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

Tcherepanova I, Harris J, Starr A, Cleveland J, Ketteringham H, Calderhead D, Horvatinovich J, Healey D, Nicolette CA - PLoS ONE (2008)

Bottom Line: 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 further demonstrate that DCs electroporated with in vitro-transcribed HIV RNAs are capable of stimulating poly-antigen-specific CD8+ T cell responses in vitro.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.

View Article: PubMed Central - PubMed

Affiliation: Research and Development Department, Argos Therapeutics, Inc., Durham, North Carolina, USA. itcherepanova@argostherapeutics.com

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.

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Related in: MedlinePlus

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.
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pone-0001489-g003: 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.

Mentions: 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. More interestingly however is the observation that the number of the Nef variable sequences differed in each subject. At the nucleotide level (Figure 3, Panel A) the subject HTM 344 displayed greater diversity where out of 15 clones analyzed, 14 clones were unique, followed by subject HTM 367 with 13 unique clones and for subject HTM 349 only 6 unique clones. The subject-specific sequence clustering together with the variable number of unique clones between patients eliminates the possibility that the mutations are random mutational artifacts introduced during RT-PCR. Not every nucleotide mutation leads to an amino acid substitution, so the diversity is lower at the level of amino acid sequence (Figure 3, Panel B) with the same order of diversity trend for the three subjects. Similar analyses were performed for cDNAs encoding Gag, Rev and Vpr cDNA amplified from various subjects (data not shown). These data indicate that, as predicted, the multiplex RT-PCR is capable of capturing various quasispecies within each individual subject.


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

Tcherepanova I, Harris J, Starr A, Cleveland J, Ketteringham H, Calderhead D, Horvatinovich J, Healey D, Nicolette CA - PLoS ONE (2008)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211536&req=5

pone-0001489-g003: 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.
Mentions: 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. More interestingly however is the observation that the number of the Nef variable sequences differed in each subject. At the nucleotide level (Figure 3, Panel A) the subject HTM 344 displayed greater diversity where out of 15 clones analyzed, 14 clones were unique, followed by subject HTM 367 with 13 unique clones and for subject HTM 349 only 6 unique clones. The subject-specific sequence clustering together with the variable number of unique clones between patients eliminates the possibility that the mutations are random mutational artifacts introduced during RT-PCR. Not every nucleotide mutation leads to an amino acid substitution, so the diversity is lower at the level of amino acid sequence (Figure 3, Panel B) with the same order of diversity trend for the three subjects. Similar analyses were performed for cDNAs encoding Gag, Rev and Vpr cDNA amplified from various subjects (data not shown). These data indicate that, as predicted, the multiplex RT-PCR is capable of capturing various quasispecies within each individual subject.

Bottom Line: 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 further demonstrate that DCs electroporated with in vitro-transcribed HIV RNAs are capable of stimulating poly-antigen-specific CD8+ T cell responses in vitro.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.

View Article: PubMed Central - PubMed

Affiliation: Research and Development Department, Argos Therapeutics, Inc., Durham, North Carolina, USA. itcherepanova@argostherapeutics.com

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.

Show MeSH
Related in: MedlinePlus