Journal: medRxiv

Article Title: Cellular and Humoral Immunity to Ebola Zaire Glycoprotein and Viral Vector Proteins Following Immunization with Recombinant Vesicular Stomatitis Virus-Based Ebola Vaccine (rVSVΔG-EBOV-GP)

doi: 10.1101/2021.11.09.21266118

Figure Lengend Snippet: Antibody responses to vesicular stomatitis virus – Indiana strain matrix protein, nucleoprotein, and glycoprotein by ELISA Optical densities for each participant from baseline and one, three, and six months post-vaccination at 1:100 dilution for A) IgG antibodies to VSV-I matrix protein, B) IgM antibodies to VSV-I matrix protein, C) IgG antibodies to VSV-I nucleoprotein, D) IgM antibodies to VSV-I nucleoprotein, and E) IgG antibodies to VSV-I glycoprotein. The positive OD threshold is indicated by a dashed line and individuals with at least one value exceeding the positivity threshold are shown in red. The VSV-I matrix protein and nucleoprotein are present in the rVSVΔG-EBOV-GP vaccine while the VSV-I glycoprotein is absent from the vaccine.

Article Snippet: In brief, Nunc Maxisorb plates (Fisher #439454) were coated with 1 µg/mL of recombinant Zaire ebolavirus glycoprotein minus trans-membrane region (IBT Bioservices 0501-015), VSV-I glycoprotein (Mudd-Summers strain, MyBioSource MBS1060254), VSV-I matrix protein (98COE North America strain, MyBioSource MBS1317470), or VSV-I nucleoprotein (Glasgow strain, MyBioSource MBS1026155) diluted with PBS and incubated at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: medRxiv

Article Title: Cellular and Humoral Immunity to Ebola Zaire Glycoprotein and Viral Vector Proteins Following Immunization with Recombinant Vesicular Stomatitis Virus-Based Ebola Vaccine (rVSVΔG-EBOV-GP)

doi: 10.1101/2021.11.09.21266118

Figure Lengend Snippet: Representative IgG-producing memory B cell ELISpots at baseline and 6 months post-rVSVΔG-EBOV-GP vaccination A: A representative ELISpot from an Ebola vaccine-naïve participant demonstrating a new IgG-producing memory B cell (MBC) response at six months post-vaccination to EBOV GP only. B: A representative ELISpot from a heterologous Ebola vaccine-experienced participant with IgG-producing MBC responses to EBOV GP both at baseline and six months post-rVSVΔG-EBOV-GP vaccination. C: A representative ELISpot from an Ebola vaccine-naïve participant demonstrating new IgG-producing MBC responses at six months post-vaccination to EBOV GP, VSV-I M protein, and VSV-I NP.

Article Snippet: In brief, Nunc Maxisorb plates (Fisher #439454) were coated with 1 µg/mL of recombinant Zaire ebolavirus glycoprotein minus trans-membrane region (IBT Bioservices 0501-015), VSV-I glycoprotein (Mudd-Summers strain, MyBioSource MBS1060254), VSV-I matrix protein (98COE North America strain, MyBioSource MBS1317470), or VSV-I nucleoprotein (Glasgow strain, MyBioSource MBS1026155) diluted with PBS and incubated at 4°C.

Techniques: Enzyme-linked Immunospot

Journal: medRxiv

Article Title: Cellular and Humoral Immunity to Ebola Zaire Glycoprotein and Viral Vector Proteins Following Immunization with Recombinant Vesicular Stomatitis Virus-Based Ebola Vaccine (rVSVΔG-EBOV-GP)

doi: 10.1101/2021.11.09.21266118

Figure Lengend Snippet: Intracellular cytokine staining for IFN-γ, IL-2, and TNF-α from CD4+ and CD8+ T cells Peripheral blood mononuclear cells from vaccinees at baseline and one month following rVSVΔG-EBOV-GP vaccination were assessed for production of IFN-γ, IL-2, and TNF-α from CD4+ or CD8+ T cells by intracellular cytokine staining using pooled peptides spanning the entire Zaire ebolavirus glycoprotein (A, B), VSV-I matrix protein (C, D), or VSV-I nucleoprotein (E,F). ns signifies p≥0.05, * signifies p<0.05, ** signifies p<0.001.

