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CLINICAL SCIENCE: CONCISE COMMUNICATIONS

Levels of intracellular HIV-DNA in patients with suppressive antiretroviral therapy

Cuzin, Lisea; Pugliese, Pascalb; Sauné, Karinec,d; Allavena, Clotildee; Ghosn, Jadef,g; Cottalorda, Jacquelineh; Rodallec, Audreyi; Chaix, Marie Laurej; Fafi-Kremer, Samirak; Soulié, Cathial,m,n; Ouka, Marlèneo; Charpentier, Charlottep,q,r; Bocket, Laurences; Mirand, Audreyt; Guiguet, Margueriteu for the Dat’AIDS study group

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doi: 10.1097/QAD.0000000000000723
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Abstract

Introduction

Despite the development of potent antiretroviral therapies (ARTs), it remains impossible to date to eradicate HIV, mainly because of its persistence in resting CD4+ T cells [1–3]. These cells retain the ability to produce virions when stimulated and thus they represent a viral reservoir [4]. An easy and reproducible way to assess this reservoir is by the quantification of cell-associated HIV-DNA in peripheral blood mononuclear cells (PBMCs) [2].

HIV reservoirs are established very early during the acute phase of HIV infection [5] and HIV-DNA levels have been shown to be independently associated with disease progression [6]. When ART is initiated at the chronic phase of infection, HIV-DNA levels decrease modestly with a blunted decrease after 4–5 years of uninterrupted ART [7–9]. Viral load peak, CD4+ cell count nadir, as well as timing of ART initiation have been associated with HIV-DNA levels [10–12]. Low HIV-DNA levels increased the likelihood of virological success in studies using single-drug ART regimens [13]. Moreover, viral replication control in blood plasma after ART discontinuation may even be possible in some patients with low HIV-DNA levels [14–16].

A better understanding of the determinants of HIV-DNA level in ART-treated HIV-infected patients is needed to design future studies targeting the reservoirs. The objective of our study was to identify factors associated with HIV-DNA levels in a large population of chronically infected patients on long-term suppressive ART.

Patients and methods

We designed a cross-sectional analysis of adult HIV-1 infected patients receiving suppressive ART (defined by all viral load measurements below 50 copies/ml over the past 2 years, with at least two assessments per year for at least 3 years), with last CD4+ T-cell count value more than 350 cells/μl, in 10 HIV reference centers in France. The patients were searched for in prospectively maintained databases of all patients in care in the study centres [17]. According to French law, the study has been approved by the consultative committee on personal information use for research purposes (CCTIRS); the patients were prospectively informed about the study and gave consent to their participation. All those who had a scheduled medical appointment in one of the centres in 2 given weeks at the end of 2013 and did not decline participation were prospectively included. Routine viral load and CD4+ T-cell count were performed on the appointment day, as to check for inclusion criteria. HIV-2 infected patients, patients who initiated ART during acute HIV infection and patients infected through vertical transmission were excluded.

The HIV-DNA level in PBMCs was quantified in whole blood, on the same sample obtained for routine measurement of viral load, by the Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ANRS) real-time PCR method (Biocentric, Bandol, France) as previously described [18]. HIV-DNA measure results were expressed in copies/106 PBMCs and classified into three groups following the distribution: low (<150), intermediate (150–1000) or high level (>1000 [19]). Demographics, clinical characteristics, antiretroviral history, viral load values and CD4+ T-cell counts were extracted from the databases. History of ART failure was based on notification in the databases of any ART modification for immunological or virological failure.

Median levels of HIV-DNA were compared between levels of risk factors using Wilcoxon nonparametric test, and categories of HIV-DNA were compared using Chi-square. For continuous risk factors, Pearson correlation measure was used to study their association with HIV-DNA (log10 copies/106 PBMCs). Factors associated with being in the low or high level by comparison with the intermediate category were investigated using multinomial logistic regression. Factors associated with HIV-DNA levels in univariate analysis (P < 0.15) were included in a multivariate model (sex, coinfection with hepatitis B or C, CDC class C, peak viral load, history of failure). All two-by-two interactions were tested. An interaction between peak viral load and history of failure was found statistically significant and a three-level variable was constructed (peak viral load <5 log10 copies/ml and no history of failure, peak viral load <5 log10 copies/ml and any history of failure, peak viral load ≥5 log10 copies/ml). A backward elimination procedure was used to determine the final model.

