Second, multiple stop codons were introduced into the central em Eco /em RI- em Eco /em RI (nucleotides 4647 to 5742) fragment covering the integrase gene by primary PCR followed by overlap PCR using synthetic primers. and long-lasting HIV-specific cellular immunity is expected to control virus replication in infected individuals. Cytotoxic T lymphocytes (CTL) are associated with the control of viremia in human immunodeficiency virus type 1 (HIV-1)-infected patients (21, 29) and simian immunodeficiency virus (SIV)-infected monkeys (9, 13, 41). However, currently available therapeutic approaches do not induce HIV-1-specific immunity. On the contrary, CD4+- and CD8+-mediated T-cell responses decline in time in patients treated BMS 777607 with highly active antiretroviral therapies (14, 30, 31). The absence of HIV-1-specific cellular immunity contributes to treatment failures and to viral rebound after interruption of therapy. Encouraging results recently indicated that both HIV-1- and SIV-specific T-cell responses could be enhanced with early treatment of acute infection and that potent T-cell immunity was associated with immune control of virus after interruption of therapy (21, 23, 36). Although these results provided a rationale to explore immunotherapeutic approaches, the use of wild-type HIV-1 for autoimmunization raised safety concerns. Therefore, novel therapeutic vaccine approaches that are able to Tmem5 induce HIV-specific cellular responses are required to control virus replication. Here we describe a new technology to induce potent HIV-specific T-cell responses using genetically modified dendritic cells (GMDC). MATERIALS AND METHODS Construction of plasmid DNA. The replication- and integration-defective viral vector pLW/int was derived from a dualtropic primary HIV-1 isolate, laboratory worker (LW) (22). The mutant HIV-1 vector contains six stop codons and one BMS 777607 deletion in the reading frame and one stop codon and one deletion in the second frame. First, the deletion was made. Second, multiple stop codons were introduced into the central em Eco /em RI- em Eco /em RI (nucleotides 4647 to 5742) fragment covering the integrase gene by primary PCR followed by overlap PCR using synthetic primers. The mutant fragment was cloned into a subclone with a deletion of the relevant fragment of the wild-type provirus. The authenticity of the final clone was checked by DNA sequencing. Culture and HIV-1 infection of primary lymphocytes, macrophages, and DC. Peripheral blood lymphocytes and monocyte-derived macrophages were isolated from human and macaque peripheral blood as described earlier (6). Human and monkey dendritic cells (DC) were cultured for 7 days in complete culture medium (RPMI 1640 with 10% fetal calf serum) supplemented with 1,000 U of granulocyte-macrophage colony-stimulating factor and 700 U of interleukin 4 (IL-4) as described elsewhere (40). The wild-type virus, LW, and the integrase mutant (LW/int) viral vector were produced by transfection of 293 T cells with BMS 777607 the corresponding plasmid DNA. Supernatants were collected 2 days after transfection and normalized for p24 before infection of primary human cells. Electron microscopic examination of the pLW- and pLW/int-transfected 293 T cells demonstrated comparable levels of viral particle production (data not shown). Transduction of DC with plasmid DNA. DC (2 105/well) were plated in 150 l of Optimem culture medium (Gibco) in a 96-well plate. Polyethylenimine (PEI) or PEI-mannose was used at an N/P ratio of 5 equivalents to complex about 2 g of plasmid DNA (4). Cells were incubated for 4 h at 37C. Half of the medium was replaced with fresh RPMI 1640 medium containing 10% fetal calf serum and cytokines (granulocyte-macrophage colony-stimulating factor and IL-4). Transduction of DC was routinely done in parallel wells. HIV-1 expression was monitored by a p24 antigen capture assay (Coulter). Analysis of in vitro T-cell priming. DC (stimulator cells) were cocultured with autologous peripheral lymphocytes (responder cells) in a BMS 777607 1:10 ratio. Aliquots of this culture were analyzed after 3 days for gamma interferon (IFN-) production and after 7 days for CTL activity. For restimulation, an autologous B-lymphoblastoid cell line (B-LCL) primed with peptides or Zn-inactivated HIVMN or microvesicle (control supernatant obtained from the same cell line in the absence of HIVMN) was used. For flow cytometry, cells were incubated with 2 g of brefeldin A per ml for 3 h and harvested for cytokine staining. Cells (5 105) were washed twice with phosphate-buffered saline (PBS) containing 1% bovine serum albumin at room temperature and stained with PC5-CD3+ (UCHT1; Immunotech) and fluorescein isothiocyanate-CD8+ (B9.11; Immunotech) antibodies for 30 min on ice. Cells were washed twice.