Article Snippet: In brief, Nunc Maxisorb plates (Fisher #439454) were coated with 1 µg/mL of recombinant Zaire ebolavirus glycoprotein minus trans-membrane region (IBT Bioservices 0501-015), VSV-I glycoprotein (Mudd-Summers strain, MyBioSource MBS1060254), VSV-I matrix protein (98COE North America strain, MyBioSource MBS1317470), or VSV-I nucleoprotein (Glasgow strain, MyBioSource MBS1026155) diluted with PBS and incubated at 4°C.

Techniques: Staining

Journal: medRxiv

Article Title: Cellular and Humoral Immunity to Ebola Zaire Glycoprotein and Viral Vector Proteins Following Immunization with Recombinant Vesicular Stomatitis Virus-Based Ebola Vaccine (rVSVΔG-EBOV-GP)

doi: 10.1101/2021.11.09.21266118

Figure Lengend Snippet: Fold change in Ebola IgG antibody optical densities at 1 month compared to baseline according the detectability of T cells recognizing VSV-I M (A) or NP (B) peptide pools at baseline. Median values with 95% intervals are shown.

Article Snippet: In brief, Nunc Maxisorb plates (Fisher #439454) were coated with 1 µg/mL of recombinant Zaire ebolavirus glycoprotein minus trans-membrane region (IBT Bioservices 0501-015), VSV-I glycoprotein (Mudd-Summers strain, MyBioSource MBS1060254), VSV-I matrix protein (98COE North America strain, MyBioSource MBS1317470), or VSV-I nucleoprotein (Glasgow strain, MyBioSource MBS1026155) diluted with PBS and incubated at 4°C.

Techniques:

Journal: medRxiv

Article Title: Cellular and Humoral Immunity to Ebola Zaire Glycoprotein and Viral Vector Proteins Following Immunization with Recombinant Vesicular Stomatitis Virus-Based Ebola Vaccine (rVSVΔG-EBOV-GP)

doi: 10.1101/2021.11.09.21266118

Figure Lengend Snippet: Pie charts representing the functionality of CD4+ or CD8+ T cells at one month post-rVSVΔG-EBOV-GP vaccination in response to peptides from Zaire ebolavirus glycoprotein, VSV-I matrix protein, or VSV-I nucleoprotein. Pie charts representing the mean functionality of CD4+ T cells (A, C, E, G) and CD8+ T cells (B, D, F, H) at one month following rVSVΔG-EBOV-GP vaccination to EBOV GP for Ebola vaccine-naïve (A, B, n=21) or Ebola vaccine-experienced (C, D, n=11) participants, and to VSV-I matrix protein (E,F, n=32) and VSV-I NP (G, H, n=32) among all participants. Due to rounding, the total mean percentage of cells of each functionality may not add up precisely to 100%.

Article Snippet: In brief, Nunc Maxisorb plates (Fisher #439454) were coated with 1 µg/mL of recombinant Zaire ebolavirus glycoprotein minus trans-membrane region (IBT Bioservices 0501-015), VSV-I glycoprotein (Mudd-Summers strain, MyBioSource MBS1060254), VSV-I matrix protein (98COE North America strain, MyBioSource MBS1317470), or VSV-I nucleoprotein (Glasgow strain, MyBioSource MBS1026155) diluted with PBS and incubated at 4°C.

Techniques:

Journal: Cell Reports

Article Title: SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization

doi: 10.1016/j.celrep.2021.109017

Figure Lengend Snippet:

Article Snippet: The upper portion of the membrane was probed with anti-HA tag antibody (mouse, Sigma-Aldrich, H3663) diluted 1:1,000 in 5% skim milk solution, while the lower portion of the membrane was probed with anti-VSV matrix protein antibody (Kerafast, EB0011; loading control) diluted 1:2,500 in 5% skim milk solution.

