HIV-1 DNA proviral load in treated and untreated HIV-1 seropositive patients

ORIGINAL ARTICLE VIROLOGY HIV-1 DNA proviral load in treated and untreated HIV-1 seropositive patients M. C. Re 1,2, F. Vitone 1, C. Biagetti 3, P. Schiavone 1, F. Alessandrini 1, I. Bon 1, E. de Crignis 1 and D. Gibellini 1 1) Department of Haematology and Oncologic Science, Microbiology Section, 2) Interuniversity Consortium, National Institute Biostructure and Biosystem (INBB), Rome, and 3) Department of Internal Medicine, Geriatric Medicine and Nephrology, Section of Infectious Diseases, University of Bologna, Italy Abstract As proviral human immunodeficiency virus type 1 (HIV-1) DNA can replenish and revive viral infection upon activation, its detection might offer significant therapeutic information, complementing the input provided by plasma RNA determination in the follow-up of infected individuals. A selected group of acutely infected subjects was studied to verify both total and 2-long terminal repeat (2-LTR) DNA proviral load during the acute phase of infection and thereafter. Patients were divided in two sex- and age-matched groups: 19 naive individuals who did not receive antiretroviral therapy during the observation period and 20 subjects treated according to current guidelines. Total and 2-LTR HIV-1 DNA proviral load, in addition to RNA viral load and CD4 cell count, were determined in peripheral blood mononuclear cells (PBMC) at baseline, 6 and 12 months after the first sampling. Total and 2-LTR HIV-1 DNA proviral load exhibited no significant variation at any time in the naive patients (total HIV-1 DNA ranging from 896 ± 731 to 715 ± 673 copies/ 10 5 PBMC and 2-LTR HIV-1 DNA ranging from 94 ± 105 to 65 ± 44 copies/10 5 PBMC), whereas a significant reduction in both total HIV-1 DNA (ranging from 997 ± 676 to 262 ± 174 copies/10 5 PBMC) and 2-LTR HIV-1 DNA proviral load (ranging from 116 ± 55 to 26 ± 35 copies/10 5 PBMC) was detected in highly active antiretroviral therapy (HAART) patients, together with a CD4 + T cell count increase and RNA load decrease. HAART negatively affects both the labile HIV burden and the integrated proviral DNA, at least in the initial period of successful treatment, suggesting that quantification of HIV-1 DNA proviral load may be an important parameter in monitoring HIV infection. Original Submission: 25 August 2008; Revised Submission: 5 January 2009; Accepted: 5 January 2009 Editor: E. Gould Article published online: 2 September 2009 Clin Microbiol Infect 2010; 16: 640 646 10.1111/j.1469-0691.2009.02826.x Corresponding author and reprint requests: M. C. Re, Department of Haematology and Oncologic Science, Microbiology Section, University of Bologna, Italy E-mail: mariacarla.re@unibo.it Introduction Although highly active antiretroviral therapy (HAART) can decrease virus load in plasma to undetectable levels, infection cannot be eradicated due to the perpetuation of viral reservoirs [1,2] established early in the infection. Human immunodeficiency virus (HIV) is persistently stored in a stable form in some specific cell lineages [2 5] and is protected from biochemical decay. The origin of viral rebound is still not completely elucidated, but is tightly linked to the persistence and transcriptional activation of HIV proviral DNA in cellular reservoirs and anatomical compartments [6 11]. Some activated T cells physiologically revert to a resting state due to the reduction or loss of key cellular factors involved in HIV replication. Hence, HIV enters a latent cycle that may be re-activated to a full replication cycle when the resting memory cell is stimulated by specific antigens [1,5]. This process proceeds during chronic infection throughout the lives of infected individuals, creating a complex archive of wild-type and mutant viruses generated throughout active viral replication [2,5,12]. All these observations suggest that the analysis of HIV DNA proviral load, in addition to RNA viral load, could Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases

CMI Re et al. HIV-1 DNA load in HIV-1 seropositive patients 641 be considered a useful marker during the follow-up of infected individuals, to evaluate reservoir status and the antiretroviral efficacy of new, more potent therapeutic regimens. Most studies have focused on total DNA proviral load characterized by quantitative determination of the overall HIV DNA pool [13 22]. The HIV DNA provirus is formed by reverse transcription of viral RNA into double-stranded linear DNA in the cytoplasm. The resulting linear DNA molecule is actively transported to the nucleus as a component of the pre-integration complex, the immediate precursor for the subsequent integration process. Proviral forms are stable structures that remain indefinitely in the host cell genome and serve as templates for viral transcription [5]. Besides the integrated forms, unintegrated forms such as episomal or linear HIV DNA have been observed: episomal HIV DNA is formed after completion of cdna even though unintegrated HIV-1 DNA may assume several monomeric and multimeric forms. This full-length cdna is transported to the nucleus, where a proportion undergoes circularization to form episomes containing one or two long terminal repeats (LTRs). Several groups [23 29] have attempted to address the possible function, activity, and importance of unintegrated episomal HIV DNA during the life cycle of retroviruses and the course of retroviral-associated diseases. It has been shown that the 2-LTR HIV DNA form might be considered a molecular determinant of infectivity [25 29]. Despite a growing number of studies involving the quantification of HIV DNA in HAART-treated patients, few data [14,28,30,31] have been published on HIV DNA in patients before treatment or untreated during the initial phase of infection. In this report, we used quantitative analysis of episomal 2-LTR and total HIV DNA load to investigate the dynamics of the HIV-1 DNA reservoir in naive or subsequently HA- ART-treated patients with acute HIV-1 infection, to establish the possible role of both integrated and 2-LTR DNA load in the evaluation of HIV infection. Materials and Methods Blood samples Blood samples collected from 39 acute HIV-1 seroconverters were included in the study after informed consent was obtained according to the Helsinki Declaration (1975) guidelines. Seroconverters were defined as patients presenting HIV enzyme immunoassay antibody results that evolved from negative/equivocal to positive in closely consecutive samples. The virological and immunological evaluation was performed at the first evidence of HIV-1 seropositivity [samples collected within 6 months after the estimated seroconversion (baseline sample: Time 1), 6 months later or 5 weeks after the beginning of therapy (Time 2) and at the end of the observation period (up to 12 months from baseline: Time 3)]. Intensive medical evaluation ruled out a history of drug abuse and a sexual transmission route was established in all HIV-1-infected subjects. Patients were divided into two groups: untreated group A comprised 19 HIV-1-naive patients who never received TABLE 1. Antiretroviral treatment [two nucleotide reverse transcriptase inhibitors (NRTI) and one non-nucleotide reverse transcriptase inhibitors (NNRTI)] in HIV-1 patients (group B) Antiretroviral treatment NRTI Patient N 1 FTC+TDF EFV Patient N 2 FTC+TDF EFV Patient N 3 ABC+3TC NVP Patient N 4 3TC+TDF NVP Patient N 5 3TC+ABC EFV Patient N 6 AZT+3TC NVP Patient N 7 FTC+TDF EFV Patient N 8 AZT+3TC EFV Patient N 9 3TC+ABC NPV Patient N 10 FTC+TDF EFV Patient N 11 3TC+TDF EFV Patient N 12 AZT+3TC NVP Patient N 13 3TC+ABC EFV Patient N 14 3TC+TDF NVP Patient N 15 FTC+TDF EFV Patient N 16 ABC+3TC NVP Patient N 17 FTC+TDF EFV Patient N 18 AZT+3TC EFV Patient N 19 AZT+3TC EFV Patient N 20 3TC+ABC EFV NNRTI FTC, emtricitabine; TDF, tenofovir; ABC, abacavir; 3TC, lamivudine; AZT, zidovudine; EFV, efavirenz; NVP, nevirapine.

