A cytostatic drug improves control of HIV-1 replication during structured treatment interruptions: a randomized study
García, Felipea; Plana, Montserratb; Arnedo, Mireiac; Ortiz, Gabriel Md; Miró, José Ma; Lopalco, Luciae; Lori, Francof; Pumarola, Tomásc; Gallart, Teresab; Gatell, José Ma
From the aInfectious Diseases Unit, the bImmunology and the cMicrobiology Laboratories, Clinic Institute of Infectious Diseases and Immunology, Institut d'Investigacions Biomèdiques August Pi I Sunyer, Hospital Clínic, Faculty of Medicine, University of Barcelona, Spain, the dGladstone Institute of Virology and Immunology, University of California, San Francisco, USA, the eDepartment of Biological and Technological Research and Infectious Diseases Clinic, San Raffaele Scientific Institute, Milan, Italy and the fResearch Institute for Genetic and Human Therapy, Washington DC, USA and Pavia Italy.
Requests for reprints to: Dr F. García, Infectious Diseases Unit, Hospital Clínic, Villarroel, 170, 08036 Barcelona, Spain.
Received: 14 March 2002; revised: 20 September 2002; accepted: 30 September 2002.
Objective: To study the effect of highly active antiretroviral therapy (HAART) with and without hydroxyurea (HU) on changes in plasma viral load (VL) set-point, and on HIV-1-specific responses, after five cycles of structured treatment interruptions (STI).
Methods: A group of 20 patients taking HAART for chronic HIV infection with VL < 20 copies/ml were randomized to continue HAART or HAART plus HU for 24 weeks followed by five STI cycles. HU was also stopped in cycles 1–3 but continued in cycles 4 and 5. The number of individuals maintaining a VL set-point < 5000 copies/ml during the fifth interruption were determined.
Results: VL remained < 5000 copies/ml in eight out of nine patients in the HU group and in four out of ten patients in the HAART group after a median 48 weeks of follow-up after the fifth interruption (P = 0.039). By STI cycle 5, there was a significant increase in the neutralizing activity (NA), in both magnitude and breadth of the total cytotoxic T lymphocyte (CTL) response and in lymphoproliferative response (LPR) from baseline. No significant differences were observed between HAART and HU groups in NA, CTL and LPR at any time-point. There were no differences in the NA titers at any time-point between responder and non-responder patients. There was a trend for higher CTL and LPR levels in responder patients (P = 0.10).
Conclusions: In this randomized, controlled study of STI with cycles of HAART or HAART plus HU, a lower peak VL rebound and a lower VL set-point was achieved in patients continuing HU while other drugs were discontinued. HU did not blunt anti-HIV-1-specific responses; however, control of VL did not correlate with anti-HIV-1-specific cellular immune responses.
It has been hypothesized that cyclic interruptions of highly active antiretroviral therapy (HAART) may boost HIV-1-specific immune responses and thus constitute an immune-based therapy for HIV-1 infection [1–7]. However, it seems that structured treatment interruption (STI) could only reset VL in approximately 20% of chronic HIV-infected patients, ranging from 10 to 40% [8,9]. Whether or not this resetting is associated consistently with an increase in the HIV-1-specific cellular immune responses remains to be elucidated [9–12]. In addition, these immune-specific responses seem to be weaker than those achieved when STI is performed in patients who started HAART immediately after acute infection .
Hydroxyurea (HU) has been used for years in HIV-1 infection because of its antiviral and cytostatic effects [13,14]. The antiviral effect is mediated through reduction in the levels of deoxynucleotide trisphosphates (dNTP), which inhibit HIV reverse transcription  and viral replication  in both quiescent lymphocytes  and terminally differentiated cells, such as macrophages  or dendritic cells . In activated lymphocytes, HU does not exert antiretroviral activity but potentiates the activity of certain nucleoside analogues, such as didanosine. Regarding the immunomodulatory effect, the main cellular consequence of inhibiting dNTP is a cytostatic effect . The impact of this cytostatic action on the immune system remains unknown . We hypothesized that, since stopping HAART induces sudden antigenic activation with high peaks of viral replications, which could destroy antigen-reactive clones , the use of a cytostatic drug would help T lymphocytes to remain quiescent, becoming refractory to productive HIV-1 infection and thus avoiding high peaks of viral replication without blunting HIV-1-specific immune responses.
