. . Table 3. List of samples tested for HIV RNA amplification. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... sample viral load volume of coies of RNA ... number RNA/mL plasma used clade per RT reaction . ... 1 7,413 3 B 2220 . ... 2 14,791 3 B 2220 ... 3 15,849 2 C 1580 . ... 4 16,596 3 B 2480 . ... 5 18,197 3 B 5460 .... 6 22,155 3 B 3320 ... 7 22,909 2 AG 2300 . ... 8 28,840 3 B 8640 .... 9 33,884 3 B 10160 ... 10 38,663 3 B 5800 . ... 11 38,905 3 B 5840 . ... 12 45,709 3 B 6860 ... 13 48,627 1 Nd 2440 . ... 14 50,070 1 Nd 2500 . ... 15 50,119 3 B 7520 .... 16 50,119 3 B 7520 ... 17 53,334 2 B 5340 . ... 18 53,703 2 C 5360 .... 19 53,725 3 B 8060 ... 20 72,865 1 Nd 3640 . ... 21 72,978 1 nd 3640 . ... 22 95,637 2 B 9560 .... 23 117,490 3 B 17620 ... 24 131,826 3 B 19780 . ... 25 134,000 3 B 20100 .... 26 146,148 1 B 7300 ... 27 154,882 3 AG 23240 . ... 28 158,489 3 B 23780 . ... 29 244,000 1 B 12200 ... 30 513,000 3 Nd 38480 . ... 31 1,138,560 3 B 170780 . ... 32 1,479,108 3 Nd 221860 .... 33 3,221,835 1 B 161100 . . .. Samples acquired as indicated in various geographical locations; nd: data not . . available; viral load test was estimated by approved clinical laboratory methods . .. (Amplicor HIV assay Roche or bDNA Bayer); copies of RNA per RT reaction .. . calculated assuming no loss of HIV RNA during isolation procedure. All samples .. obtained from randomly selected patients. .. . doi:10.1371/journal.pone.0001489.t003 First, different loci within HIV genomes are targeted in primary PCR reactions and secondly, each primer group is composed of primers which are complementary to target sequences as well as additional primers carrying compensatory mutations. During the annealing step the most complementary primer-template combina- tion gives rise to a primary product which is then amplified further in a secondary PCR reaction. We believe that because the template is first amplified in a course of primary PCR, mismatches between primary PCR fragments and secondary PCR primers is more forgiving and allows for productive amplification. Therefore greater numbers and combinations are required for the primary PCR reactions. This study demonstrates the feasibility of target region amplification from infectious plasma encompassing various HIV virus clades as well as diverse viral loads. We also demonstrate that RT-PCR for Autol DC Therapy this process captures various quasispecies within a single patient. We also observed that the diversity of Nef sequences found in subject HTM-349 is dramatically reduced compared to the other two subjects analyzed. It would be interesting to determine whether the low diversity of Nef sequences is due to a lack of Nef-specific CTLs within that patient consistent with the hypothesis that immune pressure drives the evolution of Nef variants. We believe that the ability to capture autologous sequences of HIV virus and all quasispecies arising as a result of the CTL pressure is the foundation for the successful anti HIV immunotherapy. While translated protein from the RNAs encoding the four individual antigens was studied in the DC, we were unable to devise a universal method of detection or identify an antibody which could cross-react with all subject-specific amplified material. Also, the sensitivity of these methods is insufficient to detect protein expression when a relatively low amount of RNA (1 mg or less) is delivered to the DCs. Instead we elected to study presentation of antigens by RNA- electroporated DC with detection of induced T-cell responses as a surrogate assay for protein expression using PBMCs derived from a successfully HAART-treated HIV-infected donor. Autologous RNA rather than consensus-sequence- based reagents (vectors, peptides, etc.) was used to transfect DCs to maximize the probability of antigen recognition Flow cytometric analysis of T cell- DC co-cultures demonstrated that DCs electroporated with the four HIV antigen-encoding RNAs successfully induced specific proliferation and effector function (IFN-c activity) to all four antigens within the responder CD8+ T cell subset in this experiment. Both qualitative and quantitative issues contribute to the reported immune responses. The RNAs used in this experiment have different molecular weights: 1.5 kb for Gag and 0.3 kb for Vpr thus the number of molecules of each RNA is inversely proportional to the mass used. Under the conditions used in the experiment shown in Figure 4, the relative number of molecules delivered to the DC is greater for Vpr and lower for Gag. Figure 4C shows the correlation of antigen reactivities (as measured by IFN-c release) supporting the idea that for this donor, the relative epitope densities presented on the surface of the DC is proportional to the number of RNA transcripts electroporated for each antigen. The preferential targeting of Vpr with CTLs was also reported by others [20]. However, it is well documented that different patients can have qualitatively different preferential epitopes reactivities resulting in non-linear relationships with antigen expression levels or epitopes densities on antigen presenting cells. This relationship is further impacted by differences in TCR avidity for specific epitopes or proteosome processing preferences which can vary from patient to patient and whether the predominant infecting virus species encode mutated epitopes [27]. A study of monozygotic twins infected at the same time with the same virus demonstrated that while some CD8 T cell specific responses were the same, some discordinant T cell responses were also found [28]. The discordinant responses cannot be simply explained by virus phenotype or HLA epitope restriction and suggests that additional factors play a role in selection of epitope recognition. We also noted that the majority of the cells responding to HIV antigens with proliferation and IFN-c production exhibited a CD28+/CD45RA- ‘effector/memory’ phenotype which has been linked to long-term non-progression [5,6]. Collectively, these data provide strong evidence that, at least in vitro, HIV antigens encoded by RNA can be translated and presented by DC to induce poly- antigen immunity. Such activity is presumed to be an essential aspect of an immunotherapeutic designed to control HIV viral escape. In addition, no specific activity was recorded within the CD4+ subset, consistent with the inability of antigen encoded by RNA to efficiently PLoS ONE | www.plosone.org 6 January 2008 | Issue 1 | e1489