Decreased recovery of replication-competent HIV-1 from peripheral blood mononuclear cell-derived monocyte/macrophages of HIV-positive patients after 3 years on highly active antiretroviral therapy

We studied the release of p24 antigen from peripheral blood mononuclear cell-derived monocyte/macrophages obtained from 13 HIV-positive patients receiving highly active antiretroviral therapy (HAART). Although HIV-infected monocyte/macrophages were detected in 80% of patients after 36 months of continuous treatment, additional exposure to HAART reduced the chance of detecting HIV-releasing monocyte/macrophages. Therefore, after more than 3 years of HAART, recently infected monocytes may play a less important role as a source of emerging HIV-1 upon HAART interruption. A sustained reduction in viral replication can be achieved in HIV-infected patients after treatment with combinations of drugs that inhibit the viral reverse transcriptase and protease (highly active antiretroviral therapy; HAART) [1]. However, replication-competent virus can still be recovered from latently infected resting memory CD4 T lymphocytes [2–4]. Moreover, episomal HIV infection intermediates revealing ‘covert’ virus replication have been demonstrated in patients who experienced reductions in plasma viraemia to levels below the limit of detection of the most sensitive polymerase chain reaction assays [5,6]. In most studies, preferential attention has been given to latent resting CD4 T lymphocytes as a source of HIV persistence [7,8]; however, insufficient suppression of HIV replication could also lead to viral re-emergence after HAART interruption. Outgrowth of HIV from circulating CD4 lymphocytes that were co-cultured with phytohaemagglutinin-activated HIV-negative peripheral blood mononuclear cells (PBMC) [6] and the presence of 2-long terminal repeat DNA circles [5,6] have been considered to be markers of ‘covert’ viral replication in CD4 T lymphocytes. According to these criteria, patients under HAART have been stratified into different subgroups [6]. In addition to CD4 lymphocytes, other host cells could also contribute to maintaining persistent HIV replication after HAART [9,10]. Long-lived resident macrophages can act as reservoirs of HIV, because the concentration of protease inhibitors required to inhibit HIV replication in tissue macrophages is higher than that required for T cells [11]. Blood monocytes also constitute a source of infectious virus after prolonged treatment, as replication-competent virus can still be recovered from monocyte/macrophages obtained from HIV-positive patients who had been treated for 3–30 months [9,12]. In view of the demonstration of circular viral DNA (2-long terminal repeat) and spliced RNA, it has been suggested that the presence of HIV in the monocyte/macrophages of patients under HAART is the result of recent and transcriptionally active infection rather than of latent infection [9]. Establishing the origin of re-emerging HIV in patients under HAART upon treatment interruption is important in order to design optimal treatment schemes. Therapeutic strategies aimed at reducing the number of latently infected cells involve immune activation with IL-2 or other stimulatory factors in the presence of antiretroviral drugs [13]. The elimination of replication-competent virus would require the intensification of HAART or the use of antiretroviral drugs achieving an effective concentration at the site of HIV replication. We have recently described a technique that allows the recovery of replication-competent HIV from non-stimulated PBMC-derived monocyte/macrophages from HIV-positive haemophilic patients who had successfully responded to prolonged HAART, achieving plasma viral RNA levels below 50 copies/ml [12]. All patients became infected with HIV through the transfusion of contaminated factor VIII plasma concentrates before 1985 (1983–1985) [14], and conventional methods of HIV recovery such as co-culture with phytohaemagglutinin/IL-2-activated normal lymphocytes [15] or with the CXCR4-positive MT2 cell line failed to detect replication-competent virus in their PBMC. The new assay is based on allowing spontaneous, undisturbed interaction of HIV-positive PBMC during culture, in the absence of exogenous stimuli. HIV replication is localized mainly in monocyte/macrophages and becomes detectable after 12–25 days of culture [12]. As a result of the short life-span of the circulating monocytes that give rise to HIV-producing monocyte/macrophages during in-vitro culture, this assay probably involves recently infected monocytes. Using this technique [12], we have re-examined these patients after different periods of continuous HAART. Fig. 1a shows that the suppression of active HIV replication was acheived after 1–2 years of treatment. As expected, CD4 cell counts steadily increased over time. In approximately 60% of patients, plasma viral load levels fell below 50 HIV RNA copies/ml after one year of uninterrupted HAART. However, active HIV replication persisted in 80% of patients even after 30 months of HAART (Fig. 1b). In contrast, after 36 months of uninterrupted treatment, no p24 antigen release was observed in PBMC-derived monocyte/macrophages from 86% of patients, suggesting that the source of HIV infection of monocytes had been significantly reduced.Fig. 1.: HIV replication in peripheral blood monocyte cell-derived monocyte/macrophages from HIV-positive patients under continuous highly active antiretroviral therapy. (a) Plasma viral load (standard and ultrasensitive reverse-transcriptase–polymerase chain reaction; Roche Diagnostics, Branchburg, NJ, USA) was measured in 13 patients who were followed up for 48 months of uninterrupted highly active antiretroviral therapy (HAART) (––▪––). The CD4 cell counts were determined by flow cytometry using a FACScalibur (Becton Dickinson, San Jose, CA, USA) (––▵––). (b) HIV replication in patient's monocyte/macrophages after non-stimulated peripheral blood mononuclear cell culture was evaluated by measuring p24 antigen in the cell-free supernatants (SN p24) by enzyme-linked immunosorbent assay (Organon Teknika, Buenos Aires, Argentina). The bar graph represents the proportion of patients with p24 antigen-positive results in supernatants (> 15 pg/ml) at different treatment duration periods. The contiguous table expresses the mean ± SEM p24 antigen concentrations detected in p24-positive supernatants at different treatment duration periods.It has been established that the kinetics of HIV reduction in plasma after HAART is composed of a rapid initial decline followed by a slow decrease, probably related to HIV release from different HIV reservoirs (memory CD4 lymphocytes that escape latency, macrophage reservoirs with low key replication, cells in sites that did not achieve an adequate dose of antiretroviral drugs, etc.) [16]. The rate of decay of the latent reservoir is very low and HAART has little impact on it, at least in short and medium-term periods [8]. Upon HAART interruption plasma viraemia increases, the source of the re-emerging virus still being a matter of debate. As latently infected long-lived CD4 lymphocytes become activated upon encountering their cognate antigens, HIV replication may be initiated. Residual HIV replication in tissue macrophages or insufficiently supressed HIV in CD4 T lymphocytes could also contribute to the recovery of HIV virus after HAART interruption. Patrolling blood monocytes can pick up HIV from CD4 cells that escaped latency, as well as from sites in which low-level HIV replication has continued in spite of HAART [9]. They could thus be a source of virus, even after prolonged HAART [9]. Our results suggest that continuous long-term HAART progressively reduces the HIV burden at the sites in which monocytes encounter replicating HIV and become infected. Therefore, their weight as a source of replication-competent HIV decreases as the time of treatment increases (Fig. 1). As latently infected cells may be generated every time replication occurs [4,8], the observed reduction in HIV replication in cultured PBMC-derived monocyte/macrophages after long-term HAART is good news, as it reveals the reduction of one of the sources of new latently infected cells. Liliana Belmontea Patricia Baréa Gaston R. Picchioa Raul Perez Biancoa Miguel de Tezanos Pintoa Marcelo Cortib María F. Villafañeb María M. E. de Braccoa Beatriz H. Ruibal-Aresa