The present study tests this hypothesis by examining the effects of adding HU to HAART on changes in viral load (VL) set-point, as well as on HIV-1-specific neutralizing activity (NA), cytotoxic T lymphocyte (CTL) response and CD4 cell lymphoproliferative response (LPR) after five cycles of STI.
Study design and patients
The study group were 20 patients with chronic HIV infection from the Spanish EARTH-2 study; they all had a baseline CD4 T lymphocyte count of > 500 × 106 cells/l and baseline VL > 5000 copies/ml prior to any antiretroviral treatment  and had been treated for ≥ 6 months with stavudine (30–40 mg/12 h based on body weight) plus lamivudine (150 mg/12 h) plus indinavir (800 mg/8 h) for 52 weeks with VL < 20 copies/ml for at least 32 weeks. The patients were then all given stavudine (30–40 mg/12 h based on body weight) plus didanosine (150–200 mg twice daily based on body weight) plus indinavir (800 mg/8 h) and were randomized to this basic HAART group (n = 10) or to the HU group (n = 10), who also received HU (500 mg/12 h). Plasma VL, NA of serum, LPR to HIV-1 antigens, and HIV-1-specific CTL responses were assessed.
Treatment interruptions occurred in five cycles separated by periods of 8 weeks on the same HAART. In the first STI, HAART was reintroduced if VL increased > 200 copies/ml; the median duration of this interruption of therapy was 3 weeks [interquartile range (IQR), 3–4.5]. HAART was discontinued during a fixed period of 2 weeks in the second, third and fourth STI. In the fifth STI, HAART was discontinued until the VL reached a set-point. HU was discontinued during the periods off HAART in the first, second and third STI and maintained during the fourth and fifth STI as well as during follow-up. Medical visits were scheduled at day 0, and weekly thereafter. Plasma HIV-1 RNA viremia, lymphocyte immunophenotyping, LPR to mitogens and HIV-1 antigens, and HIV-1-specific CTL responses were assessed weekly after each interruption, and bimonthly after reintroduction of HAART. NA of the serum was measured at baseline before any HAART, the day when STI was initiated and at week 24 after the fifth STI.
Plasma HIV-1 RNA levels were determined using the Amplicor HIV-1 Monitor Ultra Sensitive Specimen Preparation Protocol Ultra Direct Assay (Roche Molecular Systems, Somerville, New Jersey, USA) with a limit of quantification of 20 copies/ml. Those samples below the detection limits of this test were retested with a lower limit of detection of 5 copies/ml . The primary endpoint was to assess the number of individuals controlling HIV at < 5000 copies/ml (responders) in the two groups.
The study was explained to all patients in detail, and all gave written informed consent. The study was approved by the institutional ethical review board.
Neutralizing activity assays
Peripheral blood mononuclear cells (PBMC) from healthy blood donors were isolated by Ficoll–Hipaque centrifugation and stimulated for 48 h with 3 μg/ml phytohemagglutinin (PHA; Sigma-Aldrich, Steinheim, Germany) and 100 U/ml recombinant interleukin-2 (Amersham, Amersham, UK). Serum samples were serially diluted (fourfolds) and 75 μl samples were incubated for 1 h with 75 μl of a primary virus dilution [median effective dose adjusted to 25 for HIV45 (amphitrophic R3, R5, X4 strain that uses CCR3, CCR5, and CXCR4 as coreceptors)  and to 16.7 for HIV40 (R5 strain that uses CCR5 as its coreceptor ]. This was then added to 2 × 105 cells activated PBMC resuspended in 75 μl RPMI 1640 (Sigma, St Louis, Missouri, USA) in wells of 96-well microplates. Cultures were incubated for 2 h, washed and resuspended in PHA- and interleukin-2-containing medium. HIV-1 p24 antigen in the supernatants was measured relative to a non-treated control.
