Skip Navigation

Long-Term Decay of the HIV-1 Reservoir in HIV-1–Infected Children Treated with Highly Active Antiretroviral Therapy

  1. Marisa Zanchetta1,
  2. Sarah Walker3,
  3. Nicoletta Burighel1,
  4. Domenico Bellanova1,
  5. Osvalda Rampon2,
  6. Carlo Giaquinto2 and
  7. Anita De Rossi1
  1. 1Department of Oncology and Surgical Sciences, Unit of Viral Oncology, AIDS Reference Center, and
  2. 2Department of Pediatrics, University of Padova, Padova, Italy;
  3. 3Medical Research Centre Clinical Trials Unit, London, United Kingdom
  1. Reprints or correspondence: Dr. Anita De Rossi, Dept. of Oncology and Surgical Sciences, Sect. of Oncology, AIDS Reference Center, Via Gattamelata 64, 35128, Padova, Italy (anita.derossi{at}unipd.it)
 
Next Section

Abstract

To investigate the decay of the human immunodeficiency virus type 1 (HIV-1) reservoir in children receiving highly active antiretroviral therapy (HAART), we measured HIV-1 DNA in peripheral blood mononuclear cells from 14 children who achieved and maintained suppression of plasma viremia up to 48 months after the initiation of HAART. Levels of intracellular unspliced and multiply spliced HIV-1 RNA were used as markers of residual viral replication. During the first month of HAART, there were significant decays in levels of both plasma HIV-1 RNA and multiply spliced HIV-1 RNA, yet unspliced HIV-1 RNA persisted in most of the children. Greater HIV-1 DNA decay during the first month of HAART correlated with a higher concomitant increase in CD4+ cell counts (P=.028) and a smaller subsequent HIV-1 DNA decay (P=.0012). Furthermore, HIV-1 DNA decayed faster from 1 to 9 months of HAART (median half-life, 5 months) than during the subsequent follow-up period (median half-life, 30 months). Moreover, after 9 months of HAART, HIV-1 DNA tended to decay more slowly in children with detectable levels of unspliced HIV-1 RNA. These findings suggest that clearance of the viral reservoir in HAART-treated children may be influenced by immune repopulation and residual viral replication and may help in refining long-term treatment strategies

Highly active antiretroviral therapy (HAART) has reduced HIV-1 disease progression and mortality in children and adults [1, 2], but it cannot eradicate the virus. Sensitive assays have demonstrated that resting memory CD4+ cells retain replication-competent viral DNA that may be reactivated to produce virus even in patients with prolonged suppression of plasma viremia [3]. In addition, ongoing low levels of viral transcription and replication may occur in a fraction of patients, despite suppression of plasma viremia, as has been demonstrated by the detection of cell-associated HIV-1 RNA [4, 5] and the evolution of viral sequences [6, 7]

Because an understanding of the clearance of viral reservoirs in HIV-1–infected individuals may help in refining long-term treatment strategies, clearance kinetics of virus populations have been extensively studied in adults who have achieved and maintained plasma virus suppression with HAART. By using limiting dilution cocultures to quantify cellular reservoirs of potentially infectious HIV-1, the half-life of the CD4+ cell latent reservoir was estimated to be 6 months in a cohort that included both chronically and acutely infected patients [8], but longer half-lives have been reported in adults who initiated HAART during chronic infection [9]. Ongoing residual replication of HIV-1 may refill the latent reservoir and affect clearance kinetics [8]. The quantification of HIV-1 DNA levels may provide a more sensitive measurement of the size of the viral reservoir. Using this approach, researchers studying HIV-1–infected adults have estimated that, after the initial clearance of productively infected cells, HIV-1 DNA decays with a median half-life of 4–6 months, which is similar to the rate of decay of memory CD4+ cells [10] in both chronically [11] and acutely [12] infected patients. However, studies in individuals treated with long-term HAART have suggested that the HIV-1 reservoir decays with a longer half-life. In a study of patients who initiated HAART during primary infection, HIV-1 DNA was found to decay with a median half-life of 20 weeks from weeks 4 to 48 of therapy and of 70 weeks during the subsequent 3 years [13]. In chronically infected individuals, HIV-1 DNA has been found to decay with a half-life of 18 weeks during the first 16 weeks of HAART and a half-life of 104 weeks thereafter [14]

Studies of viral decay in HIV-1–infected children receiving HAART are scarce, and, thus far, they have been limited to patients with a short period of undetectable plasma viremia [15, 16]. The extrapolation of results from adult to pediatric populations can be problematic—several studies have outlined differences between adults and children in both their virological and immunological response to therapy. Children receiving HAART have a greater capacity for CD4+ cell recovery, likely because of more-active thymopoiesis [15, 17–19], but they are also known to have a higher rate of incomplete suppression and/or rebound of HIV-1 RNA in plasma [20, 21]. This higher rate of virologic failure in children makes it difficult to study the long-term decay of viral reservoirs

In the present study, we estimated clearance of the viral reservoir by measuring HIV-1 DNA in 14 chronically HIV-1–infected children who showed long-term control (up to 4 years) of HIV-1 replication while they were receiving HAART. Rates of clearance of HIV-1 DNA were compared with the dynamics of CD4+ cell recovery and residual viral replication as evaluated by the quantitative detection of intracellular HIV-1 RNA

