Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Intensification of Antiretroviral Therapy with a CCR5 Antagonist in Patients with Chronic HIV-1 Infection: Effect on T Cells Latently Infected

  • Carolina Gutiérrez ,

    Contributed equally to this work with: Carolina Gutiérrez, Laura Díaz

    Affiliation Infectious Diseases Department, Hospital Universitario Ramón y Cajal, and IRYCIS, Madrid, Spain

  • Laura Díaz ,

    Contributed equally to this work with: Carolina Gutiérrez, Laura Díaz

    Affiliation Inmunobiology Laboratory, Hospital General Universitario Gregorio Marañón, Madrid, Spain

  • Alejandro Vallejo,

    Affiliation Infectious Diseases Department, Hospital Universitario Ramón y Cajal, and IRYCIS, Madrid, Spain

  • Beatriz Hernández-Novoa,

    Affiliation Infectious Diseases Department, Hospital Universitario Ramón y Cajal, and IRYCIS, Madrid, Spain

  • María Abad,

    Affiliation Infectious Diseases Department, Hospital Universitario Ramón y Cajal, and IRYCIS, Madrid, Spain

  • Nadia Madrid,

    Affiliation Infectious Diseases Department, Hospital Universitario Ramón y Cajal, and IRYCIS, Madrid, Spain

  • Viktor Dahl,

    Affiliation Karolinska Institute, Stockholm, Sweden

  • Rafael Rubio,

    Affiliation Infectious Diseases Unit, Hospital General Universitario Doce de Octubre, Madrid, Spain

  • Ana M. Moreno,

    Affiliation Infectious Diseases Department, Hospital Universitario Ramón y Cajal, and IRYCIS, Madrid, Spain

  • Fernando Dronda,

    Affiliation Infectious Diseases Department, Hospital Universitario Ramón y Cajal, and IRYCIS, Madrid, Spain

  • José Luis Casado,

    Affiliation Infectious Diseases Department, Hospital Universitario Ramón y Cajal, and IRYCIS, Madrid, Spain

  • Enrique Navas,

    Affiliation Infectious Diseases Department, Hospital Universitario Ramón y Cajal, and IRYCIS, Madrid, Spain

  • María Jesús Pérez-Elías,

    Affiliation Infectious Diseases Department, Hospital Universitario Ramón y Cajal, and IRYCIS, Madrid, Spain

  • Javier Zamora,

    Affiliation Biostatistics Department, Hospital Universitario Ramón y Cajal, and IRYCIS, Madrid, Spain

  • Sarah Palmer,

    Affiliation Karolinska Institute, Stockholm, Sweden

  • Eduardo Muñoz,

    Affiliation Immunology Department, Universidad de Córdoba, Córdoba, Spain

  • María Ángeles Muñoz-Fernández,

    Affiliation Inmunobiology Laboratory, Hospital General Universitario Gregorio Marañón, Madrid, Spain

  •  [ ... ],
  • Santiago Moreno

    smoreno.hrc@salud.madrid.org

    Affiliation Infectious Diseases Department, Hospital Universitario Ramón y Cajal, and IRYCIS, Madrid, Spain

  • [ view all ]
  • [ view less ]

Intensification of Antiretroviral Therapy with a CCR5 Antagonist in Patients with Chronic HIV-1 Infection: Effect on T Cells Latently Infected

  • Carolina Gutiérrez, 
  • Laura Díaz, 
  • Alejandro Vallejo, 
  • Beatriz Hernández-Novoa, 
  • María Abad, 
  • Nadia Madrid, 
  • Viktor Dahl, 
  • Rafael Rubio, 
  • Ana M. Moreno, 
  • Fernando Dronda
PLOS
x

Abstract

Objective

The primary objective was to assess the effect of MVC intensification on latently infected CD4+ T cells in chronically HIV-1-infected patients receiving antiretroviral therapy.

Methods

We performed an open-label pilot phase II clinical trial involving chronically HIV-1-infected patients receiving stable antiretroviral therapy whose regimen was intensified with 48 weeks of maraviroc therapy. We analyzed the latent reservoir, the residual viremia and episomal 2LTR DNA to examine the relationship between these measures and the HIV-1 latent reservoir, immune activation, lymphocyte subsets (including effector and central memory T cells), and markers associated with bacterial translocation.

Results

Overall a non significant reduction in the size of the latent reservoir was found (p = 0.068). A mean reduction of 1.82 IUPM was observed in 4 patients with detectable latent reservoir at baseline after 48 weeks of intensification. No effect on plasma residual viremia was observed. Unexpectedly, all the patients had detectable 2LTR DNA circles at week 24, while none of them showed those circles at the end of the study. No changes were detected in CD4+ or CD8+ counts, although a significant decrease was found in the proportion of HLA-DR+/CD38+ CD4+ and CD8+ T-cells. LPS and sCD14 levels increased.

Conclusions

Intensification with MVC was associated with a trend to a decrease in the size of the latent HIV-1 reservoir in memory T cells. No impact on residual viremia was detected. Additional studies with larger samples are needed to confirm the results.

