Differential kinetics of cell-associated HIV load in purified cellular subsets from primary HIV infected individuals (PHI) upon one year follow up.

M. Malnati°, P. Biswas*, A. Galli*, S. Piergiovanni°, A. Lazzarin*, G. Tambussi*

° Unit of Human Virology, *Clinic of Infectious Diseases, San Raffaele Scientific Institute, Milano (Italy)

Background: Decrease of plasma viremia upon HAART presents a biphasic decay with a rapid elimination of free virus and loss of productively infected cells during the first weeks followed by a slower second phase decay. Aim of our study is to evaluate the kinetics of HIV-DNA in sequentially purified cell subsets, including peripheral blood mononuclear cells (PBMC), CD14+ monocytes and CD8-depleted PBMC (CD4 lymphocyte enriched) from PHI patients. Material and Methods: Patients (pts): we studied 12 individuals who attended our Clinic for an acute retroviral syndrome. The first time point evaluated is prior to beginning of an antiretroviral therapy (t0), followed by 2 (t2), 6 (t6) and 12 (t12) months post-therapy. Cell purification: an aliquot of PBMC was pelletted, the rest underwent sequential steps of purification with CD14 and CD8 coated magnetic beads. All pellets were stored at –80°C until processed for HIV viral load. Proviral HIV DNA was measured with two combined TaqMan “real-time PCR” assays. The first was specifically developed to accurately quantitate all clades belonging to the HIV-1 M group; the second, which uses as target a highly conserved sequence of the CCR5 gene, served to normalize the cellular genome content of each sample. Results: In all pts the CD4-enriched fraction harbored a higher viral load than the PBMC. However, a consistent viral load was measured also in the circulating CD14+ subset in the majority (9/11) of pts already at t0. The HIV copy number/106 cells ranged from 100 to approximately 10000 at t0. Upon treatment a strong decrease was observed, especially at the t2 time point in the CD8-depleted cells, except in two patients who refused antiretroviral therapy. In most cases the CD14+ mirrored the kinetics of the CD8-depleted fraction, with three exceptions. A remarkable increase of HIV load was observed in 3 pts after one year of an effective HAART treatment (plasma viremia < 80 copies/ml). Interestingly, one these pts showed an increase of proviral load higher in the CD14+ cells than in the CD4-enriched population. Data of each patient will be discussed in detail and compared with plasma viremia and CD4+ T cell count. Conclusion: After the start of an effective antiretroviral treatment the proviral DNA showed a biphasic decay in all three cellular fractions. Although the CD8-depleted cells (CD4-enriched lymphocytes) constitute the favourite target of HIV, the circulating CD14+ fraction represents since the earliest phases of HIV infection a relevant viral reservoir.