Multiplex RT-PCR amplification of HIV genes to create a completely autologous DC-based immunotherapy for the treatment of HIV infection

Abstract

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Amplification of autologous HIV sequences using multiplex PCR.

Panel A. Sequence alignment of multiple HIV isolates, revealed a region of relative conservation with variable residues in positions 7847 and 7848. Primer REVF7830 is perfectly complimentary to consensus sequence B, whereas primers REVF7830.1 and REVF7830.2 encode compensatory mutations in the 3′ region of the primer, indicated in bold. ···denotes deletions, -sequence identity, letters indicate alternative bases in the corresponding positions relative to consensus sequence B. Consensus sequences for common HIV clades as well as less frequent isolates are denoted in bold. Panel B. Schematic overview of the Rev RNA amplification strategy. Open bar denotes regions outside of open reading frame of interest, hatched bar denotes RNA region exon 2 Rev, grey bar represent DNA intermediate products durig amplification process. For details on primer design and amplification refer to Method section.

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.

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.

Figure 4. Panel A: CFSE-low cells expressed as a percentage of total PBMCs.

Mature DCs (CD209: 96%; CD14: 0%; CD80: 100%; CD83: 91%; CD86: 100%; HLA-DR: 96%; and HLA-I: 100%) were electroporated with 4 HIV antigen-encoding RNAs (hatched bar) or eGFP (solid bar) were cultured with CFSE-labeled PBMCs for 6 days. Frequency of CD8+ CFSE-low were cells determined by flow cytometry. Panel B: CD28/CD45RA phenotype of CD8+ cells induced to proliferate (CFSE-low) by DC electroporated with 4 HIV antigen-encoding RNAs (left panel), as compared to the frequency of CD8+ CFSE-low cells induced by eGFP-RNA loaded control DC (right panel), as determined by flow cytometry. Panel C: Frequency of IFN-γ positive cells within the CD8+ CFSE-low subset induced by 4 hr re-stimulation with DC expressing individual HIV antigen-encoding RNAs, or eGFP control RNA, as determined by intracellular staining and flow cytometry. The background response for single HIV RNA stimulators (1ug HIV RNA/106 DC) was calculated at 0.38% from GFP RNA-electroporated DC (1ug GFP RNA/106 DC) and is indicated by the horizontal dashed line.