route with 1/10th of the human dose of ALVAC and i.p. and chemokine secretion. gene encoding for the entire gag protein, the sequence of the gene encoding the protease, and a synthetic polynucleotide encompassing several known human CTL epitopes from your and gene products. It also contains sequences encoding the E3L and K3L vaccinia computer virus proteins in the C6 site. ALVAC Placebo (Sanofi-Pasteur) contains a mixture of 10?mM TrisCHCl buffer pH 9.0, computer virus stabilizer and freeze-drying medium. Both Delsoline ALVAC and Placebo were re-constituted in sterile 0.9% saline prior to injection. Recombinant HIV Tat protein from your HIV-1IIIB isolate and glycoprotein B (gB) from cytomegalovirus (CMV) were purified from bacterial culture at Sanofi-Pasteur. All vaccines used in this study were clinical grade and were verified to be free of lipopolysaccharide (LPS) and other impurities. 2.3. The adjuvant effect of ALVAC Groups of 10 Balb/c, 129/Sv, IFN-R knockout, or IFN-R knockout mice were immunised i.m. in the quadriceps following anaesthesia with Imalgene 500 (Merial, Lyon, France) with either gB (2?g/dose), Tat (20?g/dose), alone or in combination with Placebo or ALVAC (1/10 human dose). Delsoline Mice were boosted at day 21 and serum and spleen samples taken at day 35. Anti-Tat and gB antibody titres and specific cellular responses were then analysed as explained below. 2.4. Specific anti-Tat and anti-gB antibody titration by ELISA Maxi-Sorp ELISA plates (Nunc, Wiesbaden, Germany) were coated with 1?g/ml of either Tat or gB in PBS overnight at 4?C. Plates were blocked with 1% non-fat milk (Gibco, Paisley, UK) at 37?C for 1?h, after washing serial dilutions of serum samples and requirements were added for 1?h at Delsoline 37?C. Plates were then washed and the detection antibody, goat-anti-mouse IgG-HRP (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was added for a further hour at 37?C. Plates were washed, and a tetramethlybenzidine substrate answer added Delsoline (Tebu Bio-Labs, Le Perray-en-Yvelines, France), the enzyme reaction was stopped with the addition of phosphoric acid (1?M), and OD values at 450?nm were determined. Antibody titres were calculated from your linear regression curve of a standard specific serum originating from the same animal species as the samples, present on each ELISA plate (Softmax software?, Molecular Devices, Sunnyvale, CA). The titre of the standard had been previously decided in 15 impartial experiments. 2.5. IFN- ELISPOT MultiScreen HTS plates IL6R (Millipore, Bedford, MA) were coated overnight at 4?C with anti-IFN- capture monoclonal antibody (mAb) (Clone No. R4-6A2, BD Pharmingen, San Diego, CA), washed and blocked. Responder spleen cells were added at a concentration of 2??105 cells/well in RPMI 1640 (Gibco) supplemented with 10% FCS (Hyclone, Logan, UT), Penicillin-Streptomycin (Gibco), 200?mM l-glutamine (Gibco), and 50?mM -mercaptoethanol (Gibco). Cells were stimulated in triplicate with 1?g/ml of 9 or 15 mer Tat or gag peptides (Neosystem, Strasbourg, France), medium alone as negative control and PMA (SigmaCAldrich, St. Louis, MO)/anti-CD3 (BD Pharmingen) as positive control. ALVAC specific immune responses were measured by adding P815 cells (Mouse lymphoblast-like mastocytoma cell collection) that had been infected with a multiplicity of contamination (MOI) of 20 of parental computer virus (CpPP) immediately. Cultures were incubated overnight at 37?C/5% CO2. The following day, detection using mAb IFN–biotin (clone No. XMG1.2, BD Pharmingen), followed by Streptavidin-HRP (Southern Biotech, Birmingham, AL) was visualized using 3-amino-9-ethylcarbazole (AEC) substrate (SigmaCAldrich). IFN- generating spots were counted using an automated ELISPOT reader equipped with Spot? software (MicroVision Devices, Evry, France). 2.6. In vivo IFN- capture assay ALVAC’s ability to induce early circulating IFN- following i.m. injection was evaluated using an in vivo IFN- detection assay as explained by the manufacturer (BD Pharmingen). Briefly, prior to i.m. injection with ALVAC or Placebo groups of Balb/c mice were given an intraperitoneal (i.p.) injection with 10?g/dose of a biotin-IFN- mAb, serum samples were removed at various time-points and frozen at Delsoline ?20?C until analysis.
route with 1/10th of the human dose of ALVAC and i