The efficacy of highly active antiretroviral therapy (HAART) on virologic response varies with the potency of the combination of drugs and the previous exposure to antiretroviral therapy.1 In antiretroviral-naive patients, HAART results in a sustained and substantial reduction of plasma HIV-1 RNA level in a high proportion of patients.2 Despite the decay of plasma HIV-1 RNA below the lower limit of detection of the most sensitive assays currently available, a low level of viral replication may persist as the result of the presence of HIV-1 mRNA and DNA in peripheral blood mononuclear cells (PBMCs), which is capable of producing infectious virus.3-6 The level of total cellular HIV-1 DNA measures integrated and nonintegrated forms of HIV-1 DNA, circular or linear, and reflects the number of infected cells in the peripheral blood, with a minor proportion of integrated replication-competent virus.7,8 It has also been shown to reflect the number of latently infected cells in other compartments, such as the lymphoid tissues.9 With an effective antiretroviral treatment, the level of total HIV-1 DNA decreases significantly, whereas the fraction of integrated HIV-1 DNA is poorly affected because of the low impact of the treatment on this stable form of viral DNA.10,11
In untreated HIV-1-infected patients, the cellular HIV-1 DNA level, which is established at an early stage of primary infection,12 has been reported to be a predictive marker of disease progression independently of plasma HIV-1 RNA load,13 because patients who did not progress exhibited lower levels than patients who did.14,15 During antiretroviral therapy, the cellular HIV-1 DNA level decrease has been shown to be greater in patients treated with potent multiple therapy than in those treated with suboptimal regimens using 1 or 2 drugs as well as in patients initiating HAART early in the course of primary infection rather than in the chronic phase of the infection.16-19 The predictive value of cellular HIV-1 DNA on the virologic response to HAART has only been evaluated retrospectively in cross-sectional and longitudinal studies but not prospectively in studies designed to compare the antiviral efficacy of various regimens.5,20-22
The objective of our study was thus to evaluate the potential interest of total cellular HIV-1 DNA quantification, in addition to plasma HIV-1 RNA level measurement, to discriminate the potency of antiretroviral regimens further, notably beyond the limits of sensitivity of the assays currently available for plasma HIV-1 RNA, which yield quantitative censored data in a high proportion of patients under therapy. We investigated this viral marker in a prospective randomized study, the ANRS 081 (“Trianon”) trial, which compared the efficacy of 2 HAART regimens, including 3 drugs from 2 or 3 classes over a 72-week period of treatment.23
MATERIALS AND METHODS
Study Population and Design
Clinical and biologic data of the present study have been extracted from the ANRS 081 Trianon clinical trial,23 a randomized prospective study that compared the combination of indinavir (800 mg administered 3 times daily), stavudine (40 mg administered 2 times daily), and lamivudine (150 mg administered 2 times daily) (group 1, n = 72), a 2-drug class combination, with that of indinavir (1000 mg administered 3 times daily), stavudine (40 mg administered 2 times daily), and nevirapine (200 mg administered daily for 14 days and 200 mg administered 2 times daily thereafter) (group 2, n = 73), a 3-drug class combination, over 72 weeks. Between November 1997 and August 1998, the trial enrolled 145 patients, of whom 115 (79%) were antiretroviral naive and 30 had been previously treated with zidovudine alone or in combination with didanosine or zalcitabine. Samples of PBMCs were prospectively collected in all patients to assess cellular HIV-1 DNA. Samples of frozen PBMCs were available for the analyses in a total of 141 patients at baseline and 125 patients at week 72. Intermediate measurements were also performed 4, 8, 16, and 24 weeks after treatment initiation in a subset of 89 patients randomly selected among the 141 patients with a baseline measurement so as to investigate the early kinetics of the HIV-1 DNA response to HAART.
Cellular HIV-1 DNA Quantification
PBMCs were isolated by standard Ficoll-Hypaque density gradient centrifugation, frozen in aliquots of 5 × 106 PBMCs, and stored at −80°C. The cellular HIV-1 DNA level was measured using a quantitative polymerase chain reaction method derived from the Amplicor HIV-1 Monitor test (version 1.5, Roche Diagnostics Systems, Meylan, France) and an internal HIV-1 DNA standard (Roche). Total DNA was determined with Hoechst dye (bisbenzimide) as recommended by the manufacturer (Pharmacia, Uppsala, Sweden).24 HIV-1 DNA level was expressed as copies per microgram of total DNA, and results were expressed as numbers of HIV-1 DNA copies per million PBMCs using the following conversion: 1 μg of DNA = 150,000 PBMCs.25 The lower limit of quantification of the assay was 5 copies/106 PBMCs. All samples from each patient were tested in the same assay.
