Journal: Viruses

Article Title: Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient

doi: 10.3390/v11010019

Figure Lengend Snippet: The recall ZIKV plasmablast response is highly cross-reactive against DENV. Heat map showing the binding potency of the ZK016 and ZK018 mAbs against ZIKV and DENV. The mAbs generated from single-cell sorted plasmablasts were tested for reactivity against ZIKV and DENV1-4 recombinant E protein (E) and whole virion by ELISA. The mAbs that are fusion loop (FL)-specific are indicated with asterisks. In terms of scale, brown represents the highest and white the lowest potency of binding based on minimum effective concentration. The minimum effective concentration is the minimum mAb concentration required to obtain three times the background signal. The results plotted are mean values from two or more independent ELISA experiments.

Article Snippet: The ZIKV recombinant E and NS1 proteins were purchased from Meridian Life Science (Memphis, TN, USA; R01635, R01636).

Techniques: Binding Assay, Generated, Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Interference of the Zika Virus E-Protein With the Membrane Attack Complex of the Complement System

doi: 10.3389/fimmu.2020.569549

Figure Lengend Snippet: Binding of terminal complement proteins to ZIKV E. Constant amounts (5 µg/ml; A ) or serial dilutions ( B , for significance – not always depicted due to limits in space – see text) of purified complement proteins were coated onto ELISA plates and incubated with 10 µg/ml ZIKV E. To visualize binding, an E-specific Ab (4G2) followed by a HRP-goat-anti-mouse Ab and TMB as a substrate were added. To test whether already generated TCC is interacting with ZIKV E too (C) , the recombinant viral protein was coated into ELISA plates and incubated with serial dilutions of NHS. BSA and ΔC9 NHS served as controls. Binding to TCC was determined by incubation with neoepitope-specific anti-C9 (WU 13-15) followed by HRP-goat-anti-mouse Ab. Again, TMB was used as a substrate. Optical density (OD) was measured at a wavelength of 650 nm. Experiments were repeated three times and were performed in duplicates. For statistical analysis GraphPad Prism software was used ( A , 1-way ANOVA; B, C , 2-way ANOVA, respectively). *< 0.05, ** < 0.01 and *** < 0.001.

Article Snippet: ZIKV recombinant E and NS1 proteins were provided by Aviva Systems Biology (San Diego, CA); E protein PMA04848-1MG and Biozol (Eching, Germany); E protein MBS596001, and Zika NS1 MBS596002).

Techniques: Binding Assay, Purification, Enzyme-linked Immunosorbent Assay, Incubation, Generated, Recombinant, Software

Journal: Frontiers in Immunology

Article Title: Interference of the Zika Virus E-Protein With the Membrane Attack Complex of the Complement System

doi: 10.3389/fimmu.2020.569549

Figure Lengend Snippet: Inhibition of C9 polymerization after induction via C5b6 in the presence of ZIKV E. To trigger generation of the terminal pathway of complement, A549 cells were incubated with purified C5b6 (5 µg) for 2 h at 37°C. After washing, 5% NHS was added as a source of C7 to C9 in the presence of different amounts of ZIKV E. After incubation at 37°C for 50 min and additional washing steps, cells were lysed and loaded on a SDS gels under non-reducing conditions. Lysates were blotted and visualized as described in the Material & Method section. A representative Western blot out of three independent experiments is shown (A) . As a control, the effect of BSA on the C9 polymerization was used (B) .

Article Snippet: ZIKV recombinant E and NS1 proteins were provided by Aviva Systems Biology (San Diego, CA); E protein PMA04848-1MG and Biozol (Eching, Germany); E protein MBS596001, and Zika NS1 MBS596002).

Techniques: Inhibition, Incubation, Purification, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Interference of the Zika Virus E-Protein With the Membrane Attack Complex of the Complement System

doi: 10.3389/fimmu.2020.569549

Figure Lengend Snippet: Inhibition of C9 polymerization after induction of the classical pathway in the presence of ZIKV E. For the induction of complement activation, A549 cells were incubated with sublytic amounts of anti-MHC-1 (1:1,000). After washing, cells were incubated with 5% NHS, which was pre-incubated with different amounts of ZIKV E. After washing, cells were blotted and oligomerization of C9 was analyzed as described in . A representative Western blot out of three independent experiments is shown.

Article Snippet: ZIKV recombinant E and NS1 proteins were provided by Aviva Systems Biology (San Diego, CA); E protein PMA04848-1MG and Biozol (Eching, Germany); E protein MBS596001, and Zika NS1 MBS596002).

Techniques: Inhibition, Activation Assay, Incubation, Western Blot

Journal: Frontiers in Immunology

Article Title: Interference of the Zika Virus E-Protein With the Membrane Attack Complex of the Complement System

doi: 10.3389/fimmu.2020.569549

Figure Lengend Snippet: Inhibition of MAC formation by ZIKV E: Viral proteins were pre-incubated with C7 to C9 and added to sheep erythrocytes pre-coated with C5b6. As a control vitronectin (VN) was used, a known inhibitor of the MAC. In the positive control (no viral protein) formation of the MAC was undisturbed and set at 100%. Hemolysis was determined by measuring the release of hemoglobin in the supernatant at an OD of 415 nm. Omission of C9 served as background control. Experiments were repeated three times performed in duplicates and analyzed by 1-way ANOVA. n.s., not significant. *** < 0.001.

Article Snippet: ZIKV recombinant E and NS1 proteins were provided by Aviva Systems Biology (San Diego, CA); E protein PMA04848-1MG and Biozol (Eching, Germany); E protein MBS596001, and Zika NS1 MBS596002).

Techniques: Inhibition, Incubation, Control, Positive Control

Journal: Frontiers in Immunology

Article Title: Interference of the Zika Virus E-Protein With the Membrane Attack Complex of the Complement System

doi: 10.3389/fimmu.2020.569549

Figure Lengend Snippet: Reduction of complement mediated lysis by ZIKV: Sensitized sheep erythrocytes were incubated for 30 min at 37°C with serial dilutions of NHS. As expected, lysis of the cell decreased with decreasing dilutions of NHS and was not affected by DMEM, the buffer used as mock control. Hemolysis was determined by measuring the release of hemoglobin in the supernatant at 415 nm. Experiments were performed three times in triplicates. Significance was calculated by 2-way ANOVA. *** < 0.001.

Article Snippet: ZIKV recombinant E and NS1 proteins were provided by Aviva Systems Biology (San Diego, CA); E protein PMA04848-1MG and Biozol (Eching, Germany); E protein MBS596001, and Zika NS1 MBS596002).

Techniques: Lysis, Incubation, Control