Journal: Journal of Virology

Article Title: Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles

doi: 10.1128/JVI.00343-17

Figure Lengend Snippet: Intracellular distribution of EBOV GP, EBOV VP40, and TAFV NP expressed by recombinant MVAs. HeLa cells infected with MVA-BN-EBOV-VLP (expressing EBOV GP, EBOV VP40, and TAFV NP), MVA-BN-EBOV-GP (expressing EBOV GP alone), or wt MVA were fixed and permeabilized 6 h p.i. and incubated with either anti-EBOV GP MAb 6D8 (green), polyclonal rabbit anti-EBOV VP40 antibody (magenta), or polyclonal rabbit anti-TAFV NP antibody (cyan). Antigen-bound primary antibodies were detected with Alexa Fluor-conjugated secondary antibodies, and the cells were analyzed with an inverse confocal laser scanning microscope. MVA vector-infected cells were detected by either EGFP or RFP fluorescence (shown in gray).

Article Snippet: Rabbit polyclonal anti-EBOV NP (0301-012) and anti-EBOV VP40 (0301-010) antibodies were obtained from IBT Bioservices, Gaithersburg, MD, USA.

Techniques: Recombinant, Infection, Expressing, Incubation, Laser-Scanning Microscopy, Plasmid Preparation, Fluorescence

Journal: Journal of Virology

Article Title: Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles

doi: 10.1128/JVI.00343-17

Figure Lengend Snippet: TEM analysis of EBOV VLPs in MVA-BN-EBOV-VLP-infected cells and of concentrated VLP preparations. (A and B) TEM analysis of ultrathin sections. HeLa cells were infected with MVA-BN-EBOV-VLP (A) or MVA-BN (B) at an MOI of 10, fixed 1 day p.i. with glutaraldehyde and osmium tetroxide, and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and analyzed by TEM. (C to H) Immuno-EM analysis of concentrated VLP preparations from supernatants of HeLa cells infected for 24 h at an MOI of 10 with MVA-BN-EBOV-VLP (C and F), MVA-BN-EBOV-GP (D and G), and MVA-BN (E and H). (C, D, and E) Samples were adsorbed to EM grids and incubated with anti-EBOV GP MAb 6D8 and subsequently with secondary anti-mouse IgG antibody coupled with 18-nm colloidal gold. (F, G, and H) Samples were adsorbed to EM grids and incubated with anti-EBOV GP MAb 6D8 and polyclonal rabbit anti-vaccinia virus B5 antibody. The antigen-bound primary antibodies were detected with anti-mouse IgG conjugated with 12-nm colloidal gold and anti-rabbit IgG antibody conjugated with 18-nm colloidal gold.

Article Snippet: Rabbit polyclonal anti-EBOV NP (0301-012) and anti-EBOV VP40 (0301-010) antibodies were obtained from IBT Bioservices, Gaithersburg, MD, USA.

Techniques: Infection, Staining, Incubation, Virus

Journal: Journal of Virology

Article Title: Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles

doi: 10.1128/JVI.00343-17

Figure Lengend Snippet: Release of MVA-expressed EBOV VLPs and incorporation of NP. (A) HEK 293T/17 cells in 12 wells were infected with MVA-BN (MOI, 5) and subsequently transfected with plasmids encoding the indicated filoviral proteins under the control of a poxviral early/late promoter. After 17 h of infection/transfection, the cell supernatants were filtered and concentrated using Amicon Ultra 100K centrifugation columns according to the manufacturer's instructions. The adherent cells were lysed in 250 μl 1× Laemmli sample loading buffer. Cell lysates (CL) and Amicon column-purified supernatants (pur-SN) were analyzed by immunoblotting using antibodies directed to either TAFV NP, EBOV VP40, or EBOV NP. For detection of TAFV NP (bottom gel), a chemiluminescent substrate with enhanced sensitivity was used. (B) For analysis of B5 expression, cells were treated as described for panel A; an uninfected mock control was included as a control. A Western blot of CL and pur-SN was probed with a polyclonal rabbit anti-vaccinia virus B5 antibody. All the Western blot images were acquired using the ChemiDoc Touch System, and the images were analyzed and the signals quantified with Image Lab Software. The data shown are representative of the results of at least three independent experiments.

