMEC. Co-localization is shown in yellow in merge image or white in co-localization panel (JACoP, ImageJ). Nuclei are stained with Hoechst (blue). Images (40X; scale bars, 10 μm) are representative of 2 individual experiments run in duplicate. NS1+, NS1 from Native Antigen Company. (F) Quantification of DENV NS1 and Rab5 colocalization in HPMEC in Fig 3E . Quantification of the amount of spatial overlap between the two signals (NS1 in red and Rab5 in green) in Fig 3E was obtained using four different frames from the maximum projections of two RGB images based on the object-based approach (JACoP) and defined by the Manders’ Coefficient as previously described . The colocalization coefficient was normalized taking into account the signal obtained from 300 cells per image. Each bar represents the mean ± SEM of MFI values in the colocalization analyses obtained from two independent experiments. NS1+, NS1 from Native Antigen Company. ns = not significant; ***, p<0.001.

Article Snippet: Confluent HPMEC monolayers grown on gelatin-coated coverslips (0.2%, Sigma) were treated with 5 μg/ml of DENV2 NS1+ from the Native Antigen Company (Oxfordshire, UK) as a positive control, WT DENV NS1 produced in-house, or the DENV NS1-N207Q mutant and incubated for 1 hour at 37°C/4°C or for 6 hours at 37°C, depending on the experiment.

Techniques: Protein Binding, Binding Assay, Incubation, Western Blot, Control, Marker, Quantitation Assay, Staining

Journal: PLoS Pathogens

Article Title: Endocytosis of flavivirus NS1 is required for NS1-mediated endothelial hyperpermeability and is abolished by a single N-glycosylation site mutation

doi: 10.1371/journal.ppat.1007938

Figure Lengend Snippet: Confluent HPMEC monolayers were exposed to 10 μg/ml of different NS1 proteins and incubated at 37°C for 30 minutes. Colocalization of NS1 proteins (red, left column), as indicated, with either (A) clathrin or (B) caveolin (green, second column) in HPMEC. Colocalization is shown in yellow in merge images (third column) or white in colocalization panels (fourth column; JACoP, ImageJ). Nuclei were stained with Hoechst (blue). Images (40X; scale bars, 5 μm) are representative of 2 independent experiments performed in duplicate. (C-D) Quantification of the amount of spatial overlap between the two signals, NS1 and clathrin (C) , or NS1 and caveolin (D) in Fig 4A and 4B , respectively. The means ± SEM of two individual experiments run in duplicate are shown. (E) Clathrin-mediated endocytosis inhibitor Pitstop 2 prevents colocalization of WT DENV NS1 with Rab5 in HPMEC. Colocalization of NS1 proteins (red, first column) on HPMEC treated with either DMSO (top row) or Pitstop 2 (bottom row) with Rab5 (green, second column) 1.5 hpt with NS1. Colocalization is shown in yellow in merge images (third column) or white in colocalization panels (fourth column; JACoP, ImageJ). Nuclei were stained with Hoechst (blue). Images (40X; scale bars, 5 μm) are representative of 2 independent experiments performed in duplicate. (F) Quantification of the amount of spatial overlap between the two signals, NS1 and Rab5 in Fig 4E . The means ± SEM of two individual experiments run in duplicate are shown. ns = not significant; **, p<0.01.

Article Snippet: Confluent HPMEC monolayers grown on gelatin-coated coverslips (0.2%, Sigma) were treated with 5 μg/ml of DENV2 NS1+ from the Native Antigen Company (Oxfordshire, UK) as a positive control, WT DENV NS1 produced in-house, or the DENV NS1-N207Q mutant and incubated for 1 hour at 37°C/4°C or for 6 hours at 37°C, depending on the experiment.

Techniques: Incubation, Staining

Journal: PLoS Pathogens

Article Title: Endocytosis of flavivirus NS1 is required for NS1-mediated endothelial hyperpermeability and is abolished by a single N-glycosylation site mutation

