Measles virus-specific antibody response to immunization.

<p>Sera collected from individual mice immunized with VEE/SIN-H or FI-MV were assessed for quantity and quality of MV-specific antibody. MV-specific total IgG (<b>A</b>), IgG1 (<b>B</b>) and IgG2a (<b>C</b>) were measured by EIA. Avidity of MV-specific antibody was evaluated by a modified EIA and data are presented as an avidity index (<b>D</b>). Fifty percent plaque reduction neutralization titers (PRNT) for the Chicago-1 strain of MV on Vero cells are expressed as geometric means (<b>E</b>). Data points represent the mean +/- S.D. of three individual mice. (* <i>P</i><0.05; Student's <i>t</i> test)</p

Measles virus-specific T cell responses after immunization.

<p>IFN-γ (<b>A</b>) and IL-4–secreting cells (<b>B</b>) from the dLN were measured at day 7 after immunization with VEE/SIN-H or FI-MV. Media-only stimulation represents <i>ex vivo</i> spontaneous secretion. This value has been subtracted from the MVL stimulation values. At days 7, 14 and 21 after immunization, cells from dLNs (<b>C, D</b>) and the spleen (<b>E, F</b>) were evaluated for IFNγ and IL-4 secretion after <i>ex vivo</i> stimulation with MVL antigen in ELISpot assays. Spot-forming cells are per 5×10⁵ total cells. Assays were performed in triplicate (error bars represent S.D. of assay replicates) with cells pooled from 3 mice.</p

Protein-specific antibody responses to vaccine antigens.

<p>Serum IgG specific for MV H (<b>A</b>), F (<b>B</b>) and N (<b>C</b>) proteins over a 125-day time course measured by EIA. H-specific ASCs from the bone marrow measured by ELISpot at day 125, represented as an index of total spot number multiplied by spot area (<b>D</b>). Serum antibody data points represent the mean +/− S.D. of 3 individual mice. IgG ELISpot data are generated from cells pooled from 3 mice.</p

Determination of DENV titers by F-DENPADS.

<p>F-DENPADS cells were seeded in 24-well plates. After incubation for 24 hours, the cells were infected with a 10-fold serial dilution of DENV-2. EGFP expression was examined via flow cytometry 24 (A), 48 (B), and 72 (C) hours after infection. The percentage of EGFP-positive cells versus virus titers is plotted. The hollow dot represents the percentage of EGFP-positive cells in the non-infection group and is defined as the background value. Partially enlarged views between log 0 to 3 FFU/well are presented in the upper-left within the diagram. The data shown are the mean values ± standard deviation obtained from 3 independent experiments. The dotted lines indicate the mean plus a 2-fold standard deviation of the non-infection group. (D) Virus titers of 9 DENV samples were converted by the standard curve that was obtained from F-DENPADS cells incubated for 72 hours (in Fig 3C). Virus titers were determined in parallel by an immuno-focus assay. The correlation between the two methods is shown.</p

Characterization of DENPADS.

<p>(A) The pRep-EGFP stable clone or F-DENPADS cells were transfected with pNS2B3 or infected with DENV-2 (m.o.i. = 5). Cells transfected with pCre or mock infected cells served as controls. Cell lysates were harvested and then analyzed by immunoblotting using anti-Cre and anti-β-actin antibodies 48 hours after transfection or infection. The cleavage product (solid arrow) represents the fragment cleaved from the NS4B-_N10NS5/NLS-Cre fusion protein (hollow arrow) by NS2B3 at the region between NS4B and NS5 during DENV infection or NS3 presentation. The hollow arrowhead represents the Cre protein (~38 kDa), which is encoded by pCre. (B) F-DENPADS cells were infected with 4 DENV serotypes at an m.o.i. of 5. Cells were fixed and analyzed 48 hours post-infection using fluorescence microscopy. Scale bar, 250 μm.</p

Kinetics of the germinal center response after immunization.

<p>Cells from popliteal draining lymph nodes harvested at various times after immunization with VEE/SIN-H, FI-MV, SRBCs (positive control) or PBS were analyzed for GC B cells by flow cytometry. Cells were stained with antibody to CD19 and PNA at day 7 (<b>A</b>) and periodically over an 80-day time course (<b>B</b>). All flow cytometry data points represent values from cells pooled from 2 mice.</p

Determination of DENV titers by H-DENPADS.

<p>(A) pRep-mHRP was constructed as a membrane-targeted HRP reporter module. (B) H-DENPADS cells were infected with DENV-2 for 48 hours. The expression of mHRP was examined by surface staining with anti-HRP antibodies using flow cytometry. (C) The function of mHRP was evaluated by color development after adding TMB substrate. (D) H-DENPADS cells were seeded in 96-well plates. After incubation for 24 hours, cells were infected with a 10-fold serial dilution of DENV-1, -2, -3, or -4. TMB substrate was added 24 hours after infection. The OD450 nm was examined with a spectrophotometer. The data are representative of 3 different experiments performed in hexaplicate. The dotted lines indicate the mean plus a 2-fold standard deviation of the non-infection group. (E) Virus titers of 43 DENV samples were converted by the standard curve, which was obtained from Fig 5D. Virus titers were determined in parallel by an immuno-focus assay. The correlation between the two methods is shown.</p

Construction of DENPADS plasmids and characterization of the sensor module.

<p>(A) pSen-Cre and pRep-EGFP were constructed as the sensor and reporter modules, respectively. The cleavage site of the DENV NS2B3 protease is indicated. BHK-21 cells were transfected with pSen-Cre in the presence or absence of pNS2B3. Localization of Cre (red) and (B) ER membranes (calnexin, green) or (C) NS3 (green) were detected by immunostaining. Nuclei were counterstained with Hoechst (blue). The fluorescence intensity was analyzed by LEICA Application Suite software (magnification, 100×). Cells transfected with pCre served as controls.</p

Efficiency of F-DENPADS in detection of other flaviviruses.

<p>F-DENPADS cells were infected with DENV-2, Zika virus, or JEV at an m.o.i. of 5 or transfected with pHCV NS3/4A-Flag. Cells were examined for EGFP expression 48 hours post-infection using fluorescence microscopy. Scale bar, 500 μm.</p

Determination of DENV titers by F-DENPADS.

<p>F-DENPADS cells were seeded in 24-well plates. After incubation for 24 hours, the cells were infected with a 10-fold serial dilution of DENV-2. EGFP expression was examined via flow cytometry 24 (A), 48 (B), and 72 (C) hours after infection. The percentage of EGFP-positive cells versus virus titers is plotted. The hollow dot represents the percentage of EGFP-positive cells in the non-infection group and is defined as the background value. Partially enlarged views between log 0 to 3 FFU/well are presented in the upper-left within the diagram. The data shown are the mean values ± standard deviation obtained from 3 independent experiments. The dotted lines indicate the mean plus a 2-fold standard deviation of the non-infection group. (D) Virus titers of 9 DENV samples were converted by the standard curve that was obtained from F-DENPADS cells incubated for 72 hours (in Fig 3C). Virus titers were determined in parallel by an immuno-focus assay. The correlation between the two methods is shown.</p