The use of potent combination antiretroviral therapies in the treatment of HIV-1-infected individuals can reduce plasma HIV-1 RNA to levels below the limit of detection for long periods of time [1,2]. Although a mathematical model initially suggested that 2.3-3.1 years of complete viral replication suppression could eradicate the HIV-1 infection , the presence of HIV-1 DNA and an inducible latent viral reservoir in peripheral blood mononuclear cells (PBMCs) and lymph nodes (LNs) has been documented, despite prolonged suppression of plasma viraemia [4-7]. This latent HIV-1 reservoir is mainly located in resting CD4 T cells, and represents the major obstacle to virus eradication [8,9]. Furthermore, the initiation of highly active antiretroviral therapy (HAART) early after primary infection failed to prevent the establishment of this reservoir of latently infected resting CD4 T cells .
HIV-1 proviral DNA quantification is needed to monitor highly active antiretroviral regimens aimed at virus eradication. Reduction of the unintegrated HIV-1 DNA load has been shown after different effective antiretroviral therapies [10-13]. However, little is known about the dynamics of integrated HIV-1 proviral DNA after a long period of plasma viral suppression. The importance of integrated HIV-1 proviral DNA dynamics is highlighted by the fact that is mainly located in resting memory CD4 T cells , and such cells are probably one of the main HIV-1 reservoirs after HAART [6,7,10]. Cross-sectional analyses in patients receiving HAART have recently demonstrated the presence after therapy of unintegrated and integrated HIV-1 DNA in resting CD4 T cells, even in patients treated early after primary infection [6,10]. Nevertheless, data is lacking about the long-term longitudinal dynamics of total unintegrated and integrated provirus in patients under HAART.
In the present study, we analysed the impact of long-term virus suppression on the unintegrated and integrated HIV-1 DNA loads in 10 patients receiving HAART. We longitudinally compared the unintegrated and integrated DNA viral load in peripheral blood CD4 T cells obtained at baseline and at 48 weeks of triple antiretroviral therapy.
Materials and methods
Ten drug-naive HIV-1 seropositive patients who started a triple combination therapy were selected for this study. Six patients were treated with indinavir (IND)-lamivudine (3TC)-stavudine (D4T) and the other four patients were treated with ritonavir (RTV)-3TC-D4T. The clinical characteristics of the patients are shown in Table 1.
Total RNA was extracted from plasma by the RNA extraction procedure of the Amplicor monitor assay (Roche Diagnostic Systems, Madrid, Spain). Virion-associated HIV-1 RNA was measured by the Amplicor monitor assay (Roche) at baseline and at weeks 2, 4, 8, 16, 32 and 48. In addition, samples obtained at week 48 were measured by the Ultrasensitive assay (limit of detection, 20 copies per ml); this assay was carried out as described previously [15,16].
Total PBMC DNA was prepared by suspending 2¥106 cryopreserved Ficoll-banded PBMCs in 400μl lysis buffer (100mM KCl, 10mM Tris-HCl (pH8.3), 2.5mM MgCl2, 1%Tween-20, 1% Nonidet P-40). The cell suspension was treated with proteinase K at a final concentration of 100μg/ml. After 60min at 60°C, 200μl 6% Instagene Matrix (BioRad Laboratories, Hercules, CA, USA) was added, after 15min at 60°C the proteinase K was denatured by heating the lysate at 100°C for 8min. After two phenol-chloroform extractions and ethanol precipitation, samples were resuspended in 200μl TE buffer [10mM Tris-HCl, 1mM ethylenediamine tetraacetic acid (EDTA); pH8.0] and the PBMC genomic DNA concentration was quantified by ultraviolet light absorbance.