doi: 10.1371/journal.ppat.1007938

Figure Lengend Snippet: (A) The integrity of the EGL on HPMEC was assessed by the presence of heparan sulfate surface expression (green, bottom row), as well as cathepsin L (CTSL) activity (red, top row), at 6 hpt with DENV NS1 (5 μg/ml) in the presence or absence of Pitstop 2 (12.5 μM), or Pitstop 2 alone, at 37°C, as visualized via IFA. Nuclei were stained with Hoechst (blue). Images (20X; scale bars, 50 μm) are representative of 3 independent experiments. (B) Quantification of MFI in (A) from 3 independent experiments expressed as either fold change of cathepsin L activity from Pitstop 2 control values (left) or normalized percentage of heparan sulfate from Pitstop 2 control values . Error bars indicate SEM. **, p<0.01; ***, p<0.001. (C) HPMEC were grown on Transwell semi-permeable membranes (0.4 μm pore size), and the apical chamber was treated with either DENV NS1+ (5 μg/ml) and DMSO (blue squares); the dynamin inhibitor Dynasore (25 μM) alone (purple diamonds) or Dynasore and NS1 (orange triangles); or the clathrin-mediated endocytosis inhibitor Pitstop 2 (12.5 μM) alone (light green triangles) or Pitstop 2 and NS1 (pink circles). A TEER assay was used to evaluate the effect of NS1 and inhibitors on endothelial permeability at indicated time-points over 48 hours. (^) represents change of medium. Relative TEER values from 2 independent experiments performed in duplicate are plotted. Error bars indicate standard error of the mean (SEM).

Article Snippet: Confluent HPMEC monolayers grown on gelatin-coated coverslips (0.2%, Sigma) were treated with 5 μg/ml of DENV2 NS1+ from the Native Antigen Company (Oxfordshire, UK) as a positive control, WT DENV NS1 produced in-house, or the DENV NS1-N207Q mutant and incubated for 1 hour at 37°C/4°C or for 6 hours at 37°C, depending on the experiment.

Techniques: Expressing, Activity Assay, Staining, Control, Pore Size, Permeability

Journal: PLoS Pathogens

Article Title: Endocytosis of flavivirus NS1 is required for NS1-mediated endothelial hyperpermeability and is abolished by a single N-glycosylation site mutation

doi: 10.1371/journal.ppat.1007938

Figure Lengend Snippet: (A) HPMEC were transfected with the indicated siRNA for 72 hours then treated with 5 ug/ml DENV2 NS1. The integrity of the EGL on HPMEC was assessed 6 hours post NS1-treatment by the presence of heparan sulfate (green) on the cell surface. Nuclei were stained with Hoechst (blue). Images (20X; scale bars, 50 μm). (B) Quantification of MFI in (A) from 3 independent experiments. ns = not significant, **, P < 0.01.

Article Snippet: Confluent HPMEC monolayers grown on gelatin-coated coverslips (0.2%, Sigma) were treated with 5 μg/ml of DENV2 NS1+ from the Native Antigen Company (Oxfordshire, UK) as a positive control, WT DENV NS1 produced in-house, or the DENV NS1-N207Q mutant and incubated for 1 hour at 37°C/4°C or for 6 hours at 37°C, depending on the experiment.

Techniques: Transfection, Staining

Journal: PLoS Pathogens

Article Title: Endocytosis of flavivirus NS1 is required for NS1-mediated endothelial hyperpermeability and is abolished by a single N-glycosylation site mutation

doi: 10.1371/journal.ppat.1007938

Figure Lengend Snippet: Hair was removed from the dorsal dermis of mice, and mice were allowed to recover for 3 days. On the day of the assay, retro-orbital injections of Alexa Fluor 680-conjugated dextran were administered, followed by intradermal injections of PBS plus vehicle (DMSO, top left circle), PBS plus inhibitor cocktail (top right circle), 15 μg DENV2 NS1 plus the DMSO vehicle (bottom left circle), and 15 μg DENV2 NS1 plus inhibitors (bottom right circle). The dermis from each mouse was collected and processed two hours post-injection. (A) Representative image of the dorsal dermis from 2 mice following the fluorescent dextran assay. (B) Dermises were scanned using a fluorescent detection system (LI-COR Odyssey CLx Imaging System) at a wavelength of 700 nm, and extravasated fluorescent dextran was quantified in tissue using Image Studio software (LI-COR Biosciences). Data represent the quantification of mean fluorescent intensity from mice in (A): PBS plus DMSO vehicle (closed black circles, n = 8); PBS plus inhibitors (open black circles, n = 4); DENV2 NS1–15 μg plus DMSO vehicle (closed blue diamonds, n = 8); and DENV2 NS1–15 μg plus inhibitors (open blue diamonds, n = 8). Data in (B) represent mean +/- SD and were collected from two independent experiments. All DENV NS1 proteins used in this experiment were purchased from the Native Antigen Company. A nonparametric Mann-Whitney U test was used to determine significance between treatment groups. ns = not significant, *, p < 0.05; **, p < 0.01. The cocktail of inhibitors was composed of Pitstop 2 (0.25 mg/ml) and Dynasore (0.5 mg/ml) to antagonize local clathrin-mediated endocytosis.