642 Clinical Microbiology and Infection, Volume 16 Number 6, June 2010 CMI any drug treatment during our follow-up period; treated group B comprised 20 patients treated, about 6 7 months after the evidence of acute infection, with a pharmacological cocktail based on an association of two nucleotide reverse transcriptase inhibitors and one non-nucleotide reverse transcriptase inhibitor (Table 1). The two groups were age- and sex-matched (13 men and six women vs. 15 men and five women, and 35 ± 9 vs. 38 ± 9.5 years, respectively). All patients were assessed for antibody detection and avidity index at baseline and during follow-up. RNA, DNA viral load and number of circulating CD4 lymphocytes were determined at baseline and during follow-up. Antibody detection Antibodies were assessed using immunoenzymatic (Vironostika, BioMerieux, the Netherlands) and immunoblot (Chiron Co., Emerville, CA, USA) assays. A guanidine-based avidity detection assay was used to analyse antibody avidity in all sera as previously described [32 34]. CD4 cell count and RNA viral load determination CD4 + lymphocyte count and plasma RNA viral load were determined by flow cytometry and by Quantiplex HIV-1 3.0 assay branched DNA kit (Siemens, East Walpole, MA, USA), respectively. The lowest HIV-1 RNA detection limit was determined to be 50 copies/ml. Determination of HIV-1 proviral DNA in peripheral blood mononuclear cells using SYBR Green real-time PCR Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll HystoPaque gradient separation (Amersham-Pharmacia,Uppsala, Sweden). Cell pellets (5 10 5 PBMC) were stored at )80 C. DNA was extracted and purified by QIAmpDNA mini kit (Qiagen, Hilden, Germany) and the DNA content was determined by spectrophotometric analysis at 260/280 nm. HIV-1 DNA proviral levels were determined by SYBR Green-based real-time PCR assay as previously described [2], using 300 ng of extracted DNA per sample and SK431/SK462 gag primers as previously described [2] to amplify a 142-bp gag fragment. Amplification, data acquisition and analysis were carried out using a LightCycler instrument [LIGHTCYCLER 5.3.2 software (Roche, Mannheim, Germany)]. For each run, a standard curve was generated from a dilution series of DNA extracted from 8E5LAV lymphoblastoid cell line, which contains one integrated copy of HIV-1 genome in each cell. All standard dilutions and samples from patients were run in duplicate and the average value of the copy number was employed to quantify HIV-1 DNA copies in PBMC. Quantitative data concerning the HIV-1 DNA load were expressed as number of copies per 10 5 PBMC. Determination of 2-LTR circles in PBMC using SYBR Green real-time PCR The PCR assay was performed in a final volume of 20 ll, using 2X Quantitect SYBR Green PCR Master Mix (Qiagen) containing HotStarTaq DNA polymerase, 0.5 lm of each oligonucleotide primer and 300 ng of DNA extracted from clinical samples. The sequence of 2-LTR circle junction primers is: 5 -TAG ACC AGA TCT GAG CCT GGG A-3 and 5 -GTA GTT CTG CCA ATC AGG GAA G-3. This set of primers generates a 262-bp amplicon pair. 2-LTR gene amplification was carried out as follows: one cycle of 15 min at 95 C (hot-start PCR) for initial activation, followed by 50 cycles of 95 C for 10 s, 60 C for 30 s, 72 C for 30 s and 78 C for 5 s. A standard curve was generated from scalar dilution (from 10 5 to 10 copies) of plasmid DNA containing 2-LTR circle HIV-1 DNA fragment (a kind gift of M. Capobianchi, Rome, Italy). Results are expressed as 2-LTR circles copy number/ 10 5 cells. Phylogenetic analysis HIV-1 subtypes were determined from HIV-1 pol gene sequences [35]. Subtype assignment was determined by phylogenetic analysis using reference strains of known subtype (Los Alamos database: http://www.hiv.lanl.gov). A neighbourjoining method, using the Kimura 2-parameter distance estimation method, was used to compare the sequences to pairwise distance matrices [36]. The consistency of the phylogenetic clustering was tested using bootstrap analysis with 100 replicates. Statistical analysis Student s t-test, Mann Whitney test and Pearson s correlation coefficient were used to assess the association between virological markers and other variables. Data were expressed as means ± standard deviation (SD) (Table 2) or as median ± SD (Figs 1 and 2). Results Serological evaluation of acute infected HIV-1 patients All samples derived from patients enrolled in the study were examined for classical infection parameters at baseline and subsequently monitored up to 12 months later. The first blood sample (baseline) was collected within 6 months of infection. Infection was established by a first