Lymphocyte proliferation assays
PBMC were washed twice and resuspended at 2 × 106/ml in serum-free medium X-VIVO 10 (BioWhittaker, Walkersville, Maryland, USA). Cultures were plated in 96 round-bottomed microplates (TPP, Trasadingen, Switzerland) in triplicate at 105/well for 4-day assays and at 2 × 105/well in 7-day assays. Cells were cultured in the absence or presence of 90 μg/ml PHA (Murex Biotech, Darford, UK), OKT3 10 ng/ml (Ortho Biotech, Raritan, New Jersey, USA) and 5 μg/ml HIV-1 antigens gp160, p24 and gp120 (Protein Sciences, Meriden, Connecticut, USA and Intracell, London, UK). Incorporation of [3H]-thymidine was assessed for the last 18 h of culture (Betaplate LKB Wallac, Sweden). Results were expressed as mean counts per minute (cpm). The stimulation index was calculated for each sample as: (cpm for cells with stimulus)/(cpm for cells without stimulus). Positive antigen-specific responses were defined as more than 3000 cpm and a stimulation index greater than 3.
HIV-1-specific cytotoxic T cell responses
An epitope-specific ELISPOT assay was used to measure antigen-induced interferon-gamma (IFN-γ) release from CD8 T cells. A mean of 16 (range, 3–27) different HLA class I-restricted synthetic peptides from Gag, Pol, Env and Nef proteins were tested in each individual. The wells of 96-well microtiter plates were coated overnight with a monoclonal antibody specific to human IFN-γ (mAb1-D1K, Mabtech, Nacka, Sweden). PBMC resuspended in RPMI 1640 plus 10% tetal calf serum were plated in the presence of different peptides at 4 μmol/l and incubated overnight at 37°C under 5% CO2. Plates were developed using biotinylated anti-human IFN-γ, streptavidin-bound phosphatase alkaline conjugate and chromogenic substrate (BioRad, Hercules, California, USA). Spot-forming cells (SFC) were enumerated on a dissecting microscope. After subtracting background counts obtained with PBMC in medium alone, results were normalized to SFC/106 PBMC.
For the purpose of analysis, RNA values reported at an undetectable level (< 5 copies/ml) were considered equivalent to 5 copies/ml. The HIV RNA values were log10 transformed before analysis. The doubling time of VL was calculated as described elsewhere . The baseline VL value and CD4 T cell count was defined as the average of the screening within 1 to 3 months prior to enrolment and the day 0 determination. The set-point value of VL was determined by the average of the two last stable measurements (with a difference of less than 0.3 log10 copies/ml) separated by at least 1 month after 24 weeks off therapy. Quantitative data of VL, lymphocyte subtypes, CTL responses, proliferative stimulation index and NA were compared between groups with a Students t-test for paired samples in variables with normal distribution and similar variances, or with the Wilcoxon matched pairs test for those variables without normal distribution. Spearman rank order correlations were performed on quantitative data for mean total magnitude of CD8 or CD4 T cell responses and lowest fall in VL from baseline at respective periods of follow-up.
The schedule of treatment interruptions is shown in Fig. 1. Characteristics of the patients at baseline are shown in Table 1. There were no important or statistically significant differences between the HU and HAART groups. All patients underwent five cycles of STI separated by periods of 8 weeks on the same drug regimen. HAART was stopped in each STI but HU was continued in the first, second and third STI and discontinued in the fourth and fifth. The rationale for keeping HU in for only the fourth and fifth STI was to test which hypothesis of action of HU in this setting could be more plausible. HU might have acted in two ways. First, HU could inhibit the initial wave of HIV rebound originating from reservoirs such as quiescent lymphocytes, macrophages, or dendritic cells, in which the drug has been shown to be an effective monotherapy [15,16]; in this case, the effect of HU in the viral load rebound could be observed even when HU was stopped. Second, HU could limit the subsequent wave of viral replication among activated T lymphocytes, by virtue of its cytostatic effects; in this case, the effect of control of viral replication could be observed only if HU was maintained. The median follow-up off therapy after the fifth STI was 48 weeks (IQR, 28–76). In patients who did not have a good response (VL < 5000 copies/ml) the same drug regimen was reintroduced after 24 weeks off therapy. One subject in the HU group discontinued the study because of relocation.
