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: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: Biotechnology and Bioengineering

Article Title: Multiple Use of Regenerated Depth Filters in Antibody Purification Processes

doi: 10.1002/bit.28957

Figure Lengend Snippet: Comparison of alkaline and acidic regeneration for the reuse of the synthetic depth filter XOSP (blue, equilibration buffer 150 mM acetic acid/tris pH 5.5; yellow, alkaline solution 1 M sodium hydroxide; gray, acidic solution 500 mM phosphoric acid) displaying (A) HCP removal, (B) DNA removal, and (C) main peak analysis by SE‐HPLC for product quality. (D) displays HCP removal for acidic filter regeneration performed with 500 mM phosphoric acid (gray) or 167 mM acetic acid, 300 mM phosphoric acid (green). L, load. In case of missing bars, the measured values were below the detection limit of the assay or too small for visualization. HCP, host cell proteins; SE‐HPLC, size exclusion high performance liquid chromatography.

Article Snippet: The Millistak+ HC Pro XOSP depth filter (Merck Millipore) is composed of a synthetic polyacrylic fiber matrix and silica as filter aid.

Techniques: Comparison, High Performance Liquid Chromatography

Journal: Biotechnology and Bioengineering

Article Title: Multiple Use of Regenerated Depth Filters in Antibody Purification Processes

doi: 10.1002/bit.28957

Figure Lengend Snippet: Acidic regeneration for the reuse of the synthetic depth filter XOSP (blue, equilibration buffer 150 mM acetic acid/tris pH 5.5; green, 167 mM acetic acid, 300 mM phosphoric acid) displaying (A) HCP removal, (B) DNA removal, and (C) main peak analysis by SE‐HPLC for product quality. L, load. In case of missing bars, the measured values were below the detection limit of the assay or too small for visualization. HCP, host cell proteins; SE‐HPLC, size exclusion high performance liquid chromatography.

Article Snippet: The Millistak+ HC Pro XOSP depth filter (Merck Millipore) is composed of a synthetic polyacrylic fiber matrix and silica as filter aid.

Techniques: High Performance Liquid Chromatography