Flow cytometric analysis of the percentage of HCV-specific IFN-γ+ T cell responses from isolated liver lymphocytes.

<p>Values are reported as the average percent ± SE of HCV-specific <b>A</b>) CD4+ IFN-γ+ or <b>B</b>) CD8+ IFN-γ+ T cell responses of each animal (n = 5) from each group. Significance was determined by Student's <i>t</i> test (*p<0.05, **p<0.005 and ***p<0.0005).</p

Expression and immunogenicity of pConNS4B, pConNS5A and pConNS5B.

<p><b>A</b>) Human RD muscle cells were transiently transfected with each construct. Expression of each gene product was detected using an anti-HA monoclonal antibody and confocal microscopy (250×). <b>B</b>) IFN-γ ELISpot dose response. Animals (n = 5 per group) were immunized with 5 µg, 12.5 µg or 25 µg of pConNS4B, pConNS5A and pConNS5B. Animals received a total of two intramuscular immunizations, two weeks apart followed by electroporation. Animals were sacrificed one week following the last immunization after which splenocytes were isolated and analyzed. The response of each animal to each dose was determined with IFN-γ ELISpot assays. Splenocytes were isolated and individually analyzed for NS4B-, NS5A- or NS5B-specific T cell responses. <b>C, D, E</b>) Splenocytes were intracellularly stained for IFN-γ and analyzed with flow cytometry. <b>C</b>) Representative animal from each group. The values shown are the averaged response of five individual animals from each group. <b>D</b>) and <b>E</b>) graphical representation of percent HCV-specific IFN-γ+ T cell responses from isolated splenocytes. Values are reported as the average percent ± SE of the <b>D</b>) CD4+ IFN-γ+ or <b>E</b>) CD8+ IFN-γ+ T cell responses of each animal (n = 5) from each group. Significance was determined by Student's <i>t</i> test (*p<0.05, **p<0.005 and ***p<0.0005).</p

Clearance of NS4B, NS5A or NS5B transfected hepatocytes.

<p><b>A</b>) Confocal microscopy. Representative confocal image of hepatocyte expression of NS4B, NS5A or NS5B in each group. Expression of each construct was detected with an anti-HA antibody (red). Nuclei were stained with DAPI (blue). <b>B, C</b> and <b>D</b>) Graph of MFI ratio of expression of <b>B</b>) NS4B, <b>C</b>) NS5A or <b>D</b>) NS5B as normalized to DAPI. For each group, three images were captured for each animal (n = 5). MFI values for NS4B, NS5A or NS5B (red) were calculated and normalized to the MFI value for DAPI (blue) for each image. The values shown are the averaged response ± SE of five individual animals from both the naïve and immunized groups. Significance was determined by Student's <i>t</i> test (*p<0.05, **p<0.005 and ***p<0.0005).</p