Journal: Immunology

Article Title: Non-structural protein 1-specific antibodies directed against Zika virus in humans mediate antibody-dependent cellular cytotoxicity.

doi: 10.1111/imm.13380

Figure Lengend Snippet: FIGURE 1 Isotype specificity of ZIKV-immune sera. ELISA plates were coated with ZIKV NS1 (a, c) or ZIKV VLP (b, d), and a 1:100 dilution (n = 28) (a, b) or serial dilutions (n = 11) (c, d) of ZIKV-immune sera were added to plates. Following incubation and washes, optimized concentrations of polyclonal secondary IgG, and monoclonal Abs against the hinge or Fc portion of IgG1, IgG2, IgG3 and IgG4 Abs were added to wells (a) and (b) and IgG1 hinge Abs to (c) and (d). OD values shown at 450 nm. OD values to ZIKV VLP and ZIKV NS1 using a 1:100 dilution of ZIKV-naïve sera (n = 4), <0·2 with polyclonal secondary IgG and <0·1 with IgG1 hinge, IgG1 Fc, IgG2, IgG3 and IgG4 secondary Abs. Bars represent mean with standard error of the mean of the OD values. *P < 0·05, ***P < 0·001 and ****P < 0·0001 were obtained using the non- parametric Friedman test (three or more matched groups) with Dunn's multiple comparisons test

Article Snippet: Briefly, 96- well plates were coated overnight with 20 ng/well of ZIKV virus- like particles (Suriname Z1106033) or 50 ng/ml ZIKV NS1 (Uganda MR766) (Native Antigen Company, Oxfordshire, UK).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation

Journal: Immunology

Article Title: Non-structural protein 1-specific antibodies directed against Zika virus in humans mediate antibody-dependent cellular cytotoxicity.

doi: 10.1111/imm.13380

Figure Lengend Snippet: FIGURE 2 Frequencies of ZIKV-specific IgG and IgG1 B-cell responses using fluorescently labelled ZIKV or ZIKV NS1. PBMCs from ZIKV-immune and ZIKV-naïve donors were stimulated in vitro for seven days with r848 + rIL-2. FluoroSpot plates were coated with IgG and IgG1 capture Abs. (a) Images of wells containing media, total IgG, ZIKV and ZIKV NS1 IgG- or IgG1-specific MBCs from one representative donor. (b) Frequencies of ZIKV (green) and NS1-specific (red) ASCs per 106 input cells in MBC cultures from 12 ZIKV-immune and three ZIKV- naïve donors. Values are the mean of duplicate wells for each condition. The supernatants were evaluated for isotype-specific Abs to (c) ZIKV VLPs and (D) ZIKV NS1. *P < 0·05, **P < 0·01 and ****P < 0·0001 were obtained using the non-parametric Mann-Whitney or Friedman test (three or more matched groups) with Dunn's multiple comparisons test

Article Snippet: Briefly, 96- well plates were coated overnight with 20 ng/well of ZIKV virus- like particles (Suriname Z1106033) or 50 ng/ml ZIKV NS1 (Uganda MR766) (Native Antigen Company, Oxfordshire, UK).

Techniques: In Vitro, MANN-WHITNEY

Journal: Immunology

Article Title: Non-structural protein 1-specific antibodies directed against Zika virus in humans mediate antibody-dependent cellular cytotoxicity.

doi: 10.1111/imm.13380

Figure Lengend Snippet: FIGURE 3 Opsonization and ADCC activity of a ZIKV NS1 monoclonal Ab. (a) K562 cells transfected with DC-SIGN were infected with ZIKV (moi = 0·1 and 1), and 24 h later, the expression of E and NS1 was measured with monoclonal Abs 4G2 and B4 respectively. (b) Opsonization of ZIKV NS1-transfected CEM-NKR cells with a mAb to ZIKV NS1 from Native Antigen Company. CEM-NKR cells transfected with ZIKV NS1 (c and d) or control CEM-NKR cells (e and f) were opsonized with a ZIKV NS1 mAb (d and f), added to PBMC. A standard lymphocyte gate based upon light scatter properties followed by selection of singlet viable cells and dump channel exclusion (CD3− T cells) defined the non-T-cell lymphocyte population, and Abs to CD16 and CD56 identified NK cells

Article Snippet: Briefly, 96- well plates were coated overnight with 20 ng/well of ZIKV virus- like particles (Suriname Z1106033) or 50 ng/ml ZIKV NS1 (Uganda MR766) (Native Antigen Company, Oxfordshire, UK).