Article Snippet: Rabbit polyclonal anti-EBOV NP (0301-012) and anti-EBOV VP40 (0301-010) antibodies were obtained from IBT Bioservices, Gaithersburg, MD, USA.

Techniques: Infection, Transfection, Control, Centrifugation, Purification, Western Blot, Expressing, Virus, Software

Journal: Scientific Reports

Article Title: DNA-launched RNA replicon vaccines induce potent anti-Ebolavirus immune responses that can be further improved by a recombinant MVA boost

doi: 10.1038/s41598-018-31003-6

Figure Lengend Snippet: DREP Ebolavirus constructs generated in this study. The different DREP Ebolavirus constructs that have been generated for this study are DREP-GP-Sudan (D-GP-S), DREP-GP-Zaire (D-GP-Z), DREP-GP-VP40-Sudan (D-GP-VP40-S) and DREP-GP-VP40-Zaire (D-GP-VP40-Z), expressing either the GP or GP and VP40 genes from SUDV or EBOV. A description of the 2 A peptide is included in the Methods section. In the DREP plasmids the GP or GP-2A-VP40 genes are placed under the alphaviral subgenomic promoter (SP). The full-length replicon is placed under the cytomegalovirus promoter (CMV).

Article Snippet: Expression was detected using mouse anti-GP SUDV and EBOV antibodies (IBT Bioservices), mouse anti-VP40 SUDV antibody (IBT Bioservices), rabbit anti-VP40 EBOV antibody (IBT Bioservices) and appropriate secondary horseradish peroxidase (HRP) conjugated anti-Ig antibodies (Sigma).

Techniques: Construct, Generated, Expressing

Journal: Scientific Reports

Article Title: DNA-launched RNA replicon vaccines induce potent anti-Ebolavirus immune responses that can be further improved by a recombinant MVA boost

doi: 10.1038/s41598-018-31003-6

Figure Lengend Snippet: Analysis of Ebolavirus GP and VP40 expression. ( A ) BHK-21 cells were transfected with DREP expressing either the GP or GP and VP40 genes from SUDV or EBOV. Cell lysates were collected and run on an SDS-PAGE. Western blotting was performed using anti-GP and anti-VP40 antibodies. EBOV and SUDV VLPs (Ctrl) were used as positive controls. The blots have been cropped and full blots are available in Supplementary Information. ( B ) Replicon-driven expression of SUDV and EBOV GP and GP-VP40 VLPs was analyzed by electron microscopy (EM) from cell culture supernatants from cells expressing GP and GP-VP40. EM micrographs of VLP preparations reveal pleomorphic VLPs made from GP expressing cells (left panels), and filamentous VLP structures made from GP-VP40 expressing cells (right panels). Black bars indicate 100 nm.

Article Snippet: Expression was detected using mouse anti-GP SUDV and EBOV antibodies (IBT Bioservices), mouse anti-VP40 SUDV antibody (IBT Bioservices), rabbit anti-VP40 EBOV antibody (IBT Bioservices) and appropriate secondary horseradish peroxidase (HRP) conjugated anti-Ig antibodies (Sigma).

