Expression of CD-40 and TLR-3 on DCs expressing F or core protein.

<p>DCs were infected with adenovirus containing HCV-derived F or core antigen. After 48 hours of infection, DCs were harvested and stained with antibodies against CD-40 (A, left panel, and B) and TLR-3 (A, right panel, and C). Data shown in panel A are representative of 4 repeated experiments and panels B and C show statistical data from four cumulative experiments.</p

<i>In vitro</i> priming with DCs expressing F and core proteins leads to peptide dependent proliferation of CD4⁺ T cells in secondary cultures.

<p>The counts per minute (CPMs) shown are mean ± standard error of individual CPMs subtracted with no-antigen groups from corresponding wells. The data are shown from four experiments with four different donors. A response was considered positive when the CPM in the presence of peptide was more than 5,000 after subtracting with no-antigen groups, and was higher than the control peptide group. Core peptide was used as a control in F-stimulated group and vice-versa. The bottom table summarizes the peptide specific responses from all four donors.</p

rAd-F infection of THP-1 cells induces CD-95L expression and apoptosis.

<p>THP-1 cells were infected with rAd-F at an m.o.i. of 100. After 48 hours of infection, the cells were harvested and stained using an anti-human CD-95L PE-conjugated monoclonal antibody and analyzed by flow cytometry (A left panel and B). To measure apoptosis in THP-1 cells, after 48 hours of rAd-F infection, THP-1 cells were harvested, stained with PE-Annexin V and 7-AAD, and examined by flow cytometry (A right panel and C).</p

Primary proliferative response of naive autologous CD4⁺ and CD8⁺ T cells upon stimulation by DCs expressing HCV F and core antigens.

<p>Purified CD4⁺ and CD8⁺ T cells were stimulated using DCs expressing HCV antigens at various DC:T cell ratios. The proliferation of T cells was determined by a ³H-thymidine incorporation assay. The lines on the graphs represent DCs with no antigens, DCs expressing control vector, core protein, and F protein according to the figure legends. Mean and standard deviations of triplicate wells are shown. The data are representative of 5 repeated experiments with 5 different donors.</p

F-protein induced apoptotic DCs are efficiently taken up by live DCs <i>in vitro</i>.

<p>CFSE-labelled apoptotic DCs were incubated with live immature DCs at a ratio of 1∶1 for 2 hours. Flow cytometry analyses were conducted to assess uptake of CFSE⁺ apoptotic DCs by live CD-11c⁺ DCs. Double positive cells indicate the number of live DCs that have uptaken apoptotic DCs. (A) the gating pattern in different groups: live DCs were gated based on PI exclusion and CD-11c expression and the proportion of CFSE⁺ cells was assessed among CD-11c⁺ to determine the phagocytosed DCs. (B) CFSE⁺CD-11c⁺ double positive cells are significantly higher in the F group which has more apoptotic DCs induced by rAd-F infection compared to control vector or untreated DCs. UV treated DCs were used as positive controls for apoptotic DCs. Data are representative of three different experiments.</p

Expression of CD-95 and CD-95L in DCs expressing F or core protein.

<p>DCs were infected with adenovirus containing HCV-derived F and core antigens. After 48 hours of infection, DCs were harvested and stained with antibodies against CD-95L (A, left panel) and CD-95 (A, right panel). Data shown in panel A are representative of 5 different experiments done separately in 5 different donors. Cumulative statistical analysis of CD-95L (B) and CD-95 (C) expression is shown from four different donors.</p

Cross-reactive CD4⁺ and CD8⁺ T cells obtained from Ad vector immunized mice produce cytokines upon <i>ex vivo</i> stimulation with various HCV proteins.

<p>Splenocytes obtained from Ad vector immunized mice were cultured with HCV core, NS3, NS4 or NS5 antigens at 5 μg/ml, and analyzed after 5 days for intracellular IFN-γ and IL-10 expression profile of CD4⁺ and CD8⁺ T cells by flow cytometry. Data are obtained from a pool (n = 5) of spleen cells and are representative of two independent experiments.</p

Characterization of Ad vector stock by PCR.

<p>HCV genes core, F, NS3, NS4, NS5a or NS5b are not amplified. First panel shows the DNA ladder, followed by agarose gel electrophoresis of PCR products obtained with HCV core, F, NS3, NS4, NS5a and NS5b specific primers. HEK lysate supernatant and rAd-HCV vectors were used as negative and positive controls. Data are representative of 2–3 repeated experiments.</p

Identification of cross-reactive cellular and humoral immune responses in mice immunized with Ad vector (with or without poly I:C adjuvant).

<p><b>(A)</b> Proliferation and IFN-γ production in spleen and lymph node T cells upon <i>ex vivo</i> stimulation with HCV antigens, or a pool of 5 peptides showing high homology with Ad proteins (see text for details). <b>(B)</b> Cross-reactive antibody response against HCV antigens. Data are presented as mean±standard deviation of 3–4 replicates, and represent more than three independent experiments.</p