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

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

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

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

Cross-reactive CD4⁺ and CD8⁺ T cells obtained from Ad vector immunized mice proliferate in HCV antigens-dependent manner.

<p>Splenocytes obtained from Ad vector immunized mice were stimulated <i>ex vivo</i> with various recombinant HCV antigens (core, NS3, NS4 and NS5) or their selected respective peptides (at 5 μg/ml each), and analyzed by flow cytometry. Proliferation of CD4⁺ and CD8⁺ T cells stimulated <i>ex vivo</i> with: <b>(A)</b> HCV proteins; <b>(B)</b> Representative peptides derived from HCV proteins using the CFSE-based assay (loss of CFSE due to cell division represented by shift of peak of CFSE⁺ T cells towards left). Data are obtained from a pool (n = 5) of spleen cells and are representative of two independent experiments.</p