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: 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: We generated CEM‐NK R cells stably expressing ZIKV NS1 and established assays using a ZIKV NS1‐specific human IgG1 mAb (Native Antigen Company).

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: 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 10 6 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: We generated CEM‐NK R cells stably expressing ZIKV NS1 and established assays using a ZIKV NS1‐specific human IgG1 mAb (Native Antigen Company).

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: 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‐NK R cells with a mAb to ZIKV NS1 from Native Antigen Company. CEM‐NK R cells transfected with ZIKV NS1 (c and d) or control CEM‐NK R 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: We generated CEM‐NK R cells stably expressing ZIKV NS1 and established assays using a ZIKV NS1‐specific human IgG1 mAb (Native Antigen Company).

Techniques: Activity Assay, Transfection, Infection, Expressing, 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: 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‐NK R 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: We generated CEM‐NK R cells stably expressing ZIKV NS1 and established assays using a ZIKV NS1‐specific human IgG1 mAb (Native Antigen Company).

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: Opsonization and target cell lysis using an NK‐TVA image cytometry assay. (a) Opsonization of ZIKV NS1 CEM‐NK R 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‐NK R 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: We generated CEM‐NK R cells stably expressing ZIKV NS1 and established assays using a ZIKV NS1‐specific human IgG1 mAb (Native Antigen Company).

Techniques: Lysis, Cytometry, Positive Control

Journal: Nature medicine

Article Title: Identification of small molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen

doi: 10.1038/nm.4184

Figure Lengend Snippet: Identification of PHA-690509 and Niclosamide as antiviral compounds. (a) Top, example western blot images of titration of PHA-690509 on SNB-19 cells for anti-ZIKV activity. Glioblastoma SNB-19 cells were treated with the indicated compound for 1 hour prior to inoculation with ZIKV FSS-13025, PRVABC59, or MR766 (MOI = 1) and cells were harvested 24 hours post infection for western blot analysis. Bottom, quantification of NS1/GAPDH protein band intensities. Data were normalized to that with the DMSO treatment. Values represent mean ± s.d. (n = 3 cultures; ***P < 0.001; One-way ANOVA for comparison with the DMSO treatment). Insert shows chemical structure of PHA-690509. (b) Top, example western blot images of titration of Emricasan on SNB-19 cells for anti-ZIKV activity. Similar to (a). Values represent mean ± s.d. (n = 3 cultures; P > 0.1; One-way ANOVA for comparison with the DMSO treatment). (c) Top, chemical structure of Niclosamide and example western blot images of titration of Niclosamide on SNB-19 cells for anti-ZIKV activity using the PRVABC59 strain (MOI = 1). Bottom, quantification similar to (a). Values represent mean ±s.d. (n = 3 cultures; **P < 0.01; ***P < 0.001; One-way ANOVA for comparison with the DMSO treatment). (d) Summary of ELISA quantifications of secreted ZIKV-NS1 protein in the medium of infected cells upon treatment of different doses of Niclosamide (top) or PHA-690509 (bottom) for 24 hours. Values represent mean ± s.d. (n = 3 cultures; ***P < 0.001; One-way ANOVA for comparison with the 0 μM group). (e) Summary of titration of Niclosamide and PHA-690509 on ZIKV viral production. SNB-19 cells were treated with each indicated compound at increasing concentrations for 1 hour prior to infection with PRVABC59 at MOI = 0.5. Cell culture supernatant was collected 24 hours post infection and infectious virions were quantified for focus-forming units (FFU) using Vero cells. Data represent mean ± s.d. (n = 3 cultures). Curves represent best fits for calculating IC50, and the insets report the calculated IC50 value for each compound.

Article Snippet: The anti-ZIKV NS1 ELISA kit (ZKV-NS1-EK) was obtained from BioFront Technologies (Tallahassee, FL) and used according to the manufacturer’s protocol.

Techniques: Western Blot, Titration, Activity Assay, Infection, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Nature medicine

Article Title: Identification of small molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen

