recombinant denv2 16681 e protein  (Native Antigen Inc)

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