Article Snippet: Confluent HPMEC monolayers grown on gelatin-coated coverslips (0.2%, Sigma) were treated with 5 μg/ml of DENV2 NS1+ from the Native Antigen Company (Oxfordshire, UK) as a positive control, WT DENV NS1 produced in-house, or the DENV NS1-N207Q mutant and incubated for 1 hour at 37°C/4°C or for 6 hours at 37°C, depending on the experiment.

Techniques: Injection, Imaging, Software, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Endocytosis of flavivirus NS1 is required for NS1-mediated endothelial hyperpermeability and is abolished by a single N-glycosylation site mutation

doi: 10.1371/journal.ppat.1007938

Figure Lengend Snippet: ( A ) Amino acid sequence alignment of flavivirus NS1 sequences indicate that N207 glycosylation site is highly conserved. Accession numbers for each NS1 protein sequences are listed after the corresponding flavivirus names. Sequence analysis was performed using MultAlin and recreated in Excel. The amino acid sequence of NS1 from aa 205–211 is shown. (B-C) Human brain microvascular endothelial cells (HBMEC) were grown on Transwell semi-permeable membranes (0.4 μm pore size), and WNV NS1 (B) or ZIKV NS1 (C) proteins (5 μg/ml) were added to the apical chamber (NS1+, commercially purchased NS1, blue squares; NS1-WT, produced in-house, purple diamonds; NS1-N207Q mutant, orange triangles). A TEER assay was used to evaluate the effect of these NS1 proteins on endothelial permeability at indicated time-points over 48 hours. (^) represents change of medium. Relative TEER values from 2 independent experiments performed in duplicate are plotted. Error bars indicate SEM. (D) The integrity of the EGL on HBMEC was assessed by the presence of heparan sulfate surface expression (green) at 6 hpt with WNV NS1 or ZIKV NS1 (5 μg/ml) in the presence or absence of Pitstop 2 (12.5 μM) at 37°C, as visualized via IFA. Nuclei were stained with Hoechst (blue). Images (20X; scale bars, 50 μm) are representative of 3 independent experiments. ( E ) Quantification of MFI in (D) from 3 independent experiments. ***, p < 0.001.

Article Snippet: Confluent HPMEC monolayers grown on gelatin-coated coverslips (0.2%, Sigma) were treated with 5 μg/ml of DENV2 NS1+ from the Native Antigen Company (Oxfordshire, UK) as a positive control, WT DENV NS1 produced in-house, or the DENV NS1-N207Q mutant and incubated for 1 hour at 37°C/4°C or for 6 hours at 37°C, depending on the experiment.

Techniques: Sequencing, Glycoproteomics, Pore Size, Produced, Mutagenesis, Permeability, Expressing, Staining

Journal: PLoS Pathogens

Article Title: Endocytosis of flavivirus NS1 is required for NS1-mediated endothelial hyperpermeability and is abolished by a single N-glycosylation site mutation

doi: 10.1371/journal.ppat.1007938

Figure Lengend Snippet: (A) The flavivirus NS1 proteins are secreted as hexamers and contain two glycosylation sites (DENV, ZIKV, JEV, and YFV; aa 130 and 207) or three glycosylation sites (WNV; aa 130, 175, and 207). (B) Overview of NS1 effects on endothelial cells. (B1) Both NS1-WT and the NS1-N207Q mutant bind to glycosaminoglycans (GAGs; e.g., heparan sulfate) on endothelial cells (ECs), decorating the endothelial glycocalyx layer (EGL) and the cell surface. (B2) Both WT and the N207Q mutant of NS1 trigger recruitment of clathrin triskelion proteins to the plasma membrane. However, only the WT protein can induce maturation and scission of the clathrin-coated pit in a dynamin-dependent manner, while the N207Q mutant appears to remain sequestered on the cell surface. The use of specific chemical inhibitors for clathrin (i.e., Pitstop2) and dynamin (i.e. Dynasore) and siRNA transfection with siClathrin and siDynamin prevent NS1 internalization into HPMEC. (B3) This internalization process results in NS1 colocalization with an early endosome marker, Rab5. Clathrin-mediated endocytosis of NS1 (WT) triggers remodeling of the EGL after activation of endosomal-resident proteases such as cathepsin L, which induces activation of the endoglycosidase heparanase, inducing degradation of heparan sulfate on the EGL, leading to endothelial hyperpermeability and vascular leakage.