Previous SectionNext Section

Patients, Materials, and Methods

Patient populationThe study included 14 HIV-1–infected children born to HIV-1–seropositive Italian mothers and monitored prospectively by the Pediatric Department of Padova University since birth. All children were infected with clade B virus, as determined by sequences in the pol gene. The inclusion criterion was a prolonged virological response to protease inhibitor (PI)–based HAART, defined as suppression of plasma HIV-1 RNA to undetectable levels (<50 copies/mL of plasma) within 6 months of the initiation of HAART and persistent levels of <50 copies/mL of plasma thereafter, for at least 24 and up to 48 months. All children were PI naive at the initiation of HAART. Levels of proviral HIV-1 DNA and cell-associated HIV-1 RNA were evaluated in cryopreserved samples collected at the initiation of HAART and at fixed time points during follow-up

Quantification of HIV-1 RNA in plasmaPlasma HIV-1 RNA levels were determined using the Roche Amplicor Monitor System (Roche). The lower limit of detection was 50 HIV-1 RNA copies/mL when the ultrasensitive protocol was used

Quantification of HIV mRNA in cellsTotal cellular RNA was extracted from peripheral blood mononuclear cells (PBMCs; 1×106–5×106), using the Trizol RNA extraction kit (Invitrogen), and resuspended in 30 μL of water that contained 0.01% diethylpyrocarbonate. Residual contaminating DNA was eliminated using the Deoxyribonuclease I kit (Invitrogen). Then, 24 μL of RNA was reverse transcribed into cDNA, using the Taqman Reverse Transcription assay (PE Applied Biosystems) in a final volume of 80 μL, in accordance with the manufacturer’s instructions. HIV-1 mRNA—unspliced and multiply spliced encoding tat/rev—was quantified by real-time polymerase chain reaction (PCR) using the following primers: US1 forward (5′-TTAAGTGTTTCAATTGTGGCAAAGA-3′, nt 1956–1981 in the HIVp22 genome), US2 reverse (5′-AAAAAATTAGCCTGTCTCTCAGTACAATCT-3′; nt 2059–2122), MS1 forward (5′-AAAGGGAAACCAGAGGAGCTCT-3′; nt 672–693), and MS2 reverse (5′-GCCTGTCGGGTCCCCTC-3′; nt 8438–8454) and the following probes: USP (FAM–5′-CCCCTAGGAAAAAGGGCTGTTGGAAATG-3′–TAMRA; nt 2007–2035) and MSP (FAM–5′-TCGACGCAGGACTCGGCTTGC-3′–TAMRA; nt 695–715). For the detection of unspliced RNA, the PCR assay was performed in a 50-μL final volume that contained 10 μL of cDNA, 25 μL of 2× Taqman Universal PCR Master Mix (PE Applied Biosystems), 300 nmol/L primer US1, 900 nmol/L primer US2, and 100 nmol/L probe USP. Amplifications were performed on a 96-well reaction plate (PE Applied Biosystems) in a thermal cycler (ABI Prism 7700; PE Applied Biosystems), using the following conditions: 2 min at 50°C, 10 min at 95°C, and 45 cycles each of 15 s of 95°C and 1 min at 60°C. For each run, a standard curve was generated from duplicate samples of 5-fold serially diluted known copies of the plasmid pBH10 [22]. Each sample was run in duplicate, and the mean threshold cycle (Ct) value was plotted against the standard curve to obtain the number of unspliced RNA copies in the sample. For the detection of multiply spliced RNA, the PCR assay was performed as described above, with the exception of the primer concentrations, which were 400 nmol/L for primer MS1 and 800 nmol/L for primer MS2. Samples were run in duplicate, and mean Ct values were plotted against the standard curve generated from a 5-fold dilution of an amplicon. This amplicon was obtained by amplifying cDNA obtained from 8E51 cells with the primers MS1 and MS2. Then, 12 μL of cDNA was amplified in a 50-μL final volume that contained 1× PCR Gold Buffer, 200 nmol/L each primer, 200 nmol/L dNTPs, 1.5 mmol/L MgCl2, and 2.5 U of Taq Gold (PE Applied Biosystems). Amplification was performed in a thermal cycler (9600 Perkin Elmer) for 35 cycles, each of which consisted of 1 min at 95°C, 1 min at 60°C, and 1 min at 72°C. The 242-bp amplicon was run on 2% agarose gel, purified using the NucleoSpin Extract kit (M-Medical), and quantified using spectrophotometry. Under these conditions, both assays were sensitive enough to detect 2.5 copies of HIV-1 RNA and showed a dynamic range of at least 5 log10. To normalize HIV-1 RNA, 10 μL of cDNA of each sample was amplified by real-time PCR, under the conditions described above, for the housekeeping gene GAPDH using the GAPDH assay (PE Applied Biosystems). The HIV-1 RNA copies were then expressed relative to 1×107 copies of GAPDH

