Multiplex RT-PCR Amplification of HIV Genes to Create a Completely Autologous DC-Based Immunotherapy for the Treatment of HIV Infection Irina Tcherepanova*, Jason Harris, Aijing Starr, Jaclyn Cleveland, Helen Ketteringham, David Calderhead, Joe Horvatinovich, Don Healey, Charles A. Nicolette Research and Development Department, Argos Therapeutics, Inc., Durham, North Carolina, United States of America Background. Effective therapy for HIV-infected individuals remains an unmet medical need. Promising clinical trials with dendritic cell (DC)-based immunotherapy consisting of autologous DC loaded with autologous virus have been reported, however, these approaches depend on large numbers of HIV virions to generate sufficient doses for even limited treatment regimens. Methodology/Principal Findings. The present study describes a novel approach for RT-PCR amplification of HIV antigens. Previously, RT-PCR amplification of autologous viral sequences has been confounded by the high mutation rate of the virus which results in unreliable primer-template binding. 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 and requires neither viral sequence data nor custom-designed PCR primers for each individual. We demonstrate the application of our RT-PCR process to amplify translationally-competent RNA encoding regions of Gag, Vpr, Rev and Nef. The products amplified using this method represent a complex mixture of autologous antigens encoded by viral quasispecies. We further demonstrate that DCs electroporated with in vitro-transcribed HIV RNAs are capable of stimulating poly-antigen-specific CD8+ T cell responses in vitro. Conclusion/Significance. This study describes a strategy to overcome patient to patient viral diversity enabling strain- independent RT-PCR amplification of RNAs encoding sequence divergent quasispecies of Gag, Vpr, Rev and Nef from small volumes of infectious plasma. The approach allows creation of a completely autologous therapy that does not require advance knowledge of the HIV genomic sequences, does not have yield limitations and has no intact virus in the final product. The simultaneous use of autologous viral antigens and DCs may provoke broad patient-specific immune responses that could potentially induce effective control of viral loads in the absence of conventional antiretroviral drug therapy. Citation: Tcherepanova I, Harris J, Starr A, Cleveland J, Ketteringham H, et al (2008) Multiplex RT-PCR Amplification of HIV Genes to Create a Completely Autologous DC-Based Immunotherapy for the Treatment of HIV Infection. PLoS ONE 3(1): e1489. doi:10.1371/journal.pone.0001489 INTRODUCTION Immunotherapeutic strategies for HIV-infected individuals are focused on eliciting antiviral CD8+ T cell responses to control the level of HIV virus in vivo. Evidence that cellular immune responses play an important role in controlling HIV infection is supported by several observations including: a) Frequencies of CTL inversely correlate with HIV plasma levels [1] b) Blocking CD8+ T cells with anti-CD8-specific antibodies in SIV-infected macaques correlates with loss of viral control [2], c) resolution of acute viremia in the SIV macaque model requires circulating CD8+ T [3], d) The presence of virus-specific CTL coincides with the appearance of mutant viruses which are no longer recognized by these CTL [4], and e) The presence of CD8+CD28+ T cells are reportedly associated with long-term non-progression [5,6]. Many current HIV immunotherapies utilize individual consen- sus antigens or defined epitopes derived from those reference HIV sequences. Potential therapies based on clade-specific consensus antigens have been investigated in over 80 clinical trials, however, the results demonstrate a consistent lack of efficacy [7–9]. While augmentation of immune responses to consensus sequences used for immunization was demonstrated, these therapies did not result in reduction of viral loads. Evidence suggests that the lack of HIV- protective immunity is attributed to sequence divergence between autologous and consensus antigens. The high HIV mutation rate results in novel variants which encode point mutations within CTL epitopes and escape recognition by specific T cells. Studies with overlapping peptides confirmed that CTL recognizing autologous peptides encoded within a known HIV virus did not cross react with corresponding consensus sequences [10]. Studies on humans and non-human primates correlate virus escape from CTL with progression to AIDS [11–13]. In addition, each patient creates a unique environment for its own viral evolution. Consequently, there is substantial mutational variation between the virus infecting the patient and the reference sequences upon which most HIV immunotherapies are based. Also, since virus sequence diversity defines HIV clades, therapies based on consensus antigens from one clade may have limited ability to cross control evolutionally divergent viruses from other clades. Therefore, therapies based on autologous viral antigens would have broader applicability since the therapy would be perfectly matched to the virus species infecting each subject. To date, the only successful immunotherapies for HIV-infected patients used dendritic cells loaded with autologous viral antigens. Independent clinical studies by Lu et al., and Garcia et.al., demonstrated for the first time that Academic Editor: Linqi Zhang, AIDS Research Center, Chinese Academy of Medical Sciences and Peking Union Medical College, China Received August 17, 2007; Accepted December 13, 2007; Published January 30, 2008 Copyright: 2008 Tcherepanova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: All funding was provided by Argos Therapeutics. Competing Interests: The authors have declared that no competing interests exist. * To whom correspondence should be addressed. E-mail: itcherepanova@ argostherapeutics.com PLoS ONE | www.plosone.org 1 January 2008 | Issue 1 | e1489