Recent experimental evidence has identified several obstacles which antiretroviral therapy regimens must target before eradication of HIV-1 becomes a clinical possibility. The identification of a long-lived, latently infected, resting CD4 T-cell population prolonged the estimation for the eradication of HIV-1 from the previously calculated 1–3 years of successful highly active antiretroviral therapy (HAART) [1–3] up to 10–60 years . On the other hand, long-term use of HAART may increase the risk of drug-related side-effects, including serious metabolic abnormalities and apparently irreversible fat redistribution syndromes [5,6].
Alternative strategies gaining more experimental validation are immune-based therapies aimed at controlling HIV-1 infection [7,8]. There is a growing body of evidence supporting the effectiveness of HIV-1-specific CD4 and CD8 T-cell responses [9–15]. Case reports and a pilot study of patients started on HAART during primary infection and who control viral replication after discontinuation of therapy have been described [16–19]. HIV-1-specific immune responses can be restored in patients who initiate HAART during primary infection, and this response may preclude viral rebound [14,16,20]. More commonly, rapid rebound of plasma viral load occurs after variable periods of successful HAART in subjects with acute or chronic HIV-1 infection [21,22]. These viral rebounds could be due to the lack of specific immune response against HIV-1 antigens , as both HIV-1 specific CD4 and CD8 T-cell responses decline after several months of effective HAART . It has been hypothesized that cyclic interruptions of antiretroviral therapy may boost the HIV-1 specific immune responses and be considered as an immune-based therapy for HIV-1 infection [24,25].
The objectives of this study were to analyze the dynamics of viral load rebound and the HIV-1 specific immune responses after three cycles of structured antiretroviral therapy interruption (STI) in patients with chronic HIV-1 infection. The safety of this strategy, and the virological response after reintroducing the same antiretroviral therapy were also investigated.
Study design and patients
Ten HIV-1 chronically infected patients treated with stavudine, lamivudine and ritonavir (or indinavir, when ritonavir was not tolerated) for 52 weeks, and with viral load < 20 copies/ml for at least 32 weeks were enrolled in this study. Eight of these 10 patients were from the Spanish EARTH-1 Study (subjects 12, 72, 107, 156, 157, 169, 170 and 211) and are representative of all of the patients randomized to triple therapy and recruited in the coordinating center . The other two patients (subjects 4 and 226) were from the Spanish EARTH-2 study . Inclusion criteria for these studies were chronic asymptomatic HIV-1 infection with CD4 T-cell counts > 500 × 106/l and viral load > 10 000 copies/ml (in EARTH-1) or > 5000 copies/ml (EARTH-2) in at least two determinations separated by 1–3 months. Because of concern about development of potential resistance and/or loss of efficacy of HAART, safety criteria were instituted. First, an analysis of the treatment response was performed after the first stop. HAART would be discontinued for a second time only in patients whose plasma viral load decrease to < 20 copies/ml after therapy reinitiation. The trial would be stopped if, among the 10 patients, two or more failed to suppress viremia < 20 copies/ml upon reinitiation of therapy. Secondly, the trial would be stopped if two or more individuals showed genotypic mutations conferring resistance during the first rebound of viral load. 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.
Monitoring and enrollment
The schedule of treatment interruptions is shown in Fig. 1. After each interruption, medical visits were scheduled at day 0, and weekly, thereafter. Plasma HIV-1 RNA viremia, genotyping and phenotyping for resistance, lymphocyte immunophenotyping, CD4 T-cell proliferative responses to mitogens and HIV-1 antigens, and HIV-1 specific cytotoxic T lymphocyte (CTL) responses were assessed weekly after each discontinuation, and bimonthly after reintroduction of the antiretroviral treatment. After the first stop, the same antiretroviral treatment regimen was reintroduced after the first detection of viral load > 200 copies/ml. After the second interruption, the same antiretroviral drug treatment was reintroduced 1 month after first detection of viral load > 200 copies/ml, if the viral load did not drop spontaneously. If the viral load dropped spontaneously, antiretroviral treatment was reintroduced when viral load increased or did not decrease in two consecutive determinations. After third stop treatment was reintroduced only if viral load reached a set-point similar to baseline viral load or > 10 000 copies/ml after 6 months off therapy.
Plasma HIV-1 RNA levels were determined using the Amplicor HIV-1 Monitor Ultra Sensitive Specimen Preparation Protocol Ultra Direct Assay (Roche Molecular Systems, Inc., 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 HIV-1 RNA copies/ml .