Trial Registration

ClinicalTrials.gov NCT00795444

Introduction

Antiretroviral therapy (ART) can reduce plasma HIV-1 RNA levels to <50 copies/ml [1]. However, low residual viremia, which is only detectable using ultrasensitive assays, can persist despite ART [2][4]. The origin and clinical implications of persistent low-level viremia are uncertain. While some studies postulate that it may be the result of virus released from latently infected cells [5][7], others support that it could arise from ongoing viral replication, as a consequence of incomplete inhibitory activity or penetration of antiretroviral drugs [8][13].

Although intensification of current ART with potent drugs could potentially decrease residual viremia and prevent replenishment of viral reservoirs, prior intensification studies have not demonstrated any impact on residual viremia [12], [14][17]. Only one study confirmed a transient increase in episomal 2LTR DNA circles after raltegravir intensification [18] as an indicator of recent HIV-1 replication [19][21], and another study showed a decrease in the frequency of episodes of intermittent viremia [22]. To our knowlegede, only a few studies have previously evaluated the effect of intensification of stable therapy on the latent reservoir of resting CD4+ T cells in chronically HIV-1-infected patients with controversial results. Ramratnam et al. found an accelerated decay of the HIV-1 latent reservoir after intensification therapy with abacavir with or without efavirenz [22]. Two more studies that measured the impact on the CD4+ T cell reservoir in patients receiving a four-drug combination as initial antiretroviral therapy provided discordant results, namely, a reduction in the reservoir in patients treated during acute infection [23] and no changes in chronically infected patients [24].

Bacterial translocation and a low level of ongoing viral replication have been associated with an increase in immune activation in HIV-1-infected patients [25], [26]. Evidence of increased bacterial translocation from damaged intestinal tissue was recently demonstrated in chronic HIV-1 infection [25], [27]. In acute HIV-1 infection, intestinal CD4+ T cells are rapidly depleted and T-cell activation is high. Since monitoring intestinal cells is difficult, expression of surrogate markers such as the mucosal homing receptor α4β7 integrin on circulating T cells has been proposed to correlate with loss or restoration of intestinal CD4+ T cells and could prove helpful in monitoring the success of therapeutic strategies [28][30]. Besides, recent studies have shown that HIV-1 utilizes α4β7 integrin to bind to CD4+ T cells [31].

Maraviroc (MVC) is a potent new antiretroviral agent approved for the treatment of HIV-1 infection that blocks interaction between the virus and the CCR5 co-receptor, a crucial step in the HIV-1 life cycle [32]. Previous clinical trials have demonstrated the safety, tolerability, and efficacy of MVC in both treatment-naive and treatment-experienced patients [33], [34].

We performed a prospective open-label pilot phase II clinical trial to assess the effect of MVC intensification on latently infected CD4+ T cells in chronically HIV-1-infected patients receiving antiretroviral therapy. We also analyzed residual viremia and episomal 2LTR DNA to examine the relationship between these measures and the HIV-1 latent reservoir, immune activation, lymphocyte subsets (including effector and central memory T cells), and markers associated with bacterial translocation. The hypothesis was that if these factors are causally linked, altering one of them with the intervention should result in alteration of the others.

Methods

The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1.

Study Design

We performed an open-label pilot phase II clinical trial to evaluate the effect of MVC on the cellular HIV-1 reservoir in patients receiving ART. The study was conducted at Hospital Universitario Ramón y Cajal in Madrid, Spain between 2008 and 2010. This independent clinical trial (NCT00795444) had a follow up of 48 weeks of intensification with MVC (developed and provided by Pfizer, Inc.).

Ethics Statement

The study was carried out according to the recommendations of the Declaration of Helsinki and current Spanish legislation on clinical trials. It was approved by the AEMPS (Spanish Agency for Medications and Health Products) and our Independent Ethics Committee (Hospital Ramón y Cajal, 28034 Madrid, Spain; ceic.hrc@salud.madrid.org). All patients provided their written informed consent for participation, which included sample collection and laboratory determinations.

Patients and specimen collection

Nine patients met all the following inclusion criteria: undetectable plasma viral load (pVL) by standard commercial assays (<50 copies HIV-1 RNA/ml) for at least two years; ART with three or more drugs for at least two years; CD4+ T lymphocyte count > 350 cells/mm3; R5 viral tropism testing a pretreatment sample using a phenotypic assay (Trofile®, Monogram Biosciences, C.A), and no previous treatment with MVC. Patients were recruited from two hospitals (Hospital Ramón y Cajal and Hospital Doce de Octubre), both in Madrid, Spain.

Patient's enrollment, allocation and follow up are described in figure 1.

Samples were collected at baseline and after 12, 24, 36, and 48 weeks of intensification with MVC. A total of 300 ml of heparinized whole blood was drawn to quantify the latent HIV-1 reservoir; 50 ml of whole blood with EDTA was drawn to obtain plasma and isolate peripheral blood mononuclear cells (PBMC). Samples were cryopreserved in the HIV Biobank of the Spanish AIDS Research Network (RIS) following current procedures [35].

In addition, two control groups (10 treatment-naive HIV-1-infected patients, and 10 HIV-1-negative subjects) were also analyzed for some measurements.

Latently HIV-1-infected memory T cells

Two baseline determinations (three months apart) were performed to investigate the accuracy of the method and the stability of the reservoir.

