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Prolonged valproic acid treatment does not reduce the size of latent HIV reservoir
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[BASIC SCIENCE: CONCISE COMMUNICATION]
AIDS:Volume 22(10)19 June 2008p 1125-1129
Sagot-Lerolle, Nathaliea; Lamine, Aureliaa; Chaix, Marie-Laureb; Boufassa, Faroudyc; Aboulker, Jean-Pauld; Costagliola, Dominiquee; Goujard, Cecilef; Paller, Coralieg; Delfraissy, Jean-Francoisa,f,h; Lambotte, Oliviera,f,h; for the ANRS EP39 study
From the aINSERM, U802, Bicetre, France
bEA 3620 Paris Descartes, Laboratory of Virology, CHU Necker, Paris, France
cINSERM, U822, Bicetre, France
dINSERM SC10, Paul Brousse Hospital, Villejuif, France
eINSERM UMR S720, Paris, France
fAP-HP, Department of Internal Medicine and Infectious Diseases, Bicetre Hospital, Bicetre, FrancegLaboratory of Virology, Bicetre Hospital, Bicetre, France
hUniversite Paris-Sud, U 802, Bicetre, France.
Abstract
Objective: To investigate the impact of prolonged valproic acid treatment on the HIV reservoir in patients on highly active antiretroviral therapy.
Design: In a single-center pilot study, the size of the HIV reservoir of 11 patients receiving valproic acid for seizures for more than 2 years was compared with 13 matched patients. In addition, the outcome of patients receiving valproic acid in the French clinical trials of scheduled treatment interruption was recorded.
Methods: Total and integrated HIV-1 DNA in, respectively, peripheral blood mononuclear cells and CD4 T cells of the patients were quantified by real-time PCR methods. The frequency of CD4 T cells carrying replication-competent virus was estimated by a quantitative limiting-dilution assay in which virus growth was detected by RT-PCR in culture supernatants of activated CD4 T cells.
Clinical charts of the patients included in scheduled treatment interruption trials receiving valproic acid were reviewed.
Results: Total and integrated HIV DNA were logarithmically more abundant than cells carrying replication-competent virus, but there was no significant difference in these three parameters between the two groups of matched patients.
Three patients receiving valproic acid were included in scheduled treatment interruption trials. The rebound of viral replication was similar to that of the other patients of the trials.
Conclusion: Long-term valproic acid therapy seems to be insufficient to reduce the size of the HIV-1 reservoir.
Introduction TOP
In HIV-1-infected patients, highly active antiretroviral therapy (HAART) can keep HIV-1 RNA plasma levels below the detection limit but does not eradicate the virus [1-3]. In HAART-treated patients, replication-competent virus persists in latently infected CD4+ T cells that harbor integrated viral DNA [4]. This HIV reservoir is the main barrier for the eradication of the virus. Reducing its size is a major goal and some therapeutic interventions suggest that such a reduction could be possible. Viral replication can be induced in the latently infected cells by addition of cellular activating stimuli [5]. As viral replication leads to cell death, the number of latently infected cells can decrease [4] as antiretroviral drugs prevent new infection. Targeting the chromatin structure, which regulates the host genes and viral long terminal repeat (LTR) expression, could also lead to the transcription of the viral genome and virus replication.
A single nucleosome is located at the LTR transcription start and prevents the binding of transcription factors. Histone acetylation of this nucleosome could trigger its disruption and allow viral replication [6]. The cellular histone deacetylases (HDACs) keep this nucleosome hypoacetylated and are thus involved in HIV latency. Several HDAC inhibitors (HDACis) can induce HIV-1 gene expression [7]. Valproic acid (VPA), a well known treatment of seizures, has an ancillary HDACi effect, which has been proved to reactivate in-vitro HIV-1 replication within quiescent CD4 T cells [8]. In vivo, a recent study has tested the ability of VPA, combined with HAART, to deplete the latently infected CD4 T cells. In three patients, the number of reservoir cells decreased significantly [9]. This finding, though not definitive, suggested that new approaches will allow the cure of HIV in the future. These results, however, remain controversial; in the sole study published since the work of Lehrman et al. [9], Siliciano et al. [10] did not find a decrease in the numbers of latently infected cells in nine patients treated with VPA [11].
In this setting, we carried out a pilot study to investigate whether prolonged VPA therapy could deplete the latent HIV reservoir in patients on HAART. The size of the lymphocyte reservoir was compared in patients receiving VPA for seizures and a control group of matched patients not treated with VPA.