Article Snippet: Confluent HPMEC monolayers grown on gelatin-coated coverslips (0.2%, Sigma) were treated with 5 μg/ml of DENV2 NS1+ from the Native Antigen Company (Oxfordshire, UK) as a positive control, WT DENV NS1 produced in-house, or the DENV NS1-N207Q mutant and incubated for 1 hour at 37°C/4°C or for 6 hours at 37°C, depending on the experiment.

Techniques: Glycoproteomics, Mutagenesis, Clinical Proteomics, Membrane, Transfection, Marker, Activation Assay

Journal: eLife

Article Title: Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

doi: 10.7554/eLife.52384

Figure Lengend Snippet: Monoclonal antibody (mAb) sequences were identified from single plasmablasts of DENV-infected patient 013 and patient 020, as previously described . The patient from which corresponding mAb sequences were identified is listed in the first column, followed by mAb clonal family ID, mAb name, and gene usage, % nucleotide (nt) somatic hypermutation, and CDR3 amino acid (aa) length for the variable heavy (VH) and light (VL) chain genes. VH and VL sequences were cloned into IgG1 expression vectors and transfected into mammalian cells. Neat crude IgG1-containing culture supernatant was tested for binding to recombinant DENV2 recombinant soluble E protein (rE) and DENV2 reporter virus particles (RVP) by ELISA, and for neutralizing activity against the indicated related flavivirus RVPs. Antibodies 3H5-1 (2 µg/mL), EDE2 B7(2 µg/mL) and EDE1 C10 (10 µg/mL), and CR4354 (2 µg/mL) were used as controls. Antibody binding activity is expressed as fold-change in absorbance values over negative control wells containing media only. The heatmap (light to dark blue) indicates strength of binding, as defined in the key below the table. A value of 1 indicates no increase in binding relative to negative control wells. Percent neutralization was calculated using the formula: (% infection in the absence of IgG1 - % infection in the presence of IgG1) / (% infection in the absence of IgG1) x 100. The heatmap (yellow to red) indicates the range of neutralization potencies as indicated in the key below the table. Results are representative of 2 independent experiments. Under the crude IgG column, a value of <0.0005 indicates undetectable levels of IgG1 in crude culture supernatant. Antibodies selected for further characterization are shown in bold. ‘n/a,’ not applicable; ‘nc,’ not successfully cloned; ‘nd,’ not determined.

Article Snippet: High-binding 96-well plates (Cat# CLS3361; Millipore Sigma, Burlington, MA) were either coated directly with 500 ng/well of recombinant DENV2 16681 E protein (Cat# DENV2-ENV-500; The Native Antigen Company, Oxford, UK) or with 300 ng/well of murine antibody 4G2 for capture of concentrated and partially purified reporter DENV2 particles.

Techniques: Infection, Clone Assay, Expressing, Transfection, Binding Assay, Recombinant, Virus, Enzyme-linked Immunosorbent Assay, Activity Assay, Negative Control, Neutralization

Journal: eLife

Article Title: Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

doi: 10.7554/eLife.52384

Figure Lengend Snippet: A single dilution of the antibodies indicated on the x-axis was tested for binding to DENV2 ( A ) soluble E protein and ( B ) reporter virus particles at room temperature (RT) and 37°C by ELISA. The y-axis shows absorbance values at 450 nm (A450). Error bars indicate the range of values obtained in duplicate wells. Data are representative of 3 independent experiments. The dotted horizontal line in (B) indicates the average A450 values obtained for negative control WNV-specific antibody CR4354 at 37°C. Representative dose-response binding curves of the indicated antibodies to DENV2 ( C ) soluble E protein and ( D ) reporter virus at room temperature. The y-axis shows binding signal intensity in arbitrary units (AU). Data points and error bars indicate the mean signal intensity and standard deviation (SD) of triplicate spots within one well of the microarray, respectively. Binding curves are representative of two independent experiments.

Article Snippet: High-binding 96-well plates (Cat# CLS3361; Millipore Sigma, Burlington, MA) were either coated directly with 500 ng/well of recombinant DENV2 16681 E protein (Cat# DENV2-ENV-500; The Native Antigen Company, Oxford, UK) or with 300 ng/well of murine antibody 4G2 for capture of concentrated and partially purified reporter DENV2 particles.

