Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211536&req=5

pone-0001489-g002: 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.

Mentions: Amplification of specific HIV genome regions is complicated by the high sequence diversity of the HIV genome. This sequence diversity prevents the design of a single universal primer pair for each gene of interest. To overcome this, we designed pools of forward and reverse primers for each target gene (i.e., Gag, Rev, Vpr and Nef) such that most virus strains will react with at least one forward and one reverse primer. Schematic representation of the primer design and strategy for the HIV RNA amplification are presented in Figure 1. This strategy provides for reliable amplification of intended target antigen genes, as well as the co-amplification of existing HIV quasispecies. A list of individual primers is given in Table 1 and the composition of primer groups is given in Table 2. The number of amplification reactions for each HIV antigen was as follows: 6 for Gag, 4 for Vpr, 3 for Rev, and 2 for Nef. We amplified the four antigens from archived frozen plasma infected with diverse clades of HIV: B, C and AG (Figure 2, panels A–C). 2–3 mL of plasma were used to isolate HIV RNA and the titers of these three samples were of 53,334, 53,703 and 154,882 copies/mL, respectively. 2.5 µL of each eluted RNA was used in an RT-PCR for each antigen irrespectively of the initial viral load. PCR resulted in a productive amplification of DNA fragments of expected size for each antigen from all three samples.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001489-g002: 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.
Mentions: Amplification of specific HIV genome regions is complicated by the high sequence diversity of the HIV genome. This sequence diversity prevents the design of a single universal primer pair for each gene of interest. To overcome this, we designed pools of forward and reverse primers for each target gene (i.e., Gag, Rev, Vpr and Nef) such that most virus strains will react with at least one forward and one reverse primer. Schematic representation of the primer design and strategy for the HIV RNA amplification are presented in Figure 1. This strategy provides for reliable amplification of intended target antigen genes, as well as the co-amplification of existing HIV quasispecies. A list of individual primers is given in Table 1 and the composition of primer groups is given in Table 2. The number of amplification reactions for each HIV antigen was as follows: 6 for Gag, 4 for Vpr, 3 for Rev, and 2 for Nef. We amplified the four antigens from archived frozen plasma infected with diverse clades of HIV: B, C and AG (Figure 2, panels A–C). 2–3 mL of plasma were used to isolate HIV RNA and the titers of these three samples were of 53,334, 53,703 and 154,882 copies/mL, respectively. 2.5 µL of each eluted RNA was used in an RT-PCR for each antigen irrespectively of the initial viral load. PCR resulted in a productive amplification of DNA fragments of expected size for each antigen from all three samples.

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