Impact of 5 years of maximally successful highly active antiretroviral therapy on CD4 cell count and HIV-1 DNA level

OBJECTIVES: To evaluate the impact on CD4 cell count and HIV-1 DNA level in peripheral blood mononuclear cells (PBMC) of long-term highly active antiretroviral therapy (HAART) in the setting of maximal success, i.e., constant plasma HIV-1 RNA load suppression. DESIGN: Retrospective analysis of patients selected for a constantly undetectable plasma HIV-1 RNA load since HAART initiation. METHODS: HIV-1 DNA was measured in PBMC using a real-time polymerase chain reaction assay. Loess estimates and regression analysis were used for modelling the variations of the CD4 cell count and HIV DNA level over time. RESULTS: The study included 41 patients chronically infected with HIV-1 who had been taking HAART for a median duration of 60.4 months and had an undetectable plasma HIV RNA load ever since the first 6 months of HAART; 25 were tested for HIV-1 DNA. The mean CD4 cell count increase was high during the first 18 months on therapy (168 x 10 cells/l per year), much lower afterwards (38 x 10 cells/l per year), independently of the baseline CD4 cell count. Most of the patients (73.2%) reached a CD4 cell count constantly > or = 400 x 10/l during follow-up. HIV-1 DNA showed a mean decrease of 0.48 log10 copies/10 PBMC during the first year, of 0.18 log10 copies/10 PBMC per year during the 2nd and 3rd years, but no significant decrease afterwards. CONCLUSIONS: These results question the benefit of very long-term maintenance of HAART in terms of CD4 gain and HIV-1 DNA reduction.     

Proviral HIV-1 DNA in subjects followed since primary HIV-1 infection who suppress plasma viral load after one year of highly active antiretroviral therapy

OBJECTIVE: An assessment of the impact of one year potent antiretroviral treatment initiated during primary HIV infection on the cell-associated viral burden. DESIGN AND METHODS: Proviral HIV-1 DNA was quantified in serial peripheral blood mononuclear cell (PBMC) samples from 19 patients enrolled in the French prospective PRIMO Cohort for whom plasma HIV RNA was suppressed to undetectable levels after one year of triple therapy; that is, plasma HIV-1 RNA was maintained below 200 copies/ml. Results were compared with those observed in 19 patients with chronic HIV-1 infection presenting the same degree of virus suppression after 12 months of treatment. RESULTS: At study entry, PRIMO subjects presented heterogeneous levels of proviral HIV-1 DNA: 2-3.92 log10 copies/10(6) PBMC and plasma HIV RNA: 2.3-6.5 log10 copies/ml. One year of effective highly active antiretroviral therapy (HAART) resulted in a median diminution of proviral DNA of -0.78 log10/10(6) PBMC in PRIMO subjects. The median decline in chronic-phase patients was -0.32 for those who were pre-treated and -0.52 for those previously naive of treatment. CONCLUSION: The decline in cell-associated HIV DNA observed throughout one year treatment indicated that HAART reduces the proviral HIV-DNA load more effectively when initiated during the primary rather than the chronic phase of HIV infection. These findings therefore tend to lend support to the early initiation of treatment. Nevertheless, heterogeneous baseline values observed for CD4 cell count, plasma HIV RNA and proviral HIV DNA in PRIMO subjects, raise the question of whether treatment should be delayed in some to spare early adverse effects of HAART.     

Highly active antiretroviral treatment initiated early in the course of symptomatic primary HIV-1 infection: results of the ANRS 053 trial.

Highly active antiretroviral treatment (HAART) was given early to 64 patients with symptomatic primary human immunodeficiency virus (HIV)-1 infection. At the time of analysis, patients had been followed up for 9-21 months. No patient had died or developed an AIDS-defining event. Survival analysis showed that by month 21 the proportion of patients with plasma HIV-1 RNA <50 copies/mL was 72% (95% confidence interval, 58%-95%) in intention-to-treat analysis. After 18 months of treatment, 50% of the patients with undetectable plasma HIV-1 RNA also had undetectable HIV-1 RNA in peripheral blood mononuclear cells (PBMC). Only 1 of 3 patients had undetectable HIV-1 RNA in lymphoid tissue, while all patients had quantifiable HIV-1 DNA both in PBMC and lymphoid tissue. The median CD4 lymphocyte increase from baseline was 230 cells/microL. These preliminary results support the use of HAART in patients with primary HIV-1 infection.     

Long-term decay of the HIV-1 reservoir in HIV-1-infected children treated with highly active antiretroviral therapy

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.     

Quantification of integrated and total HIV-1 DNA after long-term highly active antiretroviral therapy in HIV-1-infected patients.