Techniques: Binding Assay, Virus, Enzyme-linked Immunosorbent Assay, Negative Control, Standard Deviation, Microarray

Journal: eLife

Article Title: Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

doi: 10.7554/eLife.52384

Figure Lengend Snippet: ( A ) J9, ( B ) J8, ( C ) C4, and ( D ) EDE1 C10 were tested as Fab fragments or full-length IgG for neutralization of DENV2 reporter virus. Dose-response neutralization curves represent three independent experiments, each performed in duplicate. Data points and error bars indicate the mean and range of duplicate wells, respectively. ( E ) Mean IC 50 values of the indicated IgG or Fab fragment from three independent experiments represented by data points. Error bars represent the SD. Values at the dotted horizontal line indicates that 50% neutralization was not achieved at the highest concentration of Fab tested. Fabs were tested at 2x excess molar concentration relative to IgG.

Article Snippet: High-binding 96-well plates (Cat# CLS3361; Millipore Sigma, Burlington, MA) were either coated directly with 500 ng/well of recombinant DENV2 16681 E protein (Cat# DENV2-ENV-500; The Native Antigen Company, Oxford, UK) or with 300 ng/well of murine antibody 4G2 for capture of concentrated and partially purified reporter DENV2 particles.

Techniques: Neutralization, Virus, Concentration Assay

Journal: eLife

Article Title: Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

doi: 10.7554/eLife.52384

Figure Lengend Snippet: ( A ) Dose-response neutralization curves for the indicated antibodies against DENV2 reporter virus prepared under standard conditions (Std) or in the presence of overexpressed furin to generate mature (Furin) virus particles. Data points and error bars indicate the mean and range of infectivity in duplicate wells, respectively. ( B ) Mean IC 50 values of antibodies against standard or mature reporter viruses from three independent experiments depicted by data points. Error bars indicate the SD. P-values were obtained from two-tailed paired t-tests.

Article Snippet: High-binding 96-well plates (Cat# CLS3361; Millipore Sigma, Burlington, MA) were either coated directly with 500 ng/well of recombinant DENV2 16681 E protein (Cat# DENV2-ENV-500; The Native Antigen Company, Oxford, UK) or with 300 ng/well of murine antibody 4G2 for capture of concentrated and partially purified reporter DENV2 particles.

Techniques: Neutralization, Virus, Infection, Two Tailed Test

Journal: eLife

Article Title: Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

doi: 10.7554/eLife.52384

Figure Lengend Snippet: Representative dose-response curves for neutralization of DENV2 16681 reporter virus pre- (filled symbols, solid lines) or post-attachment (open symbols, dotted lines) to Raji-DCSIGNR cells by the antibodies indicated above each graph. Results are representative of at least two independent experiments each performed in duplicate. Data points and error bars indicate the mean and range of duplicate wells, respectively.

Article Snippet: High-binding 96-well plates (Cat# CLS3361; Millipore Sigma, Burlington, MA) were either coated directly with 500 ng/well of recombinant DENV2 16681 E protein (Cat# DENV2-ENV-500; The Native Antigen Company, Oxford, UK) or with 300 ng/well of murine antibody 4G2 for capture of concentrated and partially purified reporter DENV2 particles.

Techniques: Neutralization, Virus

Journal: eLife

Article Title: Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

doi: 10.7554/eLife.52384

Figure Lengend Snippet: Serial dilutions of the antibodies indicated above each graph were pre-incubated with ( A ) DENV2, ( B ) ZIKV or ( C ) WNV reporter virus for 1 hr at room temperature prior to infection of K562 cells, which express FcγR and are poorly permissive for direct infection in the absence of antibodies. The y-axis shows the percentage of infected GFP-positive cells quantified by flow cytometry. Data points and error bars indicate the mean and range of infection in duplicate wells, respectively. Bar graphs represent average antibody concentrations at peak enhancement of ( D ) DENV2, ( E ) ZIKV or ( F ) WNV infection obtained from 2 to 3 independent experiments, each represented by a data point. Where indicated, error bars represent the SD in panels D-F .

Article Snippet: High-binding 96-well plates (Cat# CLS3361; Millipore Sigma, Burlington, MA) were either coated directly with 500 ng/well of recombinant DENV2 16681 E protein (Cat# DENV2-ENV-500; The Native Antigen Company, Oxford, UK) or with 300 ng/well of murine antibody 4G2 for capture of concentrated and partially purified reporter DENV2 particles.