CMI Re et al. HIV-1 DNA load in HIV-1 seropositive patients 643 TABLE 2. CD4 + T cell count (mean value) (2a) and HIV-1 viral load (mean value) (2b) expressed as RNA copies/ml (mean value) at baseline (T1) and afterwards (T2 and T3) in naive (group A) and treated (group B) patients 2a CD4+ T cells/ll (mean value) Time 1 Time 2 Time 3 Group A patients 698 ± 228 684 ± 231 630 ± 181 Group B patients 413 ± 250 466 ± 256 612 ± 439 2b HIV-1 RNA viral load (mean value) copies/ml Time 1 Time 2 Time 3 Group A patients 1 10 5 ±l 10 5 5 10 4 ±5 10 4 4 10 4 ± 3x10 4 Group B patients 2.3 10 5 ± l.7 10 5 1.2 10 3 ± 3.1 10 3 8.3 10 1 ±1 10 2 FIG. 1. Proviral load total DNA at T1, T2 and T3 in patients belonging to group A (untreated) and B (treated). Total DNA data were expressed as median of log 10 DNA copies/1 10 5 peripheral blood mononuclear cells. Group A total DNA at baseline (T1): median 2.86 log 10 ; at 6 months (T2): median 2.57 log 10 ; at the end of observation period (T3): median 2.72 log 10. Group B total DNA at baseline (T1): median 2.72 log 10 ; at 6 months (T2): median 2.61 log 10 ; at the end of observation period (T3): median 2.24 log 10. serological positive test following a negative analysis performed not more than 6 months earlier. All the samples showed a low avidity index, confirming a recent infection, with a median OD value of 0.59 ± 0.15 at Time 1 and a clear increasing value up to the end of the observation period (0.76 ± 0.1 and 0.86 ± 0.1 at Time 2 and Time 3, respectively). In parallel, Western blotting (WB) showed a serum reactivity to at least one envelope protein associated with two gag HIV-1 proteins in 36 out of 39 patients (classified as positive), and a serum reactivity to gp41 alone in three out of 39 subjects (classified as indeterminate by WB analysis but positive for the presence of provirus by qualitative PCR DNA) (data not shown). Western blots were evaluated following the international criteria for HIV-1 serological interpretation (CDC, 1989). FIG. 2. 2-Long terminal repeat (2-LTR) DNA at T1, T2 and T3 in patients belonging to group A (untreated) and B (treated). 2-LTR data were expressed as median of log 10 2-LTR copies/1 10 5 peripheral blood mononuclear cells. Group A 2-LTR HIV DNA at baseline (T1): median 1.47 log 10 ; at 6 months (T2): median 1.0 log 10 ; at the end of observation period (T3): median 1.75 log 10. Group B 2-LTR HIV DNA at baseline (T1): median 1.74 log 10 ; at 6 months (T2): median 1.40 log 10 ; at the end of observation period (T3): median 1.0 log 10. CD4 cell count and RNA viral load and phylogenetic analysis of HIV-1 coding sequences from plasma-derived virus At baseline patients belonging to untreated group A (Table 2a) showed a mean value of 698 ± 228 CD4 + T cells/ll and patients in treated group B (Table 2b) showed a mean value of 413 ± 250 CD4 + T cells/ll (p 0.00). A constant value (p 0.31) from baseline (T1) onwards was observed in untreated patients (group A), whereas a rescue of CD4 + cells by the end of the observation period (T3) (p 0.08) was recorded in patients who received HAART therapy (group B) during the course of our observation period. HIV-1 RNA viral load analysis of group A showed stable values (Table 2b) over time. As expected, the HIV-1 RNA load of HAART-treated patients (group B) showed a clear decrease at 5 weeks after

644 Clinical Microbiology and Infection, Volume 16 Number 6, June 2010 CMI the start of therapy (T2; p <0.05) (Table 2b) and a further reduction was detectable at 12 months [from 2.3 10 5 HIV RNA copies/ml to very low levels (T3; p <0.05)], concurrent with the increase in CD4 + T cell count already described above. Phylogenetic analysis of HIV-1 pol gene (RT and PR) sequences amplified directly from plasma revealed that all patients were infected with subtype B virus (data not shown) Total HIV-1 DNA proviral and HIV-1 2-LTR DNA proviral load Total HIV-1 DNA proviral load did not exhibit any appreciable difference (Fig. 1) from T1 to T3 (median 2.86 log 10 (T1); 2.57 log 10 (T2); and 2.72 log 10 (T3), p >0.1) in naive patients (group A). On the other hand, PBMC total HIV-1 DNA proviral load in HAART-treated patients (group B) decreased significantly between T1 and T3 (median 2.72 log 10 vs. 2.24 log 10; p 0.00). In parallel, assaying the 2-LTR DNA episomal form in naive patients gave median values of 1.47 log 10, 1.0 log 10 and 1.75 log 10 at T1, T2 and T3, respectively, (T1 = median) and a progressive and significant decrease between T1 and T3 (median 1.74 log 10 vs. 1.00 log 10 ; p 0.02) over time in HAART-treated patients (Fig. 2). Interestingly, 13 out of 20 subjects belonging to group B showed undetectable amounts of HIV-1 2-LTR episomal DNA at T3 (the sensitivity limit of the technique is 10 copies/1 10 5 PBMC). PBMC HIV-1 2-LTR DNA load showed a significant correlation with RNA load (p < 0.001) and only an inverse trend with CD4 cell count (p < 0.009). Although both treated and untreated patients showed a decrease in 2-LTR DNA between T1 and T2, the quantification of the 2-LTR circular HIV DNA showed that treated patients showed a lower level of 2-LTR DNA content and a net decrease of >0.5 log 10 (1.74 log 10 vs. 1.00 log 10 ) at the end of our observation period. Discussion HIV-1 proviral DNA load is an important marker for monitoring the HIV-1 reservoir. Low levels of ongoing, persistent