Techniques: Produced, Recombinant, Plasmid Preparation, Software, Imaging

Journal: Nature Communications

Article Title: RNA replicon vaccination confers long-lasting protection against H5N1 avian influenza in 23 zoo bird species

doi: 10.1038/s41467-025-64301-5

Figure Lengend Snippet: a Genome maps of VSV and VSVΔG(H5) vector. VSV contains five genes (N, P, M, G, and L). In VSVΔG(H5), the G gene is replaced by the HA gene of A/Dalmatian Pelican/Bern/1/2022 (H5N1) encoding either a polybasic (pb) or monobasic (mb) cleavage site. b Infectious virus yield on BHK-G43 helper cells infected with VSVΔG(H5 mb ) at m.o.i. 0.05 f.f.u./cell. At 20 h p.i., virus particles were concentrated from 200 mL of cell culture supernatant by ultracentrifugation (UC) and resuspended in 20 mL PBS. Infectious titers before and after concentration were determined on BHK-21 cells. The infectious titers of n = 12 vaccine batches are shown (blue triangles). The height of the bars indicates the geometric mean value. The error bars show the 95% confidence interval. Statistical significance was tested by the unpaired, two-sided Student’s t test (df = 22; t = 4.732). c Western blot of VSVΔG(H5 pb ) and VSVΔG(H5 mb ) particles after ultracentrifugation. Viral proteins were separated by SDS-PAGE under reducing conditions, stained with colloidal Coomassie (left panel), or immunostained with chicken polyclonal anti-H5 serum. The relative molecular mass (in kDa) according to the migration of molecular weight markers is indicated on left hand side. A representative experiment out of two performed is shown. d Surface H5 antigen was detected with bovine α-H5 serum. VSV matrix (M) protein was stained in permeabilized cells using mAb 23H12 (α-VSV M). VSV*ΔG-infected cells visualized by GFP. Nuclei stained with DAPI. Scale bar = 20 μm. A representative experiment out of two performed is shown. e Multicycle virus replication on MDCK cells. Cells were infected with the indicated viruses (m.o.i. = 0.0001) and cell culture supernatant sampled at 1, 24, and 48 h p.i. Infectious titers were determined on BHK-21 cells (mean ± SD of 3 infection experiments). The detection limit (25 f.f.u. mL -1 ) is indicated (dashed line). The two-way ANOVA with Tukey’s multiple comparison test compared VSVΔG(H5 P :N1 P :GFP) to VSVΔG(H5 pb ) and VSVΔG(H5 mb ). P values are indicated in the graph. f Immunofluorescence analysis of MDCK cells at 20 h p.i. with the indicated viruses (m.o.i. = 0.02). Cells infected with VSVΔG(H5 P :N1 P :GFP) were detected by GFP fluorescence. Cells infected with either VSVΔG(H5 mb ) or VSVΔG(H5 pb ) were detected by indirect immunofluorescence using a monoclonal antibody directed to the VSV M protein. Cell nuclei were stained with DAPI. The bar is equivalent to 100 μm. A representative experiment out of three performed is shown. Source data are provided as a Source Data file.

Article Snippet: The cells were washed with PBS, permeabilized for 5 min with 0.25% (vol/vol) of Triton X-100 in PBS and then incubated for 1 h with a monoclonal antibody directed to VSV matrix (M) protein (mAb 23H12, KeraFast, Boston, MA, cat. no. EB0011), diluted 1:25 in PBS.

Techniques: Plasmid Preparation, Virus, Infection, Cell Culture, Concentration Assay, Western Blot, SDS Page, Staining, Migration, Molecular Weight, Comparison, Immunofluorescence, Fluorescence

Journal: medRxiv

Article Title: Cellular and Humoral Immunity to Ebola Zaire Glycoprotein and Viral Vector Proteins Following Immunization with Recombinant Vesicular Stomatitis Virus-Based Ebola Vaccine (rVSVΔG-EBOV-GP)

doi: 10.1101/2021.11.09.21266118

Figure Lengend Snippet: Antibody responses to vesicular stomatitis virus – Indiana strain matrix protein, nucleoprotein, and glycoprotein by ELISA Optical densities for each participant from baseline and one, three, and six months post-vaccination at 1:100 dilution for A) IgG antibodies to VSV-I matrix protein, B) IgM antibodies to VSV-I matrix protein, C) IgG antibodies to VSV-I nucleoprotein, D) IgM antibodies to VSV-I nucleoprotein, and E) IgG antibodies to VSV-I glycoprotein. The positive OD threshold is indicated by a dashed line and individuals with at least one value exceeding the positivity threshold are shown in red. The VSV-I matrix protein and nucleoprotein are present in the rVSVΔG-EBOV-GP vaccine while the VSV-I glycoprotein is absent from the vaccine.