Genotyping for resistance
For the analysis of genotyping for resistance, viral RNA was extracted from plasma and reverse transcribed into complementary DNA and subsequently amplified by a single-tube reverse transcription–PCR using the TruGene HIV-1 assay (Visible Genetics, Inc. Toronto, Canada). This step produced a 1.3 kb amplicon including the protease gene and the first 318 codons of the reverse transcriptase gene of HIV-1. Amplification products were sequenced simultaneously in three gene portions: the whole protease and reverse transcriptase codons, 39–155 (beginning) and 135–247 (middle). Each sequencing reaction was loaded into a MicroGene Clipper sequencer. The resulting sequences for each sample was compared with a database containing known drug resistance mutations.
The analysis of phenotypic resistance was performed by the Antivirogram method (Virco Laboratories Inc, Mechelen, Belgium).
Lymphocyte proliferation assays
Peripheral blood mononuclear cells (PBMC) were washed twice and resuspended at 2 × 106cells/ml in serum-free medium X-VIVO 10 (BioWhittaker, Maryland, USA). Cultures were plated in triplicate at 1 × 105/well in 4-day assays and at 2 × 105/well in 7-day assays, in 96 round-bottomed microplates (TPP, Trasadingen, Switzerland). Cells were cultured in the absence or presence of phytohemagluttinin 1% 90 μg/ml (Murex, Biotech Ltd, UK), OKT3 10 ng/ml (Ortho Biotech Inc., 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 tritium-labeled thymidine was assessed for the last 18 h of culture (Betaplate, LKB Wallac, Turku, Finland). Results were expressed as mean counts per minute (c.p.m.). The stimulation index (SI) was calculated for each sample as: c.p.m. for cells with stimulus/cpm for cells without stimulus. Positive antigen-specific responses were defined as > 3000 c.p.m. and SI > 3.
HIV-1 specific CD8 T-cell responses
Enzyme linked immunoassay (ELISPOT)
The method for quantifying the release of cytokines from antigen-specific CD8 T cells has been described previously . A vaccinia virus-based ELISPOT assay was used to measure antigen-induced interferon (IFN)-γ release from CD8 T cells. Briefly, 96-well microtiter plates (Multiscreen-HA, Millipore, Bedford, Massachusetts, USA) were coated overnight with a monoclonal antibody specific to human IFN-γ (1-D1K, Mabtech, Stockolm, Sweden). PBMC were resuspended in RPMI containing 1% pooled human serum. The HIV-1 specific responses were measured from cryopreserved PBMC samples infected with control vaccinia or vaccinia vectors encoding IIIBenv, IIIBgag, IIIBpol and IIIBnef at a multiplicity of infection of 2 plaque forming units per cell. Plates were developed using biotinylated anti-human IFN-γ (Mabtech), avidin-bound biotinylated horseradish peroxidase (Vector Laboratories, Burlingame, California, USA) and stable diaminobenzidine (Research Genetics, Huntsville, Alabama, USA). The number of spot forming cells (SFC) from each vaccinia control well (< 25 SFC) was subtracted. Responses greater than 15 spots per 106 PBMC were considered to be significant. The total HIV-1 specific CD8 T-cell response was calculated by summing the magnitudes to the four HIV-1 antigens tested. A descriptive range of strength of HIV-1 specific CD8 T-cell responses has been used as follows: low (10–200/1 × 106 PMBC), moderate (201–500 SFC/1 × 106 PBMC), high (> 501 SFC/1 × 106 PMBC) .
51Cr release assay
Polyclonal stimulation [anti-CD3 monoclonal antibody 12F6 (from J. Wong, Massachusetts General Hospital) at 0.1 μg/ml in R15 plus 50 U/ml interleukin-2] and irradiated feeder cells were used to establish bulk cultures. Bulk cultures were maintained by replacing half of the volume with fresh R15 plus 50 U/ml interleukin-2 twice weekly. A standard 51Cr release assay was performed on bulk cultures, using autologous B lymphocyte cell lines infected with either vaccinia control or recombinant vaccinia expressing IIIBenv, IIIBgag, IIIBpol and IIIBnef as targets. Spontaneous release was < 30%. Responses were considered positive if specific lysis was > 10% above control at more than one effector : target ratio.