Techniques: Incubation, Virus, Infection, Flow Cytometry

Journal: eLife

Article Title: Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

doi: 10.7554/eLife.52384

Figure Lengend Snippet: ( A ) The E protein ectodomain residues of representative DENV1-4, ZIKV, and WNV, Japanese encephalitis virus (JEV), and Yellow Fever virus (YFV) strains were aligned using ClustalW2. Red, yellow, green, and blue bars above the alignment indicate residues within E protein DI, DII, DII fusion loop, and DIII, respectively. Squares above colored bars indicate residues selected for mutagenesis and generation of DENV2 reporter virus variants tested for sensitivity to J9 neutralization: gray squares = no effect on neutralization sensitivity; black squares = reduced sensitivity to J9 neutralization. Yellow squares indicate residues that are conserved across flaviviruses and are important for binding by antibody I7 in . The sequence used for ZIKV H/PF/2013 differs at two amino acids (residue 246 K > R and 345 M > I from GenBank accession number AHZ13508.1, as previously described; ). ( B ) Ribbon structure of the DENV2 E dimer (PDB 1OAN) with 34 individually mutated residues shown as gray spheres. E protein domains are color-coded as in ( A ). ( C ) Ribbon structure of the DENV2 E dimer (PDB 1OAN) with one monomer shown in black, and the other in gray. Colored spheres indicate the locations of paired mutations: K47T+V151T (red); L56V+Q211E (orange); E85Q+Q86S (green); H149S+V151T (blue); Y178F+M287V (cyan); N194S+E195D (purple); Q316L+K394S (yellow). The effect of the indicated mutations in ( B ) and ( C ) on antibody neutralization potency is shown in .

Article Snippet: High-binding 96-well plates (Cat# CLS3361; Millipore Sigma, Burlington, MA) were either coated directly with 500 ng/well of recombinant DENV2 16681 E protein (Cat# DENV2-ENV-500; The Native Antigen Company, Oxford, UK) or with 300 ng/well of murine antibody 4G2 for capture of concentrated and partially purified reporter DENV2 particles.

Techniques: Virus, Mutagenesis, Neutralization, Binding Assay, Sequencing, Residue

Journal: eLife

Article Title: Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

doi: 10.7554/eLife.52384

Figure Lengend Snippet: Infectious titers of DENV2 reporter virus encoding ( A ) single or ( B ) double E protein mutations. For each graph, white bars show the infectious titer of WT DENV2, and red, yellow, and blue bars represent mutations in DI, DII, and DIII, respectively. In ( B ), the purple bar represents a paired mutation at one residue in DI (Y178F) and another in DIII (M297V). Titers are based on one or two independent virus preparations, as indicated by data points. Where indicated, error bars represent the range of infectivity from 2 to 3 independent virus preparations.

Article Snippet: High-binding 96-well plates (Cat# CLS3361; Millipore Sigma, Burlington, MA) were either coated directly with 500 ng/well of recombinant DENV2 16681 E protein (Cat# DENV2-ENV-500; The Native Antigen Company, Oxford, UK) or with 300 ng/well of murine antibody 4G2 for capture of concentrated and partially purified reporter DENV2 particles.

Techniques: Virus, Mutagenesis, Residue, Infection

Journal: eLife

Article Title: Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

doi: 10.7554/eLife.52384

Figure Lengend Snippet: We screened a panel of DENV2 reporter virus variants encoding single (left) or double (right) E protein mutations for sensitivity to neutralization by ( A ) J9, ( B ) J8, ( C ) C4, ( D ) EDE1 C10, and ( E ) EDE2 B7. Bar graphs represent the average fold-change in IC 50 relative to WT DENV2 obtained from at least two independent experiments, as indicated by data points. Error bars indicate the range (n = 2) or SD (n > 2). The dotted line represents a 4-fold increase in IC 50 relative to DENV2 WT. On the left panel, red, yellow, and blue bars indicate mutations at residues in DI, DII, and DIII, respectively. For each antibody, neutralization of WT ZIKV reporter virus is included as a control. The location of single and paired mutations is shown in , respectively.

Article Snippet: High-binding 96-well plates (Cat# CLS3361; Millipore Sigma, Burlington, MA) were either coated directly with 500 ng/well of recombinant DENV2 16681 E protein (Cat# DENV2-ENV-500; The Native Antigen Company, Oxford, UK) or with 300 ng/well of murine antibody 4G2 for capture of concentrated and partially purified reporter DENV2 particles.