Results

Following the inclusion criteria, HIV-DNA levels were assessed in 522 patients during the study period. Their characteristics at the time of inclusion are summarized in Table 1. Median HIV-DNA level was 323 copies/106 PBMCs (IQR25%, 129–717) with a low, intermediate and high level observed in 28.3, 55.4 and 16.3%, respectively. A difference in HIV-DNA levels was observed according to sex (Table 1). Among patients with a hepatitis B (HBV) or hepatitis C (HCV) coinfection (24 HBV, 60 HCV, five HBV/HCV), 21% presented a high HIV-DNA level. An association was observed between HIV-DNA and the peak of viral load, but not with the CD4+ cell count nadir. In particular, a peak of viral load higher than 5 log10 copies/ml was associated with a high level of HIV-DNA [median 421 copies/106 PBMCs (IQR25%, 202–836) versus 246 copies/106 PBMCs (IQR25%, 91–565) in case of lower viral load peak; P < 0.0001]. Patients with no history of virological failure had lower HIV-DNA levels (median 294 copies/106 PBMCs; IQR25%, 100–663) than those with failures (median 377 copies/106 PBMCs; IQR25%, 178–873; P = 0.01). The duration of ART was not associated with the HIV-DNA levels (Pearson test, P = 0.07), but lower HIV-DNA levels were observed with an increasing duration of suppressed viremia (Pearson test, P = 0.003).

T1-11
Table 1:
Patients characteristics at the time of HIV-DNA assessment and relationship with HIV-DNA levels.

In multinomial logistic regression, four characteristics were associated with low or high HIV-DNA levels (Table 2). Women had a higher probability of reaching low HIV-DNA levels. Each additional year with uninterrupted suppressed viremia increased the likelihood of low HIV-DNA level. Compared with patients with peak viral load below 5 log10 copies/ml having no past virological failure, those with peak viral load above 5 log10 copies had a lower probability of low HIV-DNA level. On the contrary, each additional year with uninterrupted suppressed viremia decreased the likelihood of high HIV-DNA level. Patients with peak viral load below 5 log10 copies/ml who have had virological failure as well as patients with peak viral load above 5 log10 copies/ml were at a higher risk to have high HIV-DNA level than patients with viral load peak below 5 log10 copies/ml who never had any episode of past virological failure.

T2-11
Table 2:
Factors associated with low (<150 copies/106 peripheral blood mononuclear cells) or high (>1000 copies/106 peripheral blood mononuclear cells) levels compared with intermediate (150–1000 copies/106 peripheral blood mononuclear cells) level of HIV-DNA.

Discussion

In this multicentre, cross-sectional study of a large population of chronically HIV-infected adult patients receiving potent ART with sustained viral load suppression for at least 2 years, we found that 28.3% of the patients had HIV-DNA levels below 150 copies/106 PBMCs, while 16.3% had HIV-DNA levels above 1000 copies/106 PBMCs. Peak viral load below 5 log10 copies/ml and longer duration of virological suppression were associated with low levels of HIV-DNA. For patients with peak viral load below 5 log10 copies/ml, a past episode of virological failure was associated with an increased risk of high HIV-DNA levels. Our results are in keeping with previous studies that already showed relations between peak viral load, duration of virologic suppression and HIV-DNA levels [10–12,20]. The role of virological rebound has been described in a different setting, with lower HIV-DNA values in patients with viral load permanently below 50 copies/ml versus those with at least one measure over 50 copies/ml [21].

Women were more likely to achieve low levels of HIV-DNA. To the best of our knowledge, this has not been shown by others; maybe due to the small proportion of women in previous studies, nevertheless an increased proportion of women has been described in HIV-controller cohorts [22,23]. It has been previously described that women not receiving ART had lower viral load set point when than men [24,25], partly due to interactions between sex-steroid hormones and the immune system [26], although X-linked genetic differences could also be involved. In women, TLR-7 stimulated plasmacytoid dendritic cells produce more intereferon-alpha (IFN-α) than in men [27,28], which could contribute to a better control of virus replication during the acute phase of HIV-infection, and thus yielding lower HIV-DNA levels in women. Recently, it has been shown that the enhancement of some host restriction factors such as p21, schlafen 11 and PAF1 do modulate HIV persistence in the setting of viral suppression [29], but unfortunately in this study, women were underrepresented; thus, the sexual effect was not analysed. The interplay between sex and cell-intrinsic immune responses should be specifically explored.

Previous studies have reported that CD4+ cell count nadir but not peak viral load was associated with HIV-DNA level [10], or that both CD4+ cell count nadir and peak viral load were predictive of HIV DNA level [11]. In our study, the CD4+ T cells nadir was correlated with the peak viral load. After a median duration of ART of 13 years, the peak of HIV-RNA still was associated with HIV-DNA level, but not the CD4+ T cells nadir.

In posttreatment controllers, a population of patients treated at the acute phase of infection, HIV-DNA level was in median at 51 copies/106 PBMCs [30]. Our results show that it is possible to reach such low HIV-DNA levels even when ART is initiated during the chronic phase of infection, with greater frequency when peak viral load is below 5 log10 copies/ml. As it is of paramount importance to avoid any virological rebound, improving ART tolerance and adherence are major objectives. Low HIV-DNA levels may allow some ART alleviation [13], and the patients with low HIV-DNA levels might be the ideal population for future functional cure trials [31]. On the contrary, high HIV-DNA levels have been found to be associated with intermittent HIV shedding in semen of ART-treated men despite sustained viral load suppression in blood plasma [32], thus compromising the preventive efficacy of ART at a population level.