HIV-1 DNA quantificationQuantitative determination of HIV-1 DNA was performed on lysed PBMCs [23] by real-time PCR, exactly as described elsewhere [15]

ImmunophenotypingCD4+, naive CD4+CD45+RA, and memory CD4+CD45+RO cell counts were evaluated by cytometry, as reported elsewhere [15]. Naive and memory cell counts were assessed only in a subset of children

Statistical methodsChanges in levels of HIV-1 DNA, unspliced HIV-1 RNA, HIV-1 RNA in plasma, and CD4+ cells were estimated using standard normal linear regression, log10 transformed where appropriate, and adjusted for multiple measurements in individual children by use of robust regression [24]. Values below the limit of detection were analyzed using normal interval regression in which the undetectable value was replaced by the interval in which the true value could lie (i.e., the interval up to the cutoff) [25]. In view of significant differences in HIV-1 DNA decay rates during different periods after the initiation of HAART, we used piecewise linear models to describe these variations, with slope change points chosen after a visual inspection of mean values (see Results). The half-life of HIV-1 DNA was calculated using the formula t1/2 = −log10/slope. Spearman&amp;rank correlations were used to estimate pairwise associations

Previous SectionNext Section

Results

Baseline characteristics of the study populationAt baseline, the median age of the study population was 9 years (range, 3–14 years), the median CD4+ cell count was 219 cells/μL (range, 9–611 cells/μL), the mean ± SD plasma HIV-1 RNA level was 4.75±0.59 log10 copies/mL, and the mean ± SD HIV-1 DNA level was 2.85±0.66 log10 copies/1×106 cells (table 1). Intracellular unspliced and multiply spliced HIV-1 RNA was detectable in 13 and 12 children, respectively, at means ± SDs of 2.85±0.76 and 2.00±0.55 log10 copies/1×107 copies of GAPDH

Virological changes after the initiation of HAARTHIV-1 RNA levels in plasma decayed by 1.73 log10 copies/mL (95% confidence interval [CI], 1.25–2.22 log10 copies/mL) and 2.83 log10 copies/mL (95% CI, 2.53–3.14 log10 copies/mL) at 1 and 3 months, respectively, after the initiation of HAART. Thereafter, HIV-1 remained undetectable in plasma (<50 copies/mL) throughout the follow-up period (figure 1A). HIV-1 DNA levels also decayed substantially by 0.17 log10 copies/1×106 cells (95% CI, 0.02–0.32 log10 copies/1×106 cells), 0.34 log10 copies/1×106 cells (95% CI, 0.16–0.52 log10 copies/1×106 cells), and 1.10 log10 copies/1×106 cells (95% CI, 0.78–1.43 log10 copies/1×106 cells) after 1, 3, and 48 months of HAART, respectively (figure 1A). At the last follow-up, HIV-1 DNA was still detectable in all but 2 children (A3 and A13) (figure 2). Although no blips of plasma viremia were detected, a low level of residual viral replication cannot be excluded. We measured levels of intracellular HIV-1 RNA as markers of ongoing viral replication. In addition to unspliced HIV-1 RNA, the multiply spliced HIV-1 RNA encoding tat/rev plays a central role in the retroviral life cycle [26, 27], and its detection has been associated with newly infected cells [28–30]. We found that the decay of multiply spliced RNA was more rapid than the decay of unspliced RNA. Multiply spliced HIV-1 RNA was undetectable after 1 month of HAART in 6 of 8 tested children and had decayed to undetectable levels after 3 months in all but 1 child (A12) (figure 2). After this initial decay, blips of multiply spliced HIV-1 RNA levels were detected in only 2 children (A1 and A5) (figure 2). In contrast, levels of unspliced HIV-1 RNA decayed more slowly and variably. Only 3 of 8 children after 1 month of HAART and 4 of 14 children after 3 months of HAART had undetectable levels of unspliced HIV-1 RNA; these children had ⩾1 blip of detectable levels of HIV-1 RNA during follow-up. Unspliced HIV-1 RNA was detectable in most of the samples (67%), although at significantly lower levels than at baseline, during the first 9 months of therapy and in 34% of the samples thereafter (figure 2)

Figure 1
View larger version:
Figure 1

Changes in HIV-1 and CD4+ cells after the initiation of highly active antiretroviral therapy (HAART). A Change in no. of log10 HIV-1 RNA copies per milliliter of plasma (white squares) and log10 HIV-1 DNA copies per 1×106 cells (white circles). B Changes in nos. of CD4+, naive CD4+CD45+RA, and memory CD4+CD45+RO cells per microliter. Mean values and 95% confidence intervals (CIs; vertical bars) are shown. Naive and memory cells were evaluated only in a subset of children; mean changes in CD4+ cell count in all children and in the subsets of children with and without naive/memory immunophenotyping were quite similar

Figure 2
View larger version:
Figure 2

Kinetics of HIV-1 DNA, unspliced HIV-1 RNA (RNAus), and multiply spliced HIV-1 RNA (RNAms) in children. Viral parameters were estimated by real-time polymerase chain reaction at baseline and subsequently during highly active antiretroviral therapy. The identification codes of patients are reported within each panel

