Intensification of successful antiretroviral therapy (ART) with one or more drugs has been proposed as a potentially useful strategy to help achieve the eradication or a functional cure of HIV infection. The rationale for such intervention is based on the assumption that HIV replication persists in patients receiving effective ART [1–6].
Indeed, a number of reports point in this direction, including the evidence provided by treatment intensification clinical trials , suggesting the gut-associated lymphoid tissue (GALT) as the site wherein HIV replication is more likely to persist . As different intensification trials have failed to demonstrate a reduction in residual HIV found in plasma using ultrasensitive methods [5,8–12], other authors have argued against persistent replication of HIV and the utility of treatment intensification [13–15].
We and others have shown that intensification of antiretroviral treatment with different drug classes (reverse transcriptase inhibitors, CCR5 antagonists, integrase inhibitors) can lead to a reduction in the size of the latent cellular reservoir, as measured by the coculture assay, with no effect on residual viremia [16–18]. The most plausible mechanism for these findings would be the inhibition of ongoing viral replication in sites with suboptimal efficacy of the regimens, possibly due to subinhibitory concentrations of some of the drugs as suggested by other studies [3,4]. It is unknown, however, whether the effect achieved by treatment intensification persists after discontinuation of the intensifying drug. This could be important in order to better understand the mechanisms underlying the persistence of the HIV-1 cellular latent reservoir as well as for practical purposes.
To provide additional insight into the effects and dynamics of the cellular viral reservoir after treatment intensification, we amended two ongoing intensification clinical trials. The intensifying drugs used during these studies were discontinued to evaluate, after 24 weeks, whether the cellular reservoir was replenished or, alternatively, the reduction observed after intensification persisted. Together with the size of the HIV-1 cellular reservoir, we evaluated other markers of viral replication including the changes in the HIV-1 episomal DNA with 2-long terminal repeat (2-LTR) and markers of immune activation.
Materials and methods
We previously conducted two pilot open-label phase II clinical trials that evaluated the effect of two intensifying drugs [maraviroc (MVC) or raltegravir (RAL)] on the cellular HIV-1 reservoir in patients receiving ART [17,18]. The present study analyses the global effect after discontinuation of the intensifying drug (MVC or RAL). Both protocols were amended to include a measurement after 24 weeks of drug discontinuation (Fig. 1).
Both clinical trials (NCT00795444 and NCT00807443) were conducted at the Hospital Universitario Ramón y Cajal in Madrid, Spain, between 2008 and 2011 with an intensification period of 48 weeks, and a follow-up of 24 weeks after drug discontinuation. MVC was developed and provided by Pfizer, Inc, (New York, New York, USA) and RAL by Merck Sharp and Dome, (Whitehouse Station, New Jersey, USA).
The two clinical trials were conformed according to the principles of the Declaration of Helsinki and the Good Clinical Practice Guidelines and were approved by the AEMPS (Spanish Agency for Medications and Health Products) and by the local Ethics Committee. All patients gave their written informed consent to participate in both trials once amended.
Patients and specimen collection
Sixteen patients (seven from MVC trial and nine from RAL trial) completed the intensification phase of the study; however, one individual from RAL trial declined to participate in the follow-up after drug discontinuation, so finally 15 patients completed the follow-up period of 24 weeks. The inclusion criteria were identical for the two clinical trials and included undetectable plasma viral load (pVL) by standard commercial assays (<40 copies HIV-1 RNA/ml) for at least 2 years; ART with three or more drugs for at least 2 years; CD4+ T-cell count > 350 cells/μl; R5 viral tropism using a phenotypic assay (Trofile; Monogram Biosciences, San Francisco, California, USA) in a pretreatment sample in the case of the MVC trial, and no previous treatment with any of the intensifying drugs (MVC or RAL). Patients were recruited from two hospitals (Hospital Ramón y Cajal and Hospital Doce de Octubre), in Madrid, Spain.
Blood samples were collected at baseline, after 48 weeks of intensification and after 12 and 24 weeks of drug discontinuation, apart from sampling for other determinations (data not shown). A total of 300 ml of heparinized whole blood was drawn to quantify the latent HIV-1 reservoir. Fifty millilitres of whole blood with EDTA were also drawn to isolate plasma and peripheral blood mononuclear cells (PBMCs).