Techniques: Virus, Neutralization

Journal: eLife

Article Title: Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

doi: 10.7554/eLife.52384

Figure Lengend Snippet: ( A ) Ribbon structure of the DENV2 E dimer (PDB: 1OAN) with one monomer in black and the other in gray. The conserved DII fusion loop is shown in green. Colored spheres indicate the location of individual mutations at residues that contribute to J9 recognition based on our screen in and summarized in ( B ). Bar graphs depict the mean fold change in IC 50 values against DENV2 reporter virus encoding E protein mutations indicated on the x-axis relative to wildtype DENV2 for antibodies ( B ) J9, ( C ) J8, ( D ) C4, ( E ) EDE1 C10, and ( F ) EDE2 B7. For each antibody, wildtype ZIKV was included as a control. Mean values were obtained from 2 to 7 independent experiments represented by data points. Error bars indicate the standard deviation (n > 2 experiments) or range (n = 2 experiments). Bar colors correspond to those of spheres in ( A ) to indicate location of individual mutations within the E dimer. The locations of paired mutations are shown in . The dotted horizontal line indicates a 4-fold increase in IC 50 value relative to wildtype DENV2.

Article Snippet: High-binding 96-well plates (Cat# CLS3361; Millipore Sigma, Burlington, MA) were either coated directly with 500 ng/well of recombinant DENV2 16681 E protein (Cat# DENV2-ENV-500; The Native Antigen Company, Oxford, UK) or with 300 ng/well of murine antibody 4G2 for capture of concentrated and partially purified reporter DENV2 particles.

Techniques: Virus, Standard Deviation

Journal: eLife

Article Title: Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

doi: 10.7554/eLife.52384

Figure Lengend Snippet: Neutralizing activity of antibody J9 and longitudinal serum samples from patient 013 collected 4, 8, and 22 days after fever onset were tested against DENV2 reporter virus variants encoding E protein mutations that reduced J9 neutralization potency. WT ZIKV reporter virus was included as a control. Error bars indicate the range of infectivity in duplicate wells normalized to infection levels in the absence of antibody. Dose-response neutralization curves are representative of 3 independent experiments, each performed in duplicate.

Article Snippet: High-binding 96-well plates (Cat# CLS3361; Millipore Sigma, Burlington, MA) were either coated directly with 500 ng/well of recombinant DENV2 16681 E protein (Cat# DENV2-ENV-500; The Native Antigen Company, Oxford, UK) or with 300 ng/well of murine antibody 4G2 for capture of concentrated and partially purified reporter DENV2 particles.

Techniques: Activity Assay, Virus, Neutralization, Infection

Journal: eLife

Article Title: Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

doi: 10.7554/eLife.52384

Figure Lengend Snippet: For each antibody indicated on the x-axis, bars represent mean IC 50 values obtained from 3 to 4 independent experiments indicated by data points against ( A ) DENV1, ( B ) DENV2, ( C ) DENV3, and ( D ) DENV4 reporter viruses. Error bars show the SD. Values at the dotted horizontal line in each graph indicates that 50% neutralization was not achieved at the highest IgG concentration tested (10 µg/ml). IC 50 values for fully mature J9 and J8 are shown in red and blue bars, respectively. J8J9_full germ: germline J8/J9 VH paired with germline J8/J9 VL; J9_VHgerm: J9 germline VH paired with J9 mature VL; J9VL_germ: J9 mature VH paired with J9 germline VL; J8_VHgerm: J8 germline VH paired with J8 mature VL; J8_VLgerm: J8 mature VH paired with J9 germline VL; J9_VH5mut: J9 VH with five mutations indicated in yellow in paired with J9 mature VL; J8_VH5mut: J8 VH with five mutations indicated in yellow in paired with J8 mature VL; J9VH_J8VL: J9 mature VH paired with J8 mature VL; J8VH_J9VL: J8 mature VH paired with J9 mature VL; J9 -G: J9 VH with a single glycine deletion in FR2 paired with mature J9 VL.

Article Snippet: High-binding 96-well plates (Cat# CLS3361; Millipore Sigma, Burlington, MA) were either coated directly with 500 ng/well of recombinant DENV2 16681 E protein (Cat# DENV2-ENV-500; The Native Antigen Company, Oxford, UK) or with 300 ng/well of murine antibody 4G2 for capture of concentrated and partially purified reporter DENV2 particles.