Techniques: Activity Assay, Transfection, Infection, Expressing, Control, Selection

Journal: Immunology

Article Title: Non-structural protein 1-specific antibodies directed against Zika virus in humans mediate antibody-dependent cellular cytotoxicity.

doi: 10.1111/imm.13380

Figure Lengend Snippet: FIGURE 4 NK cell activation and degranulation in the presence of ZIKV- immune sera. 1:100 dilution and 1:1600 dilution of polyclonal sera from ZIKV- immune individuals were evaluated for their ability to mediate ADCC of ZIKV NS1 CEM-NKR cells using intracellular cytokine assays. Shown are the frequencies of NK cells that (a) secrete IFN-γ, (B) express CD107 or (C) express both CD107 and IFN-γ in the presence of ZIKV-immune sera. Background levels were subtracted

Article Snippet: Briefly, 96- well plates were coated overnight with 20 ng/well of ZIKV virus- like particles (Suriname Z1106033) or 50 ng/ml ZIKV NS1 (Uganda MR766) (Native Antigen Company, Oxfordshire, UK).

Techniques: Activation Assay

Journal: Immunology

Article Title: Non-structural protein 1-specific antibodies directed against Zika virus in humans mediate antibody-dependent cellular cytotoxicity.

doi: 10.1111/imm.13380

Figure Lengend Snippet: FIGURE 5 Opsonization and target cell lysis using an NK-TVA image cytometry assay. (a) Opsonization of ZIKV NS1 CEM-NKR cells by ZIKV convalescent sera. (b) Representative images of wells acquired by an ImmunoSpot S5 Analyser of an NK-TVA assay where ethanol, ZIKV NS1 or IgG Ab was added to the wells containing effector and target cells. (c) Using an optimized number of target cells, effector PBMC as a source of NK cells and different dilutions of naïve sera (n = 5), positive control sera that opsonized ZIKV NS1 CEM-NKR targets (n = 5) and experimental sera (n = 12), the loss of target cells at the single-cell level was measured by the NK-TVA image cytometry assay and calculated as a percentage of lysed target cells. *P < 0·05 and **P < 0·01 were obtained using a Kruskal–Wallis test with Dunn's multiple comparisons test

Article Snippet: Briefly, 96- well plates were coated overnight with 20 ng/well of ZIKV virus- like particles (Suriname Z1106033) or 50 ng/ml ZIKV NS1 (Uganda MR766) (Native Antigen Company, Oxfordshire, UK).

Techniques: Lysis, Cytometry, Positive Control

Journal: The Journal of Infectious Diseases

Article Title: Role of Transmembrane Protein 16F in the Incorporation of Phosphatidylserine Into Budding Ebola Virus Virions

doi: 10.1093/infdis/jiy485

Figure Lengend Snippet: Ebola virus (EBOV)–induced extracellular expression of phosphatidylserine (PtdSer) is dependent on transmembrane protein 16F (TMEM16F) function. Huh7 cells were transduced with lentiviral vectors encoding scrambled short hairpin RNA (shRNA) (Scr) or shRNAs specific for TMEM16F and infected with EBOV at a multiplicity of infection of 1 plaque-forming unit/cell. A, B, Translocation of PtdSer to the outer leaflet of the plasma membrane in EBOV-infected Huh7/shScramble (A) or Huh7/shTMEM16F (B) cells. Targeted knockdown of TMEM16F results in decreased levels of plasma membrane-associated PtdSer (scale bar represents 30 μm). C–E, Imaging flow cytometry analysis of mock-infected and EBOV-infected shRNA cell lines. C, Representative images of mock-infected and EBOV-infected Huh7/shScramble and Huh7/shTMEM16F cell lines. The merged image is based on glycoprotein (GP), annexin V, and 4′,6-diamidino-2-phenylindole (DAPI). D, Relative levels of EBOV GP (left) and annexin V staining (right) of mock-infected and EBOV-infected Huh7/shScramble and Huh7/shTMEM16F cell lines. See Supplementary Figure 2B for gating strategy. Panels A and B are representative of 1 of 7 and panels C and D are representative of 1 of 3 independent experiments. Abbreviations: BF, bright field; SSC, side scatter.