In addition, the charts of the patients receiving VPA in the Agence National de Recherche pour le Sida et les hepatites virales (ANRS) scheduled treatment interruption (STI) clinical trials were reviewed to look for an unusual course of viral replication after STI.
Patients and methods
Patients
All the patients were treated in the Infectious Diseases Department of Bicetre Hospital. The inclusion criteria were a stable HAART regimen with virologic suppression for more than 2 years (plasma viral load <50 copies/ml, Amplicor Monitor; Roche) and VPA treatment for more than 2 years. For each patient on the VPA regimen, one HAART-treated patient meeting the same inclusion criteria, but who never received VPA, was matched for sex, age and duration of infection, except for two for whom two control patients were found. All participants provided informed consent and 50 ml of blood was collected for each patient. An assay of plasma VPA was performed the same day.
Quantification of total viral DNA
Total viral DNA was quantified in peripheral blood mononuclear cells (PBMCs) using a quantitative PCR as previously described [12].
Quantification of integrated HIV-1 DNA by Alu-LTR PCR in CD4 T cells
CD4 T cells were isolated as previously described [13]. DNA was extracted from 2 X 106 total CD4 T cells (Macherey Nagel, Germany). Genomic DNA was serially diluted from 105 to 102 cell equivalents. We used a semi-quantitative Alu-LTR PCR, adapted from [14], to detect specifically integrated HIV-1 DNA. The following concentrations were tested in the Alu-PCR: 105, 5 X 104, 104, 5 X 103, 103, 5 X 102, and 102 cell equivalents. We could determine the smallest positive dilution and thereby estimate the frequency of cells harboring integrated viral DNA. A positive reaction means that there is at least one copy of integrated HIV DNA in the sample. To avoid nonspecific amplification with the LTR primer, a control PCR was systematically performed without the Alu primer as recommended [14]. The limit of detection of the Alu-PCR assay was six viruses within 50 000 cell equivalents. Results were compared with a kinetic PCR assay for GAPDH DNA. All samples were tested in duplicate.
Quantification of CD4 T cells carrying replication-competent virus
The freshly drawn CD4 T cells were cultured in duplicate in a series of dilutions from 2 X 106 to 103 cells/well. The cells were activated as previously described [15] and viral RNA was quantified in the culture supernatants on day 14. In such a limiting-dilution assay, what is important is the last cell density at which virus production is still detected.
Statistical analysis
Analysis was performed using the Fisher exact and Wilcoxon tests.
Scheduled treatment interruption trials
We screened eight STI trials looking for patients receiving VPA: ANRS trials 'INTERVAC', 'WINDOW', 'VACCITER', 'PRIMSTOP', 'PRIMOVAC', 'VACCIL-2', 'SALTO', and 'ILIADE'. The charts of the patients identified were reviewed focusing on changes in plasma viral loads.
Results
Among 1300 HIV-1-infected patients followed up in our Infectious Diseases Department, 12 met the inclusion criteria and 11 accepted the study. The reasons for valproate therapy were opportunistic brain infections (five patients) or non-HIV-related epilepsy (six patients). Thirteen patients were included in the control group (only one matched patient was found for nine VPA-treated patients). The baseline characteristics of the patients are reported in Table 1. There were no significant differences among the groups. The median duration of HIV infection was 17 years in both groups. Patients from both groups received a median of five combinations of antiretroviral drugs. No patient received 'salvage' multidrug therapy of partial efficacy. The median duration of HAART with undetectable viremia was 4 years in both groups, that of VPA treatment, 10 years. Only three patients (two controls and one on VPA) had transient episodes of detectable viremia, one only in the last 2 years.
In the two groups of patients, the total amount of viral DNA was quantified in PBMCs. There was no significant difference between the number of total viral DNA copies in PBMCs in the valproate group (median: 2.9 log/106 PBMCs; range 2.3-3.2) and the control group (median: 2.6 log/106 PBMCs; range 2.1-3.4; P = 0.54) (Fig. 1a).
Fig. 1. Prolonged VPA treatment does not significantly reduce the size of the HIV reservoir, as the total amount of viral DNA (expressed as copies per 106 PBMCs) does not differ between the group of patients receiving VPA and the control group (a), or the amount of integrated viral DNA estimated by the lowest number of CD4 T cells in which a positive Alu-PCR is obtained (b), or the number of CD4 T cells carrying replication-competent virus (c) estimated by the lowest number of cells in the supernatant of which viral RNA can be detected after activation. The black dots are the extreme values. PBMCs, peripheral blood mononuclear cells; VPA, valproic acid.