We carried out a previously described co-culture assay [36], [37]. Briefly, PBMC were isolated from 300 ml of heparinized whole blood using Ficoll density gradient centrifugation (Lymphocytes Isolation Solution, Rafer, S.L Zaragoza, Spain). Resting CD4+ T cells (CD4+/HLA-DR-/CD25-) were isolated and purified using magnetic beads according to the manufacturer's recommendations (Miltenyi Biotec, S.L. Bergisch Gladbach, Germany). CD4+ T-cell purity greater than 99% with less than 0.4% activation was confirmed by flow cytometry. The isolated resting CD4+ T cells were plated in replicate limiting dilutions and co-cultured with allogenic irradiated PBMC from seronegative donors and phytohemagglutinin. The activated cell culture was fed once a week with PBMC from healthy donors after depletion of CD8+ T cells. On days 15 and 21, culture supernatants were tested for HIV-1 replication using the HIV-1 p24 antigen assay (Innogenetics Diagnostica Iberia, S.L Tarragona, Barcelona, Spain). Infected cell frequencies were determined by the maximum likelihood method and expressed as infectious units per million (IUPM) resting CD4+ T cells [38]. The millions of purified resting CD4+ T cells obtained from the total PBMCs conditioned the number of replicates of each dilution. The assay was performed to each point as duplicate fivefold dilution series.

From baseline to week 12 of follow-up, the limit of detection of the assay was 0.12 IUPM resting CD4+ T cells. Thereafter, the limit of detection decreased to 0.023 IUPM, since the total blood extraction volume was increased after approval of the amended protocol by the IEC.

Residual viremia

Residual viremia was measured with internally controlled an ultrasensitive quantitative real-time RT-PCR (single copy assay), as reported elsewhere [39]. The optimal HIV-1 RNA detection threshold was 0.3 copies/ml when 5 to 7 ml of starting plasma was available. We used a median of 6.5 ml (IQR: 4.6 to 7).

HIV-1 episomal 2LTR DNA circles

The presence of HIV-1 episomal 2LTR DNA circles was detected by qualitative PCR. To maximize the recovery of 2LTR DNA circles and overcome the lack of sensitivity of this technique [8], [18], [40] enriched 2LTR episomal DNA circles were extracted selectively from ∼5 million PBMC using QIAprep Spin Miniprep (Qiagen, Valencia, California, USA) following the manufacturer's protocol for low-copy-number plasmids, as previously described [8].

A nested PCR flanking the episomal 2LTR DNA circle junction was designed. In the first round, 5–20 µL of episomal DNA were amplified in a 50-µL reaction with the following primers: forward, 5′-TAAGATGGGTGGCAAGTGGTCA; and reverse, 5′-TCTACTTGTCCATGCATGGCTT.

The second PCR round was performed using 1 to 2 µL of the first reaction product as the template and primers spanning the unique junction formed by ligation of 5′ and 3′ LTR sequences (forward, 5′-AATCTCTAGCAGTACTGGAAG; reverse, 5′-GCGCTTCAGCAAGCCGAGTCCT). PCR products were analyzed on a 1% agarose gel stained with GelRed (Biotium, Hayward, California, USA).

Immune activation and lymphocyte subsets

Fresh EDTA anticoagulated whole blood was used to analyze CD4+ and CD8+ T cell activation with the following antibody combination: CD3-allophycocyanin (APC)-Cy7, CD4-peridinin chlorophyll protein complex (PerCP), CD8-phycoerythrin (PE)-Cy7, CD38-phycoerythrin (PE), and HLA-DR-allophycocyanin (APC). Lymphocyte subpopulations were defined as naïve (CD45RA+CCR7+), T central memory (CD45RA-CCR7+, TCM), T effector memory (CD45RA-CCR7-, TEM), and T effector memory RA+ (CD45RA+CCR7-, TemRA). To analyze lymphocyte subsets this antibody combination was used: CD3-allophycocyanin (APC)-Cy7, CD4-peridinin chlorophyll protein complex (PerCP), CD8-phycoerythrin (PE)-Cy7, CD45RA- phycoerythrin (PE), and CCR7-allophycocyanin (APC), β7-allophycocyanin (APC). Antibodies were from Becton Dickinson (Becton Dickinson, NJ, USA), and an unstained control was performed for all samples. Briefly, 100 µL of blood were lysed with 200 µl of FACS Lysing solution (Becton Dickinson) for 30 min at room temperature, incubated with the antibodies during 20 min at 4°C, washed and resuspended in PBS containing 1% azida. Cells were analyzed in a Gallios flow cytometer (Beckman-Coulter, CA, USA). At least 15000 CD3+ cells were collected for each sample and analyzed with Kaluza software (Beckman-Coulter) initially gating lymphocytes according to morphological parameters. The gating was always the same between different time points.

Bacterial translocation

Two commercial assays were used to evaluate bacterial translocation from plasma samples. Plasma bacterial lipopolysaccharide (LPS) was measured using QCL-1000 Limulus Amebocyte Lysate (Lonza®, Basel, Switzerland) according to the manufacturer's protocol. This test quantifies endotoxins from the cell wall of gram-negative bacteria. Plasma sCD14 was quantified using the Quantikine® Human sCD14 Immunoassay (R&D Systems, Minneapolis, Minnesota, USA), according to the manufacturer's instructions. Samples were run in duplicate in all cases.