Kinetics of HIV-1 DNA decayThe rate of HIV-1 DNA decay during HAART was not simply exponential—it was faster immediately after the initiation of therapy, which is in agreement with previous observations [11, 31]. The mean changes in HIV-1 DNA levels (figure 1A) suggested that the overall change from baseline could be approximated by a piecewise linear model that included a slope to 1 month (to allow for immediate redistribution effects), from 1 to 9 months, and from 9 months onward. When this model was used, the estimated overall rate of HIV-1 DNA decay per month was 0.258 log10 copies/1×106 cells (95% CI, 0.030–0.486 log10 copies/1×106 cells) in the first month of HAART, 0.054 log10 copies/1 × 106 cells (95% CI, 0.026–0.082 log10 copies/1×106 cells) from 1 to 9 months of HAART, and 0.008 log10 copies/106 cells (95% CI, 0.005–0.022) after 9 months of HAART, with decays in HIV-1 DNA level from 1 to 9 months and after 9 months differing significantly (P=.005). Although fitting this model to the data from each child separately demonstrated that the kinetics of HIV-1 DNA decay were quite heterogeneous, the means of individual child-specific slope were very similar to those obtained from the model, as expected (table 2). From 1 to 9 months of therapy, individual slopes of HIV-1 DNA decay ranged from 0.155 to 0.111 log10 copies/1×106 cells/month, with a mean value of 0.046 log10 copies/1×106 cells and a median value of 0.065 log10 copies/1×106 cells, corresponding to a mean (median) half-life of HIV-1 DNA decay of 7 (5) months. After 9 months of therapy, individual slope values ranged from 0.087 to 0.038 log10 copies/1×106 cells/month, with a mean value of 0.016 log10 copies/1×106 cells and a median value of 0.010 log10 copies/1×106 cells, corresponding to a mean (median) half-life of HIV-1 DNA decay of 19 (30) months. Furthermore, greater decays in HIV-1 DNA levels during the first month of HAART were followed by smaller decays during the subsequent period (r=-0.771; P=.001) (figure 3)

Figure 3
View larger version:
Figure 3

Relationship between slopes of HIV-1 DNA decay (in log10 copies/1×106 cells/month) during the first month of highly active antiretroviral therapy (HAART) and slopes of HIV-1 DNA decay from 1 to 9 months of HAART

Figure 4
View larger version:
Figure 4

A Relationship between slopes of HIV-1 DNA decay and increases in CD4+ cell count during the first month of highly active antiretroviral therapy (HAART). B Relationship between slopes of HIV-1 DNA decay from 1 to 9 months of HAART and increase in CD4+ cell count during the first month of HAART. Because there is a natural but substantial decline in absolute CD4+ cell counts in uninfected children during the first years of life, the relationship between HIV-1 DNA decay and increases in CD4+ cell count was determined using both the absolute CD4+ cell count and the age-adjusted CD4+ cell z score

Table 1
View larger version:
Table 1

Baseline characteristics of the study population

Table 2
View larger version:
Table 2

Decay of HIV-1 DNA

CD4+ cell immune repopulation influence on short-term HIV-1 DNA decayCD4+ cell counts had increased by 518 cells/μL (95% CI, 366–670 cells/μL) at 24 months after the initiation of HAART; thereafter, the CD4+ cell count remained stable (figure 1B). In agreement with previous findings [15, 18, 32], naive CD45+RA cells comprised most of the increase in CD4+ cell counts (figure 1B). The mean slope of the increase in CD4+ cell count per month was 53 cells/μL (95% CI, 6–101 cells/μL) and 43 cells/μL (95% CI, 24–63 cells/μL) during the first month and from 1 to 9 months of HAART, respectively. Thereafter, the mean slope of the increase in CD4+ cell count was 3 cells/μL/month (95% CI, 0.4–6 cells/μL/month). The increase in CD4+ cell count during the first month of HAART correlated with the level of HIV-1 DNA decay during the same period (P=.0289)—that is, the greater the increase in CD4+ cell count, the greater was the HIV-1 DNA decline, but the slope of this correlation was less steep from 1 to 9 months of HAART (P=.067) (figure 4A and 4B). Because there is a natural but substantial decline in absolute CD4+ cell count in uninfected children during the first years of life, results were confirmed using age-adjusted CD4+ cell z scores [33] (figure 4A and 4B). Thereafter, no significant relationship was found between changes in CD4+ cell count and HIV-1 DNA decay (1–9 months, r=-0.279 and P=.338; >9 months, r=0.134 and P=.648)