A VL increase was detected in all individuals during all STI. However, dynamics of VL rebound changed when the first STI was compared with the fifth. The mean doubling time of VL rebound increased from the first to the last STI [from 2.08 days (SE, 0.38) to 6.2 days (SE, 2.8) (P < 0.05) in the HU group and from 3.3 days (SE, 0.65) to 5.6 days (SE, 1.6) in the HAART group (P < 0.05)] without significant differences between the two groups. There were also no differences in VL rebound at week 2 off therapy when comparing the HU group (mean VL 3.1, 3.5, 3.5 log10 copies/ml during first, second and third STI, respectively) and the HAART group (mean VL 3.7, 3.4, 3.2 log10 copies/ml during first, second and third STI, respectively) (Fig. 2). Conversely, when HU was continued during the period of discontinuation of the other drugs in the fourth and fifth STI, the VL rebound at week 2 off therapy in the HU group was 2.2 and 2.2 log10 copies/ml, respectively (that is, 1 log10 copies/ml lower than during the previous STI periods in the same group), whereas the VL rebound did not change in the HAART group (mean VL 3.2 and 3.1 log10 copies/ml during the fourth and fifth STI, respectively) (Fig. 2).
After the fifth interruption of therapy, the peak of VL rebound was lower in the group maintaining treatment with HU. In this group, a peak VL >5000 copies/ml was observed in only two out of nine compared with eight out of ten patients in the HAART group (P = 0.0185). More importantly, VL remained < 5000 copies/ml in eight out of nine patients in HU group and in four out of ten patients in HAART group after a median of 48 weeks of follow-up after the fifth interruption of therapy (P = 0.039) (Fig. 3). The mean difference between baseline VL before any therapy and the set-point determined by the average of the two last stable measurements (with a difference of less than 0.3 log10 copies/ml) separated by at least 1 month after 24 weeks off therapy during the fifth STI was −1.4 and −0.6 log10 copies/ml in the HU and HAART groups, respectively.
NA was analyzed in 15 individuals who had available serum at baseline before any antiretroviral therapy (seven from the HU group and eight from HAART group). By STI cycle 5, there was a significant increase in the HIV45-specific NA from baseline (P = 0.003) but no increase in HIV40-specific NA. No significant differences were observed between the HAART and HU groups in NA at any time-point. There were no differences in the NA titers at any time-point between responder and non-responder patients.
At baseline, only 7 out of 19 patients showed a moderate to strong CTL response. Both the magnitude and the breadth of the total CTL response progressively improved during STI. The median total CTL response at baseline was 570 SFC/106 PBMC and during STI cycles 1–5 was 698, 582, 1080, 1429 and 2814 SFC/106 PBMC, respectively (P = 0.0001). The breadth of CTL response increased from a median of 3 peptides (range, 0–8) at baseline to 6 peptides (range, 4–16) at STI cycle 5. A weak LPR to HIV-1 p24 protein was detected at baseline in 3 out of 19 patients and after STI cycle 5 in 14 out of 19 patients (P = 0.0003). No significant differences were observed between the HAART and HU groups in CTL and LPR at any time-point. There was a trend for higher CTL and LPR levels in responder patients (P = 0.10 for both) measured at the moment that VL reached the set-point after the fifth discontinuation of antiretroviral therapy. However, three out of eight responder patients in the HU group did not show CTL response or LPR. Conversely, three out of six non-responder patients in the HAART group showed strong CTL and LPR responses. These increases in individual patients were analysed to see if they correlated with greater lengthening of the doubling time of VL rebound from the first to the last STI. The increase in CTL responses was not correlated with the lengthening of the doubling time of VL rebound (r = −0.11; P = 0.68); however the increase in LPR was strongly correlated with lengthening of the doubling time of VL rebound (r = 0.73; P = 0.003). Moreover, the LPR at the moment that VL reached the set-point after the fifth discontinuation of antiretroviral therapy was also strongly correlated with lengthening of the doubling time of VL rebound (r = 0.95; P = 0.0001).
