RT-PCR for Autol DC Therapy Figure 3. Capture of HIV quasispesies using the developed multiplex RT-PCR approach. Phylogenetic relationships of nucleotide sequences of isolated full-length Nef clones (Panel A) and amino acid sequences (Panel B). Horizontal scale indicates the number of nucleotide mutations or amino acid substitutions on each clone relative to neighbor clones. doi:10.1371/journal.pone.0001489.g003 target the endosomal pathway [29]. We speculate that there may be specific advantages to being able to induce antiviral CD8+ T cell immunity without concomitant expansion of CD4+ T cells which might serve as a reservoir for virus and facilitate enhanced viremia. The breadth of the CTL repertoire has been shown to correlate with anti-HIV-protective immunity. In an animal model compar- ing immune responses in groups of animals immunized with either a gag-pol-encoding DNA vector, codon optimized Gag-Pol, or multivalent Expression Library Immunization (ELI) therapeutics, the induced immune responses were up to 10-fold higher in the group immunized with the multivalent (ELI) therapeutic than the other two constructs tested [30]. Also, improved protection against simian AIDS was reported in a study with an experimental immunotherapy directed against six antigens (Gag, Pol, Env, Rev, Tat, Nef) compared to one directed against only three antigens (Gag, Pol, and Env) [21]. In addition, immunotherapies targeting multiple regions of the viral genome have the potential to force the accumulation of multiple mutations as a consequence of polyvalent CTL pressure which can drive the virus into a state of poor replicative fitness [31]. This study establishes the feasibility of multiplex RT-PCR- mediated amplification of autologous RNA encoding HIV antigens from small volumes of infectious plasma. We also demonstrate that this approach captures multiple quasispecies present within a given subject which could allow the induction of multivalent CTL responses, a critical factor in minimizing the chances of rapid emergence of viral escape variants. Furthermore, we demonstrate ability of DCs transfected with autologous amplified HIV antigen RNA to induce antigen-specific CD8+ T cells. METHODS Design of primers for strain-independent amplification of HIV antigens The strategy used to design primer pools for PCR amplification for the selected HIV antigens, using the Rev sequence as an example, is summarized in Figure 1, Sequences of HIV isolates were aligned using BLAST analysis with sequence NC_001802 serving as an arbitrary reference [32,33]. Nucleotide regions which appeared to have sequence conservation were selected for primer . .. . Table 4. Selective utilization of primers by RT-PCR from various subjects materials. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... Utilization in RT-PCR in various subjects . ... Primer name Primer sequence 59-39 HTM 344 HTM 349 HTM 367 . ... T7 Nef F 8343 TAATACGACTCACTATAGGGAGACCACCATGGGTGGCAAGTGGTCAAAAAG 1 0 10 . ... T7 Nef F 8343.1 TAATACGACTCACTATAGGGAGACCACCATGGGTGGCAAGTGGTCAAAACG 12 0 0 . ... New TAATACGACTCACTATAGGGAGACCACCATGGGTGGCAAGTGGTCAAAAGG 2 15 5 .... TAATACGACTCACTATAGGGAGACCACCATGGGTGGCAAGTGGTCAAAAAT ... 64T Nef R 9069 (T)64CCAGTACAGGCAAAAAGC 7 8 11 . ... 64T Nef R9069.1 (T) 64CAGTACAGGCGAAAAGC 6 2 4 . ... 64T Nef R9069.1 (T) 64CCAGTACAGGCAAGAAGC 2 5 0 . . ... The Nef cDNA sequences were analyzed in the regions corresponding to the regions defined by the primers and identity of the primers was identifies by sequence. .. Total of 15 Nef clones were analyzed for subjects HTM344, HTM 349 and HTM 367. The number in the right three columns represents how many clones contained the .. identified primer. . .. doi:10.1371/journal.pone.0001489.t004 PLoS ONE | www.plosone.org 7 January 2008 | Issue 1 | e1489