Article Snippet: Rabbit immune serum against EBOV viruslike particles (VLPs) (Integrated BioTherapeutics) were diluted at 1:100 in stain buffer and used to stain for glycoprotein (GP).

Techniques: Virus, Expressing, Transduction, shRNA, Infection, Translocation Assay, Clinical Proteomics, Membrane, Knockdown, Imaging, Flow Cytometry, Staining

Journal: The Journal of Infectious Diseases

Article Title: Role of Transmembrane Protein 16F in the Incorporation of Phosphatidylserine Into Budding Ebola Virus Virions

doi: 10.1093/infdis/jiy485

Figure Lengend Snippet: Blocking transmembrane protein 16F (TMEM16F) function reduces the levels of viral membrane-associated PtdSer leading to decreased infectivity. A, Huh7/short hairpin (sh)Scramble, Huh7/shTMEM16F and XK-related protein 8 (XKr8) cell lines were mock infected or infected with Ebola virus (EBOV) at a multiplicity of infection (of 0.3 plaque-forming unit /cell for 48 hours. Cells were stained for EBOV glycoprotein (GP) to determine the relative number of infected cells. (Scale bar represents 40 μm). B, Cells were infected with EBOV expressing green fluorescent protein (GFP) and analyzed by flow cytometry to determine the relative percentages of infected (GFP+) cells at 24 and 48 hours. C, Amount of viral proteins in lysates and supernatants determined at 48 hours in Huh7/shScramble or Huh7/shTMEM16F shRNA cell lines. D, Supernatants from Huh7/shScramble and Huh7/ shTMEM16F cells were added to Vero E6 cells for 20 hours to determine the relative production of infectious virus. Means shown in graphs are from triplicate samples, with standard deviations and are representative of 3 independent experiments, *P < .001 (Student t test). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; Scr, scrambled shRNA.

Article Snippet: Rabbit immune serum against EBOV viruslike particles (VLPs) (Integrated BioTherapeutics) were diluted at 1:100 in stain buffer and used to stain for glycoprotein (GP).

Techniques: Blocking Assay, Membrane, Infection, Virus, Staining, Expressing, Flow Cytometry, shRNA

Journal: The Journal of Infectious Diseases

Article Title: Role of Transmembrane Protein 16F in the Incorporation of Phosphatidylserine Into Budding Ebola Virus Virions

doi: 10.1093/infdis/jiy485

Figure Lengend Snippet: Blocking transmembrane protein 16F (TMEM16F) function reduces the levels of viral membrane-associated PtdSer, leading to decreased infectivity. A, Representative images from imaging flow cytometry analysis of virions purified from Huh7/short hairpin (sh)Scramble and Huh7/shTMEM16F Ebola virus (EBOV)–infected cells. The merged image is based on GP and annexin V. B, Relative glycoprotein (GP) expression profiles. C, Relative annexin V incorporation in GP+ population. Abbreviations: BF, bright field; Scr, scrambled shRNA.

Article Snippet: Rabbit immune serum against EBOV viruslike particles (VLPs) (Integrated BioTherapeutics) were diluted at 1:100 in stain buffer and used to stain for glycoprotein (GP).