Statistical analysis

This is an exploratory pilot study that is trying to prove a concept. A treatment group of 10 patients has been considered adequate, based on data from previous studies published by other authors on the same subject.

Continuous variables were expressed as the median and interquartile range 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 non-normally distributed continuous variables. Categorical variables were described as proportions. The association between categorical variables was evaluated using the chi-square test. A Spearman correlation was used. Statistical analysis was performed using SPSS software 16.0 (Inc., Chicago, Illinois, USA).

Results

Patient characteristics

The baseline characteristics of the patients are summarized in table 1. The median age was 46 years and 8 patients were male. The median baseline CD4+ and CD8+ T-cell counts were 711 and 784 cells/mm3, respectively. All patients were receiving nucleoside reverse transcriptase inhibitor (NRTI)–containing regimens combined with non-nucleoside reverse transcriptase inhibitors (NNRTIs) in two cases (22%), with protease inhibitors (PIs) in five cases (56%), and with a third nucleoside in two cases (22%). The median duration of ART before study entry was 75 months.

MVC was well tolerated. Two patients interrupted the study early. Data from these patients were included only until the study interruption. None of them was included in the final analysis at week 48.One patient, who was coinfected with hepatitis C virus, had a significant increase in liver enzymes after the visit at week 12; the other discontinued all antiretroviral drugs due to poor adherence after the visit at week 12. The remaining patients maintained an HIV viral load <50 copies/ml until the end of the study.

Effect of MVC intensification on latent HIV-1 reservoir

The number of latently HIV-1-infected memory CD4+ T cells decreased after 48 weeks of MVC intensification, although the difference was not significant (p = 0.068) (figure 2). Three patients showed a reservoir below the limit of detection before receiving MVC (figure 2A), and this remained undetectable after 48 weeks despite a transient increase in two patients. The remaining patients, all of whom had a quantifiable reservoir at study entry, showed a decrease in the IUPM after 48 weeks of intensification (figure 2B) or after 12 weeks of intensification (figure 2C, two patients who discontinued the study after week 12). A mean reduction of 1.82 IUPM was found in the 4 patients with detectable latent reservoir at baseline after 48 weeks of intensification with MVC.

thumbnail
Figure 2. Effect of MVC intensification on HIV-1 latently infected memory CD4+ T cells.

(a) Patients who had a latent reservoir below the limit of detection at baseline, and remained undetectable after 48 weeks of intensification. (b) Patients with quantifiable latent reservoir at baseline, and decreased after 48 weeks of intensification, (c) patients with quantifiable latent reservoir at baseline but discontinuated the study after w12. Square symbols: detection limit of 0.12 IUPM. Diamond symbols: detection limit of 0.023 IUPM. Open symbols: under the limit of detection.

http://dx.doi.org/10.1371/journal.pone.0027864.g002

Effect of MVC intensification on residual viremia

The impact on the cellular reservoir was not associated with significant changes in residual viremia. At baseline, 8 of the 9 patients had undetectable residual viremia using the single copy assay. A significant increase in residual viremia was observed after 12 weeks of MVC intensification compared to baseline (p = 0.027). Afterwards, residual viremia decreased progressively until the end of the study, with no significant differences compared to baseline (p = 0.08, table 2).

thumbnail
Table 2. Effect of MVC intensification on residual viremia and episomal DNA circles.

http://dx.doi.org/10.1371/journal.pone.0027864.t002

Effect of MVC intensification on episomal 2LTR DNA circles

At baseline, episomal 2LTRs DNA circles were undetectable in all patients. However, at week 12 they were detected in 5 patients (p = 0.037, compared to baseline) and at week 24 in all 9 patients (p = 0.012, compared to baseline). At week 48, episomal 2LTR DNA circles were again undetectable in all patients (table 2).

Effect of MVC intensification on T-cell activation

The level of activation of CD4+ T cells decreased significantly after 12, 24, and 36 weeks of MVC intensification (p = 0.028, p = 0.027, and p = 0.028, respectively), only to increase after 48 weeks, although the difference with baseline was not significant (p = 0.6) (figure 3). Compared to HIV-negative subjects, CD4+ T-cell activation at baseline was significantly higher (p<0.001), with no significant differences at weeks 12 and 24 (p = 0.950 and p = 0.181, respectively).

thumbnail
Figure 3. Immune activation during MVC intensification.

CD4+ and CD8+ T cell activation of the patients during the follow up is shown. A group of HIV-1 negative individuals are also shown.

http://dx.doi.org/10.1371/journal.pone.0027864.g003

The level of activation of CD8+ T cells also decreased significantly after 12 weeks and until the end of the study (p = 0.043, p = 0.025, p = 0.028, and p = 0.046, at weeks 12, 24, 36, and 48, respectively) (figure 3). The level of cell activation at baseline was higher than in the HIV-negative subjects (p = 0.001). Only at week 48 was this difference not significant compared to the HIV-negative group (p = 0.181).