Plasma HIV-1 RNA Quantification
Plasma HIV-1 RNA was measured with the Amplicor HIV-1 Monitor test (version 1.5; Roche Diagnostics Systems) in all patients in a central laboratory 4 weeks before enrollment in the trial (week-4); at baseline (week 0); and 4, 8, 16, 24, 32, 40, 48, 56, 64, and 72 weeks after treatment initiation. Samples that yielded plasma HIV-1 RNA levels less than 200 copies/mL were retested using the ultrasensitive specimen preparation protocol, which provided a detection limit of 20 copies/mL.
Data were analyzed using the SAS statistical analysis software (SAS Institute, Cary, NC). The cellular HIV-1 DNA level change was described over the 72-week treatment period. Slopes of changes over time in cellular HIV-1 DNA log copy numbers per million PBMCs were estimated using a mixed model. This method allowed us to take into account all available data, even from patients with some missing measurements. Baseline characteristics known to contribute to the virologic response of patients were included as effects in the model. Models including a single or 2 phases of decay were compared using the likelihood ratio test to select which of the 2 models better fitted the change over time in cellular HIV-1 DNA level. Similarly, junction points at weeks 4, 8, and 16 were successively tested in the 2-phase model, and corresponding results were compared using log-likelihoods. Cellular HIV-1 DNA half-life was calculated using the following formula: T1/2 = −log10(2)/Slope.
Within-patient variability in plasma HIV-1 RNA, corresponding to the aggregate of measurement error and biologic variation, was estimated from data collected on the 2 pretherapy visits at weeks −4 and 0. Similarly, within-patient variability in cellular HIV-1 DNA was estimated from measurements at 2 close visits. Because the sampling of cells was not feasible on 2 separate occasions before trial entry, week 4 and week 8 measurements were used instead in a variance component analysis that isolated the contribution of therapy in the variation between these 2 time points and subtracted it from the within-patient variability estimate.
The residual error of the variance component analysis was taken as the mean within-patient variance of cellular HIV-1 DNA measurement. The derived square root was then used as an estimate of the SD of the measurement error plus individual biologic variation.
The cellular HIV-1 DNA level and changes over time from baseline were compared between the 2 treatment groups and also between antiretroviral-naive and pretreated patients. Baseline levels and changes from baseline in cellular HIV-1 DNA level were compared between patients achieving and not achieving a complete virologic response, defined as a plasma HIV-1 RNA level at week 72 below the limit of quantification of 20 copies/mL. Baseline cellular HIV-1 DNA levels were also compared between patients achieving and not achieving a plasma HIV-1 RNA level at week 8 below the limit of quantification (early virologic response).
Comparisons were performed on an intent-to-treat approach, analyzing the subjects in the groups to which they were randomized, irrespective of subsequent changes. The Mann-Whitney U test or sign test was used in comparisons of continuous variables, and the Fisher exact test was used in comparisons of proportions. Associations between continuous variables were assessed by nonparametric Spearman correlation coefficients. A logistic regression model was used to determine which of the baseline characteristics were associated with a decrease in cellular HIV-1 DNA level from baseline value greater than 2-fold the estimated mean within-patient variation. All reported tests results are 2-sided, with a significance level fixed at 0.05.
Study population characteristics are reported in Table 1. These characteristics were well balanced between the 2 treatment groups. At study entry, cellular HIV-1 DNA was detectable in all patients, with a median [interquartile range] value of 3.05 [2.79; 3.36] log10 copies/106 PBMCs. The distribution was bell shaped, with no significant departure from normality (Kolmogorov-Smirnov test: P = 0.15; Fig. 1A). The cellular HIV-1 DNA level was positively correlated with plasma HIV-1 RNA level (r = 0.57, P < 0.0001; Fig. 2A) and negatively correlated with CD4 cell count (r = −0.25, P = 0.0025; see Fig. 2B) in contrast with the plasma HIV-1 RNA level, which was not significantly correlated with the CD4 cell count in this population (r = −0.14, P = 0.10).
