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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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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.
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pone-0001489-g004: 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.

Mentions: The goal of active HIV immunotherapy is to stimulate the preferential expansion of antiviral effector T cells. To demonstrate that HIV RNAs generated by our approach can express antigens capable of inducing CD8+ T-cell immunity, we prepared DC electroporated with all four autologous HIV antigens encoded as RNAs. 1 µg Gag RNA, 0.25 µg Nef RNA, 1 µg Rev RNA, and 1 µg Vpr RNA were electroporated along with 1 µg of CD40L RNA per 106 DC. Since cells were fully matured by overnight incubation in the presence of TNFα, INFγ and PGE2 the maturation status of the DCs did not change after electroporation with the RNAs (Data not shown). These cells were co-cultured with autologous PBMCs pre-labeled with CFSE. After 6 days of co-culture, the frequency and phenotype of proliferating cells was detected by residual CFSE fluorescence within the CD8+ T cell population with effector (CD45RA+/CD28−) or effector/memory (CD45RA−/CD28+) phenotypes. After 6 days of co-culture, the CD8+ T-cell population was stimulated with either eGFP RNA-transfected DC (negative control) or HIV RNA-transfected DC. The frequency of CFSE-low cells stimulated with GFP RNA-loaded DC was 3.75% while those stimulated with HIV RNA-loaded DC had a frequency of 7.41% (Figure 4 Panel A). No proliferation above the negative control background was observed within the CD4+ T cell subset, with all DC populations inducing ∼1% CD4+ CFSE-low cells within total PBMCs (data not shown). Within the proliferating CFSE-low CD8+ T cell subset stimulated with HIV RNA-loaded DC, 24.7% of cells exhibited a phenotype consistent with fully differentiated effector cells (CD8+CD28−CD45RA+) versus 54.8% of cells had a phenotype indicative of effector/memory cells (CD8+CD28+CD45RA−) (Figure 4 Panel B).


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211536&req=5

pone-0001489-g004: 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.
Mentions: The goal of active HIV immunotherapy is to stimulate the preferential expansion of antiviral effector T cells. To demonstrate that HIV RNAs generated by our approach can express antigens capable of inducing CD8+ T-cell immunity, we prepared DC electroporated with all four autologous HIV antigens encoded as RNAs. 1 µg Gag RNA, 0.25 µg Nef RNA, 1 µg Rev RNA, and 1 µg Vpr RNA were electroporated along with 1 µg of CD40L RNA per 106 DC. Since cells were fully matured by overnight incubation in the presence of TNFα, INFγ and PGE2 the maturation status of the DCs did not change after electroporation with the RNAs (Data not shown). These cells were co-cultured with autologous PBMCs pre-labeled with CFSE. After 6 days of co-culture, the frequency and phenotype of proliferating cells was detected by residual CFSE fluorescence within the CD8+ T cell population with effector (CD45RA+/CD28−) or effector/memory (CD45RA−/CD28+) phenotypes. After 6 days of co-culture, the CD8+ T-cell population was stimulated with either eGFP RNA-transfected DC (negative control) or HIV RNA-transfected DC. The frequency of CFSE-low cells stimulated with GFP RNA-loaded DC was 3.75% while those stimulated with HIV RNA-loaded DC had a frequency of 7.41% (Figure 4 Panel A). No proliferation above the negative control background was observed within the CD4+ T cell subset, with all DC populations inducing ∼1% CD4+ CFSE-low cells within total PBMCs (data not shown). Within the proliferating CFSE-low CD8+ T cell subset stimulated with HIV RNA-loaded DC, 24.7% of cells exhibited a phenotype consistent with fully differentiated effector cells (CD8+CD28−CD45RA+) versus 54.8% of cells had a phenotype indicative of effector/memory cells (CD8+CD28+CD45RA−) (Figure 4 Panel B).

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