Influence of unspliced HIV-1 RNA decay on long-term HIV-1 DNA decayUnspliced HIV-1 RNA was either persistently or seldom detected during the first 9 months of HAART in all but 1 child, who tested negative at baseline. The mean slope of unspliced HIV-1 RNA decay per month was 0.673 log10 copies/1×107 copies of GAPDH (95% CI, 0.114–1.232 log10 copies/1×107 copies of GAPDH) during the first month of therapy and 0.103 log10 copies/1×107 copies of GAPDH (95% CI, 0.029–0.176 log10 copies/1×107 copies of GAPDH) from 1 to 9 months of HAART. No significant relationship was observed between the decay of HIV-1 DNA and that of unspliced HIV-1 RNA during the first month or from 1 to 9 months of therapy in individual children. Three children (A2, A9, and A13) (figure 2) were persistently negative for unspliced HIV-1 RNA after 9 months of HAART. Although numbers were small, the rate of HIV-1 DNA decay after 9 months tended to be higher in these 3 children than in 11 children with detectable unspliced HIV-1 RNA after 9 months of HAART (mean decay, 0.048 [range, 0.087–0.006] vs. 0.007 [range, 0.032–0.037] log10 copies/1×106 cells/month; P=.029, Student’s t test; P=.31,&amp;rank sum test). Thus, after 9 months of HAART, HIV-1 DNA tended to decay with a mean half-life of 6 months in the children with no sign of detectable unspliced HIV-1 RNA and a mean half-life of 43 months in children with signs of residual HIV-1 replication. Three additional children (A3, A4, and A7) (figure 2) remained persistently negative after 12 months of HAART; a weak tendency toward greater HIV-1 DNA decay was observed in the 6 children with persistently suppressed unspliced HIV-1 RNA after 12 months (mean decay, 0.028 vs. 0.007 log10 copies/1×106 cells/month in the 8 children with detectable unspliced HIV-1 RNA after 12 months; P=.22, Student’s t test; P=.51,&amp;rank sum test)

Previous SectionNext Section

Discussion

The present article describes the decay of the HIV-1 reservoir in children with long-term sustained suppression of plasma viremia. We evaluated the HIV-1 reservoir by quantifying total HIV-1 DNA in PBMCs. Given the presence of defective genomes and linear and circular forms of unintegrated viral DNA [34, 35], these HIV-1 DNA levels may provide an upper limit for the number of infected cells that exceeds the number of replication-competent provirus genomes. Although the rate of HIV-1 DNA decay after the initiation of HAART varied widely from child to child, we found that the first phase of the to decay in HIV-1 DNA levels, during the first month, was more rapid than the subsequent rate of decay. This is in agreement with our previous observation [31] and with the notion that the clearance of most productive infected cells and labile linear preintegration forms of viral DNA occurs soon after the initiation of HAART [11, 36]. The observed rapid clearance of multiply spliced HIV-1 RNA, a surrogate marker of productive infected cells [37], supports this concept. This initial phase of HIV-1 DNA decay was influenced by the dynamics of increases in CD4+ cell counts. In particular, a large increase in CD4+ cell counts during the first month was associated with a concurrent large amount of HIV-1 DNA decay but with a less steep decay in HIV-1 DNA levels during the immediate follow-up period, from 1 to 9 months of HAART. During HAART, children have a greater and faster capacity for CD4+ cell recovery than adults, mainly because of more active thymopoiesis, and they reconstitute CD4+ cells mainly with naive thymically derived cells [15, 18, 32], which HIV-1 infects poorly [38]. This rapid repopulation by newly uninfected cells is likely to initially dilute the number of HIV-1 DNA–positive cells. Nevertheless, during the first months of HAART, most children still had detectable, albeit decreasing, circulating infectious virus in plasma. In this milieu, circulating antigens may activate naive CD4+ cells to differentiate into memory cells, which are more susceptible to HIV-1 infection [39]. The increase in numbers of CD4+ cells suitable for new cycles of viral infection might negatively affect the subsequent HIV-1 DNA decay. This is in agreement with our previous observation that the decline in HIV-1 DNA levels was influenced negatively by the increase in thymically derived cells in children who did not achieve a sustained suppression of HIV-1 RNA in plasma during antiretroviral treatment [21]

After the initial rapid clearance of productive infected cells, the HIV-1 reservoir in patients with sustained suppression of plasma viremia is mainly composed of latently infected memory T cells [3, 9]. In adults, it has been estimated that this reservoir decayed with a median half-life of 4–6 months [8, 11, 12], which is close to the estimated rate of decay of memory T lymphocytes [10]; however, slower rates of decay have been reported in other studies [9]. All of these studies were performed under the assumption that clearance of the latent HIV-1 reservoir was constant over time. Recently, studies in adults treated with HAART for 3–4 years have indicated that HIV-1 DNA decays more slowly after 1 year (median half-life, 70 weeks) than during the first 1–12 months of therapy (median half-life, 20 weeks) [13]. In agreement with this result, we also observed that decay of the HIV-1 reservoir after the first month is not simply exponential—it is more rapid during the first 1–9 months of HAART (median half-life, 5 months) than after 9 months (median half-life, 30 months). This is consistent with the finding that cellular reservoirs contain cells that clear at different rates and with a progressive increase over time of more-stable, long-lived, latently infected cells to these reservoirs [40]

