RT-PCR for Autol DC Therapy Figure 2. Successful clade-independent amplification of HIV RNA encoding for antigens from infectious plasma. Panel A: Agarose gel electrophoresis analysis of PCR fragment obtained from three diverse plasma. Amplification from subject plasma infected with Clade B sample. M: 100 bp DNA ladder (Invitrogen). Panel B: Amplification from subject plasma infected with Clade C virus. M: 100 bp DNA ladder (Invitrogen). Panel C: Amplification from subject plasma infected with Clade AG virus. M: AmpliSize DNA ladder (BioRad). Analysis of products obtained after the secondary PCR reaction for Gag, Vpr, Rev, and Nef as marked on the top. Panel D. cDNA obtained in preparative secondary PCR reaction corresponding to Gag, Vpr, Rev, and Nef antigens. M: 100 bp DNA ladder (Invitrogen). The molecular weight of representative DNA bands is indicated on the left. Panel E. RNA corresponding to Gag, Vpr, Rev, and Nef antigens obtained by in vitro transcription using amplified PCR products from subjects plasma. M: molecular weight RNA ladder (Promega), representative marker sizes are indicated on the left. G, V, R, N: in vitro transcribed RNAs for Gag, Vpr, Nef and Nef respectively. doi:10.1371/journal.pone.0001489.g002 with individual HIV antigen-encoding RNA-electroporated DC all expressed less than 0.15% IFN-c activity within the CD8+ CFSE-low subset (data not shown). DISCUSSION The strategy of the HIV therapy using autologous DC loaded with autologous antigen has been successfully tested in two clinical trials [14,15]. The HIV therapy described here exploits similar therapeutic principles of the autologous approach but with added advantages in terms of potential safety and applicability to a broader HIV-infected patient population. Our approach requires only a small amount of patient plasma, which may be pivotal for eventual commercial-scale manufactur- ing. We demonstrated amplification from samples containing as little as 263 or 370 copies of cDNA in a single PCR reaction. Many RT-PCR methods such as HIV sequence detection methods detect as low as 50 copies or even a single copy [22–24]. However, such methods developed for high sensitivity detect only small amplicons and do not require an intact HIV genome. To clone longer HIV fragments, 1,000 copies of the HIV genome are required for use in the initial RT reaction [25]. Therefore, the HIV RNA copy requirements described here is comparable to those reported in the literature. Due to the fact that RNA is amplified, first through RT-PCR, and then by in vitro transcription, milligram-scale RNA masses can be achieved, sufficient to transfect large numbers of autologous DCs. The complete coding regions for p55 Gag and Nef and partial products for Rev and Vpr were amplified. The full length Rev mRNA is formed in the course of a trans-splicing reaction which is not possible to reproduce in vitro. Since the exon 1 of Rev encodes only 25 amino acids we designed primers to amplify exon 2 only. The ATG initiation codon which enables translation of that RNA is introduced in the context of a secondary PCR primer. In the case of Vpr, primers were designed to amplify the sequence coding for amino acids 1–71 only. All of the predicted epitopes for CTL recognition within the Vpr protein are located between amino acids 12–70 (http://www.hiv.lanl.gov/content/immunology). The approach of expressing functionally inactive HIV genes which are still able to successfully induce immunity have been documented by others [26]. Our ability to amplify HIV sequences in a clade-independent manner rests on the principles of multiplex RT-PCR technology. PLoS ONE | www.plosone.org 5 January 2008 | Issue 1 | e1489