Specific correlates and predictors of productive HIV-1 infection in peripheral blood mononuclear cells after cessation of antiretroviral therapy.

 

Multiply spliced HIV-1 RNA predict and mirror productive HIV-1 infection in peripheral blood mononuclear cells after cessation of antiretroviral therapy.

 

Multiply spliced  but not intracellular unspliced cell-associated HIV RNA reflect productive HIV-1 infection in PBMC after early cessation of antiretroviral therapy.

 

Extracellular, virion-associated and multiplyspliced HIV-RNA are specific correlates of productive infection after early cessation of antiretroviral therapy.

 

*M. Fischer¹, B. Joos¹, B. Hirschel, R. Weber¹ and H. Günthard¹

¹University Hospital Zurich Switzerland; ²UC-San Diego

Background: To determine residualHIV-1 may replicate at low levels replication in patients (pts) successfully treated with HAART, but quantification of residual replication remains difficult remaines difficult. To identify potential correlates of ongoing HIV-1 replication, we studied kinetics of a panel of cell-associated multiply spliced and unspliced HIV-1 RNAs in PBMC during early viral rebound in pts patients which underwent structured treatment interruptions (STI) of successful long-term HAART.

To identify potential correlates of ongoing HIV-1 replication which may persist despite of highly active antiretroviral therapy (HAART), the kinetics of a panel cell-associated of multiply spliced and unspliced HIV-1 RNAs were assessed in PBMC during early viral rebound in patients which underwent structured treatment interruptions (STI).

Methods: Cell-associated HIVviral nucleic acids were measured in PBMC of HIV-1  infected pts patients (n = 28) at day 0 (baseline), 4, 8, and 14 during STIbefore and during 2-week STIs. Multiply spliced HIV-1 RNAs spanning exons 4 and 7 encoding rev, tat and nef (HIV-MsRNA-4tat/7-revt/r/n) and transcripts spanning exon 5 and 7 lacking the the initation codon of rev encoding solely nef (HIV-MsRNA5/7-nefMsRNA-n) were assessed by highly ultrasensitive real-time PCR assays (with detection limits of < >5  copies/million 10⁶ cells). PBMC-associated unspliced HIV-RNA was dissected into it's an intracellular intracellularfraction (HIV-UsRNA-intra) and an extracellular- virion virion-associated encapsidated fraction (HIV-UsRNA-extra) by differential specific eextraction protocols. HIV-UsRNAs and HIVHIV-DNA were quantified by modifieda modification of the Roche Amplicor tests with sensitivities of < 2 copies/10⁶ million cells.

Results: At baseline on HAART, HIV-DNA and intracellular HIVHIV-UsRNA-intra persisted in all 100% of the specimens tested. HIV-MsRNA_5/7-nef MsRNA-n was detectable in 43% of the samples whereasand MsRNA-t/r/n HIV-MsRNA_4/7-rev was present in 78%wh andereas e-xtracellular HIV-UsRNA-extra was present only in a minority of the specimens (in7% and 87%). Upon STI, systemic viral rebound as assessed by plasma viral reboundemia was followed by significant increases (p < 0.006) of cell-associated HIV-1 RNAs with a lag time of around 4 days but not by significant changes of HIV-DNA. Rebound of plasma viremia, was correlated best (p < 0.0001) with increases of extracellular HIV-UsRNA-extra A (r² = 0.68), HIV-MsRNA_5/7-nef MsRNA-n (r² 2= 0.51) and HIV-MsRNA_4/7-rev MsRNA-t/r/n (r² = 0.48), whereas correlation with changes of PBMC associated intra-cellular HIV-UsRNA-intra was less pronouncedweaker (p = 0.004; r² = 0.30). Furthermore, iIncreases of of e-xtracellular HIV-UsRNA-extra  correlated best with increases of HIV-MsRNA_4/7-rev MsRNA-t/r/n (p < 0.0001; r² = 0.73).

Baseline levels of HIV-MsRNA_5/7-nef MsRNA-n were predictive for increases in plasma viremia, ex-tracellular HIV-UsRNA-extra and HIV-MsRNA_4/7-rev MsRNA-t/r/n (p = 0.014; p = 0.003 and p = 0.02, respectively).

Conclusions: HIV-MsRNA_4/7-rev MsRNA-t/r/n and PBMC-associated extra-xtracellular HIV-UsRNA were identified to be specific correlates of productively infected cells and ongoing HIV-replication as indicated by their rare detectabilitytheir low frequency of detection at baseline and the tight correlation of their increases with rebound of plasma viremia.

 Increases of plasma viremia, extracellular UsRNA and MsRNA-t/r/n were significantly predicted by levels of Notably, PBMC associated HIV-UsRNA consisted of a major fraction of nascent viral particles as indicated by it's highly significant correlation to intracellular HIV-transcription. In contrast, intracellular HIV-UsRNA and HIV-DNA showed modest or no correlation with rebound of plasma viremia and were observed at a frequency of 100% at baseline. Thus a majority of these latter viral nucleic acids appeared to stem from nonprod