Effect of MVC intensification on T-cell subsets

No differences in the absolute number or percentage of CD4+ or CD8+ T cells were found during follow-up. A significant increase in CD8+ effector memory and TemRA T cell count was found during the first 24 and 36 weeks after MVC intensification, respectively. Thereafter, the differences compared to baseline were not significant (figure 4). On the other hand, a significant decrease in CD8+ central memory T cells was observed at week 12 (p = 0.002). The level of CD8+ naive T cells was similar during follow-up (p>0.05 at all time points). No significant differences were observed in any CD4+ T cell subsets during follow-up (data not shown).

thumbnail
Figure 4. CD8

+ T cell subpopulations after MVC intensification. The proportion of naïve, central memory (TCM), effector memory (TEM), and effector memory RA+ (TemRA) cells is shown.

http://dx.doi.org/10.1371/journal.pone.0027864.g004

The proportion of CD8+ naïve and memory T cells expressing a β7 receptor significantly increased after 12 weeks of intensification (p = 0.037 and p = 0.007, respectively) (figure 5A and 5B respectively). The expression of β7 receptor also increased in activated CD8+ T cells at week 12 compared to baseline (p = 0.046), and a trend was observed during the rest of the follow up (figure 5C).

thumbnail
Figure 5. Proportion of CD8

+ T cells expressing β7 receptor. A) naïve T cells, B) memory T cells, and C) activated T cells.

http://dx.doi.org/10.1371/journal.pone.0027864.g005

Effect of MVC intensification on bacterial translocation

A significant increase in sCD14 level was observed after 24, 36 and 48 weeks of intensification with MVC compared to baseline (p = 0.015, p = 0.028 and p = 0.028, respectively) (figure 6). These levels were significantly lower than those of naive HIV-1-infected patients at all time points. LPS levels were also higher at weeks 24, 36, and 48 of intensification than at baseline (p = 0.006, p = 0.006, and p = 0.021, respectively) (figure 6). The level of LPS at baseline was significantly lower that that of treatment-naive HIV-1-infected patients; during follow-up, these levels were no different from those of the treatment-naive patients.

thumbnail
Figure 6. Bacterial translocation.

Levels of sCD14 and LPS during the follow up are shown. The levels of a group of HIV-1 positive naïve for ART are also included.

http://dx.doi.org/10.1371/journal.pone.0027864.g006

A significant direct correlation was found between sCD14 levels and LPS levels at baseline (p = 0.035, r = 0.743) while a similar trend was observed during the rest of the follow up. Furthermore, the LPS level correlated directly with both the proportion of CD4+ T cells expressing a β7 receptor and the level of naive CD4+ T cells expressing β7 receptor at week 24 (p = 0.047, r = 0.817). Similarly, the level of LPS at week 36 correlated directly with that of CD8+ effector memory T cells (p = 0.045, r = 0.818).

Discussion

In this pilot study, intensification of treatment with MVC in patients chronically infected with HIV-1 receiving successful long-term ART was associated with a trend to an accelerated decay in the pool of latently infected CD4+ T cells. No impact on residual viremia was observed.

Intensification of successful antiretroviral therapy has been explored in two different ways. Some studies have evaluated its role for clinical purposes, that is, to achieve better virological control or a greater increase in CD4+ T-cell counts, or, more recently, to reduce immune activation. With the exception of one study that showed a decrease in HIV RNA levels after abacavir intensification, none of the studies has been able to show significant improvements with intensified therapy compared to the standard three-drug regimens [11], [12], [14][17], [41]. As a consequence, most clinicians feel that no further research in this direction is necessary. Other studies have evaluated intensification as a strategy to help to eradicate HIV. The rationale for this approach is that suppression of putative replication could reduce residual viremia and replenishment of cellular reservoirs leading to a decrease in the total latent reservoir [8][18], [41]. The results of all the studies are uniform in that no single group has shown a significant reduction in plasma HIV RNA as a result of intensification. This finding could be interpreted as confirmation of the lack of residual viral replication, thus supporting the hypothesis that residual viremia is the result of viral release from stable reservoirs and not the consequence of new rounds of viral replication [5][7]. An alternative hypothesis would be that the effect of intensification takes place mainly outside plasma sites and, therefore, the measurement of plasma viral load only could not detect this effect. Some of the studies that included measurements other than plasma HIV RNA seem to support this hypothesis [42][44].

We observed a reduction in the size of the latent reservoir after intensification of treatment with MVC, although this reduction was not significant, probably due to the small sample size. This finding is consistent with other studies that included patients with different baseline characteristics [22], [23], although some authors do not support it [24]. The decline in the latent reservoir that we found does not appear to be secondary to a decrease in residual viral replication in plasma, since there were no detectable changes in the measurements of residual viremia. It has been suggested that a dilutional effect could account for the reduction observed, but the lack of an increase in the absolute count and the percentage of both CD8+ and CD4+ T cells strongly argues against this possibility.