For the purpose of analysis, RNA values reported as undetectable (< 5 copies/ml plasma) were considered equivalent to 5 copies/ml. The HIV RNA values were log10 transformed before analysis. The baseline value of viral load and CD4 T-cell count was defined as the mean of the screening within 1–3 months prior to enrolment and day 0 determination. The set-point value of viral load was considered to be the average of the two last stable measurements (with a difference of < 0.3 log10) separated by ≥ 1 month after more than 6 months off therapy. The doubling time of plasma viral load was calculated as described elsewhere . Quantitative data of lymphocyte subtypes, CD8 T-cell responses and proliferative SI were compared between different points with Student's t test for paired samples in variables with normal distribution and similar variances, or with Wilcoxon's matched pairs test for those variables without these criteria. Spearman rank order correlations were performed on quantitative data for mean total magnitude of CD8 or CD4 T-cell responses and lowest drop in viral load from baseline at respective periods of follow-up.
We studied 10 chronically HIV-1 infected patients with stable baseline CD4 T-cell counts > 500 × 106 cells/l (in at least two determinations separated by 1–3 months) and stable baseline viral load > 5000 copies/ml (in at least two determinations separated by 1–3 months) who agreed to undergo cyclic treatment interruptions (Table 1). These subjects were enrolled in the triple therapy arm of two open multicenter, randomized trials comparing double versus triple versus no therapy [26,27]. After 1 year of triple therapy all 10 patients had plasma viral loads < 20 copies/ml. These subjects underwent three cycles of STI of the same HAART regimen separated by 6 months (Fig. 1). Subject 156 was lost to follow-up after the first stop. As a control we selected 20 subjects matched for CD4 T-cell count and viral load and enrolled in the no therapy arm of the same two randomized multicenter trials.
Dynamics of viral load rebound
Viral load rebound was detected in all subjects at each STI. However, there were qualitative and quantitative attenuations in the dynamics of viral load rebound that became more pronounced by the third interruption cycle in the majority of subjects (Figs 2 and 3). In four subjects, viral load rebounded to a level above baseline viral load (> 0.5 log10) after the first and second interruption of therapy (subjects 12, 157, 169, and 170). However, after the third interruption, only subject 157 had viral load rebound above baseline. In this subject, at the second interruption, viral load rebound was accompanied by symptoms resembling the symptomatic primary infection 4 years earlier, including lymph node enlargement, rash and low grade fever. The symptoms resolved following reintroduction of the same therapy and viral load suppression. In five additional subjects (subjects 4, 72, 107, 211, and 226), viral load rebounded to similar or lower levels than baseline after the first, second and third interruptions. Finally, subject 169 was lost to follow-up after the first stop (Fig. 2).
Interestingly, in four out of nine subjects during the second stop (subjects 4, 72, 107, and 226) rebound viral load dropped spontaneously and was sustained below the baseline value in the absence of HAART (difference between lowest viral load during the second stop and baseline, −1.10, −2.05, −0.81, and −2.07 log10, respectively). Likewise, after the third stop, viral load dropped to lower values than baseline for six out of nine subjects. At the third stop, subjects 4, 72, 107, 170, 211, and 226 had a difference between set-point viral load [defined as the mean of the two last values separated at least by 1 month after a median off therapy of 52 weeks (range, 24–62 weeks)] and baseline viral load of −1.3, −1.0, −0.8, −0.7, −0.6, 1.3 log10, respectively. Moreover, four out of these six subjects maintained viral load < 5000 copies/ml after a median of 52 weeks off therapy (Table 1 and Fig. 2). In the 20 chronically HIV-1 infected control patients enrolled in the untreated study arms, viral load remained stable or increased during the year of follow-up (see Fig. 4). No sustained decreases in viral load were observed in untreated control subjects. Similar results have been reported in other studies of untreated HIV-1 infected subjects .