Techniques: Expressing, Transfection, SDS Page, Western Blot, Electron Microscopy, Cell Culture

Journal: Scientific Reports

Article Title: DNA-launched RNA replicon vaccines induce potent anti-Ebolavirus immune responses that can be further improved by a recombinant MVA boost

doi: 10.1038/s41598-018-31003-6

Figure Lengend Snippet: Anti-GP Ebolavirus antibody responses. Anti-SUDV-GP ( A , C ) and anti-EBOV-GP ( B , D ) antibody endpoint titers were determined in immunized mice after prime ( A , B ) and after boost ( C , D ). Mice were immunized with 5 µg of DREP Ebolavirus constructs (D-GP-S, D-GP-Z, D-GP-S/Z; n = 12/group, D-GP-VP40-S, D-GP-VP40-Z; n = 6/group) or GP protein (P-S/Z; n = 6/group) and bled 21 days after the immunization and individual serum samples were assayed by ELISA using plates coated with SUDV or EBOV GP (A and B, respectively). In panels C and D, mice (n = 6/group) were primed as above and boosted on day 28 and blood was collected on day 49 individual serum samples were assayed by ELISA using plates coated with SUDV or EBOV GP (C,D, respectively). Statistical analysis was performed using Kruskal Wallis test followed by Dunn’s test for multiple comparisons, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001. Comparisons that were non significant are not indicated.

Article Snippet: Expression was detected using mouse anti-GP SUDV and EBOV antibodies (IBT Bioservices), mouse anti-VP40 SUDV antibody (IBT Bioservices), rabbit anti-VP40 EBOV antibody (IBT Bioservices) and appropriate secondary horseradish peroxidase (HRP) conjugated anti-Ig antibodies (Sigma).

Techniques: Construct, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: DNA-launched RNA replicon vaccines induce potent anti-Ebolavirus immune responses that can be further improved by a recombinant MVA boost

doi: 10.1038/s41598-018-31003-6

Figure Lengend Snippet: Relationship between binding antibody titers and neutralizing antibody titers against EBOV.

Article Snippet: Expression was detected using mouse anti-GP SUDV and EBOV antibodies (IBT Bioservices), mouse anti-VP40 SUDV antibody (IBT Bioservices), rabbit anti-VP40 EBOV antibody (IBT Bioservices) and appropriate secondary horseradish peroxidase (HRP) conjugated anti-Ig antibodies (Sigma).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Infectious Diseases

Article Title: Suspected Exposure to Filoviruses Among People Contacting Wildlife in Southwestern Uganda

doi: 10.1093/infdis/jiy251

Figure Lengend Snippet: Sudan Virus (SUDV), Ebola Virus (EBOV), and Bundibugyo virus (BDBV) Prevalence, by Demographic Characteristic, Among Individuals With Wildlife Contact Who Presented to Healthcare Facilities in the Bwindi Region of Uganda During March–June 2013

Article Snippet: The following antigens were used: EBOV, SUDV, and MARV strain Musoke recombinant virus GPs (IBT Bioservices, Gaithersburg, MD) and Bundibugyo virus (BDBV) strain Uganda 2007 recombinant, partial virus envelope GP1 (Ile 33–Gln 304; Acro Biosystems, Newark, DE).

Techniques: Virus

Journal: Analytical and Bioanalytical Chemistry

Article Title: Antibody affinity as a driver of signal generation in a paper-based immunoassay for Ebola virus surveillance

doi: 10.1007/s00216-021-03317-4

Figure Lengend Snippet: Ebola GP structure with highlighted antibody-binding epitopes. a Protein construct of Ebola GP, with GP1 in cyan and GP2 in dark blue. Disulfide bonds are shown linking GP1 and GP2, and the GP1 glycan cap and mucin-like domain are highlighted. Of note, the C-terminal transmembrane domain has been removed. b Crystal structure of Ebola GP trimer with mucin-like domains modeled in gray (PDB: 5JQ3) . Binding epitopes for monoclonal antibodies 13F6, 4F3, and c6D8 (light blue); 13C6 (yellow); and 2G4, 4G7, and KZ52 (overlapping, red) are highlighted

Article Snippet: We purchased the following mAbs from IBT Bioservices (Rockville, MD): chimeric anti-EBOV GP mAb c6D8, chimeric anti-EBOV GP mAb h13F6, and mouse anti-EBOV GP mAb 4F3.