Other findings in our study may help explain the effect on the latent reservoir. Unexpectedly, 2LTR DNA circles were transiently detectable during MVC intensification. Due to its mechanism of action, MVC is not expected to increase 2LTR DNA circles, even when inhibiting persistent viral replication. The increase in levels of this marker suggests that treatment with MVC in patients with undetectable viral load may induce viral replication. Preclinical studies showed that MVC had no agonistic activity on activated CD4+ T cells and, therefore, could not induce viral replication [32]. Later reports, however, have shown that the possibility of acting as a partial agonist on CCR5 could be dependent on cell type [45]. Therefore, MVC could activate CCR5 in resting CD4+ T cells and, through intracellular signalling, increase transcriptional activation of the latent virus. As an alternative hypothesis, CCR5 blockade by MVC could lead natural ligands or chemokines to bind to other receptors and induce latent HIV transcriptional activation through distinct signalling pathways. Both hypotheses would be consistent with the reduced number of latently infected memory T cells found in this study, the transient increase in the number of copies of HIV RNA seen in some patients, and the increased bacterial translocation plus migration of α4β7 cells to the gut. This possibility is further supported by the increased immune activation in intestinal tissue biopsies that was observed in another MVC intensification study [46]. Finally, trafficking of cells to peripheral blood from some other compartment cannot be discarded to explain some of the results, including the increase in 2 LTR circles.

Neither of the above hypotheses can account for the diminished immune activation that we detected. MVC has been shown to decrease immune activation in clinical trials, and it could be assumed that a different mechanism drives the impact on immune activation [47]. The decrease in immune activation does not correlate with a decrease in bacterial translocation, which in fact increased during the study, thus supporting increased viral replication and activation in the intestine resulting from the release of proinflammatory cytokines [48].

The transient increase in TEM and TemRA CD8+ T cell counts parallels the increase in bacterial translocation. These increments are balanced by a transient decrease in central memory CD8+ T cell counts, while naive CD8+ T cell levels remain stable [49]. In addition, the finding of increased β7 receptor levels on CD8+ T cells and especially on activated CD8 T cells support the idea of migration of these cells to the intestine, where bacterial translocation takes place. This migration of activated cells to the gut could account for the reduced immune activation observed in the periphery. In this sense, immune events detected in peripheral blood may not reflect what takes place in the intestine, as is the case for the huge CD4+ T-cell destruction that occurs in the intestine of the SIV rhesus macaque during acute infection, which is not reflected in lymph nodes or peripheral blood [50].

Our work has a series of limitations. First, it is a pilot study with a small sample size, thus limiting the strength of the conclusions. Second, no control group was included. The study was designed as a proof of concept with no guarantee of any spontaneous effect in the sample. Finally, the methodological tools used for the different measurements and their interpretations are still imperfect.

In summary, our study shows positive effects of MVC intensification on latently HIV-1-infected CD4+ T cells. These effects are not reflected in reduced residual plasma viremia and may be associated with the activity of the drug in an anatomical reservoir, most likely the intestine. The mechanism by which MVC decrease the HIV-1 reservoir is under investigation. The decrease we observed, however, is by no means large enough to eradicate HIV. New therapeutic strategies, probably based on anti-latency drugs, are necessary to eradicate viral reservoirs. In this sense, complete inhibition of viral replication at all sites can be presumed to be necessary before these new treatment strategies can be applied. The exact role of treatment intensification in eradication remains undefined, and a large, comparative trial with adequate methodology is needed to provide a definitive answer to this relevant question.

Supporting Information

Protocol S1.

Trial Protocol.

doi:10.1371/journal.pone.0027864.s001

(DOC)

Checklist S1.

CONSORT Checklist.

doi:10.1371/journal.pone.0027864.s002

(DOC)

Acknowledgments

We would like to thank Carmen Page, Raquel Lorente and Ester Domínguez for 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.

Author Contributions

Conceived and designed the experiments: CG LD AV BH-N SP. Performed the experiments: CG LD AV BH-N MA NM VD. Analyzed the data: SM CG LD AV BH-N JZ. Contributed reagents/materials/analysis tools: CG LD AV BH-N SP. Wrote the paper: CG LD AV BH-N SM. Approved of the final version to be published: CG LD AV BH-N MA NM VD RR AM FD JC EN MP-E JZ SP EM MM-F SM. Conceived and design the study: SM MM-F EM. Inclusion and patients follow up: RR AM FD JC EN MP-E.