HIV-1 cellular reservoir quantification: detection of cells carrying replication-competent HIV-1 virus
The detection of cells carrying replication-competent virus was determined using a previously described enhanced culture assay of highly enriched resting CD4+ T cells that constitute the principal reservoir for the virus [19,20] with some modifications [17,18].
Briefly, PBMCs were isolated by Ficoll density gradient centrifugation (Lymphocytes Isolation Solution; Rafer, S.L, Zaragoza, Spain) from 300 ml of heparinized whole blood. Resting CD4+ T cells were isolated from these total PBMCs by a negative selection of CD3+/CD4+/HLA-DR−/CD25− cells using magnetic beads according to the manufacturer's recommendation (Miltenye Biotec, S.L. Bergisch Gladbach, Germany). The isolated resting cells (as minimum 63 × 106) yielded a purity greater than 99%.
Cells were plated in a duplicate five-fold serial dilution cultures from 25 × 106 to 320 cells. Ten-fold of allogenic irradiated PBMC from healthy donors was added to each culture with phytohaematoglutinin (PHA, 1 μg/ml) and recombinant interleukin 2 (IL-2, 100 U/ml) for efficient cell activation.
The maximum number of resting cells that were put into one well of a six-well plate was 106 in a final volume of 8 ml.. Two individual wells of a six-well plate were labelled as 106 dilution plate. The 5 × 106 dilution was split into five wells with 1 × 106 cells/well and treated as a group, whereas the 25 × 106 dilution was split into 25 wells with 1 × 106 cells/well and again treated as a group. Cell concentrations below 1 × 106 were cultured in 24-well tissue culture plates with the appropriate number of resting cells, that is 2 × 105, 4 × 104, 8 × 103, 1.6 × 103 and 320 in a final volume of 2 ml.
Plates were placed at 37°C in a humidified 5% CO2 incubator. On day 2, supernatants containing PHA were removed from the wells and replaced with fresh culture media (without PHA). Then, CD8+ T-cell depleted PBMCs from healthy donors, prepared 2–3 days before in the presence of PHA (1 μg/ml), were added once a week to each culture. Depletion of CD8+ T cells was performed using a positive selection method (CD8 positive selection; Miltenyie Biotec). These cells were obtained from different healthy donors each week and used fresh to avoid possible cell viability problems due to cryopreservation. Also, these cells were tested for fully permisivity for viral infection using a mixture of NL4-3 and IIIB HIV-1 strains. A total of 6 × 106 cells were added to each well of a six-well plate and 1 × 106 cells to each well of a 24-well plate.
On days 15 and 21, culture supernatants were tested for the presence of HIV-1 antigen using an HIV-1 p24 antigen assay kit (Innogenetics Diagnostica Iberia, S.L. Tarragona, Barcelona, Spain). Infected cell frequencies were determined using the maximum likelihood method and expressed as infectious units per million (IUPM) of resting CD4+ T cells, with a limit of detection of 0.023 IUPM .
HIV-1 episomal DNA 2-long terminal repeat circles determination
The presence of 2-LTR circles was detected by a qualitative PCR, as described previously [17,18]. To maximize the recovery of 2-LTR circles and to overcome the lack of sensitivity of this technique [1,22,23], enriched 2-LTR circles were extracted selectively from approximately 5 million PBMCs using QIAprep Spin Miniprep (Qiagen, Valencia, California, USA) following the manufacturer's protocol for low copy number plasmids .
Immune activation and lymphocyte subsets
Fresh EDTA anticoagulated whole blood was used to analyse CD4+ and CD8+ T-cell activation using the following antibody combination: CD3-allophycocyanin (APC)-Cy7, CD4-peridinin chlorophyll protein complex (PerCP), CD8-phycoerythrin (PE)-Cy7, CD38-phycoerythrin (PE) and HLA-DR-APC, as previously described . T-cell activation was characterized by HLADR+CD38+ expression. Antibodies were from Becton Dickinson (Becton Dickinson, Franklin Lakes, New Jersey, USA), and an unstained control was performed for all samples. At least 105 CD3+ T cells were isolated for each sample and analysed with Kaluza software (Beckman-Coulter, Brea, California, USA) initially gating lymphocytes according to morphological parameters. Gating was similar between different time points.