Safety and immune system changes
No patients had to discontinue HAART or HU because of side-effects. VL fell to undetectable levels after reinitiating therapy in all patients at all cycles (Fig. 2). Seven patients had to change therapy because of side-effects (all of them during the period before STI), mostly nephrolithiasis and gastrointestinal complaints (Table 2). In six out of seven patients who changed triple therapy because of side-effects, indinavir was replaced by nelfinavir, and in one case (polyneuropathy) didanosine was changed to lamivudine. Mean fasting cholesterol, triglycerides and glucose levels did not change significantly after 1 year of follow-up. Triglycerides increased to a pathological level in three patients. In two of these, levels went back to normal after discontinuation of HAART. There were four patients with clinical lipodystrophy. None of the patients in whom HU was maintained reported any side-effects during the period off HAART. The proportion of patients with side-effects did not differ between the HU and HAART groups.
From the day of randomization to the day of STI initiation, the absolute number and percentage of CD4 T cells did not change significantly in either the HAART group or the HU group (Fig. 4). The mean CD4 T cells at day 0 before STI in the HAART and HU groups was 957 × 106 cells/l (SE, 69) and 1001 × 106 cells/l (SE, 155), respectively. The mean percentage CD4 T cells at day 0 before STI was 48% (SE, 3) and 40 (4) in the HAART and HU groups, respectively. Non-significant falls in CD4 T cell count were observed from the day of initiation of STI to the day before the fifth STI [mean from 957 × 106 cells/l (SE, 69) to 849 × 106 cells/l (SE, 83) (P = 0.37) in the HAART group and from 1001 × 106 cells/l (SE, 155) to 883 × 106 cells/l (SE, 183) (P = 0.38) in the HU group]. Significant falls in CD4 T cell percentage were observed from the day of initiation of STI to the day before the fifth STI [mean from 48% (SE, 3) to 36% (SE, 2) in the HAART group (P = 0.01) and from 40% (SE, 4) to 34% (SE, 3) in the HU group (P = 0.05)]. Neither absolute numbers nor percentages changed significantly during the follow-up of the fifth STI in both groups. CD4 T lymphocytes did not fall below baseline (500 × 106 cells/l) in any patient at any time.
Current treatment guidelines for HIV infection recommend a relatively late initiation of HAART [26,27] because of adherence problems to a sometimes very complex therapy , the unlikely possibility of eradication of HIV-1 with HAART alone, [25,29–31] and the risk of drug-related side-effects, including serious metabolic abnormalities and apparently irreversible fat redistribution syndromes [32–34]. Moreover, it has not been demonstrated that an earlier initiation of therapy (i.e., at a CD4 T cell count above 350–500 × 106 cells/l) can offer any advantage with regard to further immunological improvement, avoiding development of resistance, control of viral replication, reduction of clinical events or death when compared with starting HAART at a later stage [35,36].
However, these guidelines raise a number of questions that are yet unsolved. First, what should be recommended to those patients who started therapy when their CD4 T cell counts were above the current recommendation or to those patients whose CD4 T cell counts increased and stabilized even for years above a high level (i.e., 500 × 106 cells/l) after initiating HAART? Second, not treating patients in earlier stages of HIV-1 infection means that they will await immune system deterioration without any intervention. It would be helpful to implement strategies with less side-effects than the current ‘HAART for life’ to avoid the natural evolution of the disease in patients with high CD4 T cell counts . Lastly, some patients who have not been treated until later stages of the disease will have a high level of VL, which could increase the risk of transmission and cause a public health problem. Alternative strategies gaining more experimental validation are immune-based therapies aimed at controlling HIV-1 replication [38,39]. It has been hypothesized that cyclic interruptions of antiretroviral therapy may boost HIV-1-specific immune responses, reset VL and be considered a modality of immune-based therapy for HIV-1 infection, at least when initiated immediately after acute infection [4,6]. These immune-specific responses seem to be weaker, however, when STI is performed during chronic HIV infection. Moreover, there is contradictory evidence on the possibility of the association of these immune responses with the reset of the VL set-point, occurring in a low proportion of patients (approximately 20% of patients with chronic HIV infection) [8–12].