Techniques: Binding Assay, Construct, Glycoproteomics, Bioprocessing

Journal: Analytical and Bioanalytical Chemistry

Article Title: Antibody affinity as a driver of signal generation in a paper-based immunoassay for Ebola virus surveillance

doi: 10.1007/s00216-021-03317-4

Figure Lengend Snippet: Binding parameters for EBOV mAbs to GP as determined by biolayer interferometry. K D is calculated as the ratio of k off to k on

Article Snippet: We purchased the following mAbs from IBT Bioservices (Rockville, MD): chimeric anti-EBOV GP mAb c6D8, chimeric anti-EBOV GP mAb h13F6, and mouse anti-EBOV GP mAb 4F3.

Techniques: Binding Assay

Journal: Analytical and Bioanalytical Chemistry

Article Title: Antibody affinity as a driver of signal generation in a paper-based immunoassay for Ebola virus surveillance

doi: 10.1007/s00216-021-03317-4

Figure Lengend Snippet: Paper-based immunoassay results for all mAbs. a Representative scans of test zones from 0 to 40 μg mL −1 for mAbs KZ52, 13C6, and 13F6 demonstrating strong, moderate, and no signal, respectively. b Quantitative results from ImageJ analysis for each mAb in the first batch of assays (KZ52, 13C6, 4F3, c6D8, 13F6), n = 3. c Representative scans of test zones from 0 to 40 μg mL −1 for mAbs KZ52, 4G7, and 2G4. d Quantitative results from ImageJ analysis in the second batch of assays (KZ52, 2G4, and 4G7), n = 3. Error bars indicate standard deviation

Article Snippet: We purchased the following mAbs from IBT Bioservices (Rockville, MD): chimeric anti-EBOV GP mAb c6D8, chimeric anti-EBOV GP mAb h13F6, and mouse anti-EBOV GP mAb 4F3.

Techniques: Standard Deviation

Journal: Analytical and Bioanalytical Chemistry

Article Title: Antibody affinity as a driver of signal generation in a paper-based immunoassay for Ebola virus surveillance

doi: 10.1007/s00216-021-03317-4

Figure Lengend Snippet: Trends in binding parameters (k on , k off , K D ) and corresponding results from paper-based immunoassays

Article Snippet: We purchased the following mAbs from IBT Bioservices (Rockville, MD): chimeric anti-EBOV GP mAb c6D8, chimeric anti-EBOV GP mAb h13F6, and mouse anti-EBOV GP mAb 4F3.

Techniques: Binding Assay

Journal: PLoS Pathogens

Article Title: Asymmetric antiviral effects of ebolavirus antibodies targeting glycoprotein stem and glycan cap

doi: 10.1371/journal.ppat.1007204

Figure Lengend Snippet: A - C , expression of NK cell activation markers CD107A ( A ), IFNγ ( B ), MIP-1β ( C ), and phagocytic activity of THP-1 monocytes ( D ) or neutrophils ( E ) in response to incubation with BDBV GP immune complexes. Bars show mean values of triplicates ± SE from a single NK cell donor and are representative of two independent experiments across four NK cell donors ( A - C ), or mean values of duplicates ± SE, and are representative of two independent experiments ( D , E ). P values were calculated by factorial ANOVA (Fisher LSD test), compared to the irrelevant mAb b12, which is a HIV-specific human IgG1. The EBOV GP-specific mAb, c13C6 (IgG1), was included as a specificity control. F - I , analysis of glycan content in the Fc fragments of mAbs: the relative abundance of galactose ( F ), fucose ( G ), bisecting N-acetylglucosamine ( H ), or sialic acid ( I ). Bars show fraction of specific carbohydrate residue-containing molecules in a total antibody pool. Schematic structures of N297-associated bi-antennary glycan with analyzed sugar moieties highlighted with rectangles are indicated at the right.

Article Snippet: The control EBOV-specific mAb c13C6 was purchased from IBT Bioservices.

Techniques: Expressing, Activation Assay, Activity Assay, Incubation