The rates of clearance of HIV-1 DNA that we observed in children were slower than those estimated in adults [8, 10–14]. Notably, an overall decay of HIV-1 DNA with a median half-life of 14 months was also reported in children with ∼2 years of HIV-1 RNA suppression in plasma [16]. Several factors may influence the rates of clearance of the latent HIV-1 reservoir, including residual viral replication. Although HIV-1 RNA was undetectable in plasma in all measurements after its initial decline and multiply spliced HIV-1 RNA was rarely found during HAART, unspliced HIV-1 RNA was frequently detected during the first 9 months, and blips in levels of unspliced RNA were detected in 11 and 8 children after 9 and 12 months of therapy, respectively. Similar findings of residual unspliced HIV-1 RNA have been reported in both PBMCs [4, 5, 41] and lymphoid tissue [37] in patients receiving effective antiretroviral therapy, whereas RNA species encoding tat/rev were undetected. It cannot be excluded that low levels of multiply spliced HIV-1 RNA, below the detection limit, were present in samples, and the persistence of unspliced HIV-1 RNA might be viewed as a sign of an ongoing productive infection. The detection of unspliced HIV-1 RNA may also reflect ongoing cellular activation with an incomplete pattern of viral transcription and a lack of virion production. Stimulation by antigens may provide an opportunity for latently infected cells to become activated and to initiate the program of viral genome transcription. HIV-1–infected memory CD4+ cells that recognize common antigens may be preferentially activated and cleared during the first phase of latent HIV-1 reservoir decay, whereas other cells, including those that recognize uncommon antigens and other cell types [42–44], may be activated slowly and eventually eliminated at a slower clearance rate. Although further studies on separate cell subsets are needed to clarify the varying contributions of different cell types to the residual viral reservoir, we estimated that this residual reservoir was cleared with a half-life of 6–43 months. A slower rate of decay may be expected when unspliced HIV-1 RNA is present, which suggests that residual viral transcription and replication nearly balance viral clearance. These findings indicate that the eradication of HIV-1 infection is not feasible in chronically infected children. The results of our study should be useful in refining long-term therapeutic strategies

Previous SectionNext Section

Acknowledgments

We thank Pierantonio Gallo for artwork and Lisa Smith for editorial assistance

Previous SectionNext Section

Footnotes

  • ↵Potential conflicts of interest: none reported

    Financial support: Istituto Superiore Sanitá, Progetto AIDS (grants 40F.31 and 45F.13)