Continuous variables were expressed as median and interquartile range (IQR) and discrete variables as percentages. The t-test for independent samples was used to compare normally distributed continuous variables and the Mann–Whitney test to compare nonnormally distributed continuous variables. Categorical variables were described as proportions. The association between categorical variables was evaluated using the chi-square test. The Spearman correlation coefficient was used to analyse the correlations between continuous variables. Statistical analysis was performed using IBM SPSS software 16.0 (IBM, Armonk, New York, USA).
Baseline characteristics of patients are summarized in Table 1. Median age was 46 years (IQR 41–50) and no significant differences were found between CD4+ and CD8+ T-cell counts at baseline [676 cells/μl (IQR 522–828) and 678 cells/μl (IQR 567–1066), respectively, P = 0.7] and at the end of 24-week follow-up [642 cells/μl (IQR 552–722) and 647 cells/μl (IQR 439–886), respectively, P = 0.1].
All were receiving nucleoside reverse transcriptase inhibitor (NRTI)-containing regimens combined with nonnucleoside reverse transcriptase inhibitors (NNRTIs) in seven cases (46.6%), with protease inhibitors in six cases (40%), and with one-third NRTI in two cases (13.3%). During follow-up, no changes were made in previous ART regimens. All individuals remained virally suppressed during the period of the study.
Effect of discontinuation of the intensifying drug on the HIV-1-cellular latent reservoir
The median size of the HIV-1-cellular latent reservoir at baseline including the 15 patients from the two clinical trials was 1.1 IUPM (IQR 0.023–3.2). At the end of the 48-week intensification period, the number of latently HIV-1 infected memory CD4+ T cells decreased significantly, with a median of 0.0 IUPM (IQR 0.0–0.27) (P = 0.004). This effect in reducing the size of the HIV-1 latent reservoir was maintained at the end of the 24-week period after drug discontinuation, with a median of 0.0 IUPM (IQR 0.0–0.0), with no statistical differences with respect to the 48-week intensification period study point (P = 0.9), but significantly lower than at baseline (P = 0.008) (Fig. 2).
Effect of discontinuation of the intensifying drug on episomal 2-long terminal repeat DNA circles
All individuals showed undetectable episomal 2-LTR DNA circles at baseline and after the 48-week intensification period. As described, a transient increase was observed during intensification in patients in the MVC group at week 12 and 24 (P = 0.037 and P = 0.012, respectively, compared with baseline). After discontinuation, 2-LTR circles remained undetectable in all patients except two, without reaching a statistical significance compared with baseline and to the end of the 48-week intensification period (P = 0.1)
Effect of discontinuation of the intensifying drug on T-cell counts and T-cell activation
Globally, no differences were observed in the total CD4+ and CD8+ T-cell counts either after the 48 weeks of intensification or after the 24 weeks of discontinuation with respect to baseline.
We have previously reported a trend to decreased immune activation after intensification with both MVC and RAL. This effect was maintained at week 24 after discontinuation, with a significant decrease in CD4+and CD8+ T-cell activation compared with baseline (P = 0.003 and P = 0.042, respectively), and a significant decrease in CD4+ T-cell activation compared with week 48 after intensification (P = 0.020) (Fig. 3a).
Then, we analysed the differences in immune activation according to the intensifying drug. Although no significant decrease in CD4+ T-cell activation was observed at week 48 after intensification in the MVC group, these levels were significantly lower at week 24 after discontinuation than at baseline (P = 0.028). The effect of intensification with MVC in reducing CD8+ T-cell activation was maintained at week 12 and 24 after discontinuation compared with baseline (P = 0.028 and 0.028, respectively) (Fig. 3b). In the RAL group, no significant differences were observed in immune activation between baseline and week 48 after intensification, but only the levels of CD8+ T-cell activation were significantly lower at week 24 after discontinuation than at baseline (P = 0.042) (Fig. 3c).