As total viral DNA includes both unintegrated and integrated HIV DNA, an Alu-LTR PCR was used to detect integrated HIV-1 DNA in a limiting-dilution assay. As HIV latent infection is established in CD4 T cells, the assay was performed in DNA extracted from purified CD4 T cells. This assay, successfully performed in all patients except one in each group in which HIV DNA was below the detection limit, showed that the frequency of CD4 T cells harboring integrated viral DNA was similar in the two groups: 2.7 X 10-4 in the valproate group (range 3 X 10-5-10-3) and 1.6 X 10-4 in the control group (range 3 X 10-5-2 X 10-3, P = 0.23), in median (Fig. 1b).
Integrated viral DNA can be defective and the viral DNA quantification methods do not discriminate between replication-competent and defective virus. Therefore, limiting-dilution cultures of activated CD4 T cells were performed to induce the replication of the latent viruses. The last positive dilution culture indicates the presence of at least one cell carrying replication-competent virus. This could be performed only in 10 patients in each group because of limited amounts of CD4 cells. The frequency of CD4 T cells carrying replication-competent virus did not differ between the two groups, with a median of respectively 10-6cells/well (range 0-2 X 10-6 cells) in the valproate group and 10-6cells/well in the control group (range 0-10-5, P > 0.99) (Fig. 1c). In four patients (one only in the VPA group), no replication-competent virus-carrying cell was found, probably because the number of cells tested was too low, whereas integrated DNA was detected in the CD4 T cells.
In addition, we looked for a delayed rebound of HIV viremia in the patients included in STI trials who received VPA for seizures. Among 1003 patients, three patients received VPA. In the patient from the 'WINDOW' study, plasma HIV RNA increased from fewer than 400 copies to values from 13 000 to 60 000 copies/ml after each 8 weeks off-regimen. The patient from the 'VACCIL-2' study had rebound of viral replication after each treatment interruption. The 'SALTO' patient had detectable viremia 2 months after HAART interruption. The course of viral replication in these patients was similar to the general data observed in these studies.
Discussion
Present antiretroviral therapies do not reduce the HIV lymphocyte reservoir and HIV eradication will only be achieved if the latent reservoirs are targeted directly [16]. VPA has been suggested to be a promising drug [9] because of its ancillary HDACi activity. This preliminary result, however, has not been reproduced and has been the matter of discussion [11]. Here, we show that the size of the HIV lymphocyte reservoir was not significantly different in a group of HAART-treated patients receiving prolonged treatment with VPA and in a matched control group.
As reported in [9,11], VPA use does not result in decrease in total viral DNA or in integrated DNA. A possible explanation is that a large number of CD4 T cells carrying integrated DNA are infected with nonreplication-competent virus. This is supported by the striking difference we observed in the frequencies of cells containing either integrated DNA or replication-competent virus (respectively at least one integrated DNA copy per 103-104 CD4 T cell and one cell carrying replication-competent virus per 106 cells), as previously reported [1]. Our results appear to differ from those of Lehrman et al. [9] concerning the decrease in the number of replication-competent virus-infected cells. Potential explanations are differences in the assays used to detect latently infected cells, the lack of control group, and the use of enfuvirtide to suppress residual viral replication in the latter study. Indeed, VPA could induce viral replication in latently infected cells in vivo, but intensification of HAART could be required to avoid new cell infections.
This study has two limitations: (i) we did not study precisely the latently infected resting DR-CD4 T cells, which are the cornerstone of the HIV reservoir, but all the CD4 T cells, (ii) some of the viral RNA detected in the supernatants could be noninfectious. We did not, however, note a change in any of our assays between the two groups of patients. Our results are supported by the lone report since the work of Lehrman et al. [9] studying the HIV reservoir in patients treated with VPA [11]. This study did not show a decrease in the numbers of latently infected cells, despite the lack of control-matched group.
In addition to this ex-vivo study, we have in-vivo data about the outcome of the three patients treated with VPA included in STI trials. We did not find differences in the time or intensity of the rebound of the plasma viremia in these three patients compared with the other patients of the trials. This aspect has not been previously reported and is in accordance with the data from a single patient who decided to stop HAART himself [17].
In conclusion, our results do not support a role of VPA alone as a therapeutic agent able to reduce the size of HIV reservoir. Trials should be designed with more potent drugs that act by different mechanisms than current HAART, added to intensified therapy.
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