The immunological and virological long-term benefits of a continuous viral suppression with antiretroviral therapy (ART) are still unknown. First, there is still a debate on whether ART allows a CD4 T-cell recovery after more than 5 years [1–4]. Second, ART had been shown to rapidly restore memory T-cell responses to recall or opportunistic antigens , but only partially to HIV itself [6–8], and it is not known whether long-term ART can restore such functions. Third, the HIV reservoir in peripheral blood mononuclear cells (PBMCs) had been shown to decrease mostly during the first 3 years of HIV suppression , but the effects of long-term ART remain poorly characterized. Finally, it is not known whether long-term ART can allow advanced stage patients to reach a status close to Elite controllers. We studied the impact of a strict viral control throughout 10 years of ART on HIV RNA using ultrasensitive technique, cell-associated HIV DNA, CD4 cell counts and HIV-specific T-cell responses in 16 long-term ART-treated (LTT) progressors. They were compared with short-term ART-treated (STT) patients and Elite HIV controllers.
From the 601 Pitié-Salpêtrière patients treated for 10 years, HIV-1 infected LTT patients were selected on the basis of an undetectable HIV RNA at each time point, with at least one CD4 and HIV RNA measurement/year and more than 20 time points overall. Patients with history of ART interruptions, ‘blips’ of viral replication or immunomodulatory therapies were excluded. Sixteen patients receiving ART for 9.8 (9.3–10.6) years, with a median age of 54 (38–71) years fulfilling these criteria, were included. Before ART initiation, median CD4 cell count was 200 (16–519) cells/μl, median HIV RNA was 5.2 (4.2–6) log copies/ml and five patients had an AIDS-defining clinical condition. Only one patient was HLA B57. No clinical event occurred during their treatment. Thirteen STT-matched patients with similar criteria but with shorter ART duration [median 3.3 (2.3–4.4) years] and similar clinical characteristics at ART initiation (data not shown) were included as controls. In addition, we compared LTT and STT patients to 11 HIV-infected untreated Elite HIV controllers from the French ALT ANRS CO-15 cohort  with undetectable HIV RNA at every time point during a 10-year follow-up and a median age of 38 (32–65) years, significantly younger than LTT (P < 0.001, Mann–Whitney test). Plasma HIV-1 RNA levels were determined with the Amplicor-Monitor v1.5 and Cobas AmpliPrep/Cobas Taqman HIV-1 assays (Roche-Diagnostic Systems; threshold 200 and 40 copies/ml, respectively). Ultrasensitive plasma HIV-1 RNA (threshold 1 copy/ml) and HIV-1 cell-associated DNA in PBMCs were quantified [10,11]. Lymphocyte proliferation assays against HIV-p24 were performed on fresh PBMCs using 3H-thymidine and BrdU incorporation assays . ELISpot interferon (IFN)-γ assays with thawed PBMCs used 15 pools of 15-mer overlapping HIV peptides covering gag and RT , and responses were expressed as the sum of positive responses to each peptide pool.
In the 16 LTT patients, the CD4 cell counts (678 measurements) showed a continuous increase from a median baseline of 200 (16–519) to 712 (349–1144) cells/μl at year 10 (Fig. 1a) in the following three slopes: the first rapid slope of +191 cellsD4/μl per year (IQR = 47.2) from day 0 to year 1, the second slower slope of +81 cells/μl per year (IQR = 27.1) from years 1 to 3  and the third even slower but still positive slope of +22 cells/μl per year (IQR = 9) between years 3 and 10. These three positive slopes significantly differed from zero at each time point (P < 0.02, Wilcoxon test). At the time of analysis, the CD4 cell counts did not differ between STT and Elite HIV controllers patients (Mann–Whitney test): 560 (124–1338) and 715 (559–902) cells/μl, respectively. HIV plasma viral loads were below 1 copy/ml in 14/16 LTT and 8/13 STT patients, whereas they were detectable in 9/11 Elite HIV controllers [median 46 (1–530) copies/ml] (Fig. 1b). The median cell-associated HIV DNA in the 16 LTT patients was 157 (5–632) copies/106 cells, lower but not significantly lower than that in the eight STT patients tested [353 (126–2240) copies/106 cells] (P = 0.05, Mann–Whitney test) but significantly higher than that in the Elite HIV controllers [50 (20–170) copies/106 cells] (P < 0.01) (Fig. 1c). Stronger proliferative T-cell responses to HIV-p24 were detected in LTT with higher stimulation indexes [median 12 (2–80)] than in both STT [median 3 (1–53)] (P = 0.03) and Elite HIV controllers [median 3 (1–74)] (P = 0.13, Mann–Whitney test) (Fig. 1d). These responses were mediated by CD4+ T cells as shown in the BrdU proliferation assay for seven LTT patients tested (data not shown). HIV-specific T cells producing IFN-γ ex vivo were significantly lower in LTT than in Elite HIV controllers [median 227 (0–4067) and 2594 (160–11 167) spot-forming cells/106 PBMCs, P < 0.01] (Fig. 1e).
