The HIV reservoir is established early, through proviral DNA integration into the host cell genome. This latent infection prevents viral eradication, despite suppressive antiretroviral therapy.
HIV-1 RNA reverse transcription generates linear cytoplasmic HIV DNA species. After complex processing involving the viral integrase, this linear HIV DNA is partly integrated into the host genome, and is referred to as proviral DNA. Circularization of the remaining extrachromosomal DNA creates episomes with one or two long-terminal repeats (1 or 2-LTR circles), depending on the mechanism of LTR ligation. Together, these different forms compose total HIV DNA. In studies of human T-cell lines, 2-LTR episomes have been identified as labile products whose presence is indicative of recent cell infection events .
Integrase inhibitors are designed to prevent viral DNA integration into the host cell genome. In cell culture models, integrase inhibitors lead to a rapid increase in 2-LTR circles and prevent viral DNA integration . In animal models, 2-LTR circles accumulate (up to seven-fold) in the spleen early during integrase inhibitor treatment .
Total HIV DNA measured in peripheral blood mononuclear cell (PBMC) after long-term viral suppression largely corresponds to integrated HIV genomes and, thus, to the cellular virus reservoir . Integrase inhibitors may have the potential to deplete the HIV reservoir and/or to prevent its replenishment. In addition, these drugs could serve as molecular tools for probing the origin of residual viremia and the persistence of the HIV reservoir.
Clinical trials of raltegravir, the first approved integrase inhibitor, showed rapid and sustained viremic control both in previously untreated patients and in patients on failing regimens [5–7]. The EASIER-ANRS 138 randomized trial confirmed that switching to raltegravir was virologically noninferior to continued enfuvirtide therapy in multidrug-resistant HIV-1-infected patients on suppressive antiretroviral therapy . Here we analyzed the effect of raltegravir on total HIV-1 DNA and 2-LTR circle levels for up to 48 weeks in patients enrolled in the EASIER trial.
Patients and methods
The EASIER-ANRS 138 study was a noninferiority randomized multicenter trial of enfuvirtide substitution by raltegravir in 169 highly treatment-experienced patients who, at baseline [week (W0)], had plasma HIV-1 RNA below 400 copies/ml after at least 3 months on the same enfuvirtide-based regimen. At W24, patients in the enfuvirtide arm were invited to switch to a raltegravir-based regimen and all accepted. These patients continued on the same raltegravir-based regimen until W48. The primary endpoint for efficacy was the cumulative proportion of patients with plasma viral load (pVL) above 400 copies/ml at W24. Switching to raltegravir was found to be virologically noninferior to continuing enfuvirtide therapy. The proportion of patients with pVL below 50 copies/ml was 85 and 88% at W0 and 89 and 88% at W24 in the raltegravir and enfuvirtide groups, respectively. Changes in the CD4 cell count were also similar, with median increases of 15 and 11 cells/μl between W0 and W24 in the enfuvirtide and raltegravir groups, respectively .
HIV-1 DNA was quantified at W0 and W24 in the first 30 patients enrolled in each arm, and also at W48 in the raltegravir group. Total HIV-1 DNA was extracted from whole blood (MagnaPure, Roche Meylan, France) and amplified with primers annealing in the LTR region . HIV-1 2-LTR circles were quantified with primers spanning the 2-LTR circle junction, as previously described [1,10]. Real-time PCR was performed in triplicate on each sample. To express the results as log10 copies per million PBMCs, the number of cells was determined by quantifying the albumin gene, and the number of PBMC was determined according to the whole blood count.
Changes in the HIV-1 total DNA level over time were compared between the groups by using the Wilcoxon signed-rank test, with an alpha risk (two-sided) of 0.05. SAS software version 8.2 (SAS Institute Inc., Cary, North Carolina, USA) was used for all analyses. The results are expressed as medians [interquartile range (IQR)].