  • Received September 28, 2005.
  • Accepted January 25, 2006.
Previous Section
 

References

  1. ↵
    1. Palella FJ Jr.,
    2. Delaney KM,
    3. Moorman AC,
    4. et al
    . Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N Engl J Med 1998;338:853-60.
    CrossRefMedlineWeb of Science
  2. ↵
    1. Gibb DM,
    2. Duong T,
    3. Tookey PA,
    4. et al
    . Decline in mortality, AIDS, and hospital admissions in perinatally HIV-1 infected children in the United Kingdom and Ireland. BMJ 2003;327:1019-25.
    Abstract/FREE Full Text
  3. ↵
    1. Chun TW,
    2. Carruth L,
    3. Finzi D,
    4. et al
    . Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 1997;387:183-8.
    CrossRefMedline
  4. ↵
    1. Furtado MR,
    2. Callaway DS,
    3. Phair JP,
    4. et al
    . Persistence of HIV-1 transcription in peripheral blood mononuclear cells in patients receiving potent antiretroviral therapy. N Engl J Med 1999;340:1614-22.
    CrossRefMedlineWeb of Science
  5. ↵
    1. Fischer M,
    2. Wong JK,
    3. Russenberger D,
    4. et al
    . Residual cell-associated unspliced HIV-1 RNA in peripheral blood of patients on potent antiretroviral therapy represents intracellular transcripts. Antivir Ther 2002;7:91-103.
    MedlineWeb of Science
  6. ↵
    1. Zhang L,
    2. Ramratnam B,
    3. Tenner-Racz K,
    4. et al
    . Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy. N Engl J Med 1999;340:1605-13.
    CrossRefMedlineWeb of Science
  7. ↵
    1. Gunthard HF,
    2. Frost SD,
    3. Leigh-Brown AJ,
    4. et al
    . Evolution of envelope sequences of human immunodeficiency virus type 1 in cellular reservoirs in the setting of potent antiviral therapy. J Virol 1999;73:9404-12.
    Abstract/FREE Full Text
  8. ↵
    1. Ramratnam B,
    2. Mittler JE,
    3. Zhang L,
    4. et al
    . The decay of the latent reservoir of replication-competent HIV-1 is inversely correlated with the extent of residual viral replication during prolonged anti-retroviral therapy. Nat Med 2000;6:82-5.
    CrossRefMedlineWeb of Science
  9. ↵
    1. Finzi D,
    2. Blankson J,
    3. Siliciano JD,
    4. et al
    . Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med 1999;5:512-7.
    CrossRefMedlineWeb of Science
  10. ↵
    1. Hellerstein M,
    2. Hanley MB,
    3. Cesar D,
    4. et al
    . Directly measured kinetics of circulating T lymphocytes in normal and HIV-1-infected humans. Nat Med 1999;5:83-9.
    CrossRefMedlineWeb of Science
  11. ↵
    1. Perelson AS,
    2. Essunger P,
    3. Cao Y,
    4. et al
    . Decay characteristics of HIV-1-infected compartments during combination therapy. Nature 1997;387:188-91.
    CrossRefMedline
  12. ↵
    1. Yerly S,
    2. Perneger TV,
    3. Vora S,
    4. Hirschel B,
    5. Perrin L
    . Decay of cell-associated HIV-1 DNA correlates with residual replication in patients treated during acute HIV-1 infection. AIDS 2000;14:2805-12.
    CrossRefMedlineWeb of Science
  13. ↵
    1. Strain MC,
    2. Little SJ,
    3. Daar ES,
    4. et al
    . Effect of treatment, during primary infection, on establishment and clearance of cellular reservoirs of HIV-1. J Infect Dis 2005;191:1410-8.
    Abstract/FREE Full Text
  14. ↵
    1. Morand-Joubert L,
    2. Marcellin F,
    3. Launay O,
    4. et al
    . Contribution of cellular HIV-1 DNA quantification to the efficacy analysis of antiretroviral therapy: a randomized comparison of 2 regimens, including 3 drugs from 2 or 3 classes (TRIANON, ANRS 081). J Acquir Immune Defic Syndr 2005;38:268-76.
    MedlineWeb of Science
  15. ↵
    1. Ometto L,
    2. De Forni D,
    3. Patiri F,
    4. et al
    . Immune reconstitution in HIV-1-infected children on antiretroviral therapy: role of thymic output and viral fitness. AIDS 2002;16:839-49.
    CrossRefMedlineWeb of Science
  16. ↵
    1. Saitoh A,
    2. Hsia K,
    3. Fenton T,
    4. et al
    . Persistence of human immunodeficiency virus (HIV) type 1 DNA in peripheral blood despite prolonged suppression of plasma HIV-1 RNA in children. J Infect Dis 2002;185:1409-16.
    Abstract/FREE Full Text
  17. ↵
    1. Chavan S,
    2. Bennuri B,
    3. Kharbanda M,
    4. Chandrasekaran A,
    5. Bakshi S
    . Evaluation of T cell receptor gene rearrangement excision circles after antiretroviral therapy in children infected with human immunodeficiency virus. J Infect Dis 2001;183:1445-54.
    Abstract/FREE Full Text
  18. ↵
    1. De Rossi A,
    2. Walker AS,
    3. Klein N,
    4. et al
    . Increased thymic output after initiation of antiretroviral therapy in human immunodeficiency virus type 1–infected children in the Paediatric European Network for Treatment of AIDS (PENTA) 5 Trial. J Infect Dis 2002;186:312-20.
    Abstract/FREE Full Text
  19. ↵
    1. Resino S,
    2. Galan I,
    3. Perez A,
    4. et al
    . HIV-infected children with moderate/severe immune-suppression: changes in the immune system after highly active antiretroviral therapy. Clin Exp Immunol 2004;137:570-7.
    CrossRefMedlineWeb of Science
  20. ↵
    1. Walker AS,
    2. Doerholt K,
    3. Sharland M,
    4. Gibb DM
    . Response to highly active antiretroviral therapy varies with age: the UK and Ireland Collaborative HIV Paediatric Study. AIDS 2004;18:1915-24.
    CrossRefMedlineWeb of Science
  21. ↵
    1. De Rossi Walker S,
    2. De Forni D,
    3. Klein N,
    4. Gibb D
    . Relationship between changes in thymic emigrants and cell-associated HIV-1 DNA in HIV-1-infected children initiating antiretroviral therapy. Antivir Ther 2005;10:63-71.
    MedlineWeb of Science
  22. ↵
    1. Hahn BH,
    2. Shaw GM,
    3. Arya SK,
    4. Popovic M,
    5. Gallo RC