Specific HIV-1 immune responses
To determine whether spontaneous decreases in viral load following therapy discontinuation were associated with HIV-1 specific cellular immune responses, we measured the HIV-1 specific CD4 T-cell response by proliferation assay, and quantified HIV-1 specific CD8 T-cell activity by ELISPOT and 51Cr release assay. At baseline before HAART, the patients had neither detectable anti-HIV-1 specific CTL nor helper responses. Before and during the first interruption of therapy, no subject had detectable CD4 T-cell proliferative responses against HIV-1 p24 (Fig. 3) or envelope antigens (gp120 and gp160; data not shown). All nine subjects reaching the second interruption had viral load < 20 copies/ml, and eight out of nine had undetectable HIV-1 specific cellular immune responses (Fig. 3). Subject 226 had a strong CD4 T-cell proliferative response against HIV-1 p24, but had no Env-specific CD4 T-cell proliferative response, or HIV-1 specific CD8 T-cell responses (Fig. 3a). After the second stop, four out of nine subjects (subjects 4, 72, 107, and 226) developed CD4 T-cell proliferative responses against HIV-1 p24 (Fig 3a and Fig. 3b) but not to Env antigens. Overall, in five out of nine patients (the same four subjects 4, 72, 107, 226, plus an additional subject 211), a moderate to strong HIV-1 specific CD8 T-cell response was detected with ELISPOT after the second interruption (Fig. 3). After 3 months of reinitiating antiretroviral therapy all subjects except 226 lost HIV-1 specific proliferative responses. After the third stop, eight out of nine subjects (4, 12, 72, 107, 169, 170, 211, and 226) developed measurable, although weak, CD4 T-cell responses (Fig. 3). A significantly higher CD4 T-cell proliferative response was measured in subjects during the third versus the first stop (one-sided Wilcoxon matched pairs t test, P = 0.078). A moderate to strong HIV-1 specific CD8 T-cell response was detected in seven out of eight subjects after the third interruption (subjects 4, 12, 72, 107, 169, 170, and 211; in patient 226 it was not measured;Fig. 3). CTL lytic activity measured with the 51Cr release assay was detected only at time points with strong CD8 T-cell responses in the ELISPOT (data not shown). HIV-1 specific CD8 T-cell responses were maintained while off therapy in subjects having spontaneous decreases in viral load. CD4 T-cell responses specific to p24 were intermittently positive in these subjects (Fig. 3). None of the 20 patients in the untreated control group had detectable CD4 T-cell proliferative responses during the year of follow-up. HIV-1 specific CD8 T-cell responses were moderate in one out of the seven patients in the untreated control group in whom CD8 T-cells were measured.
Relationship between dynamics of viral load changes and changes in HIV-1 specific cellular immune responses during the three cycles of therapy interruption
Significant quantitative attenuation in plasma viral load rebound was detected. Doubling times increased significantly between the first and third interruption (Wilcoxon matched pairs t test, P = 0.008;Table 1), and highest peak in viral load after each rebound was significantly lower in the third versus the second interruption (Wilcoxon matched pairs t test, P < 0.05). Cycles of STI also resulted in spontaneous decreases in viral load set-point in four out of nine subjects at the second interruption and six out of nine subjects at the third interruption. Moreover, four of these six subjects remained with a plasma viral load < 5000 copies/ml after a median of 12 months off therapy.
At the second and third stops, the increase in the magnitude of HIV-1 specific cellular immune responses from the average level before discontinuation of therapy was significantly greater in subjects having a spontaneous decrease in plasma virus load set-point (subjects 4, 72, 107, and 226 at second stop and subjects 4, 72, 107, 170, 211, and 226, at third stop) versus those having no change or increase (rank sum t test;P = 0.02 for CTL responses and P = 0.0001 for CD4 T-cell responses;Fig. 5). In addition, the average magnitude of the cellular immune response detected over the period of interruption was inversely correlated with the point of largest drop in viral load after peak viremia relative to baseline viral load at the second and third stops (Spearman rank order correlation;r, −0.60, P < 0.02 for total HIV-1 specific CD8 T cells and r, −0.81, P < 0.01 for p24-specific CD4 T-cell response).
Safety and immune system changes
Known mutations associated with resistance to reverse transcriptase or protease inhibitors or evidence of phenotypic resistance were not detected after the first, second or third stop (data not shown). Moreover, after the first and second stops, viral load dropped below 20 copies/ml in all patients within 1 month of reintroduction of the same antiretroviral regimen, and it remained below 20 copies/ml after 6 months on treatment after the first and second stop. Patients 12, 157, 170 and 211 reinitiated therapy after the third stop and all of them maintained a viral load below 20 copies/ml after 6 months on therapy.
Decreases in CD4 T-cell count were observed immediately after each stop. CD4 T-cell counts dropped from a mean (SE) of 934 × 106 (106 × 106) to 672 × 106 (59 × 106) cells/l during the first stop, 1039 × 106 (112 × 106) to 678 × 106 (78 × 106) cells/l during the second stop, and 989 × 106 (100 × 106) to 754 × 106 (80 × 106) cells/l (P = 0.05) during the third stop. However, the value after 12 months off therapy was significantly higher than the level before starting any antiretroviral therapy [754 × 106 (78 × 106) cells/l at month 12 off therapy versus 609 × 106 (51 × 106) cells/l before starting therapy;P = 0.004]. Proliferative responses to mitogens also showed a decrease during the treatment interruptions [reduction of SI for phytohemagglutinin from 34 (SE, 8) to 17 (9), P = 0.06 during the first stop; from 44 (6) to 24 (6), P = 0.008 during the second stop; and from 40 (5) to 23 (4) during the third stop, P = 0.03]. Reintroduction of the same treatment led to complete recovery of these parameters by 6 months after the first and second stop (data not shown).