The baseline characteristics of the 60 patients included in this substudy were similar to those of the overall study population  (Table 1). Sixty percentage of the substudy patients were at CDC stage C. They had been on highly active antiretroviral therapy (HAART) for 14 years (11.7–15.2) and on an enfuvirtide-containing regimen for the previous 2.5 years (1.7–3.3). Baseline and nadir CD4 cell counts were 400/μl (270–543) and 37/μl (10–118), respectively. Baseline pVL was below 50 copies/ml in 87% of patients, whereas the remaining 13% of patients had low-level viremia (50–400 copies/ml). A boosted protease inhibitor was included in the regimen containing enfuvirtide or raltegravir in all but one of the patients.
At W0 the total HIV-1 DNA level was 3.6 log10 (3.4–3.9) per million PBMCs in both groups (Fig. 1). At W24 the CD4 cell count was 416 (280–576)/μl overall, and 88% of patients had pVL below 50 copies/ml. The total HIV-1 DNA level at W24 was 3.6 (3.1–4.0) and 3.7 (3.5–3.9) log10/106 PBMCs in the enfuvirtide and raltegravir groups, respectively. At W48, among the 29 patients with available plasma samples in the raltegravir arm, the CD4 cell count was 446 (288–587)/μl, 93% of patients had pVL below 50 copies/ml, and the total HIV-1 DNA level was 3.5 (3.1–3.9) log10/106 PBMC. No significant change in HIV-1 total DNA was observed between W0 and W24 in either group [0.01 (−0.27;0.30) and 0.02 (−0.17;0.25) log10 per million PBMCs in the enfuvirtide and raltegravir arms, respectively (P = 0.71)]. No significant change in HIV-1 total DNA was observed in the raltegravir group between W0 and W48 [−0.21 (−0.37;0.24)] or between W24 and W48 [−0.18 (−0.49;0.04)]. No significant change in HIV-1 total DNA was observed in the subset of eight patients with low-level viremia at inclusion.
HIV-1 2-LTR circles were detectable in six patients at W0 (two in the enfuvirtide arm and four in the raltegravir arm), with a median of 89 (36–129) copies per million PBMCs. 2-LTR circles were no longer detectable in any of these six patients at W24, but they became detectable in three new patients (two in the enfuvirtide arm and one in the raltegravir arm). None of the patients had detectable 2-LTR circles at W48. Among the nine patients in whom 2-LTR circles were detected during the study period, three had at least one pVL value between 50 and 400 copies/ml during follow-up [one patient in the enfuvirtide group at W8 (167 copies/ml), and two patients in raltegravir group: one at W48 (100 copies/ml) and the other at both W8 (64 copies/ml) and W24 (220 copies/ml)]. Baseline characteristics did not differ significantly between the nine patients with detectable 2-LTR circles and the other 51 patients.
This is the first study to examine the impact of raltegravir on the time course of HIV DNA forms among highly treatment-experienced patients with controlled viremia participating in a randomized clinical trial. The main finding is that total HIV-1 DNA load was similar after 24 weeks on raltegravir and enfuvirtide-containing regimens in patients with sustained plasma HIV-1 RNA below 400 copies/ml. No significant individual changes were seen either after 48 weeks of raltegravir therapy.
The impact of raltegravir intensification on the HIV reservoir has recently been studied in patients on effective HAART . After 24 weeks, total and proviral HIV-1 DNA loads were similar in 45 patients with and 24 patients without raltegravir intensification. Similar data had previously been reported during long-term maintenance of standard HAART in patients with less advanced HIV disease : total HIV-1 DNA fell by an average of 0.48 log10 copies/106 PBMCs during the first year of treatment, then by 0.18 log10 per year during the second and third years, with no further decrease thereafter.
Compared to values reported in patients at an earlier stage of HIV infection (median 2.7 log10/106 PBMC, ), total HIV-1 DNA levels in our heavily pretreated patients (median 3.6 log/106 PBMC) were higher, a finding possibly related to their low CD4 cell nadir (median 37/μl)  and to long-lasting viral replication before effective control.
