The patients were divided into three dose groups, 167 ��g (n = 3), 500 ��g (n = 3), and 1,500 ��g (n = 6) of DNA. Each patient received four monthly vaccinations with the same dose. Since this was a first-in-man study, with this a DNA vaccine Pacritinib against HCV delivered by in vivo EP, 2 weeks were allowed between the enrollments of each patient to monitor safety. After passing screening, patients were admitted to hospital for 8 hours after the first vaccination and then phoned back at 24 hours. The local reaction at the site of vaccination was recorded during the first week. Venous blood was sampled before each vaccination, 6 hours after, and then every second week until treatment week 16. A final sample was taken 24 weeks after last vaccination, which was performed at week 12.
These samples were tested for blood biochemistry, hematology, and HCV RNA. Samples were tested by a qualitative (COBAS AmpliPrep/COBAS AMPLICOR HCV Test, v2.0; Roche Diagnostics, Pleasanton, CA) and a quantitative assay (sensitivity 15 IU/ml, COBAS AmpliPrep/COBAS TaqMan HCV Test; Roche Diagnostics) kit for HCV RNA. PBMCs for analysis of immune responses were isolated by Ficoll-Paque gradient density centrifugation at week 0, 2 weeks after each vaccination, and at week 39. PBMC were aliquoted and frozen in liquid nitrogen until tested. Immunological analysis. All T cell assays were performed on frozen PBMC at ImmuSystems (Munich, Germany) as described previously.48 All samples were tested for proliferation by 3H-thymidine incorporation and for IFN�� production by ELISpot.
48 Peptides corresponding to known HLA class I epitopes and to the complete NS3/4A sequence of the vaccine were generated by standard techniques.49 The following peptide pools were generated: 1: CD4 peptide pool (SPVFSDNSSPPAVPQSYQVA, AQGYKVLVLNPSVAATMG, GRHLIFCHSKKKCD, TTVRLRAYMNTPGLPVCQDH, ENLPYLVAYQATVCARAQ, SGKPAIIPDREVLYREFDEM), 2: CD8 peptide pool, HLA-A2 (CINGVCWTV, YLVTRHADV, LLCPAGHAV, TGSPITYSTY, KLVALGVNAV, YLVAYQATV, VLAALAAYCL), 3: CD8 peptide pool, HLA-A non-A2 (ATDALMTGF (A1), TMGFGAYMSK (A11), GAYMSKAHGI (A11), TLTHPVTK (A11), AYAQQTRGL (A24), MYTNVDQD (A24), TYSTYGKFL (A24), MGFGAYMSK (A3), LIFCHSKKK (A3)), 4: CD8 peptide pool, HLA-B and HLA-C (HPNIEEVAL (B35), TPAETTVRL (B35), IPDREVLY (B35), CHSTDATSIL (B38), HSKKKCDEL (B8), ELAAKLVAL (B8), LIRLKPTL (B8), EVTLTHPVTKYIMTCMSA (B8), AYAAQGYKVL (C3)), 5: nine peptide pools containing 15 overlapping peptides of HCV NS3 and NS4A (pool 11026-1110, pool 21101-1185, pool 31176-1260, pool 41251-1335, pool 51326-1410, pool 61401-1485, pool 71476-1560, pool 81551-1635, and pool 91626-1710).
In the proliferation assay, PBMCs (5��104/well) were incubated in 96-well U-bottom plates (TPP, Trasadingen, Switzerland) in triplicates for 5 days in the presence of different concentrations (3, GSK-3 1, and 0.