To date, the pool of latently infected resting CD4+ T cells has been the most profoundly analysed HIV-1 reservoir and is widely recognized as one of the major barriers to achieving eradication or a functional cure of HIV-1 [24–27]. In recent years, the absence of consensus on the causes of the stability of the latent viral reservoir originated a storm of controversy in relation to the possibility that residual HIV replication in subsets of CD4+ T cells in the lymphoid tissue may contribute to replenishment of HIV-1 reservoir [8,13,15,28,29]. More recently, different groups have drawn attention to the GALT as a major HIV-1 reservoir in individuals receiving ART, as CD4+ T-cell recovery is poorer in GALT and viral replication remains higher with respect to peripheral blood. This persistent viral replication in GALT probably contributes to maintenance of the reservoir despite peripheral viral suppression [7,8,30–32]. Therefore, intensification of conventional ART could further reduce the low-level ongoing viral replication and, subsequently, the reservoir replenishment.
Herein, we provide the first evidence that ART intensification can generate a significant and stable reduction of latently infected resting CD4+ T cells that is maintained after discontinuation of the intensifying drug. Of note, in this pooled analysis of two ART intensification clinical trials of successful ART, participants showed a small size of the HIV-1 reservoir (median IUPM = 0.8 IUPM) at baseline, and low levels of CD4+ and CD8+ T-cell activation. Thus, this population had an optimal immunovirological status before the intervention, probably driven by the long-standing virological suppression [median 62 months (IQR 41–109)]. Even so, ART intensification was effective in reducing the size of the HIV-1 reservoir after 48 weeks, and strikingly, this effect was maintained after 24 weeks of discontinuation of the intensifying drug, with IUPM below the threshold of detection, and with undetectable 2-LTRs circles in the majority of patients. It could be argued that an increase in adherence could drive most of the impact of ART intensification on the size of the reservoir . In our opinion, this seems unlikely based in two facts. First, in addition to long-standing virological suppression with no clinical rebound at any time, pharmacy records of adherence were excellent for patients included in the two studies of intensification. Only one patient had bad adherence during the MVC intensification clinical trials due to serious personal problems and he developed virological failure. Secondly, there was a clear relationship between the intensification of ART and the decrease of the reservoir.
Along with this efficacy in reducing the pool of latently infected resting CD4+ T cells, a significant reduction in immune activation was also observed at the end of the follow-up, suggesting at least an indirect relation between CD4+ and CD8+ T -cell activation and the persistence of HIV-1 in the cellular reservoir.
So far, only three studies, including our clinical trials with MVC and RAL, have evaluated the effect of intensification strategies on the HIV-1-cellular latent reservoir in patients with virological suppression under ART by using the coculture method [16–18], which is currently considered the gold-standard technique to identify latently infected resting CD4+ T cells. Importantly, all three studies revealed a decay of the reservoir. The fact that all three experimental strategies used drugs with different mechanisms of action (abacavir with/without efavirenz, MVC and RAL) imply that the reduction of the reservoir might be a consequence of a shrinkage in the viral replication in the anatomical reservoirs – such as the GALT –  rather than be driven by a specific drug effect of abacavir, MVC or RAL on the cellular reservoir. Thus, the rationale for ART intensification strategies would be an increase in drug concentrations up to inhibitory levels in sites with suboptimal efficacy of conventional ART regimens, as suggested previously [1–6,34].
In the light of these data, an important question remained to be answered. The proof that intensification strategies are effective in reducing the HIV-1 reservoir by inhibition of residual viral replication would be a decrease in IUPM after intensification and a subsequent rebound of IUPM and T-cell activation after discontinuation of the intensifying drug, due to an increase of the low-level viral replication and a replenishment of the HIV-1 reservoir [1–6,9–11,23]. This study was designed to provide new insight into this question. Although we found a decrease in IUPM and T-cell activation after intensification with two different drugs, the reservoir persisted undetectable and percentages of T-activated cells remained significantly lower after discontinuation of MVC/RAL with respect to baseline, contrarily to what we had hypothesized and expected. These surprising results deserve some considerations. The straightforward explanation is that replenishment of the HIV-1 reservoir might take more than 24 weeks after interruption of an intensification strategy. Against this reasoning, most immunovirological events such as plasma HIV-1 load rebound or CD4 cell count decline occur within the first weeks after ART interruptions [6,35]. Alternatively, intensification with MVC/RAL could have suppressed residual HIV-1 replication in tissues to an extent that may allow the immune response to exert some sort of control and prevent replenishment of the HIV-1 reservoir. The maintenance of low T-cell activation after MVC/RAL interruption supports this hypothesis. As IUPM and T-cell activation declined in parallel after intensification, an increase in T-cell activation after treatment discontinuation would have suggested an increase in tissue HIV-1 load. However, both IUPM and T-cell activation remained at low levels after intensification interruption.
