immunization with inactivated whole autologous HIV virus- loaded DC therapy can lead to durable control of viral load [14,15]. Although these clinical studies demonstrated the potential utility of an autologous DC therapy, the choice of whole inactivated HIV virus as an immunogen is not ideal and may have significant safety and practical limitations. In the present study we report on strain-independent RT-PCR amplification of four HIV antigens to generate templates for in vitro- transcribed RNA. Previously a major obstacle to RT-PCR amplification of autologous viral sequences was designing functional PCR primers due to the high mutation rate of the virus. To resolve this problem, we developed a multiplex RT-PCR strategy that allows reliable strain-independent amplification of highly polymorphic target antigens from any patient without the requirement for first knowing the viral sequence and custom-designing of PCR primers for each individual. The amplified products contain a complex mixture of autologous antigens encoded by viral quasispecies. We further demonstrate that in vitro-transcribed RNA can be delivered to DCs where the encoded antigens are expressed, processed, and presented by MHC class I molecules on the cell surface. Regions for Gag, Vpr, Rev and Nef were selected for amplification to generate this completely autologous RNA- transfected DC therapy which lacks infectious virus in the final formulation, thereby circumventing potential safety concerns. The choice of antigens targeted for amplification was based on the several criteria: a) Substantial regions of the target gene had to be amenable to PCR amplification using our primer design strategy, b) Antigen expression should not adversely affect dendritic cell function and c) Antigens had to induce functional CTLs [16–21]. The simultaneous use of autologous viral antigens and DCs may provide for a broad patient-specific immune response that could potentially provide better control of residual virus or rebound of virus following the cessation of antiretroviral therapy. RESULTS Successful amplification of multiple HIV RNA antigens using the devised multiplex RT-PCR protocol Amplification of specific HIV genome regions is complicated by the high sequence diversity of the HIV genome. This sequence diversity prevents the design of a single universal primer pair for each gene of interest. To overcome this, we designed pools of forward and reverse primers for each target gene (i.e., Gag, Rev, Vpr and Nef) such that most virus strains will react with at least one forward and one reverse primer. Schematic representation of the primer design and strategy for the HIV RNA amplification are presented in Figure 1. This strategy provides for reliable amplification of intended target antigen genes, as well as the co- amplification of existing HIV quasispecies. A list of individual primers is given in Table 1 and the composition of primer groups is given in Table 2. The number of amplification reactions for each HIV antigen was as follows: 6 for Gag, 4 for Vpr, 3 for Rev, and 2 for Nef. We amplified the four antigens from archived frozen plasma infected with diverse clades of HIV: B, C and AG (Figure 2, panels A–C). 2–3 mL of plasma were used to isolate HIV RNA and the titers of these three samples were of 53,334, 53,703 and 154,882 copies/mL, respectively. 2.5 mL of each eluted RNA was used in an RT-PCR for each antigen irrespectively of the initial viral load. PCR resulted in a productive amplification of DNA fragments of expected size for each antigen from all three samples. The example of purified products from preparative PCR is shown in Figure 2, Panel D. The reactions were set exactly as RT-PCR for Autol DC Therapy the secondary PCR reaction described in the Methods, but in identical replicates to generate sufficient mass of cDNA for the in vitro transcription reaction. Sequence analysis of these fragments confirmed that the amplified cDNAs correspond to Gag, Vpr, Rev and Nef. Products from the nested PCR reactions were transcribed in vitro to generate RNA and all four antigens were transcribed successfully (Figure 2, Panel E). Because of the HIV genome diversity and presence of deletion and insertions within the open reading frames of interest, the molecular weight of cDNA is expected to vary. We performed a detailed analysis of cDNA and in vitro-transcribed RNA molecular weights for all four antigens amplified from 10 distinct infectious plasma samples. The size of the cDNA was measured by migration on an agarose gel relative to molecular weight markers. The observed size distribution of the cDNA analyzed by non- denaturing agarose gel electrophoresis was 15726150 for Gag, 293625 for Vpr; 496625 and 841650 for Nef. The observed size distribution for amplified RNA analyzed by denaturing agarose gel electrophoresis was 1761654 for Gag, 338637 for Vpr, 427631 for Rev and 801652 for Nef. The range of molecular weights for each antigen observed with these 10 samples is indicative of the high degree of subject-to-subject antigen sequence diversity. Summary of samples from 33 patients with diverse viral load from which all four antigens were amplified is given on Table 3. Whenever possible, a greater volume of viral plasma was used for HIV RNA extraction to achieve higher yields of viral RNA. The sample with the lowest viral load examined was sample 1. For this sample the first cDNA synthesis reaction contained 2220 HIV RNA copies. Due to the multiplex design of the amplification, the RT reaction is divided into multiple PCR reactions. Since the Gag single strand cDNA is divided between 6 PCR reactions, the actual copy number in each PCR reaction is 370. A similar result was obtained for sample 3 with a higher viral load but smaller volume of available plasma. For this sample, each RT reaction contained 1580 copies of RNA and each PCR reaction for Gag contained 263 copies of cDNA. The calculation of final recovered HIV RNA concentration assumes no loss during the extraction procedure. With losses, the absolute copy requirement would be even lower. Overall these data demonstrate consistently successful amplification of all four antigens from plasma samples with diverse viral loads. Multiplex PCR method amplifies diverse HIV quasispecies An advantage of this approach for antigen generation is its ability to capture HIV mutations which evolve under dynamic host CTL pressure [11]. It is broadly applicable to the general HIV-infected population irrespective of Clade designation, but also anticipates that it would capture various quasispecies present in a given subject. This is the cornerstone of our novel therapeutic paradigm which enables targeting, not only of dominant viruses, but also newly emerging virus populations which evolve as a result of immune pressure. To test our hypothesis that multiple quasispecies are co- amplified from a given subject, PCR fragments encoding full length Nef cDNA amplified from Clade B samples HTM-349, HTM-367 and HTM-344 (viral load 513,000; 53,334 and 95,637 copies per mL respectively) were cloned, sequenced and analyzed using phylogenetic tree analysis. A total of 15 clones were analyzed for each subject (Figure 3). The analysis demonstrated that the cDNA population did indeed capture genes encoded by various HIV quasispecies. Phylogeneic tree analysis demonstrated that each subject’s Nef sequences grouped within other sequences from that subject and were distinct from another subjects’ sequences. PLoS ONE | www.plosone.org 2 January 2008 | Issue 1 | e1489