Changes in Cellular HIV-1 DNA Level Over Time With Therapy
The cellular HIV-1 DNA level decreased from baseline to week 72 by a median [interquartile range] of 0.50 [0.22; 0.71] log10 copies/106 PBMCs but remained detectable in all patients over the 72 weeks of follow-up.
The cellular HIV-1 DNA log10 copy number decreased according to a 2-phase linear pattern, with a steeper decay in the first phase from baseline to week 16; at that time, plasma HIV-1 RNA had decreased to less than 20 copies/mL in 63% of patients. From baseline to week 16, the median [interquartile range] decrease in HIV-1 DNA was significant and reached 0.26 [0.11; 0.51] log10 copies/106 PBMCs (P < 0.0001), which corresponded to a mean reduction of less than 10% of the initial level of total HIV-1 DNA. The second phase, from week 16 to week 72, was characterized by a much slower but still significant decay. In this latter phase, the median decrease was 0.15 [0.06; 0.41] log10 copies/106 PBMCs. At week 72, the median level was 2.58 [2.25; 2.95] log10 copies/106 PBMCs, with a shape of distribution similar to that observed at baseline (see Fig. 1B).
Factors included in the mixed models used to estimate slopes for changes from baseline in cellular HIV-1 DNA level were treatment group, time, baseline cellular HIV-1 DNA level, and CD4 cell count. A 2-phase model better fitted the data than a 1-phase model. Junction points at week 8 and week 24 provided lower likelihoods in the corresponding models than a junction point at week 16. The slope in the first decay phase, from week 0 to week 16, was estimated at −0.017 (95% confidence interval [CI]: −0.022; −0.011) log10 copies/106 PBMCs per week, corresponding to a cellular HIV-1 DNA half-life of 18 (95% CI: 14; 26) weeks. The slope of the second phase beyond week 16 was estimated at −0.0029 (95% CI: −0.0043; −0.0014) log10 copies/106 PBMCs per week, corresponding to a cellular HIV-1 DNA half-life of 104 (95% CI: 69; 210) weeks. In the antiretroviral-naive subgroup of patients, estimated slopes were: −0.017 (95% CI: −0.023; −0.011) log10 copies/106 PBMCs per week and −0.0033 (95% CI: −0.0049; −0.0017) log10 copies/106 PBMCs per week for the first and second phases, respectively, with corresponding half-lives of 18 (95% CI: 13; 27) weeks and 91 (95% CI: 61; 178) weeks.
Within-Patient Variability in Cellular HIV-1 DNA and Plasma HIV-1 RNA
From the analysis of variance model, the following decomposition for cellular HIV-1 DNA variance was obtained: within-patient variability (26%), between-patient variability (71%), and between-visit variability (3%). The mean within-patient variance was estimated at 0.062, leading to an SD of 0.25 log10 copies/106 PBMCs, which can be taken as the measurement of the within-subject variation in cellular HIV-1 DNA in this study.
Similarly, the within-patient variance of plasma HIV-1 RNA, which accounted for 17% of the total variance, was estimated at 0.067, leading to an SD of 0.26 log10 copies/mL, consistent with published studies.26
Changes in Cellular HIV-1 DNA Level According to Antiretroviral Regimen
The median decrease in cellular HIV-1 DNA level from baseline to week 72 was not significantly different between the 2 treatment groups (0.54 and 0.45 log10 copies/106 PBMCs in groups 1 and 2, respectively; P = 0.17; Table 2) in contrast to that of plasma HIV-1 RNA, which decreased to less than 20 copies/mL at week 72 in a higher proportion of patients from group 1 than from group 2 (79% and 52%; P = 0.0009).23 This difference in plasma HIV-1 RNA response was not reflected in the early decay in cellular HIV-1 DNA from baseline to week 16, which was not different between the 2 treatment groups (median decreases by 0.25 and 0.29 log10 copies/106 PBMCs in groups 1 and 2, respectively; P = 0.65; Table 2). These analyses yielded similar results when restricted to the subgroup of antiretroviral-naive patients (data not shown).
