Potent antiviral treatment only suppresses cryptic HIV-1 replication after five years of sustained control of plasma viremia

McDermott JL, Bertolotti F, Martini I, Murdaca G *, Puppo F*, Varnier OE. Institute of Microbiology, DISCAT,; *DIMI, Medical School University of Genova (Italy)

Background: During prolonged anti-retroviral therapy, HIV viremia is reduced, yet covert replication maintains a HIV reservoir. While the presence of 2LTR DNA is considered an unreliable marker of replication, the significance of the absence of 2LTR DNA in aviremic subjects has not been evaluated. Material and Methods: Total cellular DNA was obtained from 241 peripheral blood mononuclear cell (PBMC) samples collected from 22 aviremic subjects during a period of 5 years. A 142 bp of the HIV gag gene and a 120-bp region of the U3-U5 junction between the 2 LTRs of unintegrated 2LTR DNA were amplified and detected using a colorimetric assay. Results: Total HIV-1 DNA was quantified in 174 (72%) and undetected in 67 (28%) samples. The presence of 2LTR DNA was detected in 89 (37%) DNA samples. At the start of the study, unintegrated 2LTR DNA was detectable in 60% of the patients monitored. After 3 years suppressive HAART, a dramatic 4 fold decrease was observed in the number of patients with detectable 2LTR and after 5 years uninterrupted therapy 2LTR DNA was undetectable in all tested patients. As reported recently a decrease in the number of 2LTR circles present in the PBMC population monitored may occur due to the effects of dilution during cell division. In contrast to unintegrated DNA, integrated proviral DNA does not decay by dilution and therefore remains constant within the PBMC population. However, if covert HIV replication occurs, the 2LTR circles produced by this de novo infection would counter-act the decline in abundance due to cell division within the same cell population. This hypothesis could explain the constant detection of 2LTR and total HIV DNA in our patient population during the first 3 years of follow-up.  We believe that the absence of 2LTR DNA in our patients following 5 years suppressive HAART was directly due to the effects of both dilution and complete suppression of viral replication in these patients. In fact, 90% of the 2-LTR negative samples contained detectable levels of total HIV DNA, made by only integrated proviral DNA. Conclusions: Our observations suggest that the total HIV DNA and 2LTR circle assays provide distinct but useful information for the long term follow up of aviremic patients. Potent antiviral treatment only suppresses cryptic replication after five years of sustained control of plasma viremia.