Article Snippet: In brief, Nunc Maxisorb plates (Fisher #439454) were coated with 1 µg/mL of recombinant Zaire ebolavirus glycoprotein minus trans-membrane region (IBT Bioservices 0501-015), VSV-I glycoprotein (Mudd-Summers strain, MyBioSource MBS1060254), VSV-I matrix protein (98COE North America strain, MyBioSource MBS1317470), or VSV-I nucleoprotein (Glasgow strain, MyBioSource MBS1026155) diluted with PBS and incubated at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: medRxiv

Article Title: Cellular and Humoral Immunity to Ebola Zaire Glycoprotein and Viral Vector Proteins Following Immunization with Recombinant Vesicular Stomatitis Virus-Based Ebola Vaccine (rVSVΔG-EBOV-GP)

doi: 10.1101/2021.11.09.21266118

Figure Lengend Snippet: Representative IgG-producing memory B cell ELISpots at baseline and 6 months post-rVSVΔG-EBOV-GP vaccination A: A representative ELISpot from an Ebola vaccine-naïve participant demonstrating a new IgG-producing memory B cell (MBC) response at six months post-vaccination to EBOV GP only. B: A representative ELISpot from a heterologous Ebola vaccine-experienced participant with IgG-producing MBC responses to EBOV GP both at baseline and six months post-rVSVΔG-EBOV-GP vaccination. C: A representative ELISpot from an Ebola vaccine-naïve participant demonstrating new IgG-producing MBC responses at six months post-vaccination to EBOV GP, VSV-I M protein, and VSV-I NP.

Article Snippet: In brief, Nunc Maxisorb plates (Fisher #439454) were coated with 1 µg/mL of recombinant Zaire ebolavirus glycoprotein minus trans-membrane region (IBT Bioservices 0501-015), VSV-I glycoprotein (Mudd-Summers strain, MyBioSource MBS1060254), VSV-I matrix protein (98COE North America strain, MyBioSource MBS1317470), or VSV-I nucleoprotein (Glasgow strain, MyBioSource MBS1026155) diluted with PBS and incubated at 4°C.

Techniques: Enzyme-linked Immunospot

Journal: medRxiv

Article Title: Cellular and Humoral Immunity to Ebola Zaire Glycoprotein and Viral Vector Proteins Following Immunization with Recombinant Vesicular Stomatitis Virus-Based Ebola Vaccine (rVSVΔG-EBOV-GP)

doi: 10.1101/2021.11.09.21266118

Figure Lengend Snippet: Intracellular cytokine staining for IFN-γ, IL-2, and TNF-α from CD4+ and CD8+ T cells Peripheral blood mononuclear cells from vaccinees at baseline and one month following rVSVΔG-EBOV-GP vaccination were assessed for production of IFN-γ, IL-2, and TNF-α from CD4+ or CD8+ T cells by intracellular cytokine staining using pooled peptides spanning the entire Zaire ebolavirus glycoprotein (A, B), VSV-I matrix protein (C, D), or VSV-I nucleoprotein (E,F). ns signifies p≥0.05, * signifies p<0.05, ** signifies p<0.001.

Article Snippet: In brief, Nunc Maxisorb plates (Fisher #439454) were coated with 1 µg/mL of recombinant Zaire ebolavirus glycoprotein minus trans-membrane region (IBT Bioservices 0501-015), VSV-I glycoprotein (Mudd-Summers strain, MyBioSource MBS1060254), VSV-I matrix protein (98COE North America strain, MyBioSource MBS1317470), or VSV-I nucleoprotein (Glasgow strain, MyBioSource MBS1026155) diluted with PBS and incubated at 4°C.