Changes in Cellular HIV-1 DNA Level According to Prior Antiretroviral Status
The baseline cellular HIV-1 DNA median level was similar in antiretroviral-naive and pretreated patients (3.07 and 3.02 log10 copies/106 PBMCs, respectively; P = 0.85), as was the baseline plasma HIV-1 RNA median level (4.87 and 4.53 log10 copies/mL, respectively; P = 0.11). The cellular HIV-1 DNA median decrease during the first phase of response from baseline to week 16 was identical in antiretroviral-naive and pretreated patients (0.26 and 0.24 log10 copies/106 PBMCs, respectively; P = 0.92; see Table 2). In contrast, the cellular HIV-1 DNA median level reached at week 72 was significantly lower in antiretroviral-naive patients than in pretreated patients (2.50 vs. 2.71 log10 copies/106 PBMCs; P = 0.034), illustrating the continuing decay of cellular HIV-1 DNA level occurring beyond week 16 in the former but not the latter group of patients. The median overall decrease from baseline to week 72 was 0.55 and 0.23 log10 copies/106 PBMCs for antiretroviral-naive and pretreated patients, respectively (P = 0.0008; Fig. 3).
Among the 73 patients with available measurements at weeks 16 and 72, the median decrease in cellular HIV-1 DNA between these 2 time points was 0.08 log10 copies/106 PBMCs in the 13 pretreated patients compared with 0.22 log10 copies/106 PBMCs in the 60 antiretroviral-naive patients (P = 0.08), whereas plasma HIV-1 RNA was maintained at less than 20 copies/mL from week 16 to week 72 in 3 of 13 pretreated patients and 19 of 60 antiretroviral-naive patients (P = 0.74).
Factors Associated With Decrease in Cellular HIV-1 DNA Level
In multivariate analysis, a high baseline CD4 cell count and the absence of previous exposure to antiretroviral therapy were found to be predictive factors of a decrease in cellular HIV-1 DNA level greater than 0.50 log10 copies/106 PBMCs (Table 3). As illustrated in Table 4, which displays baseline values and decreases from baseline to week 72 in cellular HIV-1 DNA and plasma HIV-1 RNA according to baseline CD4 count, the magnitude of decrease in cellular HIV-1 DNA tended to be greater in patients with a higher baseline CD4 cell count than in others. In contrast, a decrease in plasma HIV-1 RNA did not seem to be dependent on the baseline CD4 cell count, and no association was found between HIV-1 RNA response at week 72 and the baseline CD4 count (P = 0.52).
Relations Between Plasma HIV-1 RNA and Cellular HIV-1 DNA Responses to Therapy
The baseline cellular HIV-1 DNA level was significantly lower in patients with undetectable plasma HIV-1 RNA at week 8 than in others (2.76 and 3.16 log10 copies/106 PBMCs, respectively; P = 0.0001). It was also marginally lower in patients with undetectable plasma HIV-1 RNA at week 72 than in others (3.03 and 3.17 log10 copies/106 PBMCs, respectively; P = 0.037). An early decrease, from baseline to week 16, in the cellular HIV-1 DNA level was not predictive of the plasma HIV-1 RNA level at week 72 (P = 0.97), and a decrease from baseline to week 72 in the cellular HIV-1 DNA level was similar in patients who achieved a plasma HIV-1 RNA level less than the limit of 20 copies/mL at week 72 and others (0.54 and 0.45 log10 copies/106 PBMCs in 92 and 33 patients, respectively; P = 0.14). The decrease in HIV-1 DNA was different according to previous exposure to therapy (see Table 2), as was the plasma HIV-1 RNA response, which was more frequent in antiretroviral-naive than pretreated patients (71% and 47%; P = 0.014).