We observed no increase in 2-LTR circles after 24 and 48 weeks on raltegravir. In contrast, Buzon et al. reported a transient and significant increase in 2-LTR circles after 2 weeks of raltegravir intensification, mainly in patients receiving a protease inhibitor-containing regimen. Such an increase in 2-LTR circle levels has been observed early after integration inhibition in cell culture, and during the first weeks of raltegravir therapy [2,3,11,15]. In our study, the lack of increase in 2-LTR circle levels after 24 and 48 weeks on raltegravir suggests that most of the HIV DNA we detected consisted of integrated forms.
The use of 2-LTR circles as a surrogate marker for ongoing viral replication is controversial [16,17]. Buzon et al. reported that patients with an increase in episomal DNAs had stronger immune activation at baseline, normalizing after raltegravir intensification. Detection of 2-LTR circles in virologically controlled patients would suggest that, despite suppressive HAART, active replication persists and drives immune activation in some patients. It would be interesting to study the emergence of antiretroviral resistance and the replenishment of the HIV reservoir in these patients, as the latter might be prevented by integrase intensification.
In two small noncomparative studies, raltegravir intensification of HAART in virologically controlled patients did not reduce residual viremia measured with a single-copy assay [18,19]. A similar lack of effect on residual viremia has been noted after intensification with other potent drugs [protease inhibitor or non-nucleoside reverse transcriptase inhibitor] , further suggesting that the main source of residual viremia is viruses release from latently infected cells rather than ongoing viral replication in persistently activated cells .
In most patients with HIV virologically controlled by potent regimens, including those comprising an integrase inhibitor, the viral reservoir, reflected by HIV-1 DNA load, is stable and nondynamic during the first year of follow-up. It remains to be shown whether these findings in chronically HIV-infected patients with controlled viremia may be extrapolated to other situations such as primary infection or initial treatment of chronic infection.
C.D., M.L.N. and F.S. executed and interpreted virological analyses, and prepared the manuscript. I.C., J.B. and J.P.A. monitored patients' data and performed statistical analyses. N.deC. and J.M.M. designed the study and participated in patients' recruitment and clinical care. P.Y. and J.G. participated in patients' recruitment and clinical care.
The study was presented in part at the XVIIIth International HIV Drug Resistance Workshop, 9–13 June 2009, Fort Myers, FL, USA (abstract #9) and at the 17th Conference on Retroviruses and Opportunistic Infections, 16–19 February 2010, San Francisco, CA, USA (abstract #281).
1. Sharkey ME, Teo I, Greenough T, Sharova N, Luzuriaga K, Sullivan JL, et al
. Persistence of episomal HIV-1 infection intermediates in patients on highly active antiretroviral therapy. Nat Med 2000; 6:76–81.
2. Hazuda DJ, Anthony NJ, Gomez RP, Jolly SM, Wai JS, Zhuang L, et al
. A naphthyridine carboxamide provides evidence for discordant resistance between mechanistically identical inhibitors of HIV-1 integrase. Proc Natl Acad Sci U S A 2004; 101:11233–11238.
3. Goffinet C, Allespach I, Oberbremer L, Golden PL, Foster SA, Johns BA, et al
. Pharmacovirological impact of an integrase inhibitor on human immunodeficiency virus type 1 cDNA species in vivo. J Virol 2009; 83:7706–7717.
4. Koelsch KK, Liu L, Haubrich R, May S, Havlir D, Gunthard HF, et al
. Dynamics of total, linear nonintegrated, and integrated HIV-1 DNA in vivo and in vitro. J Infect Dis 2008; 197:411–419.
5. Eron JJ, Young B, Cooper DA, Youle M, Dejesus E, Andrade-Villanueva J, et al
. Switch to a raltegravir-based regimen versus continuation of a lopinavir-ritonavir-based regimen in stable HIV-infected patients with suppressed viraemia (SWITCHMRK 1 and 2): two multicentre, double-blind, randomised controlled trials. Lancet 2010; 375:396–407.
6. Grinsztejn B, Nguyen BY, Katlama C, Gatell JM, Lazzarin A, Vittecoq D, et al
. Safety and efficacy of the HIV-1 integrase inhibitor raltegravir (MK-0518) in treatment-experienced patients with multidrug-resistant virus: a phase II randomised controlled trial. Lancet 2007; 369:1261–1269.
