We have previously demonstrated the power from the vaccine vectors predicated on replicon RNA from the Australian flavivirus Kunjin (KUN) to induce protective antiviral and anticancer CD8+ T-cell reactions using murine polyepitope like a model immunogen (I. induced powerful Gag-specific Compact disc8+ T-cell reactions, with one immunization of KUNVLPs inducing 4.5-fold-more CD8+ T IKK-gamma (phospho-Ser376) antibody cells compared to the number induced after immunization with recombinant vaccinia virus carrying the gene (rVVVLPs also provided significant protection against challenge with rVVpolyprotein, which may be the precursor for the inner structural proteins (matrix, capsid, nucleocapsid, and p6) from the adult virion (6). HIV Gag protein are conserved and the prospective of mix clade S/GSK1349572 immunity (8 fairly, 26). Both Compact disc4 and Compact disc8+ T-cell immunity aimed against Gag protein are thought to be important for safety (10, 15). While not thought to mediate safety, anti-Gag antibodies are elevated in HIV-infected people and by experimental vaccines including Gag, where in fact the antibody reactions may be considered one way of measuring vaccine efficiency (30). Right here we display that immunization with KUN replicons expressing the entire HIV-1 gene induced powerful Gag-specific Compact disc8+ T-cell and antibody reactions and shielded mice from problem with recombinant vaccinia disease expressing the gene. METHODS and MATERIALS Plasmids. The DNA-based and RNA-based KUN replicon vectors C20UbHDVrep and pKUNrep1, respectively (41), had been used for building of plasmids including the HIV-1 gene. Essentially, the complete HIV-1 gene was amplified by PCR from the pBRDH2-neo plasmid (an HIV-1SF2/BH10 construct) (14) with primers gene. The DNA and RNA constructs are driven by the CMV and SP6 promoters, respectively. The constructs contain the following: (i) sequences required for KUN RNA replication (5 and 3 … DNA and RNA transfections and immunofluorescence. For DNA transfection, BHK21 cells in 60-mm-diameter dishes or on glass coverslips were transfected with 2 or 0.4 g of plasmid DNA, respectively, using Lipofectamine Plus transfection reagent (Life Technologies, Melbourne, Australia), as described by the manufacturer. RNA was transcribed in vitro from the RNA were seeded into a six-well plate, and at 32 h postelectroporation, the cells were radiolabeled for 4 h with 100 Ci of [35S]methionine/cysteine in the presence of actinomycin D (Sigma, Castle Hill, Australia). Tissue culture fluid was collected, and the cells were lysed in lysis buffer (phosphate-buffered saline [PBS] containing 0.5% Nonidet P-40). Samples were incubated with anti-pr55antibody (diluted 1:50) overnight at 4C and then incubated for 1 h with 30 l of 10% protein A-Sepharose beads at 4C. Pelleted Sepharose beads were washed three times with PBS, resuspended in gel loading buffer, boiled for 5 min, and separated on a sodium dodecyl sulfate-10% polyacrylamide gel. Electron microscopy (EM). BHK21 cells electroporated with KUNRNA or S/GSK1349572 KUN vector RNA were seeded into a 60-mm-diameter dish, and the cells were then harvested at 24 and 48 h after electroporation. The cells were collected in PBS and fixed with 3% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The samples were treated with 1% osmium tetroxide, dehydrated with acetone, and embedded in Epon 612 resin as previously described (23). Sections collected on grids were stained with uranyl acetate and lead citrate. All specimens were examined on a JEOL 1010 transmission S/GSK1349572 electron microscope at 80 kV. Preparation of VLPs. VLPs were prepared essentially as described previously except that 3 106 BHK21 cells were electroporated with 30 g S/GSK1349572 of in vitro-transcribed KUNRNA. At 32 h postelectroporation, the cells were trypsinized, subjected to a second electroporation using in vitro-transcribed noncytopathic Semliki Forest virus (SFV) replicon RNA containing the Leu713-to-Pro codon substitution in the SFV nsP2 gene and encoding KUN structural proteins (derivative of SFV-MEC105) (18) (details will be described elsewhere) and incubated for 48 to 60 h before harvesting secreted VLPs. The titer of infectious VLPs was determined by infection of Vero cells with 10-fold serial dilutions of the VLPs and counting the number of NS3-positive cells by IIF analysis at 30 to 40 h postinfection. Immunization of mice. Female BALB/c (DNA (100 g) was diluted in 100 l of PBS and injected intramuscularly (i.m.) into the quadriceps muscle of each hind leg (50 l in each leg). (ii) In vitro-transcribed KUNRNA (30 g) was dissolved in 100 l of diethyl pyrocarbonate-treated PBS and injected i.m. (50 l into each leg). (iii) KUNVLPs in Dulbecco modified Eagle medium containing 5% fetal calf serum was injected intraperitoneally (i.p.) at 106 infectious units (IU) per mouse. (iv) A KUN VLP encoding the murine polyepitope (KUNmptVLP) which contains four (rVVRNA were seeded into each well of a 24-well plate. Culture fluid and cell lysate samples were harvested at.