OBJECTIVE: To assess the impact of long-term virus suppression on the peripheral blood CD4 T cells integrated and total HIV-1 DNA loads in patients receiving highly active antiretroviral therapy (HAART). METHODS: A total of 10 HIV-1-infected patients receiving a triple combination therapy (two nucleoside analogues and one protease inhibitor) were longitudinally studied to compare integrated and total HIV-1 DNA loads. HIV-1 DNA quantification was performed using a quantitative nested polymerase chain reaction (PCR) on genomic peripheral blood mononuclear cell (PBMC) DNA obtained at baseline and at 48 weeks of HAART. RESULTS: All the study patients showed an early and sustained decrease in plasma HIV-1 RNA to below the limit of detection (200 copies/ml). Concordant with the plasma viral decline, a significant increase in the CD4 T cell count was observed (P = 0.007). A statistically significant fivefold decrease in total HIV-1 DNA was detected after 48 weeks of HAART (P = 0.005). However, no statistically significant change was noted after the therapy when the integrated HIV-1 DNA copy number was compared (P = 0.333). Taken together, these results suggest that in the patients analysed the integrated HIV-1 DNA does not decay rapidly after HAART. CONCLUSION: Within the study cohort the total amount of PBMC HIV-1 DNA decreased drastically after 48 weeks of HAART. Nevertheless, the integrated HIV-1 DNA did not significantly decay during this period. Although the data presented here are limited by the number of patients analysed, our findings suggest that 48 weeks of HAART does not significantly reduce the integrated HIV-1 proviral DNA load in the latently infected CD4 T cell reservoir.     

The mutational archive in proviral DNA does not change during 24 months of continuous or intermittent highly active antiretroviral therapy

Objectives The aim of the study was to determine the modifications of the mutational archive in proviral HIV-1 DNA occurring during 24 months of intermittent or continuous highly active antiretroviral therapy (HAART).
Methods The study population included subjects enrolled in the Istituto Superiore di Sanità Pulsed Antiretroviral Therapy (ISS PART) clinical trial. All of these patients were on first-line HAART and had plasma HIV-1 RNA below 50 HIV-1 RNA copies/mL. A genotypic resistance test was performed on HIV-1 DNA extracted from peripheral blood mononuclear cells (PBMC) at baseline and after 24 months of follow-up. Resistance-associated mutations (RAMs) were defined according to the International AIDS Society (IAS) USA classification.
Results Sixty-nine subjects were included in the study [36 enrolled in arm A of the ISS PART (continuous HAART) and 33 enrolled in arm B (intermittent HAART)]. No major modifications of the mutational archive were found in either group after 24 months of follow-up, in terms of both the proportion of subjects with mutations and the total number of mutations.
Conclusions In this patient population, the mutational archive in HIV-1 DNA extracted from PBMC was stable for 24 months, irrespective of HAART modality, whether continuous or intermittent.     

Maternal HIV-1 DNA load and mother-to-child transmission

While many factors contribute to mother-to-child transmission (MTCT) of HIV-1, maternal plasma HIV-1 RNA viral load (RNA-VL) has been consistently found as the main risk factor, including when antiretroviral prophylaxis was used to prevent MTCT. However the predictive value of RNA-VL is poor. A recent study of HIV-1-positive pregnant women who did not receive antiretroviral prophylaxis reported an association between HIV-1 DNA viral load (DNA-VL) and MTCT that was stronger than the association between RNA-VL and MTCT. We sought to determine if HIV-1 DNA-VL was independently associated with MTCT of HIV in a population of women who received zidovudine prophylaxis during pregnancy and whose infants received zidovudine after birth. Patients were 33 non-breastfeeding transmitting (TR) and 33 nontransmitting mothers (NTR) from Perinatal HIV Prevention Trial (PHPT-1), a multicenter clinical trial conducted in Thailand comparing zidovudine prophylaxis durations to prevent MTCT. TR and NTR mothers were matched according to baseline RNA-VL. Maternal peripheral blood mononuclear cell (PBMC)-associated HIV-1 DNA was extracted from whole blood, and DNA-VL was established by quantitative real-time polymerase chain reaction. We found that TR had a significantly higher cell-associated HIV-1 DNA viral load than did NTR. Median TR DNA-VL was 2.54 log(10) copies per microgram PBMC DNA, while it was 2.28 log(10) copies per microgram PBMC DNA in NTR (Wilcoxon p = 0.02). In summary, HIV-1 DNA viral load was associated with MTCT in a population of women who received antiretroviral prophylaxis during pregnancy, independently from RNA viral load.     

Cellular HIV-1 DNA load predicts HIV-RNA rebound and the outcome of highly active antiretroviral therapy

OBJECTIVE: To assess whether cellular HIV-1 DNA prior to highly active antiretroviral therapy (HAART) initiation predicts its outcome. DESIGN AND METHODS: Patients included all 51 hemophiliacs of the Greek component of the Multicenter Hemophilia Cohort Study who had initiated HAART and for whom cryopreserved lymphocyte samples before HAART initiation were available. Cellular HIV-1 DNA quantification was performed by a molecular beacon-based real-time PCR assay in multiple samples per patient with a median (interquartile range) follow-up of 76 (45-102) weeks. RESULTS: The median (range) baseline HIV-1 DNA load was 297 (< 10 to 3468) copies per 1 x 10(6) peripheral blood mononuclear cells. Baseline HIV-1 DNA load did not predict initial virological response (VR). None of the patients with initial VR and baseline HIV-1 DNA load at or below the median experienced a subsequent virological rebound, while the cumulative probability of virological rebound by week 104 was 55% among those with HIV-1 DNA load greater than the median (P < 0.008). Cellular HIV-1 DNA load was the only parameter associated with sustained virological response as shown by univariate or multivariate analyses [adjusted odds ratio (95% confidence interval) 0.197 (0.048-0.801) per 1 log10 increase in DNA copies, P = 0.023]. CONCLUSION: Low cellular HIV-1 DNA load is a marker of sustained virological response in patients with initial VR and it can reliably predict the long-term success of HAART.     

