The investigators will focus on developing a human dendritic cell (DC)-based vaccine strategy to induce virus-specific cytotoxic T lymphocyte (CTL) immunity. Novel replication-defective lentiviral (HIV-1) vectors will be constructed to deliver antigenic epitopes into nonreplicating populations of mature and immature DC.
Specific Aim 1 will determine whether fully functional """"""""mature"""""""" DC cultured from blood monocytes (mo-DC) will generate HIV-specific CD8+ CTL. Short-term cultures of mo-DC can be easily generated with at least 95 percent homogeneity. Three env/nef deleted HIV-1 vectors pseudotyped with VSV(G) will be evaluated. Their LTR were shown to be productively transcribed in mo-DC. To generate a HIV-specific CTL response, CD8+ T cells from HLA-A2.1 healthy donors will be primed with virus-transduced mo-DC in vitro, using conditions established for production of greater than 10E8 peptide-specific CTL. Specificity of the CTL will be measured by lysis of virus-infected A2.1-Jurkat cells. Reactivities to known A2-1-restricted epitopes of gag and pol also will be assessed with A2.-1-Jurkat cells pulsed with appropriate peptides. If the virus-specific CTL reactivity was not directed to known epitopes, the investigators will identify the novel epitopes by a panel of nanomers (""""""""mimotopes"""""""") encoding the immunogenic domains of gag or pol proteins. Frozen transduced mo-DC may be used for repeat in vivo immunizations, or to generate CTL for adoptive T cell therapy.
Specific Aim 2 will determine whether fresh (uncultured) DC and committed DC precursors are useful targets for transduction. Fresh mature and immature DC populations from G-CSF-mobilized peripheral blood will be infected, to predict the feasibility of using lentiviral vectors to immunize in vivo. In particular, the investigators will monitor whether transduction impedes the development of antigen presenting functions of immature DC as measured by their ability to generate HIV-specific CTL and expression of appropriate costimulatory and accessory molecules. If necessary, the investigators will incorporate into the viral vectors cytokines such as flt3 ligand (FL) or IL-4, to facilitate maturation of fresh DC.
Specific Aim 3 will verify that in vivo transduction is feasible in a murine model. The investigators will first immunize mice with syngeneic DC transduced by the HIV vector with the CMV promoter, and measure virus-specific CTL activity in spleen and lymph nodes. For proof of concept of in vivo transduction, the investigators will immunize mice in vivo with the vector, after daily injections of FL designed to dramatically increase the number of DC precursors in vivo.