doi: 10.1038/nm.4184

Figure Lengend Snippet: Inhibition of ZIKV infection by Niclosamide and PHA-690509 at a post-entry step and blockade of ZIKV infection by additional CDKis. (a) Schematic illustration of time-of-addition experiment for Niclosamide and PHA-690509. (b) Top, example western blot images of SNB-19 cells treated with 10 μg/ml anti-AXL antibody, 2 μM Niclosamide, or 92 μM PHA-690509 for 1 hour prior to or 4 hours post infection with PRVABC59 (MOI = 1). Bottom, quantification of NS1/GAPDH protein band intensities. Data were normalized to that with the DMSO treatment. Values represent mean ± s.d. (n = 3 cultures; *P < 0.05; **P < 0.01; ***P < 0.001; One-way ANOVA for comparison with the DMSO treatment). (c) Inhibition of ZIKV infection in CDKi-treated cells. Left, example western blot images of SNB-19 cells treated with the indicated compound at 92 μM for one hour prior to infection with FSS13025 or PRVABC59 (MOI = 1). Right, quantification similar to (b). Values represent mean ± s.d. (n = 3 cultures; ***P < 0.01; One-way ANOVA for comparison with the DMSO treatment). (d) Effect of various CDKis on ZIKV production. Similar to Figure 2e. All data were normalized to 0 μM for each compound. Data represent mean ± s.d. (n = 3 cultures). Curves represent best fits for calculating IC50 (listed on the right panel).

Article Snippet: The anti-ZIKV NS1 ELISA kit (ZKV-NS1-EK) was obtained from BioFront Technologies (Tallahassee, FL) and used according to the manufacturer’s protocol.

Techniques: Inhibition, Infection, Western Blot

Journal: Nature medicine

Article Title: Identification of small molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen

doi: 10.1038/nm.4184

Figure Lengend Snippet: Niclosamide and PHA-690509 inhibit ZIKV infection in human astrocytes and forebrain-specific hNPCs. (a) Top, example immunostaining images of astrocytes treated with 2 μM Niclosamide, 92 μM PHA-690509, 9 μM Emricasan, or a combination of 92 μM PHA-690509 and 9 μM Emricasan for 1 hour prior to infection with PRVABC59 (MOI = 0.5). Cells were fixed 24 hours post infection and stained for ZIKVE (green) and DAPI (blue; Scale bar: 50 μm, applies to all image panels in a). Bottom, quantification of the percentage of ZIKV-infected astrocytes over DAPI. Values represent mean ± s.d. (n = 6 cultures; ***P < 0.001; One-way ANOVA for comparison with the DMSO treatment). (b) Top, example western blot images of hNPCs infected at a MOI of 0.1 and analyzed 48 hours post infection. Bottom, quantifications of NS1/GAPDH protein band intensities. Data were normalized to that with the DMSO treatment. Values represent mean ± s.d. (n = 3 cultures; ***P < 0.01; One-way ANOVA for comparison with the DMSO treatment). (c) Virus production from compound treated iPSC-derived human astrocytes. Similar to Figure 2e. Data represent mean ± s.d. (n = 3 cultures). (d–e) Summary of additive effects of a combination of Emricasan and PHA-690509 on inhibiting increased caspase-3 activity in human astrocytes infected with FSS13025-ZIKV or MR766 ZIKV (d), and on improving cell viability as measured by ATP production (e). Similar to Figure 1a. Values represent mean ± s.d. (n = 3 cultures; *P < 0.05; **P < 0.01; One-way ANOVA).

Article Snippet: The anti-ZIKV NS1 ELISA kit (ZKV-NS1-EK) was obtained from BioFront Technologies (Tallahassee, FL) and used according to the manufacturer’s protocol.

Techniques: Infection, Immunostaining, Staining, Western Blot, Derivative Assay, Activity Assay

Journal: Biosensors & Bioelectronics

Article Title: A single snapshot multiplex immunoassay platform utilizing dense test lines based on engineered beads

doi: 10.1016/j.bios.2021.113388

Figure Lengend Snippet: Operation of the MOnITOR chip for one-step indirect immunoassays. (a) In the ‘Preparation’ stage, the chip immediately started to operate. (b) Engineered bead lines were secured on the reaction zone. The scale bar represents 100 μm. (c) Red-dyed sample was loaded. Trapped gas bubbles eventually diffused through the PDMS matrix (blue arrows). Wasted volumes in the loading and dummy channels consume only 1.6% of the loaded sample. (d,e) Sequentially-loaded blue-dyed buffer flowed and filled the storage chamber. During storage chamber filling, 2nd Ab was rehydrated. Rehydrated 2nd Ab diffusion toward the buffer inlet was prevented by the delaying channel. (f) When loading sample with anti-ZIKV NS1 and anti-CHIKV E2 mAb, only the corresponding beads reacted. (g) Buffer continuously flowed toward the reaction zone by contouring the empty sample inlet. (h) Unbound 2nd Ab were washed by flowing buffer. An image of the reaction zone was acquired in this stage. The scale bar represents 1 cm. (i) In the case of (f), the transferred 2nd Ab only reacted with ZIKV, CHIKV, and + beads. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Anti-ZIKV NS1, anti-DENV NS1, anti-CHIKV E2, and anti-Mayaro virus (MAYV) E2 monoclonal antibodies (mAb) were also provided from BioNano Health Guard Research Center.

Techniques: Diffusion-based Assay