The data of the present randomized study shows that the maintenance of a single cytostatic drug (namely HU) during the discontinuation of antiretroviral drugs may significantly increase the percentage of patients able to achieve control of viral replication after five cycles of STI and a prolonged period off HAART (median 48 weeks). How can the results be explained? It did not seem that HU had any distinct effect on HIV-specific immune responses. HU might have acted in two other ways: by inhibiting the initial wave of HIV rebound originating from reservoirs such as quiescent lymphocytes, macrophages, or dendritic cells, in which the drug has been shown to be an effective monotherapy [15,16]; or by limiting the subsequent wave of viral replication among activated T lymphocytes by virtue of its cytostatic effects, as suggested by several mathematical models and by a randomized, controlled trial (for a review, see ). The fact that the final set-point and not the initial doubling time was affected by HU strongly favors the latter hypothesis. Moreover, rebound at 2 weeks was similar in both groups when both HU and HAART were discontinued during the first, second and third STI while it was much lower in the HU group when HU was maintained (in cycles 4 and 5). This further supports the hypothesis that HU controls viral replication by virtue of its cytostatic effect on CD4 T lymphocytes. It is important to note that the cytostatic action of HU had no deleterious effects on HIV-1-specific neutralizing antibodies, T-helper or CTL responses. A question that is not answered with this study is whether the same effect on viral replication could be observed if HU were added to a regimen just prior to interruption and then kept on while the antiretroviral drugs were discontinued.
These data also confirm that HIV-1-specific cellular immune responses can be augmented following cycles of therapy interruption in chronic HIV infection, consistent with previous findings [1,2,6,9]. Subjects who had no detectable responses at baseline or after the first STI progressively generated HIV-1-specific neutralizing antibodies and cellular immune responses with repeated cycles of STI. This correlated with a progressive increase in doubling time of VL rebound, however, not with a lowering in VL set-point. It is plausible that the transient, incomplete recovery of HIV-specific immune responses induced by STI in our patients, and, in general, in chronic HIV infection , is sufficient to limit the initial spread of HIV during rebound (doubling time), but it becomes subsequently overwhelmed by the virus without affecting the final set-point.
There are some safety concerns related to STI protocols. One potential problem is the development of drug resistance . In all our patients, VL dropped to undetectable levels when the same treatment was reintroduced. The most serious safety concern associated with STI in our study was a significant fall in the CD4 T cell percentage (but not absolute number) at week 40 (before the fifth and last STI). However, CD4 T cells never fell below the baseline levels (that is, before any antiretroviral therapy). Consequently, this strategy may be considered exclusively in a selected HIV-1-infected population under strict medical supervision, with an objective different from that of ‘drug holidays’ in patients with very advanced disease .
These data represent the first demonstration that control of HIV might be induced during chronic infection by adding a cytostatic drug to STI, thus overcoming one limitation of STI in this patient population. The results may indicate a novel approach, to be tested and confirmed, for the treatment of selected populations of HIV-1-infected patients, such as those who started HAART with a high level of CD4 T cells and now have to continue HAART for many years to come, or patients with high CD4 T cells and a high level of VL who could reset their set-point VL to a lower value and delay the necessity of starting continuous HAART. The combination of STI and cytostatic drugs in HIV-1 infection should proceed with caution until the impact and long-term safety has been further investigated in larger clinical trials.
We thank Julianna Lisziewicz, Elisa de Lazzari, Sebastian Bonhoeffer, and Douglas F Nixon for comments about the manuscript, Claudia Pastori for technical help with the NA assays and Sylva Petrocchi and Al Ruel for editorial assistance. We are indebted to all the participants of the study, laboratory technicians (M. J. Maleno, A. Capón, M. García, and A. García) and to Bristol-Myers-Squibb for supplying the hydroxyurea.
Sponsorship: This study was supported in part by grants FIPSE 3118/00*, FIPSE 3074/99*, SAF 98/0021, FIS 99/0289, FIS 01/1595, SAF 01/2591, FIPSE 36259/01, NIH R01 AI44595, F31 GM20068-01 and ISS/1999 40C52. (FIPSE is a non-profit foundation including the Spanish Ministry of Health, Abbott Laboratories, Boehringer Ingelheim, Bristol-Myers-Squibb, GlaxoSmithKline, Merck Sharp and Dohme and Roche.) Montserrat Plana received a grant by SEIMC (Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica). Gabriel Ortiz is a student in the Tri-Institutional MD–PhD Program supported by the NIH Medical Scientist Training Program Grant GM 0773. Bristol-Myers-Squibb supplied the hydroxyurea.
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