Techniques: Blocking Assay, Membrane, Infection, Imaging, Flow Cytometry, Purification, Virus, Expressing, shRNA

Journal: The Journal of Infectious Diseases

Article Title: Role of Transmembrane Protein 16F in the Incorporation of Phosphatidylserine Into Budding Ebola Virus Virions

doi: 10.1093/infdis/jiy485

Figure Lengend Snippet: Blocking calcium (Ca2+)–dependent signaling reduces annexin V exposure during Ebola virus (EBOV) infection. The addition of calcium chelator (BAPTA) was used to block calcium-dependent translocation of PtdSer; findings shown represent 1 of 2 independent experiments. A, The extent of PtdSer translocation analyzed by confocal microscopy (scale bar represents 10 μm). B, Histograms depicting relative annexin V levels analyzed by flow cytometry. Gate shown was used to compare expression levels. C, Graphic representation of the percentage of annexin V+ cells from flow cytometry samples in panel B. Mean values are shown for triplicate samples, with standard deviations. *P < .01 (Student t test). Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Rabbit immune serum against EBOV viruslike particles (VLPs) (Integrated BioTherapeutics) were diluted at 1:100 in stain buffer and used to stain for glycoprotein (GP).

Techniques: Blocking Assay, Virus, Infection, Translocation Assay, Confocal Microscopy, Flow Cytometry, Expressing

Journal: The Journal of Infectious Diseases

Article Title: Role of Transmembrane Protein 16F in the Incorporation of Phosphatidylserine Into Budding Ebola Virus Virions

doi: 10.1093/infdis/jiy485

Figure Lengend Snippet: Blocking transmembrane protein 16F (TMEM16F) function with R5421 reduces viral infectivity. A, Huh7 cells were treated with an increasing concentration of R5421 to determine whether blocking TMEM16F reduces plasma membrane-associated phosphatidylserine after Ebola virus (EBOV) infection. Cells were incubated for 48 hours, stained with annexin V and analyzed by flow cytometry. B, Percentages of cells expressing green fluorescent protein (GFP) at 48 hours after EBOV infection of mock-infected or R5421-treated Huh7 cells. C, Viral production from R5421-treated Huh7 cells was determined by adding cell-free supernatants to Vero E6 cells. At 20 hours after infection, cells were fixed and analyzed for GFP expression by flow cytometry. Means shown in graphs are from triplicate samples, with standard deviations; results are representative of 3 independent experiments. *P < .01; †P < .001. Abbreviation: NS, not significant (Student t test).

Article Snippet: Rabbit immune serum against EBOV viruslike particles (VLPs) (Integrated BioTherapeutics) were diluted at 1:100 in stain buffer and used to stain for glycoprotein (GP).

Techniques: Blocking Assay, Infection, Concentration Assay, Clinical Proteomics, Membrane, Virus, Incubation, Staining, Flow Cytometry, Expressing

Journal: The Journal of Infectious Diseases

Article Title: Role of Transmembrane Protein 16F in the Incorporation of Phosphatidylserine Into Budding Ebola Virus Virions

doi: 10.1093/infdis/jiy485

Figure Lengend Snippet: Schematic representation of the role of transmembrane protein 16F (TMEM16F) in the incorporation of phosphatidylserine (PtdSer) into budding virion membranes. Steps are as follows: (1) glycoprotein (GP) expression in the endoplasmic reticulum (ER) activates the ER stress response; (2) increased intracellular calcium (Ca2+) levels trigger TMEM16F activation; (3) TMEM16F mediates translocation of PtdSer from the inner to the outer leaflet of the plasma membrane; (4) PtdSer translocation and Ebola virus (EBOV) budding occur within the lipid raft regions of the plasma membrane. Also shown are the cellular targets inhibited by BAPTA and R5421. Abbreviation: PM, plasma membrane.

Article Snippet: Rabbit immune serum against EBOV viruslike particles (VLPs) (Integrated BioTherapeutics) were diluted at 1:100 in stain buffer and used to stain for glycoprotein (GP).

Techniques: Expressing, Activation Assay, Translocation Assay, Clinical Proteomics, Membrane, Virus