This study reports the benefits of 10 years of continuous viral suppression with ART on CD4 cell counts, HIV-specific T-cell responses and HIV control and reservoir. Contrasting with the finding of previous studies reporting that CD4 cell counts plateau after 3–5 years of ART [2–4,14,15], our study showed that the CD4 cell counts continue to increase 10 years after ART initiation, despite low CD4 nadirs or an advanced age in some patients. This might reflect the continuous virological control and the lack of viral blips in these highly selected patients.
Immune benefits of long-term treatments were also assessed by the restoration of stronger memory CD4+ T-cell responses to HIV-p24 than in STT, although both groups showed similar standard predictors of immune restoration, such as age, CD4 cell count nadir and plasma HIV RNA control [15–18]. Those memory responses reached levels comparable to those observed in Elite HIV controllers. These data suggest that a prolonged virological control with ART allows a continuous restoration process of HIV-specific central memory T cells. In contrast, this long-term virus control puts at rest the anti-HIV T-cell response producing IFN-γ ex vivo, which should correspond to effector or effector/memory T cells. This phenomenon reflects the prolonged clonal contraction induced by the sustained very low levels of HIV antigen stimulation in vivo. Finally, the trend toward lower plasma HIV RNA and HIV DNA in LTT than in STT suggests that HIV reservoirs can continue to decrease after 5 years of ART, although their HIV reservoirs remain higher than in untreated Elite HIV controllers. The substantial residual HIV replication in Elite HIV controllers reflects the absence of ART.
In conclusion, this sustained control of HIV over 10 years of continuous ART in advanced stage progressors induces the residual HIV replication down to undetectable levels and affects the HIV reservoirs. This allows an immune reconstitution process with near to normal CD4 cell counts and strong HIV-specific memory T cells comparable to those of Elite HIV controllers. The positive CD4 cell count slope observed until the 10th year of treatment disfavors the hypothesis that the immune system cannot achieve normalization. These findings suggest the importance of promoting continuous viral suppression with ART to achieve better immunological and virological outcomes as the duration of viral control increases.
This study was supported by the AP-HP Centre d'Investigations Biomédicales (CIB) of the Pitié-Salpêtrière Hospital and by the French Agency for AIDS Research (ANRS). The authors thank the following decamune patient's physicians: Pascal Astagneau, Philippe Bossi, Martin Danis, Luc Paris and Marc Antoine Valantin.
The DECAMUNE study group is composed of Brigitte Autran, Guillaume Breton, Vincent Calvez, Guislaine Carcelain, Dominique Costagliola, Amélie Guihot, Christine Katlama, Anne-Geneviève Marcelin, Roland Tubiana.
The ALT-ANRS CO-15 group is composed of Henri Agut, Jean-Pierre Clauvel, Dominique Costagliola, Patrice Debré, Christine Rouzioux, Didier Sicard, Ioannis Theodorou, Brigitte Autran.
This study was presented during the 15th Conference on Retroviruses and Opportunistic Infections, February 2008, Boston, MA, USA (Abstract A-190).
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