Techniques: Neutralization, Concentration Assay

Journal: Vaccines

Article Title: Recombinant Modified Vaccinia Virus Ankara Expressing a Glycosylation Mutant of Dengue Virus NS1 Induces Specific Antibody and T-Cell Responses in Mice.

doi: 10.3390/vaccines11040714

Figure Lengend Snippet: Figure 1. Construction and genetic characterisation of rMVA-D2-NS1-N207Q. (a) Schematic represen- tation of rMVA-D2-NS1-N207Q. Top shows the approximate location of the transgene (orange) and the major deletion sites (del) I–VI in the MVA genome. Bottom shows the properties of the transgene product. The N-terminal β-roll is coloured blue (amino acids 1 to 29), the α/β subdomain of the wing domain is depicted in yellow (aa 38–151), connector subdomains within the wing domain are shown in gold (aa 30–37 and 152–180), and the C-terminal β-ladder is coloured red (aa 181–352). The N207Q substitution is highlighted in white. The second transmembrane domain (TM2) of the envelope (E) protein is shown in green. The dotted line between TM2 and β-roll indicates post-translational cleavage. (b) Viral genomic DNA was extracted from stocks of non-recombinant, wild-type MVA (wtMVA), and rMVA-D2-NS1-N207Q, and analysed by del III–specific PCR. M, 1 kb DNA ladder. (c) rMVA-D2-NS1-N207Q stock (passage 1 [P1]) was passaged five times in CEFs. Viral genomic DNA was extracted from P1 and P6 viruses, and analysed by del III–specific PCR. M, 1 kb DNA ladder.

Article Snippet: Cells were blocked with 2.5% normal horse serum (NHS; Cytiva, Marlborough, MA, USA) in PBS for 30 min at RT, followed by staining with mouse monoclonal anti-DENV NS1 antibody (1:1000; clone FE8, The Native Antigen Company, Oxford, UK) and rabbit polyclonal anti-calnexin antibody (1:500; Sigma-Aldrich, St. Louis, MO, USA) for 1 h at RT.

Techniques: Recombinant

Journal: Vaccines

Article Title: Recombinant Modified Vaccinia Virus Ankara Expressing a Glycosylation Mutant of Dengue Virus NS1 Induces Specific Antibody and T-Cell Responses in Mice.

doi: 10.3390/vaccines11040714

Figure Lengend Snippet: Figure 2. Characterisation of transgene expression by rMVA-D2-NS1-N207Q. (a) HeLa cells were mock-infected or infected with wtMVA or rMVA-D2-NS1-N207Q (MOI = 3) and lysed at 24 hpi (mock and wtMVA) or at the indicated time points (rMVA-D2-NS1-N207Q). Whole-cell lysates were analysed by Western blotting with antibodies against DENV NS1 and vinculin (loading control). Relative NS1 levels were determined by densitometric analysis. Note that the 0 hpi time point was collected immediately after 1 h of virus adsorption. (b) A549 cells were mock-infected or infected with wtMVA or rMVA-D2-NS1-N207Q (MOI = 3). At 24 hpi, cells were analysed by immunofluorescence staining for DENV NS1 (green) and calnexin (red). Nuclei were stained with Hoechst 33342 (blue). Scale bars, 20 µm. (c–e) HeLa cells were mock-infected or infected with wtMVA or rMVA-D2-NS1- N207Q (MOI = 3). (c) Supernatant samples were collected at 24 hpi (mock and wtMVA) or at the indicated time points (rMVA-D2-NS1-N207Q) and analysed by Western blotting with an antibody against DENV NS1. Note that the 0 hpi time point was collected immediately after 1 h of virus adsorption. (d) Supernatant samples from cells infected with rMVA-D2-NS1-N207Q were collected at 24 hpi, boiled (or not) for 5 min at 95 ◦C prior to non-reducing SDS-PAGE, and analysed by Western blotting with an antibody against DENV NS1. (e) Supernatant samples from cells infected with rMVA-D2-NS1-N207Q were collected at 24 hpi, left untreated or digested with Endo H or PNGase F, and analysed by Western blotting with an antibody against DENV NS1.

Article Snippet: Cells were blocked with 2.5% normal horse serum (NHS; Cytiva, Marlborough, MA, USA) in PBS for 30 min at RT, followed by staining with mouse monoclonal anti-DENV NS1 antibody (1:1000; clone FE8, The Native Antigen Company, Oxford, UK) and rabbit polyclonal anti-calnexin antibody (1:500; Sigma-Aldrich, St. Louis, MO, USA) for 1 h at RT.

Techniques: Expressing, Infection, Western Blot, Control, Virus, Adsorption, Staining, SDS Page