    . Molecular cloning and characterization of the HTLV-III virus associated with AIDS. Nature 1984;312:166-9.
    CrossRefMedline
  23. ↵
    1. De Rossi A,
    2. Masiero S,
    3. Giaquinto C,
    4. et al
    . Dynamics of viral replication in infants with vertically acquired human immunodeficiency virus type 1 infection. J Clin Invest 1996;97:323-30.
    MedlineWeb of Science
  24. ↵
    1. Arnemyia T
    . Regression analysis when the dependent variable is truncated normal. Econometrica 1973;41:997-1016.
    CrossRefWeb of Science
  25. ↵
    1. Huber PJ
    . Proceedings of the Fifth Berkeley Symposium on Mathematical Statistics and Probability. Vol. 1. Berkeley: University of California Press; 1967. The behaviour of maximum likelihood estimates under non-standard conditions; p. 221-33.
  26. ↵
    1. Fisher AG,
    2. Feinber MB,
    3. Josephs SF,
    4. et al
    . The transactivator gene of HTLV-III is essential for virus production. Nature 1986;320:367-71.
    CrossRefMedline
  27. ↵
    1. Malim MH,
    2. Hauber J,
    3. Le SY,
    4. Maizel JV,
    5. Cullen BR
    . The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 1989;338:254-7.
    CrossRefMedline
  28. ↵
    1. Klotman ME,
    2. Kim S,
    3. Buchbinder A,
    4. De Rossi A,
    5. Baltimore D
    . Kinetics of expression of multiply spliced RNA in early human immunodeficiency virus type 1 infection of lymphocytes and monocytes. Proc Natl Acad Sci USA 1991;88:5011-15.
    Abstract/FREE Full Text
  29. ↵
    1. Bagnarelli P,
    2. Valenza A,
    3. Menzo S,
    4. et al
    . Dynamics and modulation of human immunodeficiency virus type 1 transcripts in vitro and in vivo. J Virol 1996;70:7603-13.
    Abstract/FREE Full Text
  30. ↵
    1. Vesanen M,
    2. Markowitz M,
    3. Cao Y,
    4. Ho DD,
    5. Saksela K
    . Human immunodeficiency virus type-1 mRNA splicing pattern in infected persons is determined by the proportion of newly infected cells. Virology 1997;236:104-9.
    CrossRefMedlineWeb of Science
  31. ↵
    1. De Rossi A,
    2. Walker AS,
    3. De Forni D,
    4. Gibb DM
    . Paediatric European Network for Treatment of AIDS (PENTA). Biphasic decay of cell-associated HIV-1 DNA in HIV-1-infected children on antiretroviral therapy. AIDS 2002;16:1961-3.
    CrossRefMedlineWeb of Science
  32. ↵
    1. Gibb DM,
    2. Newberry A,
    3. Klein N,
    4. De Rossi A,
    5. Grosch-Woerner I
    . Immune repopulation after HAART in previously untreated HIV-1-infected children. Lancet 2000;355:1331-2.
    CrossRefMedlineWeb of Science
  33. ↵
    1. Wade AM,
    2. Ades AE
    . Age related reference ranges: significance test for models and confidence intervals for centiles. Stat Med 1994;13:2359-67.
    MedlineWeb of Science
  34. ↵
    1. Nicholson WJ,
    2. Shepherd AJ,
    3. Aw DW
    . Detection of unintegrated HIV type 1 DNA in cell culture and clinical peripheral blood mononuclear cell samples: correlation to disease stage. AIDS Res Hum Retroviruses 1996;12:315-23.
    MedlineWeb of Science
  35. ↵
    1. Sanchez G,
    2. Xu X,
    3. Chermann JC,
    4. Hirsch I
    . Accumulation of defective viral genomes in peripheral blood mononuclear cells of human immunodeficiency virus type 1-infected individuals. J Virol 1997;71:2233-40.
    Abstract/FREE Full Text
  36. ↵
    1. Pierson TC,
    2. Zhou Y,
    3. Kieffer TL,
    4. Ruff CT,
    5. Buck C
    . Molecular characterization of preintegration latency in human immunodeficiency virus type 1 infection. J Virol 2002;76:8518-31.
    Abstract/FREE Full Text
  37. ↵
    1. Fischer M,
    2. Joos B,
    3. Hirschel B,
    4. et al
    . Cellular viral rebound after cessation of potent antiretroviral therapy predicted by levels of multiply spliced HIV-1 RNA encoding nef. J Infect Dis 2004;190:1979-88.
    Abstract/FREE Full Text
  38. ↵
    1. Brenchley JM,
    2. Hill BJ,
    3. Ambrozak DR,
    4. et al
    . T-cell subsets that harbor human immunodeficiency virus (HIV) in vivo: implications for HIV pathogenesis. J Virol 2004;78:1160-8.
    Abstract/FREE Full Text
  39. ↵
    1. Brenchley JM,
    2. Douek DC,
    3. Ambrozak DR,
    4. et al
    . Expansion of activated human naive T-cells precedes effector function. Clin Exp Immunol 2002;130:432-40.
    CrossRefMedline
  40. ↵
    1. Strain MC,
    2. Gunthard HF,
    3. Havlir DV,
    4. et al
    . Heterogeneous clearance rates of long-lived lymphocytes infected with HIV: intrinsic stability predicts lifelong persistence. Proc Natl Acad Sci USA 2003;100:4819-24.
    Abstract/FREE Full Text
  41. ↵
    1. Chun TW,
    2. Justement JS,
    3. Lempicki RA,
    4. et al
    . Gene expression and viral production in latently infected, resting CD4+ T cells in viremic versus aviremic HIV-infected individuals. Proc Natl Acad Sci USA 2003;100:1908-13.
    Abstract/FREE Full Text
  42. ↵
    1. Pomerantz RJ
    . Residual HIV-1 infection during antiretroviral therapy: the challenge of viral persistence. AIDS 2001;15:1201-11.
    CrossRefMedlineWeb of Science
  43. ↵
    1. Sonza S,
    2. Mutimer HP,
    3. Oelrichs R,
    4. et al
    . Monocytes harbour replication-competent, non-latent HIV-1 in patients on highly active antiretroviral therapy. AIDS 2001;15:17-22.
    CrossRefMedlineWeb of Science
  44. ↵
    1. Valentin A,
    2. Rosati M,
    3. Patenaude DJ,
    4. et al
    . Persistent HIV-1 infection of natural killer cells in patients receiving highly active antiretroviral therapy. Proc Natl Acad Sci USA 2002;99:7015-20.
    Abstract/FREE Full Text
| Table of Contents

This Article

  1. J Infect Dis. (2006) 193 (12): 1718-1727. doi: 10.1086/504264
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most