References

  1. 1. Wong JK, Hezareh M, Gunthard HF, Havlir DV, Ignacio CC, et al. (1997) Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 278: 1291–5.
  2. 2. Palmer S, Maldarelli F, Wiegand A, Bernstein B, Hanna GJ, et al. (2008) Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy. Proc Natl Acad Sci U S A. 105: 3879–84.
  3. 3. Maldarelli F, Palmer S, King MS, Wiegand A, Polis MA, et al. (2007) ART suppresses plasma HIV-1 RNA to a stable set point predicted by pretherapy viremia. PLoS Pathog 3: e46.
  4. 4. Anderson JA, Archin NM, Ince W, Parker D, Wiegand A, et al. (2011) Clonal sequences recovered from plasma from patients with residual HIV-1 viremia and on intensified antiretroviral therapy are identical to replicating viral RNAs recovered from circulating resting CD4+ T cells. J Virol. 85: 5220–3.
  5. 5. Joos B, Fischer M, Kuster H, Pillai SK, Wong JK, et al. (2008) HIV rebounds from latently infected cells, rather than from continuing low-level replication. Proc Natl Acad Sci U S A 105: 16725–30.
  6. 6. Persaud D, Siberry GK, Ahonkhai A, Kajdas J, Monie D, et al. (2004) Continued production of drug-sensitive human immunodeficiency virus type 1 in children on combination antiretroviral therapy who have undetectable viral loads. J Virol 78: 968–79.
  7. 7. Kieffer TL, Finucane MM, Nettles RE, Quinn TC, Broman KW, et al. (2004) Genotypic analysis of HIV-1 drug resistance at the limit of detection: virus production without evolution in treated adults with undetectable HIV loads. J Infect Dis 189: 1452–65.
  8. 8. Sharkey ME, Teo I, Greenough T, Sharova N, Luzuriaga K, et al. (2000) Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy. Nat Med 6: 76–81.
  9. 9. Hunt PW, Martin JN, Sinclair E, Bredt B, Hagos E, et al. (2003) T cell activation is associated with lower CD4+ T cell gains in human immunodeficiency virus-infected patients with sustained viral suppression during antiretroviral therapy. J Infect Dis 187: 1534–43.
  10. 10. Chun TW, Nickle DC, Justement JS, Large D, Semerjian A, et al. (2005) HIV-infected individuals receiving effective antiviral therapy for extended periods of time continually replenish their viral reservoir. J Clin Invest 115: 3250–5.
  11. 11. Havlir DV, Strain MC, Clerici M, Ignacio C, Trabattoni D, et al. (2003) Productive infection maintains a dynamic steady state of residual viremia in human immunodeficiency virus type 1-infected persons treated with suppressive antiretroviral therapy for five years. J Virol 77: 11212–9.
  12. 12. McMahon D, Jones J, Wiegand A, Gange SJ Kearney M, et al. (2010) Short-course raltegravir intensification does not reduce persistent low-level viremia in patients with HIV-1 suppression during receipt of combination antiretroviral therapy. Clin Infect Dis 50: 912–9.
  13. 13. Chun TW, Justement JS, Murray D, Hallahan CW, Maenza J, et al. (2010) Rebound of plasma viremia following cessation of antiretroviral therapy despite profoundly low levels of HIV reservoir: implications for eradication. AIDS 24: 2803–8.
  14. 14. Dinoso JB, Kim SY, Wiegand AM, Palmer SE, Gange SJ, et al. (2009) Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy. Proc Natl Acad Sci U S A 106: 9403–8.
  15. 15. Gandhi RT, Zheng L, Bosch RJ, Chan ES, Margolis DM, et al. (2010) The effect of raltegravir intensification on low-level residual viremia in HIV-infected patients on antiretroviral therapy: a randomized controlled trial. PLoS Med. 10. : 7 pii–:e1000321.
  16. 16. Yilmaz A, Verhofstede C, D'Avolio A, Watson V, Hagberg L, et al. (2010) Treatment intensification has no effect on the HIV-1 central nervous system infection in patients on suppressive antiretroviral therapy. J Acquir Immune Defic Syndr; 15: 590–596.
  17. 17. Wiegand A, Poethke C, Kearney M, Spindler J, ÓShea A, et al. (2010) Raltegravir intensification does not reduce persistent HIV-1 viremia in treatment-experienced patients.
  18. 18. Buzon MJ, Massanella M, Llibre JM, Esteve A, Dahl V, et al. (2010) HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects. Nat Med 16: 460–5.
  19. 19. Sharkey M, Triques K, Kuritzkes DR, Stevenson M (2005) In vivo evidence for instability of episomal human immunodeficiency virus type 1 cDNA. J Virol 79: 5203–10.
  20. 20. Chavez HH, Tran TA, Dembele B, Nasreddine N, Lambotte O, et al. (2007) Lack of evidence for prolonged double-long terminal repeat episomal HIV DNA stability in vivo. J Acquir Immune Defic Syndr 45: 247–9.
  21. 21. Chomont N, El-Far M, Ancuta P, Trautmann L, Procopio FA, et al. (2009) HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nat Med 15: 893–900.
  22. 22. Ramratnam B, Ribeiro R, He T, Chung C, Simon V, et al. (2004) Intensification of antiretroviral therapy accelerates the decay of the HIV-1 latent reservoir and decreases, but does not eliminate, ongoing virus replication. J Acquir Immune Defic Syndr 35: 33–7.
  23. 23. Chun TW, Justement JS, Moir S, Hallahan CW, Maenza J, et al. (2007) Decay of the HIV reservoir in patients receiving antiretroviral therapy for extended periods: implications for eradication of virus. J Infect Dis 195: 1762–4.
