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

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

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

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

Immunization of mice with Ad vector leads to reduced titer of Vaccinia-HCV chimeric virus.

<p>Mice immunized twice intramuscularly with Ad vector (with or without poly I:C), PBS, or HEK cell lysate, were challenged 8 days after the second immunization with wild-type Vaccinia (WT-Vac) or chimeric Vaccinia-HCV. Ovaries were harvested 5 days after challenge and viral loads in each mouse were determined by plaque-forming assay using TK-1 cells. <b>(A)</b> Challenge with HCV core-NS2-NS3 (Vac-C/NS2/NS3) or wild type vaccinia (WT-Vac). <b>(B)</b> Challenge with Vaccinia-HCV NS3-NS4-NS5 (Vac-NS3/4/5). Data are presented as mean ± standard deviation of % reduction in viral titer compared to corresponding unimmunized control group, and statistical comparison was done by two-tailed <i>t</i>-test (p<0.05 was considered significant).</p

Cytotoxic killing of target cells loaded with HCV antigens-derived peptides, by splenocytes obtained from Ad vector immunized mice.

<p>Splenocytes harvested from Ad vector immunized mice, were stimulated <i>in vitro</i> with the HCV protein antigens core, NS3, NS4 and NS5 at 5 μg/ml concentrations for 4 days. The target EL4 cells were incubated with corresponding HCV peptides each at 1 μg/ml concentration (core peptides #: 2, 14, 17, 25, 27, 28, 32; NS3 peptides #: 8, 10; NS4 peptides #: 3, 4, 8; NS5a peptides #: 1, 2, 16, 20 and NS5b peptides: 5, 19, 23, 39; or All: a mixture of the above peptides from core, NS3, NS4 and NS5) and peptide-loaded EL4 cells were cultured with effectors at 10:1 (effectors: target) ratio for 4–5 hours. Empty (no peptide loaded) EL4 targets were used as a negative control. CFSE-labeled live targets were quantified by flow cytometry, and % killed targets were calculated using the formula: % Killing = [(Average live cells in PBS control − live cells in immunized group) /Average live cells in PBS control] × 100). Data shown are mean±SD and are representative of three independent experiments.</p