7. Lennox JL, DeJesus E, Lazzarin A, Pollard RB, Madruga JV, Berger DS, et al
. Safety and efficacy of raltegravir-based versus efavirenz-based combination therapy in treatment-naive patients with HIV-1 infection: a multicentre, double-blind randomised controlled trial. Lancet 2009; 374:796–806.
8. De Castro N, Braun J, Charreau I, Pialoux G, Cotte L, Katlama C, et al
. Switch from enfuvirtide to raltegravir in virologically suppressed multidrug-resistant HIV-1-infected patients: a randomized open-label trial. Clin Infect Dis 2009; 49:1259–1267.
9. Avettand-Fenoel V, Chaix ML, Blanche S, Burgard M, Floch C, Toure K, et al
. LTR real-time PCR for HIV-1 DNA quantitation in blood cells for early diagnosis in infants born to seropositive mothers treated in HAART area (ANRS CO 01). J Med Virol 2009; 81:217–223.
10. Gueudin M, Damond F, Braun J, Taieb A, Lemee V, Plantier JC, et al
. Differences in proviral DNA load between HIV-1- and HIV-2-infected patients. AIDS 2008; 22:211–215.
11. Buzon MJ, Massanella M, Llibre JM, Esteve A, Dahl V, Puertas MC, et al
. HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects.Nat Med
12. Viard JP, Burgard M, Hubert JB, Aaron L, Rabian C, Pertuiset N, et al
. Impact of 5 years of maximally successful highly active antiretroviral therapy on CD4 cell count and HIV-1 DNA level. AIDS 2004; 18:45–49.
13. Kostrikis LG, Touloumi G, Karanicolas R, Pantazis N, Anastassopoulou C, Karafoulidou A, et al
. Quantitation of human immunodeficiency virus type 1 DNA forms with the second template switch in peripheral blood cells predicts disease progression independently of plasma RNA load. J Virol 2002; 76:10099–10108.
14. Burgard M, Boufassa F, Viard JP, Garrigue I, Ruffault A, Izopet J, et al
. Factors influencing peripheral blood mononuclear cell-associated HIV-1 DNA level after long-term suppressive antiretroviral therapy in 236 patients. AIDS 2009; 23:2165–2171.
15. Reigadas S, Andreola ML, Wittkop L, Cosnefroy O, Anies G, Recordon-Pinson P, et al
. Evolution of 2-long terminal repeat (2-LTR) episomal HIV-1 DNA in raltegravir-treated patients and in vitro infected cells. J Antimicrob Chemother 2010; 65:434–437.
16. Brussel A, Mathez D, Broche-Pierre S, Lancar R, Calvez T, Sonigo P, Leibowitch J. Longitudinal monitoring of 2-long terminal repeat circles in peripheral blood mononuclear cells from patients with chronic HIV-1 infection. AIDS 2003; 17:645–652.
17. Sharkey M, Triques K, Kuritzkes DR, Stevenson M. In vivo evidence for instability of episomal human immunodeficiency virus type 1 cDNA. J Virol 2005; 79:5203–5210.
18. Grant PM, Palmer S, Bendavid E, Talbot A, Slamowitz DC, Cain P, et al
. Switch from enfuvirtide to raltegravir in virologically suppressed HIV-1 infected patients: effects on level of residual viremia and quality of life. J Clin Virol 2009; 46:305–308.
19. McMahon D, Jones J, Wiegand A, Gange SJ, Kearney M, Palmer S, et al
. Short-course raltegravir intensification does not reduce persistent low-level viremia in patients with HIV-1 suppression during receipt of combination antiretroviral therapy. Clin Infect Dis 2010; 50:912–919.
20. Dinoso JB, Kim SY, Wiegand AM, Palmer SE, Gange SJ, Cranmer L, et al
. Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy. Proc Natl Acad Sci U S A 2009; 106:9403–9408.
21. Palmer S, Maldarelli F, Wiegand A, Bernstein B, Hanna GJ, Brun SC, et al
. Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy. Proc Natl Acad Sci U S A 2008; 105:3879–3884.