We report here the results of the first prospective study planned in a randomized trial to determine the potential interest of total cellular HIV-1 DNA quantification, in addition to plasma HIV-1 RNA level measurement, for the comparison of 2 effective antiretroviral therapies. With a dynamic range of approximately 3 log10 in our study population, a within-patient variability similar to that of the plasma HIV-1 RNA26 and a measurement most often not censored by the lower limit of detection of the assay, the cellular HIV-1 DNA level could provide additional information to this comparison. Our study included follow-up of a large number of patients whose plasma HIV-1 RNA has been reduced to, and subsequently maintained at, undetectable levels by HAART for a clinically meaningful period. A dramatic decrease in plasma viral load was observed in both study regimens, which included 3 drugs from 2 classes (nucleoside reverse transcriptase inhibitor [NRTI] and protease inhibitor [PI]) or 3 classes (NRTI, nonnucleoside reverse transcriptase inhibitor [NNRTI], and PI). Although the plasma HIV-1 RNA level was decreased and maintained below the lower limit of detection of the most sensitive assay in most patients, the trial demonstrated the virologic superiority of the 2-drug class regimen over that combining 3 classes.23 In contrast, early as well as overall changes in cellular HIV-1 DNA, which remained detectable in all PBMC samples in spite of a plasma HIV-1 RNA level suppressed to less than 20 copies/mL for extended periods, were not different between the 2 treatment groups.
In this trial, cellular HIV-1 DNA measurements thus failed not only to discriminate between the 2 regimens for their capacity to inhibit new cellular infections fully but even to account for the differences evidenced by plasma HIV-1 RNA alone. No difference has been observed in cellular HIV-1 DNA decrease in children with a plasma HIV-1 RNA level suppressed to less than 50 copies/mL and treated with 1 or 2 nucleoside inhibitors associated with efavirenz and nelfinavir at any of the intervals examined.27 Like plasma HIV-1 RNA, a higher reduction of cellular HIV-1 DNA was observed in the patients on triple therapy compared with the patients on dual therapy17 as well as between these patients compared with patients on monotherapy.16 In our study, the decrease in cellular HIV-1 DNA level at week 72 was similar in responders (plasma HIV-1 RNA less than 20 copies/mL) and nonresponders. Thus, the evolution of cellular HIV-1 DNA level under treatment is correlated with the efficacy of antiretroviral therapy28 but seems to be less discriminating than that of plasma HIV-1 RNA because of its lower variation.
Several studies suggest that the cellular HIV-1 DNA level can predict disease progression in the absence of treatment as well as virologic response to treatment.13,27,29 Saitoh et al27 showed that rapid responders (<50 copies/mL after ≤8 weeks) had lower levels of cellular HIV-1 DNA at baseline than slow responders (<50 copies/mL after >8 weeks). We observed a similar result in early virologic response measured at week 8. The baseline cellular HIV-1 DNA predictive value decreased with the duration of therapy, however, becoming marginal at week 72, and baseline results were not clinically informative, given the within-patient variability of the measure. Two recent studies20,30 showed a predictive value of cellular HIV-1 DNA on virologic response to simplified maintenance therapy or structured therapeutic interruptions in another population of previously treated patients, with sustained suppression of plasma HIV RNA.
In our study, we observed a relation between the baseline CD4 count and the cellular HIV-1 DNA level at baseline and its evolution throughout 18 months of effective treatment. The cellular HIV-1 DNA level was negatively correlated with CD4 count, contrary to the plasma HIV-1 RNA level. Consistent with the study by Andreoni et al,21 a more pronounced decay of cellular HIV-1 DNA was obtained in subjects with a higher CD4 cell count. These results emphasize the role of immunity in the control and reduction of cellular HIV-1 DNA during natural disease progression as well as during effective antiretroviral therapy.
Furthermore, cellular HIV-1 DNA levels decreased more in antiretroviral-naive patients than in pretreated ones after HAART initiation.31 Previous therapy is known to be a strong determinant of subsequent virologic response, presumably because of preexisting viral resistance.
The cellular HIV-1 DNA level was positively correlated with plasma HIV-1 RNA at baseline in accordance with previous reports.21,22,32 After the initiation of HAART, the level of cellular DNA decreased gradually over time, not only from baseline to week 16 but beyond that, whereas plasma HIV-1 RNA had been suppressed below the lower limit of detection of the assay, with an estimated half-life of cellular HIV-1 DNA of 18 weeks in the first phase of the decay and 104 weeks in the second phase.22,27,33,34 These values are similar to those reported in adults and children receiving suppressive antiretroviral therapy but greater than those reported in patients treated during acute infection.21,27,33,35 The first slope in HIV-1 DNA decay observed in our study corresponds to the time necessary to lower plasma viral RNA below the detection limit. The second slope in HIV-1 DNA decay is related to the decrease of total DNA, located mainly in resting memory CD4 T cells as well as naive cells.36,37 The modest effect on DNA level (less than 10%) highlights the weak impact of antiretroviral treatment on the viral reservoir. The level of total DNA is not completely stable, however, and continues to decrease slowly during efficient treatment.33,38,39
In conclusion, this work provides evidence that the biphasic decrease of cellular HIV-1 DNA level during HAART is positively correlated with baseline CD4 cell count and negatively correlated with previous therapy. In our study, the cellular HIV-1 DNA level did not add information to that of the plasma HIV-1 RNA level for comparison of the viral efficacy of the 2 studied regimens.