Techniques: Staining

Journal: medRxiv

Article Title: Cellular and Humoral Immunity to Ebola Zaire Glycoprotein and Viral Vector Proteins Following Immunization with Recombinant Vesicular Stomatitis Virus-Based Ebola Vaccine (rVSVΔG-EBOV-GP)

doi: 10.1101/2021.11.09.21266118

Figure Lengend Snippet: Fold change in Ebola IgG antibody optical densities at 1 month compared to baseline according the detectability of T cells recognizing VSV-I M (A) or NP (B) peptide pools at baseline. Median values with 95% intervals are shown.

Article Snippet: In brief, Nunc Maxisorb plates (Fisher #439454) were coated with 1 µg/mL of recombinant Zaire ebolavirus glycoprotein minus trans-membrane region (IBT Bioservices 0501-015), VSV-I glycoprotein (Mudd-Summers strain, MyBioSource MBS1060254), VSV-I matrix protein (98COE North America strain, MyBioSource MBS1317470), or VSV-I nucleoprotein (Glasgow strain, MyBioSource MBS1026155) diluted with PBS and incubated at 4°C.

Techniques:

Journal: medRxiv

Article Title: Cellular and Humoral Immunity to Ebola Zaire Glycoprotein and Viral Vector Proteins Following Immunization with Recombinant Vesicular Stomatitis Virus-Based Ebola Vaccine (rVSVΔG-EBOV-GP)

doi: 10.1101/2021.11.09.21266118

Figure Lengend Snippet: Pie charts representing the functionality of CD4+ or CD8+ T cells at one month post-rVSVΔG-EBOV-GP vaccination in response to peptides from Zaire ebolavirus glycoprotein, VSV-I matrix protein, or VSV-I nucleoprotein. Pie charts representing the mean functionality of CD4+ T cells (A, C, E, G) and CD8+ T cells (B, D, F, H) at one month following rVSVΔG-EBOV-GP vaccination to EBOV GP for Ebola vaccine-naïve (A, B, n=21) or Ebola vaccine-experienced (C, D, n=11) participants, and to VSV-I matrix protein (E,F, n=32) and VSV-I NP (G, H, n=32) among all participants. Due to rounding, the total mean percentage of cells of each functionality may not add up precisely to 100%.

Article Snippet: In brief, Nunc Maxisorb plates (Fisher #439454) were coated with 1 µg/mL of recombinant Zaire ebolavirus glycoprotein minus trans-membrane region (IBT Bioservices 0501-015), VSV-I glycoprotein (Mudd-Summers strain, MyBioSource MBS1060254), VSV-I matrix protein (98COE North America strain, MyBioSource MBS1317470), or VSV-I nucleoprotein (Glasgow strain, MyBioSource MBS1026155) diluted with PBS and incubated at 4°C.

Techniques:

Journal: Journal of Virology

Article Title: A Polymorphism within the Internal Fusion Loop of the Ebola Virus Glycoprotein Modulates Host Cell Entry

doi: 10.1128/JVI.00177-17

Figure Lengend Snippet: Determinants in the GP2 subunits account for the reduced EBOV2014-GP-mediated entry into nonhuman primate cells. Rhabdoviral pseudotypes harboring no glycoprotein (pCAGGS, negative control, white), VSV-G (positive control, gray), EBOV1976-GP wt (blue), EBOV2014-GP (red), and the indicated chimeras were inoculated onto different mammalian cell lines. At 18 h postinoculation, the transduction efficiency was quantified by measuring the firefly luciferase activity in cell lysates. The averages of three independent experiments (with separate pseudotype preparations) are shown. Results were normalized against transduction mediated by EBOV1976-GP wt (set as one) and are shown as x-fold changes. Error bars indicate the standard errors of the mean. A paired, two-tailed Student t test was used to assess statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance).