Data show that the HIV-1 specific cellular immune responses were augmented following cycles of therapy interruption in these HIV-1 chronically infected subjects. Subjects generating HIV-1 specific cellular immune responses at the second stop maintained these at the third stop (Fig. 3a). Subjects who did not have detectable HIV-1 specific cellular immune responses at the first or second stops developed these responses by the third stop (Fig. 3b and c). Our data corroborates recent findings showing that some chronically infected patients can mount brisk HIV-1 specific CD4 and CD8 T-cell responses with therapy interruptions [24,25,30,31]. We show that three cycles of therapy interruption were successful at boosting anti-viral cellular responses in subjects who were not capable of generating these responses after two cycles of STI (Fig. 3). However, given the low number of subjects these results should be confirmed by further testing in a larger setting.
These findings are supported by previous work reporting similar inverse relationships between viral load and the magnitude of the HIV-1 specific cellular immune responses in chronically HIV-1 infected subjects and HIV-1 infected long-term non-progressing individuals [14,15,32]. We conclude that boosted HIV-1 specific immune responses may attenuate viral load rebound and decrease viral load set-point in chronically infected subjects undergoing cycles of STI.
There are several important safety issues with STI protocols. One potential concern is the development of drug resistance [8,33]. Known mutations associated with resistance to reverse transcriptase or protease inhibitors or evidence of phenotypic resistance were not detected after each stop. Another concern was the possibility that viremia would not be re-suppressed upon re-initiation of therapy. After the first and second stops, viral load dropped below 20 copies/ml in all patients within 1 month of reintroduction of the same antiretroviral regimen, and it remained below 20 copies/ml after 6 months on treatment. These findings are in agreement with previous observations in human studies of antiretroviral therapy interruption [21,21,34]. In addition, structured therapy interruption might allow for the replenishment of viral reservoirs during rebound viremia . However, the slow decay rate of the latent reservoir would require a clinically challenging 10–60 years of full adherence to treatment before eradication of HIV-1 infection . Given the potential adverse side effects of HAART, the rigorous dosing schedule and evidence of low-level viral replication despite adherence to maximally suppressive HAART, eradication of the latent reservoir with the currently available medications is not a realistic clinical possibility [4,36]. Drug intensification combined with new immunotherapies may provide an alternative strategy for eradicating or controlling HIV-1 infection.
The most serious safety concern of STI identified in this study is the significant drop in total CD4 T-cell count observed in almost all subjects after rebound in viral load. However, these patients remained with a significantly higher level (a mean of 145 × 106 cells/l) after 12 months off therapy than before starting therapy.
The pattern of viral replication after therapy interruption in these subjects seems to differ from that of primarily infected HIV-1 patients who discontinue drugs [16,19]. Some primarily infected HIV-1 subjects lack viral load rebound > 50 copies/ml after drug cessation or after the first cycle of STI . In this study, all subjects experienced viral load rebound. Assuming that HIV-1 specific immune responses are predominantly responsible for containment of viral replication, the differences between viral load rebound in primarily HIV-1 infected or chronically HIV-1 infected subjects after STI may be explained by the degree of immunocompetence, viral sequence diversity, and the different patterns of HIV-1 specific immunity after the initiation of HAART. Chronically HIV-1 infected subjects are considerably more immunosupressed than primarily HIV-1 infected subjects [14,23,37–39] and also harbor more diverse viral sequences requiring broader immune responses for viral control. HAART initiated during primary HIV-1 infection may better preserve HIV-1 specific T-helper cell functions and CD8 T-cell responses [14,37,40], which could maintain suppression of plasma viremia < 50 copies/ml after STI in primarily HIV-1 infected subjects [16,19,41].
These data may provide support for the concept of inducing an HIV-1 specific immune response by cycles of structured therapy interruptions in chronically HIV-1 infected patients. This approach appears to be relatively safe, in terms of a quick virological response after reintroduction of the same treatment and recovery of immune parameters after 6 months of reintroducing therapy. However, STI in chronically infected patients is associated with decreases in total CD4 T-cell counts immediately after the interruption of therapy. Clinical trials of STI in advanced HIV-1 infection should proceed with caution until the impact and long-term safety of this approach has been investigated further.
The authors thank the participants of the study; and L. Perrin for help with the ultrasensitive viral load measurements.
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Keywords:© 2001 Lippincott Williams & Wilkins, Inc.
HIV; structured treatment interruptions; dynamics; specific immunity; viral load