The general aim of the project is to examine the potential of autologous dendritic cells, matured ex vivo with hepatitis C virus (HCV) antigens and peptides, to initiate a cellular immune response in normal individuals and HCV-positive patients, after autologous transfusion. There are three specific aims: 1. To examine the potential of dendritic cells (DC), matured and loaded in vitro with HCV-proteins and-peptides, to elicit a cellular immune response in vitro. 2. To examine the potential of similar DC, matured and loaded ex vivo with HCV protein and peptides, to elicit a cellular immune response in vivo, after autologous transfer. 3. To examine the effect on the HCV viral load and the accompanying liver disease in HCV carriers after autologous transfusion of similarly matured and loaded DC, and examine the immunological response in these patients. Monocyte-derived DC will be purified, the phenotype analysed and the cells exposed to HCV peptides, lipopeptides or replicon RNA encoding HCV antigens, to mature the cells. The mature DC will then be mixed with autologous lymphocytes and the ability of the DC to elicit a cellular immune response in vitro will be analysed. The most efficient protocol will thereafter be used to mature the DC and the ability of the mature DC to elicit a cellular immune response in vivo after autologous transfusion will be measured by ELISPOT to detect IFN-gamma expressing cells, chromium release to detect cytotoxic T lymphocytes and lymphocyte proliferation to detect a CD4+ Th-cell response. The study will then be extended to use the most effective method to transfer mature DC to HCV-positive carriers. The effect on the viral load in the liver disease will be measured by quantitative RT-PCR and ALT levels, respectively, and correlated with the markers of the cellular immune response described above. ? ?
| Jones, Kathryn L; Drane, Debbie; Gowans, Eric J (2007) Long-term storage of DNA-free RNA for use in vaccine studies. Biotechniques 43:675-81 |