Article Snippet: To show that equal amounts of VSVpp were used and that GP-harboring VSVpp did not contain VSV-G, a separate membrane (obtained under the same conditions as described previously) was first incubated with a mixture of antibodies against VSV-M (matrix protein, raised in mice [KeraFast]; 1:1,000) and VSV-G (I1, mouse hybridoma supernatant from CRL-2700 [American Type Culture Collection]; 1:200) and then incubated with a peroxidase-linked anti-mouse secondary antibody (Dianova; 1:5,000).

Techniques: Negative Control, Positive Control, Transduction, Luciferase, Activity Assay, Two Tailed Test

Journal: Journal of Virology

Article Title: A Polymorphism within the Internal Fusion Loop of the Ebola Virus Glycoprotein Modulates Host Cell Entry

doi: 10.1128/JVI.00177-17

Figure Lengend Snippet: EBOV2014-GP-mediated entry into nonhuman primate cell lines, human HOS cells, and human monocyte-derived macrophages and dendritic cells is less efficient compared to EBOV1976-GP. Replication-deficient VSV encoding firefly luciferase was pseudotyped with VSV-G (positive control, gray), EBOV1976-GP (blue), EBOV2014-GP (red), or no glycoprotein (pCAGGS, negative control, white) and inoculated onto different mammalian cell lines of human (HEK-293T, Huh-7, HOS), rhesus macaque (LLC-MK2, sMAGI), African green monkey (Vero, Vero E6, COS-7) or fruit bat (HypNi/1.1, RoNi/7, and EpoNi/22.1) origin (A) or onto monocyte-derived macrophages (Mϕs) and dendritic cells (moDCs) (B). At 18 h postinoculation, the transduction efficiency was quantified by measuring firefly luciferase activity in cell lysates. The average of at least three independent experiments (with separate pseudotype preparations) is shown for panel A, while for panel B the monocyte-derived macrophages and dendritic cells from three individual donors were analyzed with one pseudotype preparation that has been previously tested on HEK-293T cells for comparable transduction efficiency. For all experiments, transduction mediated by EBOV1976-GP was set as one and x-fold changes are indicated. Error bars indicate the standard errors of the mean. A paired, two-tailed Student t test was used to assess statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance).

Article Snippet: To show that equal amounts of VSVpp were used and that GP-harboring VSVpp did not contain VSV-G, a separate membrane (obtained under the same conditions as described previously) was first incubated with a mixture of antibodies against VSV-M (matrix protein, raised in mice [KeraFast]; 1:1,000) and VSV-G (I1, mouse hybridoma supernatant from CRL-2700 [American Type Culture Collection]; 1:200) and then incubated with a peroxidase-linked anti-mouse secondary antibody (Dianova; 1:5,000).

Techniques: Derivative Assay, Luciferase, Positive Control, Negative Control, Transduction, Activity Assay, Two Tailed Test

Journal: Journal of Virology

Article Title: A Polymorphism within the Internal Fusion Loop of the Ebola Virus Glycoprotein Modulates Host Cell Entry

doi: 10.1128/JVI.00177-17

Figure Lengend Snippet: EBOV-GP mutants analyzed. (A) Domain organization of EBOV-GP. The signal peptide (SP; yellow), receptor binding domain (RBD; red), glycan cap (green), mucin-like domain (MLD; blue), internal fusion loop (IFL; orange), heptad repeats 1 and 2 (HR1 and HR2; light blue), transmembrane domain (TD; brown) are indicated. Signals for N-glycosylation (circles with lines) and amino acid polymorphisms between EBOV1976-GP and EBOV2014-GP (black arrowheads) are marked above and below the GP, respectively. The furin cleavage site located between GP1 and GP2 is indicated by an arrow, and the sequence is shown. (B) EBOV-GP mutants analyzed. Substitutions of single amino acids are indicated by stars and gave rise to mutants EBOV1976-GP (A82V), EBOV2014-GP (V82A), EBOV1976-GP (I544T), and EBOV2014-GP (T544I). Domains or subunits were exchanged in mutants EBOV1976-GP (GC+MLD_2014), EBOV2014-GP (GC+MLD_1976), EBOV1976-GP (GP2_2014), and EBOV2014-GP (GP2_1976). Domains or subunits derived from EBOV1976-GP are indicated in blue, and their counterparts from EBOV2014-GP are shown in red. (C) Equal volumes of pseudotypes bearing the indicated GPs were pelleted through a 20% sucrose cushion and subjected to Western blot analysis. The GP was detected using an EBOV-GP-specific rabbit antiserum and peroxidase-coupled, rabbit-specific secondary antibodies. In addition, VSV-G and VSV-M were visualized using monoclonal primary antibodies and peroxidase-coupled, mouse-specific secondary antibodies. The numbers on the left indicate molecular masses in kilodaltons (kDa). Similar results were obtained in a separate experiment.