Quantification of integrated and total HIV-1 DNA
HIV-1 DNA quantification was performed by endpoint limiting dilution, as described previously . Duplicate fivefold serial dilutions of PBMC genomic DNA equivalent to 200000, 40000, 8000, 1600 and 320 cells were amplified by a nested polymerase chain reaction (PCR) capable of amplifying a single proviral molecule. Genomic DNA from ACH-2 cells carrying one copy of an integrated form of HIV-1 was used as control DNA. The integrated HIV-1 DNA was amplified by using the oligonucleotides, Alu-long terminal repeat (LTR) 58: 58-TCCCAGCTACTCGGGAGGC TGAGG-38 (nt 164 to 187 of the Alu consensus sequence ) and Alu-LTR 3‚‚: 58-AGGCAAGCTT TATTGAGGTTAAGC-38 (HIV-1 HXB2 positions 516 to 540). PCR protocol was as described by Chun et al. , except that cycling parameters were: 55 (30s), 72 (3min) and 95°C (30s) for 35 cycles with a final extension step at 72°C for 7min. A 5μl aliquot was reamplified in a 100μl reaction mix using the oligonucleotides NI-2 58: 58-CACACACAAGGCTACTTC CCT-38 (HIV-1 HXB2 positions 56 to 77) and NI-2 38: 58-GCCACTCCCCIGTCCCGCCC-38 (HIV-1 HXB2 positions 389 to 408). The cycling profile was: 55 (30s), 72 (30s) and 95°C (30s) for 35 cycles with a final extension step at 72°C for 7min. The total HIV-1 DNA copy number was quantified with oligonucleotides NI-2 58 and NI-2 38 for the first PCR reaction. The cycling profile was: 55 (30s), 72 (30s) and 95°C (30s) for 35 cycles with a final extension step at 72°C for 7min. A second PCR reaction was carried out with oligonucleotides NI-3 58: 58-TGGCAGAAC TACACACCAGG-38 (HIV-1 HXB2 positions 81 to 100) and NI-3 38: 58-GAAAGTCCCCAGCGG AAAGTCCC-38 (HIV-1 HXB2 positions 350 to 372); the cycle profile was as above. To verify PCR amplification, 1/20 of the nested PCR mixture was run on a 3% agarose gel. After ethidium bromide gel staining, the HIV-1 DNA copy number was determined using a Poisson probability distribution implemented by the statistical computer program QUALITY . Briefly, this computer program describes a statistical method used to estimate copy numbers from PCR limiting dilution assays and the associated standard error of the estimate.
The distribution of RNA and DNA HIV-1 loads and the CD4 T cell counts between baseline and the end time points from each patient were subjected to parametric and non-parametric statistical treatment by using the Student‚s t-test and the Wilcoxon signed rank test, respectively, included in the SPSS version 7.5 software package (SPSS, Inc., Chicago, IL, USA).
All study patients were comparable starting HIV-1 RNA levels of 4-5 log10 units/ml of plasma and CD4 T cell counts between 300 and 1140/ml (median=616 cells/ml) (Table 1). Eight out of 10 patients reached undetectable plasma HIV-1 RNA levels (<200 copies/ml) at week 8; patients D and E achieved undetectable levels at weeks 16 and 24, respectively (Table 1). Plasma HIV-1 RNA remained undetectable (<200 copies/ml) in 10 patients during the study period, only patient F showed a transient viral rebound at 32 weeks of therapy. Furthermore, at the time when HIV-1 DNA load quantification was performed (at 48 weeks from baseline), plasma samples were measured by the Ultrasensitive assay (limit of detection of 20 copies per ml). In patients E and J the HIV-1 RNA was detectable (23 and 117 copies/ml, respectively), whereas in the remaining patients it was undetectable (Table 1). A gradual significant increase in CD4 T cell counts were observed (median=937 cells/μl) (P=0.007, Wilcoxon signed rank test) after 48 weeks of antiretroviral therapy. Taken together, these results suggest that long-term viral suppression and treatment success were achieved in all the study patients.
The detection and quantification of integrated and total HIV-1 DNA in PBMCs was performed using a previously described Alu-LTR PCR method [6,10,14]. Because sequence analysis of the human DNA flanking sites of HIV-1 integration has revealed that in 59% of the integration events a human Alu element was found within 600 base pairs of the provirus , the nested PCR protocol used in this study to quantify integrated HIV-1 DNA allowed the detection of integrated HIV-1 DNA with a similar sensitivity to that found for total viral DNA. To eliminate the possibility of cross-sample contamination contributing to the positive PCR signals, the last two DNA dilution-positive PCR reactions for integrated and total DNA from each patient and each time point were sequenced. Phylogenetic analysis of all sequences showed distinct clusters of viral sequences for each patient (data not shown). Likewise, identity was not found with sequences from molecular clones used in our laboratory (data not shown).
