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246 Detection of HIV-1 RNA in Peripheral Blood Mononuclear Cells of Patients
Receiving Prolonged Highly Active Antiviral or Vaccine Therapy
J. Zhang and C. S. Crumpacker*
Beth Israel Deaconess Med. Ctr., Harvard Med. Sch, Boston, MA, USA
Background: The HIV-1 RNAs are transcribed differentially during HIV-1 infection. The early viral spliced mRNA encodes the HIV-1 accessory proteins, which include Tat, Rev, and Nef. These viral proteins accelerate the HIV-1 replication and down-regulate the host immune system, playing cardinal roles in viral pathogenesis and development of AIDS. The HIV-1 genomic RNA is transcribed after spliced mRNA reaches a peak level and accumulates in the cytoplasm as a species of 9.2 kb. We have detected HIV-1 spliced and genomic RNA in patient peripheral blood mononuclear cells who receive prolonged highly active antiviral therapy and have viral load (viral genomic RNA in plasma) below the limit of detection for greater than 2 years (< 50 copies/ml). Employing these methods to detect viral RNA expression in patient cells after a therapeutic vaccine treatment may provide a surrogate marker to evaluate the efficacy of a candidate AIDS vaccine, since the vaccine induced immunity, specially the CD8+ cell directed cell immunity, could control HIV-1 RNA expression in these cells.
Methods: Fourteen patients were enrolled in this study. The patients were asymptomatic HIV-1 seropositive with >500 CD4+ cells/ml and were on combination therapy with Indinavir, Zidovudine, and Lamivudine for >116 weeks. These patients had plasma HIV-1 RNA level < 50 copies/ml when their PBMC were examined for HIV-1 spliced and genomic mRNAs. All of these patients were enrolled in the Merck 060/ICC 004 study at Beth Israel Deaconess Med. Ctr. and Massachusetts Gen. Hosp. and have continuously suppressed plasma HIV-1 RNA below 50 copies/ml on this regimen of Indinavir, Zidovudine, and Lamivudine. RT-PCR Assay: Three RT-PCR assays were used in this study. The assay for detection of HIV-1 early spliced mRNA was used to detect the tat, rev, and nef mRNA expression in patient PBMCs. The assay to detect the HIV-1 genomic RNA was used to detect viral genomic RNA in patient plasma and in PBMCs. For detection of early viral spliced mRNA, the published primers were used. The primer pair US and ART7 spans known splice junctions to amplify the doubly spliced mRNAs for the regulatory proteins Tat, Rev, and Nef. The Roche ultrasensitive Amplicor HIV-1 monitor test was used to exam HIV-1 genomic RNA expression in patient plasma. A modification of this test was used to examine HIV-1 genomic RNA in patient PBMC specimens. An in-house RT-PCR assay was also employed to examine the HIV-1 genomic RNA expression in patient cells qualitatively.
Results: In this study, we examine viral RNA expression in 14 patients who were under highly effective anti-HIV drug therapy for more than 2.2 years. The plasma viral RNA was undetectable (< 50 copies/ml) in all these patients, however, the early viral RNA and viral genomic RNA were detected in the PBMCs of most patients despite the prolonged combination drug treatment. The HIV-1 early spliced mRNA and genomic RNA were determined in peripheral blood mononuclear cells (PBMC) of 14 patients who were under highly effective combination antiviral therapy for >116 weeks. The cell associated viral tat, rev, and nef mRNA were detected in 86% (12/14) of the patients. The cell associated viral genomic RNA was detected in 57% (8/14) of the patients. All the patients who were HIV-1 genomic RNA positive had the early viral spliced mRNA detected in their PBMCs and the copy number of the viral genomic RNA in these patients was detected as 67(4,428 copies in 2 million cells.
Conclusions: Our results show that patients receiving highly active combination antiviral treatment for greater than 2 years had actively transcribed viral spliced mRNA and viral genomic RNA in their PBMCs even when their plasma viral RNA level is undetectable. The viral genomic RNA copy number in PBMC was detected with a range of 67(4,428 copies/2 million cells, whereas the plasma HIV-1 RNA copy number was below 50 copies/ml. A previous report stated that viral spliced RNA and genomic RNA were detected in patient PBMCs in 5 patients who received HAART therapy for 80 weeks. In our study, HIV-1 spliced mRNA was detected in 12 of 14 patients (86%) and HIV-1 genomic RNA were detected in 8 of 14 (57%) patients who were under HAART therapy for >116 weeks. Our study further shows that HIV-1 transcription persists in peripheral blood mononuclear cells in patients receiving potent, extended anti-retroviral therapy. The RT-PCR assays detect viral spliced and genomic RNA in patient cells will be useful assays to evaluate the efficacy of a therapeutic vaccine treatment, since the restored immunity, especially the CD8+ cell directed cell immunity, might greatly decrease the number of cells harboring HIV-1 and protect uninfected cells.
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