The findings regarding 2-LTRs circles also merit some considerations. These circles are episomal forms of nonintegrated HIV DNA with a relatively short half-life. Therefore, detection of 2-LTRs circles is generally considered a marker of recent infection [1,22,23,36] and do not necessarily reflect the size of the HIV-1 cellular latent reservoir . As 2-LTRs were undetectable at baseline, after intensification and at the end of follow-up, the findings on 2-LTRs circles cannot lead us to any specific conclusion, other than they may indirectly reflect the absence of residual viremia in peripheral blood .
Our study is subject to a series of limitations. First, the small sample size demands a cautious interpretation; although our results were consistent across all comparisons and reached statistical significance, they should be reproduced in larger clinical trials. Second, the coculture assay is a technique of high sensitivity; however, an improvement of the threshold of detection might have enabled us to detect differences in the reservoir size between the determinations after intensification and at the end of drug discontinuation. Third, although we estimated that 24 weeks would be enough to detect a rebound of IUPM, this period might be insufficient to assess this outcome and future studies in this direction should be designed with longer periods of follow-up. The lack of a control group also limits the strength of the conclusions. However, the intensive laboratory work and high costs associated together with the fact that a spontaneous decrease of the reservoir was very unlikely made us decide not to include the control group. As stated in a previous report, the probability that the results be associated with hazard is really low (P < 0.017). Even so, the comparison with a control group would have led to more definitive conclusions.
In summary, our data provide new evidence that the effects of ART intensification with MVC or RAL in reducing the HIV-1 cellular latent reservoir persist at least 24 weeks after discontinuation of the drug. We believe that despite the great controversy on this field [1–6,9–12,23,38,39], this study shows that ART intensification strategies can alter the dynamics of the HIV-1 reservoir  and reduce its size to undetectable levels in selected patients. Thus, ART intensification strategies should be considered in future strategies – including the use of antilatency agents [40,41] – to achieve a functional cure or HIV eradication.
We would like to thank Carmen Page, Raquel Lorente, Ester Domínguez and María Coronel for the excellent technical assistance; the Radiophysics Departments at the Hospitals Ramón y Cajal and Gregorio Marañón for their help in the irradiation of cells, and the Regional Blood Transfusion Center in Madrid for providing the buffy coats from healthy donors. Specially, we thank all of our patients and their families for their participation in the study.
C.G., B.H., A.V., S.S., L.D., M.A.M.-F. and S.M. conceived and design the study and the experiments. A.M., F.D., S.S. and S.M. participated on the inclusion and follow up of the patients, and analysed clinical data. C.G., B.H., A.V., L.D., M.A. and N.M. performed all the experiments. C.G., B.H., A.V., S.S., L.D., M.A.M.-F., M.A., N.M., J.Z. and S.M. analysed all data. C.G., B.H., A.V., S.S. and S.M. contributed to the writing of the article. C.G., B.H., A.V., S.S., L.D., M.A., N.M., J.Z., A.M., F.D., M.A.M.-F. and S.M. approved the final version of the manuscript.
This work was supported in part by the Spanish AIDS Network ‘Red Temática Cooperativa de Investigación en SIDA (RD06/0006)’, the Spanish National of Health, ‘Instituto de Salud Carlos III’, grants (FIS-PI080958 and CP08/00046), and by the Foundation of Investigation and prevention of AIDS (FIPSE-36-0844/09).
Conflicts of interest
We declare that we have no conflicts of interest.
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