Isolation and amplification of HIV antigens from patient plasma HIV RNA was isolated from 1 to 3 mL of plasma from HIV patients using a NucliSens kit (BioMerieux), according to the manufacturer’s instructions and eluted in 30 mL of nuclease free water. First strand cDNA synthesis reaction contained gene- specific primers for either Gag, Vpr or Rev, and oligo dT(20) (Invitrogen) for Nef, 40 units of RNAseOut (Invitrogen), 0.5 mM of each dNTP (Clontech), and Superscript first strand buffer. After annealing at 65uC for 5 minutes, DTT to 5 mM and 400 units of Superscript III (Invitrogen) were added and the reaction was incubated at 55uC for 1 hour. 2.5 mL of the first strand cDNA reaction was then taken into a primary PCR reaction containing 5 units of PFU ultra HS, PFU buffer (Stratagene), 0.2 mM of each dNTP (Stratagene), and the corresponding group of primers at a final concentration 0.4 mM for Gag, 0.6 mM for Vpr, 0.2 mM for Rev, and 0.4 mM for Nef, in a final reaction volume of 50 mL. The PCR reaction was denatured at 95uC for 2 minutes and then run for 40 cycles as follows: 95uC for 30 seconds, 54uC for 30 seconds, and 72uC for 3 minutes and 10 minutes for the last cycle. The annealing temperature was kept at 54uC here and in the secondary PCR amplification to allow for annealing of primers to templates with a limited degree of mismatch [34] 1 mL of the primary PCR reaction was then taken into a secondary PCR reaction containing 2.5 units of PFU Ultra HS, PFU buffer (Stratagene), 0.2 mM of each dNTP (Stratagene), and gene specific T7 and 64T groups of primers, in a final reaction volume of 25 mL. The cycling parameters were the same as in the primary PCR reaction. Products of the secondary PCR reaction were purified using a QIAquick purification column (QIAGEN). For preparative PCR several (i.e. 6 or 12) reactions were established with same conditions as secondary PCR except the total volume of each reaction was 50 mL. PCR Amplification of HIV sequences from noninfectious templates Gag, Rev and Vpr were amplified from plasmid pBKBH10S and Nef was amplified from plasmid p93TH253.3 obtained from NIH AIDS Research & Reference Reagent program [35,36]. Single forward and reverse PCR primers were selected with full complementarity to the template determined by sequence analysis. All PCR conditions were exactly the same as used for amplification of the infectious material. In vitro transcription of HIV antigens Secondary PCR fragments served as templates for an in vitro transcription reaction using mMessage mMachine T7 Ultra kit (Ambion) according to the manufacturer’s instructions. The amplified RNA was purified using RNeasy columns (QIAGEN). Sequence analysis and phylogeneic relationship Sequencing analyses were performed using the UNC sequencing facility (University of North Carolina, Chapel Hill). Nucleotide sequence analysis, identity verification and phylogeneic tree analysis were performed using Lasergene software (DNAStar), the Los Alamos HIV Sequence Database (http://www.hiv.lanl. gov), and BLAST analysis [32,33]. Isolation of human dendritic cells A leukapheresis sample from a volunteer was collected on a COBE Spectra (Gambro BCT) using the AutoPBSC procedure described RT-PCR for Autol DC Therapy by Lifeblood (Memphis, TN). Peripheral blood mononuclear cells were isolated using a Ficoll density gradient (HistopaqueH-1007 Hybri-MaxH, Sigma) and cultured for 1 to 2 hours to allow adherence of the monocytes. Non-adherent cells were removed and the remaining monocytes were cultured in X-VIVO 15TM (Cambrex) medium for 6 to 7 days, supplemented with 1000 U/mL each of GM-CSF (Berlex, LeukineH liquid) and IL-4 (R&D Systems). Generation of DC for functional testing of HIV IVT RNAs for the induction of anti-HIV immunity in vitro Immature DCs were generated as described above from a successfully HAART-treated HIV donor with a viral plasma copy number of less than 200 copies per mL. To achieve DC maturation, immature DC were cultured on day 5 with 10 ng/ ml TNF-a, 1000 mg/ml IFN-c, 1 mg/ml PGE2. On Day 6, matured DCs were co-electroporated with in vitro transcribed RNA encoding CD40L at 1 mg per million of DC, and 1 mg Gag, Rev, Vpr and 0.25 mg Nef autologous HIV RNAs per million of DCs. A negative control DC stimulator was generated by transfecting DC with CD40L RNA and 3.25 mg eGFP RNA, instead of HIV RNA mix. RNA-electroporated DC were further cultured for 4 hrs in X-VIVO-15 medium without additional cytokine supplements. In vitro co-culture of DC and PBMC from an HIV- infected subject to induce anti-HIV T-cell responses to multiple HIV antigens CFSE labeling PBMCs from the HIV donor were enriched by Ficoll gradient separation, washed twice with PBS and re- suspended at 2.06107 cells per mL in PBS. CFSE was added to the cell suspension for a final working concentration of 1.0 mM and incubation for 8 minutes at room temperature. The staining was quenched by the addition of an equal volume of Human AB Serum and incubation for 2 minutes. Initial DC/PBMC co-culture Cultures of HIV RNA- electroporated mature DC, and eGFP-RNA control DC were established in parallel with CFSE-labeled PBMC at a 1:10 ratio, 1 million total cells/mL in 5% Human AB serum for 6 days at 37uC, 5% CO2. Cell surface phenotyping of proliferating CFSE ‘low’ labeled cells After 6 days of culture, PBMCs were harvested, washed once with 2 mL PBS containing 10% FBS and stained for surface antigens using CD45RA PE, CD8 PerCP-Cy5.5, CD28 APC or CD45RA PE, CD4 PerCP-Cy5.5, CD28 APC antibodies (BD Bioscience) at room temperature in the dark for 20 minutes. Samples were washed twice with cold PBS containing 10% FBS and re-suspended in 300 mL of 2% BD Cytofix (BD Bioscience) Samples were acquired on a BD FACSCalibur flow cytometer and analysed using FlowJo software (Three Star, Inc.) Analysis gates were set on the basis of FSC v. SSC to define viable lymphocytes and lymphoblasts, and the frequency of proliferating cells determined by detection of CFSE ‘low’ cells, and their associated cell surface phenotype Measurement of anti-HIV specific activity by restimulation of PBMCs with individual DC populations expressing a single HIV gene Immature DC were generated as described above, matured with TNF-a, IFN-c and PGE2 and cells split into 5 groups, allowing for DC populations to be generated expressing just a single HIV gene from the panel of four individual antigens, and a fifth DC population electroporated with eGFP RNA, as negative control. The DC populations were co- cultured in parallel with CFSE-labeled PBMC harvested from the previous 6-day co-culture described above. One hour after re- stimulation with DCs, 0.25 ml of Golgi plug (BD Bioscience) was PLoS ONE | www.plosone.org 9 January 2008 | Issue 1 | e1489