The quantification of integrated and total HIV-1 DNA load was carried out in all study patients at baseline and at 48 weeks of HAART (Table 1), and the distribution of these values was determined for each time point and for both HIV-1 DNAs (Fig. 1). The integrated and total HIV-1 DNA were detected in all patients and at the two time points analysed (Table 1). In the absence of therapy most HIV-1 DNA was unintegrated (Table 1 and Fig. 1), as shown previously [14,20]. The median of integrated HIV-1 DNA at baseline was 215 copies per 106 PBMCs, whereas the median total HIV-1 DNA was 2792 copies per 106 PBMCs. At baseline the number of copies of total HIV-1 DNA and the number of copies of integrated HIV-1 were significantly different (P=0.005). Similarly, when the plasma viral load and total HIV-1 DNA from the baseline were compared there was also a strong trend towards statistically significant correlation (P=0.005). A statistically significant fivefold decrease in total HIV-1 DNA was observed after therapy (P=0.005) (Fig. 1). The same analysis was computed by contrasting the baseline data and that obtained from four patients 20 to 64 weeks before the beginning of the therapy (Table 1). These results were shown to be statistically no different from the baseline data (P=0.273, for total HIV-1 DNA and P=0.715, for integrated HIV-1 DNA), suggesting that the above observed decrease of HIV-1 DNA was caused by HAART.
After 48 weeks of therapy, the integrated HIV-1 DNA copy number (median=110 copies/106 PBMCs) was lower than the baseline copy number (median=215 copies/106 PBMCs), but no statistically significant change was noted (Fig. 1) (P=0.333). Therefore, in the study cohort the total amount of HIV-1 DNA decreased drastically after 48 weeks of therapy. whereas the integrated DNA remained constant during this period (Fig. 1). When parametric statistics (Student‚s t-test) were used to construct confidence intervals on the observed changes in the HIV-1 DNA after the introduction of HAART, no differences were found to those shown using non-parametric statistics (data not shown). Finally, the total (median=500 copies/106 PBMCs) and integrated (110 copies copies/106 PBMCs) HIV-1 DNA load distribution after 48 weeks of HAART were compared. A statistically significant difference was found (P=0.005), suggesting that the unintegrated HIV-1 DNA, in most patients, was distinguishable from the integrated DNA after 48 weeks of plasma virus suppression.
We have shown that an overall fivefold reduction in the total HIV-1 DNA copy number occurs in patients after 48 weeks on HAART. However, this significant reduction in the total HIV-1 DNA load is not accompanied by a reduction in the integrated DNA load. Because the integrated HIV-1 DNA is mainly located in resting memory CD4 T cells , our results reveal that frequencies of latently infected CD4 T cells do not decrease appreciably with increasing time on HAART, as had been suggested previously [6,7,10]. Because of the limitations of our dataset (10 patients), additional studies are needed to assess the impact of HAART on the PBMC HIV-1 DNA in larger and more diverse populations. Recently, on the basis of the half-life decay of total proviral DNA in PBMCs, found to be 3-5 months , it has been estimated that 5-7 years of completely inhibitory therapy are required for a latent CD4 T cell HIV-1 reservoir pool size of 104 to 106 to decay to <1 . Considering the stability of the integrated HIV-1 found in the present study (Fig. 1) after 48 weeks of successful HAART, straight calculations of HIV-1 eradication have to be taken with caution. Future studies for longer periods of time will be required to ascertain whether this integrated HIV-1 DNA reservoir will significantly decrease. Therefore treatment approaches aimed at virus eradication should consider the activation of this latently infected CD4 T cell reservoir in order to remove all residual provirus. Different strategies including different cytokines or HIV-1 vaccines have been suggested [8,9]. In the current study we present an easy nested PCR protocol that can be used to monitor changes in integrated HIV-1 DNA, which is probably one of the main HIV-1 reservoirs after HAART.
In a previous cross-sectional study, which analysed the integrated and total HIV-1 DNA load in resting CD4 T cells from infected patients receiving HAART, levels of unintegrated HIV-1 DNA 28-fold higher than integrated HIV-1 DNA were found . This was correlated with persistent virus replication after HAART because unintegrated HIV-1 DNA is thought to have a short half-life in vivo and its presence is associated with ongoing virus replication. Recently, a similar result was found in resting CD4 T cells from patients treated with HAART early after primary infection . It is a matter of controversy whether active virus replication is ongoing in patients with a prolonged undetectable plasma HIV-1 RNA [6,21,22]. Although the presence of ongoing replication after 48 weeks of HIV-1 plasma suppression caused by HAART is out of the scope of the present study, we have found that, in most patients, after 48 weeks of HAART the amount of integrated HIV-1 DNA was lower than the amount of total DNA (Table 1 and Fig. 1), which difference is statistically significant (P=0.005). Whether active replication is ongoing in these patients is currently under investigation.
Quantification of HIV-1 DNA has been proposed as the next step for monitoring patients with undetectable plasma viral loads . The data presented here that account for a high reduction of total HIV-1 DNA after one year of HAART but not for a significant reduction in the HIV-1 integrated DNA suggest that clinical trials aimed to address virus eradication should include the analysis of this integrated HIV-1 DNA reservoir.
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