Hepatitis C is one of the worlds most pandemic and insidious diseases. Less than 41% of patients respond to the current treatment, and a large fraction is ineligible for therapy. Thus, there is an urgent need for new therapeutic strategies. Clearance of hepatitis C virus (HCV) is correlated with the level of HCV-specific CD4+ T cells, and viral escape mutations have been identified in immunodominant CD4+ T-cell epitopes. These results suggest that an immunotherapy designed to increase and broaden HCV-specific CD4+ T cells could provide a new therapeutic approach. Dendritic cells (DCs), the most important class of professional antigen presenting cells, possess the ability to elicit both humoral and cellular immune responses. These cells are poised to capture pathogens, migrate to draining lymph nodes, and select antigen-specific CD4+ T cells to regulate T, B, and NK cells, all of which may contribute to protective immunity. The objective of this proposal is to develop a novel vaccine strategy targeting the HCV nonstructural protein 3 (NS3) directly to DC subsets, e.g., Langerhans Cells (LCs). Recently we showed that LCs can be generated by culturing monocytes with GM-CSF+IL15. Such LCs induce significant T-cell activation in vivo. Furthermore, we have generated peptides that bind specifically to LCs or interstitial DCs from a phage display peptide library. We hypothesize that targeting NS3 directly to DCs will increase the level and duration of specific immune responses. Thus, we will target NS3 to DCs by coupling or fusing it to DC-specific peptides. We further hypothesize that NS3 can be structurally modified in order to eliminate the immunodominant epitopes and therefore recruit new T cells against HCV. Specific aims are: 1) To determine whether targeting NS3 specifically to DC subsets enhances specific immune responses against HCV by analyzing T-cell proliferation/activation in humanized SCID mice; and 2) To augment the development of IFN3 gamma-producing NS3-specific CD4+ T cells by engineering the three-dimensional structure of HCV NS3. Alternative modes of loading DC subsets will be explored, including via recombinant Lactobacillus sp. that express and secrete DC-targeted NS3. A needle-less and non-toxic immunotherapy would provide a treatment for hepatitis C patients who currently have none.