Genetic characterization of rebounding HIV-1 after interruption of HAART

E. Bulgheroni 1,2, Rusconi S 1, L Sutton 2, RT D’Aquila 2, MP De Pasquale 2.1Institute of Infectious Diseases and Tropical Medicine, University of Milan, H Sacco (Italy), 2Vanderbilt University Medical Center, Nashville TN (USA).

Background: The source of rebounding plasma rebounding virus when HAART is interrupted remains uncertain. It has been suggested that rebounding virus may emerge from latently, or actively, infected cells in blood, as well as from cells in other body compartments. Also the status of HIV replication in patients before the interruption of therapy (e.g. whether If viremia is consistently suppressed to <50 c/ml or there is evidence of residual, low-level viremia above 50 c/ml) may affect the dynamics and evolution of rebounding virus. We examined the genetics of rebounding virus in plasma and blood cells after interruption of HAART in 4 chronically infected individuals in whom successful suppression of viremia had been achieved for considerable periods of time. Material and Methods: HIV RNA was extracted from blood plasma and, culture supernatants of enhanced cultures of peripheral blood CD4+ cells and semen plasma. Proviral DNA was extracted from PBMCs. Pol was amplified, and sequenced by population- based cycle sequencing (TrueGene, VGI). Clonal analyses are still underway. Phylogenetic analysis of sequences was performed by the neighbor-joining alghoritm and by the Kimura 2-parameter distance matrix method. Genetic distances were estimated by DNADIST. Results: 3 chronically infected patients with blood viremia <50 copies/ml (Roche Amplicor Ultrasensitive) for a considerable time had their treatment interrupted. A fourth patient had viremia < 50 c//ml by bDNA and 2156 c/ml by Roche Amplicor Ultrasensitive Assay when dropped the therapy was stopped. In all of them the VL was re-suppressed to undetectable level once therapy was resumed 7-21 months later. In 3 out of 4, the rebounding virus harbored mutations associated with resistance to previous regimens, according to drug history of the patients (41L, 70R, 184V, 219Q in RT). The phylogenetic analysis revealed that the all sequences from different sources in each individual patient clustered together irrespectively of their source of origin w/owithout evidence of cross-contamination. Sequences over time while off-treatment did not show time-ordered clustering. In 2 subjects, plasma sequences from the baseline time point when treatment was stopped (T0) and from subsequent time points while off-treatment were distinguishable and divergent from the cell-associated virus DNA at T0. Others showed minimal genetic distances between pol sequences at early T0 and later off-therapy timepoints. Conclusion: During treatment interruption, in chronical infected patients with suppressed viremia sequences of rebounding virus in plasma are related to those present at the time treatment is stopped in blood cell DNA and replication competent virus recovered from blood cells in vitro. However, in some subjects, the baseline sequences in plasma and those present later during treatment interruption and blood cells diverged from those in blood cells present later during treatment interruption, suggesting that persistent virus from other tissues than blood cells can also contribute to recurrent replication.