  24. 24. Gandhi RT, Bosch RJ, Aga E, Albrecht M, Demeter LM, et al. (2010) No evidence for decay of the latent reservoir in HIV-1-infected patients receiving intensive enfuvirtide-containing antiretroviral therapy. J Infect Dis 201: 293–6.
  25. 25. Brenchley JM, Price DA, Schacker TW, Asher TE, Silvestri G, et al. (2006) Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med 12: 1365–71.
  26. 26. Baroncelli S, Galluzzo CM, Pirillo MF, Mancini MG, Weimer LE, et al. (2009) Microbial translocation is associated with residual viral replication in HAART-treated HIV+ subjects with <50 copies/ml HIV-1 RNA. J Clin Virol 46: 367–70.
  27. 27. Chege D, Sheth PM, Kain T, Kim CJ, Kovacs C, et al. (2011) Sigmoid Th17 populations, the HIV latent reservoir, and microbial translocation in men on long-term antiretroviral therapy. AIDS 25: 741–9.
  28. 28. Brenchley JM, Douek DC (2008) HIV infection and the gastrointestinal immune system. Mucosal Immunol 1: 23–30.
  29. 29. Sheth PM, Chege D, Shin LY, Huibner S, Yue FY, et al. (2008) Immune reconstitution in the sigmoid colon after long-term HIV therapy. Mucosal Immunol 1: 382–8.
  30. 30. Chun TW, Murray D, Justement JS, Hallahan CW, Moir S, et al. (2011) Relationship between residual plasma viremia and the size of HIV proviral DNA reservoirs in infected individuals receiving effective antiretroviral therapy. J Infect Dis 204: 135–8.
  31. 31. Cicala C, Martinelli E, McNally JP, Goode DJ, Gopaul R, et al. (2009) The integrin alpha4beta7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1. Proc Natl Acad Sci U S A. 106: 20877–82.
  32. 32. Dorr P, Westby M, Dobbs S, Griffin P, Irvine B, et al. (2005) Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity. Antimicrob Agents Chemother 49: 4721–32.
  33. 33. Gulick RM, Lalezari J, Goodrich J, Clumeck N, DeJesus E, et al. (2008) Maraviroc for previously treated patients with R5 HIV-1 infection. N Engl J Med 359: 1429–41.
  34. 34. Cooper DA, Heera J, Goodrich J, Tawadrous M, Saag M, et al. (2010) Maraviroc versus efavirenz, both in combination with zidovudine-lamivudine, for the treatment of antiretroviral-naive subjects with CCR5-tropic HIV-1 infection. J Infect Dis 201: 803–13.
  35. 35. García-Merino I, de las Cuevas N, Jimenez JL, Gallego J, Gómez C, et al. (2009) The Spanish HIV Biobank: a model of cooperative HIV research. Retrovirology 6: 27.
  36. 36. Chun TW, Stuyver L, Mizell SB, Ehler LA, Mican JA, et al. (1997) Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci U S A. 94: 13193–7.
  37. 37. Siliciano JD, Kajdas J, Finzi D, Quinn TC, Chadwick K, et al. (2003) Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat Med 9: 727–8.
  38. 38. Myers LE, McQuay LJ, Hollinger FB (1994) Dilution assay statistics. J Clin Microbiol 32: 732–9.
  39. 39. Palmer S, Wiegand AP, Maldarelli F, Bazmi H, Mican JM, et al. (2003) New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol 41: 4531–6.
  40. 40. Zazzi M, Romano L, Catucci M, Venturi G, De Milito A, et al. (1997) Evaluation of the presence of 2-LTR HIV-1 unintegrated DNA as a simple molecular predictor of disease progression. J Med Virol. 52: 20–5.
  41. 41. Markowitz M, Caskey M, Figueroa A, Rodriguez K, La Mar M, et al. (2011) A randomized Open-Label Trial of 5-drug vs 3-drug Standard PI-Based cART Initiated during Acute and Early HIV-1 Infection: 48-Week results.
  42. 42. Chun TW, Nickle DC, Justement JS, Meyers JH, Roby G, et al. (2008) Persistence of HIV in Gut-Associated Lymphoid Tissue despite Long-Term Antiretroviral Therapy. J Infect Dis 197: 714–20.
  43. 43. Yukl SA, Shergill AK, McQuaid K, Gianella S, Lampiris H, et al. (2010) Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy. AIDS 24: 2451–60.
  44. 44. Yukl SA, Gianella S, Sinclair E, Epling L, Li Q, et al. (2010) Differences in HIV burden and immune activation within the gut of HIV-positive patients receiving suppressive antiretroviral therapy. J Infect Dis 202: 1553–61.
  45. 45. Wu Y, Yoder A (2009) Chemokine coreceptor signalling in HIV-1 infection and pathogenesis. PLoS Pathog 5: e1000520.
  46. 46. Hunt P, Hayes T, Dahl V, Funderburg N, Adeyemi O, et al. (2011) Effects of MVC Intensification in HIV-infected individuals with incomplete CD4+ T Cell Recovery during Suppressive ART.
  47. 47. Funderburg N, Kalinowska M, Eason J, Goodrich J, Heera J, et al. (2010) Effects of maraviroc and efavirenz on markers of immune activation and inflammation and associations with CD4+ cell rises in HIV-infected patients. PLoS One 5: e13188.
  48. 48. Kotler DP (1999) Characterization of intestinal disease associated with human immunodeficiency virus infection and response to antiretroviral therapy. J Infect Dis 179: S454–6.
  49. 49. Northfield JW, Loo CP, Barbour JD, Spotts G, Hecht FM, et al. (2007) Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T(EMRA) cells in early infection are linked to control of HIV-1 viremia and predict the subsequent viral load set point. J Virol 81: 5759–65.
  50. 50. Kewenig S, Schneider T, Hohloch K, Lampe-Dreyer K, Ullrich R, et al. (1999) Rapid mucosal CD4(+) T-cell depletion and enteropathy in simian immunodeficiency virus-infected rhesus macaques. Gastroenterology 116: 1115–23.