Article Snippet: To show that equal amounts of VSVpp were used and that GP-harboring VSVpp did not contain VSV-G, a separate membrane (obtained under the same conditions as described previously) was first incubated with a mixture of antibodies against VSV-M (matrix protein, raised in mice [KeraFast]; 1:1,000) and VSV-G (I1, mouse hybridoma supernatant from CRL-2700 [American Type Culture Collection]; 1:200) and then incubated with a peroxidase-linked anti-mouse secondary antibody (Dianova; 1:5,000).

Techniques: Binding Assay, Sequencing, Derivative Assay, Western Blot

Journal: Journal of Virology

Article Title: A Polymorphism within the Internal Fusion Loop of the Ebola Virus Glycoprotein Modulates Host Cell Entry

doi: 10.1128/JVI.00177-17

Figure Lengend Snippet: A single polymorphism, T544I, in GP2 accounts for the differential ability of EBOV2014-GP and EBOV1976-GP to drive entry into nonhuman primate cells. (A) The sequences spanning the border between GP1 and GP2 were aligned for the wt and mutant GPs analyzed. Conserved amino acid residues are marked with asterisks, while residues that differ between two GPs are indicated by bold, red letters. Amino acid residues of the internal fusion loop (IFL) are highlighted by a gray box. (B) All complete EBOV-GP sequences available at NCBI database that were not linked to the West African epidemic (n = 66) were analyzed for the amino acid present at position 544. The relative distribution of threonine (orange) and isoleucine (green) is shown. (C) Rhabdoviral pseudotypes harboring wild-type (wt) EBOV1976-GP, EBOV1976-GP (I544T) (both blue), wt EBOV2014-GP, or EBOV2014-GP (T544I) (both red) were inoculated onto COS-7 cells. Pseudotypes that contained no glycoprotein (pCAGGS, white) or VSV-G (gray) served as negative and positive controls, respectively. At 18 h postinoculation, transduction efficiency was quantified by measuring firefly luciferase activity in cell lysates. The average of three independent experiments (with separate pseudotype preparations) is shown. Results were normalized against transduction mediated by EBOV1976-GP wt (set as one) and are shown as x-fold changes. Error bars indicate the standard error of the mean. (D) EBOV-based VLPs harboring either no glycoprotein (white, negative control), wt EBOV1976-GP, EBOV1976-GP (I544T), wt EBOV2014-GP, or EBOV2014-GP (T544I) were inoculated onto COS-7 cells. At 3 h postinoculation, the firefly luciferase activity in cell lysates was quantified, corrected for background activity (measured in cells inoculated with pseudotypes not bearing a glycoprotein), and normalized against luciferase activity of VLPs used for inoculation (input factor). The results of a representative experiment carried out with quadruplicate samples are shown. Error bars indicate standard deviations. Similar results were obtained in two separate experiments performed with independent VLP preparations. Paired (pseudotypes) and unpaired (VLPs) two-tailed Student t tests were used to assess statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance).

Article Snippet: To show that equal amounts of VSVpp were used and that GP-harboring VSVpp did not contain VSV-G, a separate membrane (obtained under the same conditions as described previously) was first incubated with a mixture of antibodies against VSV-M (matrix protein, raised in mice [KeraFast]; 1:1,000) and VSV-G (I1, mouse hybridoma supernatant from CRL-2700 [American Type Culture Collection]; 1:200) and then incubated with a peroxidase-linked anti-mouse secondary antibody (Dianova; 1:5,000).

Techniques: Mutagenesis, Transduction, Luciferase, Activity Assay, Negative Control, Two Tailed Test