Our study has some strength and limitations. We studied a large population seeking care in different centres, representative of the patients under care in France in 2013. The retrospective analysis of prospectively collected data is a clear limit because of some missing values or possible errors in the database. This phenomenon is limited because of permanent assessment and control of the quality of the databases [17]. Some patients’ characteristics that may be associated with HIV-DNA levels, such as ART adherence, could not be assessed. We limited those biases by restricting the analysis to patients with a sustained viral load below 50 copies/ml for at least the 2 years before HIV-DNA assessment, and indeed, the studied population had a median duration of virological suppression of 6 years. By doing this choice, we loosed the potential to analyse the role of different ART regimens on the HIV DNA levels, in this population treated with numerous and various regimens for more than 10 years. As in any observational study, we cannot infer any causality to the observed relations between HIV-DNA and patients characteristics. Total HIV-DNA levels (i.e. integrated and nonintegrated HIV-DNA) were assessed using a standardized reproducible method [18]. It has been shown that total HIV-DNA does not differ from integrated DNA in patients with prolonged virological suppression in blood plasma [33–35] and was related with both time to and magnitude of viral rebound after treatment interruption [15,36]. Thus, we do not believe that choosing a simple and reproducible method could bias our results in assessing the viral reservoir.

In conclusion, our results emphasize that among chronically HIV-infected patients with long-term suppressive ART, achieving a level of HIV-DNA less than 150 copies/106 PBMCs was common (28%). Nevertheless, one over six still presented HIV DNA above 1000 copies/106 PBMCs. After a median duration of 13 years of ART, HIV-DNA level was still associated with peak viral load, and low level of HIV-DNA was more likely to be observed with longer duration of suppressed viremia. Avoiding any rebound in viral load while on ART is paramount. Finally, the possible advantage of being women needs further research.

Acknowledgements

We thank Pr Pierre Delobel for his advice regarding the sexual specificity, and the centres technical staff for permanent data quality control.

All authors contributed significantly to the study. L.C., C.A., P.P., M.L.C. and M.G. designed the study. M.G. was responsible for the statistical analysis. K.S. was the virology coordinator. J.G., J.C., K.S., A.R., S.F.K., C.S., M.O., C.C., L.B. and A.M. performed the HIV-DNA quantifications in each centre. L.C., C.A., M.G. and J.G. wrote the first version of the article, which was amended by coauthors. All authors approved the final version.

The Dat’AIDS association provided funding for the Biocentric test kits. The databases are collected via the Nadis software, Fedialis Medica, France, developed and maintained with financial support from ViiV healthcare.

Dat’AIDS Study Group: P. Enel, V. Obry-Roguet, O. Faucher, S. Bregigeon, I. Poizot-Martin, (Marseille); B. Marchou, P. Massip, E. Bonnet, M. Obadia, M. Alvarez, L. Porte, L. Cuzin, P. Delobel, M. Chauveau, D. Garipuy, M.Mularczyk, J. Bernard, I. Lepain, M. Marcel, E. Puntis, K. Sauné (Toulouse); P. Pugliese, C. Ceppi, E. Cua, J. Cottalorda, P. Dellamonica, E. Demonchy, B. Dunais, J. Durant, C. Etienne, S. Ferrando, J. G. Fuzibet, R. Garraffo, K. Risso, V. Mondain, A. Naqvi, N. Oran, I. Perbost, S. Pillet, B. Prouvost-Keller, C. Pradier, S. Wehrlen-Pugliese, E. Rosenthal, S. Sausse, P.M. Roger (Nice); C. Allavena, C. Bernaud, E. Billaud, C. Biron, B. Bonnet, S. Bouchez, D. Boutoille, C. Brunet-Cartier, N. Hall, T. Jovelin, P. Morineau, F. Raffi, V. Reliquet, H. Hue, L. Larmet, So. Pineau, V. Ferré , E. André-Garnier, A. Rodallec (Nantes); A. Cheret, P. Choisy (Tourcoing); Y. Yazdanpanah, R. Landman, C. Duvivier, M.A. Valantin, R. Agher, C. Katlama, Lortholary, V. Avettand-Fenoel, C. Rouzioux, P.H. Consigny, G. Cessot, F. Touam, R. Usubillaga, K. Benhadj (Paris); A. Cabié , S. Abel, S. Pierre-François, M. Ouka, J. Martial (Fort de France); D. Rey, E. Ebel, P. Fischer, M. Partisani, C. Cheneau, M. Priester, M.L. Batard, C Bernard-Henry, E de Mautort (Strasbourg); C. Chirouze, C. Drobacheff-Thiébaut, J.P. Faller, J.F. Faucher, A. Foltzer, H. Gil, L. Hustache-Mathieu, C. Bourdeaux (Besançon), B. Hoen(Pointe à Pitre); C. Jacomet, H. Laurichesse, O. Lesens, M. Vidal, N. Mrozek, C. Aumeran, O. Baud, J. Beytout, D. Coban, S. Casanova (Clermont-Ferrand).

Conflicts of interest

The authors do not have any conflict of interest related to this work.

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Keywords:

antiretroviral therapy; HIV-DNA; treatment failure; women

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