The authors thank the study participants and members of the scientific committee (J. Dormont and A. Bouxin-Métro [Agence Nationale de Recherches sur le SIDA], M. C. Gervais [Merck Sharp and Dhome], D. Chiche [Bristol Myers Squibb], and B. Baumelou [Boerhinger Ingelheim]) for fruitful discussion and advice. The authors also thank C. Rouzioux for comments and advice on data interpretation.
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ANRS 081 Study Group Members
ANRS 081 study group members and participating centers include the following: M. Bentata, B. Jarousse, L. Guillevin, and P. Deny (Hôpital Avicenne, Bobigny); Y. Bourezane, B. Hoen, and D. Bettinger (Hôpital Saint-Jacques, Besançon); C. Goujard, J. F. Delfraissy, and P. Nordmann (Hôpital de Bicêtre, Le Kremlin Bicêtre); J. M. Salord, C. Caulin, and M. C. Mazeron (Hôpital Lariboisière, Paris); F. Bani-Sadre, P. de Truchis, and C. Perronne (Hôpital Raymond Poincaré, Garches); N. Adda, T. H. Nguyen, W. Rozenbaum, and F. Zatla (Hôpital Rothschild, Paris); V. Chambrin, F. Boué, P. Galanaud, and D. Cointe (Hôpital Antoine Béclère, Clamart); O. Launay, P. Yeni, C. Carbon, S. Masson, S. Matheron, J. P. Coulaud, M. H. Prevot, I. Fournier, E. Bouvet, and C. Benoist-Silberstein (Hôpital Bichat-Claude Bernard, Paris); B. Silberman, D. Salmon-Céron, D. Sicard, and A. Krivine (Hôpital Cochin, Paris); D. Zucman, O. Blétry, and P. Honderlick (Center Médico-Chirurgical Foch); N. Amirat, A. Simon, S. Herson, and C. Delaugerre (Groupe hospitalier Pitié-Salpétrière, Paris); D. Bollens, M. C. Meyohas, J. Frottier, O. Picard, and J. C. Imbert (Hôpital Saint-Antoine, Paris); D. Ponscarme, J. M. Molina, and F. Ferchal (Hôpital Saint-Louis, Paris); S. Abel, G. Comlan-Mayaud, A. Cabié, G. Sobesky, and M. Ouka (Hôpital Pierre Zobda Quitman, Fort-de-France); L. Cotte, F. Bissuel, C. Trepo, and J. Ritter (Hôpital Hôtel Dieu, Lyon); V. Baillat, J. Reynes, F. Janbon, and B. Montes (Center Hospitalo-Universitaire Gui de Chauliac, Montpellier); V. Reliquet, S. Leautez, F. Raffi, and S. Poirier (Center Hospitalo-Universitaire Hôtel Dieu, Nantes); P. Clevenbergh, P. Dellamonica, and Y. Lefichoux (Hôpital de l'Archet, Nice); F. Souala, C. Michelet, and A. Ruffault (Hôpital Pontchaillou, Rennes); G. Kempf, V. Krantz, D. Rey, J. M. Lang, and M. P. Schmitt (Center Hospitalier Régional Universitaire de Strasbourg, Strasbourg); M. Obadia, P. Massip, and K. Sandres (Hôpital Purpan, Toulouse); P. Leclerc, D. Ruiz, J. P. Stahl, and A. Schmuck (Center Hospitalo-Universitaire Nord Albert Michallon, Grenoble); and P. Poubeau, C. Arvin-Berod, and A. Mixhault (Center Hospitalier Sud Réunion, Saint-Pierre, La Réunion).
Keywords:© 2005 Lippincott Williams & Wilkins, Inc.
HIV-1 DNA level; antiretroviral therapy; predictive factor