HIV replication may contribute to the longevity of the latent viral reservoir by continually fuelling new rounds of infection in resting CD4 + T cells. The quantification of HIV DNA in peripheral blood cells, in addition to RNA viral load in patients plasma, may be extremely important for disease monitoring, especially in HAART-treated patients [23,27, 37 39], when RNA viral load is undetectable by current techniques [25,30,40,41]. This report studied total and 2-LTR circular HIV-1 DNA proviral load, by SYBR Green-based real-time PCR, a useful and reliable approach for determining genome regions with a high mutation rate [41 43]. Standardized methods for the measurement of the two most significant forms of proviral DNA, total and non-integrated, are currently lacking, despite the widespread use of molecular biology techniques. In line with other reports [18,44,48], our molecular approach, performed in acutely infected patients with 1 year of follow-up, demonstrated that HAART-treated individuals show a significant decrease in both total and 2-LTR circular HIV-1 DNA proviral load in PBMC compared with naive patients (coupled with a significant increase in CD4 cell count and a drop in viral replication). These findings confirm that HIV-1 reservoir decay correlates with therapeutic effectiveness [18,49 51]. Persistence of HIV-1 reservoirs might result in viral rebound when therapy is interrupted or discontinued [16,21,37]. It is reasonable to assume that decreasing DNA loads reflect at least partial exhaustion of HIV-1 reservoirs. Interestingly, when we focused on the 2-LTR HIV-1 DNA load, which is considered to be a molecular determinant of infectivity, we also observed a significant and longitudinal decrease in the HAART group compared with the group of naive subjects. This suggests a direct role of therapy, starting very early after the estimated seroconversion. We were able to determine low levels of detectable 2-LTR HIV-1 DNA load in 13 out 20 group B subjects (65% of the population examined), in agreement with other reports [23,29]. Previous research [25,27] has demonstrated that at least 50% of individuals given long-standing treatment, and having undetectable viral loads for sustained periods of time, had detectable 2-LTR circular DNA, showing that labile replication intermediates are present in a substantial proportion of HIV-1-infected individuals. Since our study focused on a small group of patients, with follow-up to 12 months after certification of acute infection, the few patients with undetectable 2-LTR amounts could be explained by a transitory decrease due to a partial but consistent pause in viral replication. In fact, we found a decrease in both total and 2-LTR DNA in HAART-treated patients at 5 weeks after the beginning of therapy, which was associated with an increased CD4 cell count. The persistence of small amounts of 2-LTR HIV-1 DNA in PBMC from some patients demonstrates that a small rate of replication is retained even when HAART is substantially effective. These observations confirm that HAART could not eradicate the infection because HIV is able to replicate

CMI Re et al. HIV-1 DNA load in HIV-1 seropositive patients 645 even in the presence of effective therapy. Plasma-based viral RNA assays may fail to demonstrate the full extent of viral activity. By contrast, a trend of increasing 2-LTR DNA load in naive patients emphasizes that viral replication is constantly active in this group. The absence of therapy can lead to continuous viral replication with the further accumulation of proviral load and also 2-LTR labile forms. There have been contrasting reports on the implications of DNA proviral content in HIV-1 patients [30,43,45 47,51 53]. However, our data, obtained for closely controlled patients, emphasize the importance of DNA proviral quantification in HIV-1 patients. Viral persistence provides a mechanism for life-long persistence of replication-competent HIV-1, rendering hopes of virus eradication with current antiretroviral regimens unlikely. Although RNA viral load provides important information on viral replication, HIV-1 DNA proviral load can be considered an additional marker to provide crucial information. Only a thorough evaluation of both virological markers will fully characterize the course of HIV-1 infection and provide a more complete laboratory-based assessment of disease progression. In conclusion, the availability of a new standardized assay to determine DNA proviral load will be important in assessing the true extent of virological suppression and in verifying the efficacy of new immune-based therapies aimed at purging HIV-1 DNA reservoirs. Transparency Declaration This work was funded by Fondazione Cassa di Risparmio Bologna, Italy (no 2006.0035, June 2006), AIDS projects (30G.27) of the Italian Ministry of Health, the SIVIM study group for test standardization, Funds for selected research topics of the University of Bologna and MURST 60%. 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