Decay of cell-associated HIV-1 DNA correlates with residual replication in patients treated during acute HIV-1 infection

OBJECTIVES: To evaluate the decay rate of cell-associated HIV-1 RNA and DNA and to identify factors associated with residual viral load in patients treated at the time of primary HIV-1 infection. PATIENTS: A group of 15 patients adherent to highly active antiretroviral therapy (HAART) with sustained undetectable HIV-1 viremia for at least 24 months. METHODS: Viremia, cell-associated HIV-1 RNA and DNA in blood and lymph node mononuclear cells were measured using ultrasensitive assays. RESULTS: Viremia decreased rapidly in all patients; HIV RNA remained < 3 copies/ml in nine patients and fluctuated between 3 and 50 copies/ml in five patients and between 50 and 200 copies/ml in one patient. Decay rates of cell-associated RNA and DNA presented an inflexion point at 1 and 3 months, respectively: first-phase mean half-lives were 0.15 and 0.84 months, respectively, and second-phase mean half-lives were 13.7 and 6.6 months, respectively (95% confidence interval 4.4-13.8). The second phase decay rates were markedly slower, with a DNA decay rate that was highly associated with the mean levels of cell-associated RNA measured in blood from 6 to 33 months (P= 0.001) and in lymph nodes collected at 14 months (P= 0.02). CONCLUSIONS: The clearance of HIV-1 infected cells is correlated with the extent of viral replication as measured by cell-associated RNA levels in both blood and lymph nodes. Quantification of cell-associated RNA and DNA further defines treatment efficacy in 'aviremic' patients.     

Peripheral blood Dendritic cells are not a major reservoir for HIV type 1 in infected individuals on virally suppressive HAART

Dendritic cells (DCs) are potent antigen-presenting cells, and their physiological localization in tissues that interact with the external environment is important as a first barrier against pathogens such as human immunodeficiency virus type I (HIV-1). Several models have been proposed to explain the possible role of DCs as a reservoir for HIV-1 in patients on virally suppressive highly active antiretroviral therapy (HAART). However, the low yield of cell isolates has made this evaluation a difficult task. The present study analyzes whether peripheral blood DCs from HIV-1-infected individuals on virally suppressive HAART, with plasma HIV-1 RNA levels of less than 50 copies/ml, carry either HIV-1 provirus and/or HIV-1 virions. Peripheral blood DCs were isolated from a cohort of 10 HIV-1-seropositive men taking suppressive HAART. In five patients, plasmacytoid and myeloid dendritic cells were isolated to attempt to identify their respective roles in HIV-1 residual disease. Viral out-growth assays were performed in vitro, as well as gag and R/U5 polymerase chain reaction (PCR) amplification of viral RNA and DNA, respectively, from DC and peripheral blood mononuclear cell (PBMC) extracts. Fluorescence activated cell-sorting (FACS) data revealed cellular yields from 85.90 to 95.18%, of relatively pure DCs isolated from patients' PBMCs. Although HIV-1 RNA gag and DNA RU/5 were detected in all PBMC samples isolated from the patients, proviral DNA and viral RNA forms were not detected in any of the DC isolates. In addition, no replication-competent virus was demonstrated in DC coculture assays, while virus was isolated from each patients' CD8+ T-lymphocyte-depleted PBMC cocultures. Furthermore, HIV-1 gag proviral DNA was not detected in either plasmacytoid or myeloid DC subfractions. The current study suggests that in HIV-1-infected individuals treated with suppressive HAART, peripheral blood DCs do not carry HIV-1 proviral DNA or viral particles attached to their surface. These populations of peripheral blood DCs are likely not a major HIV-1 reservoir in patients on HAART with clinically undetectable plasma viral RNA.     

HIV-1 resistant strains acquired at the time of primary infection massively fuel the cellular reservoir and persist for lengthy periods of time

OBJECTIVE: Characterization of the early establishment of the viral reservoir in patients acquiring resistant strains at primary HIV-1 infection (PHI), and longitudinal analysis of resistance mutations in circulating virions and intracellular HIV strains. PATIENTS AND METHODS: Drug-resistance was compared between HIV RNA and peripheral blood mononuclear cell (PBMC)-HIV DNA at the time of PHI in 44 patients enrolled in the Primo Cohort and harbouring plasma HIV-1 resistant to at least one antiretroviral drug. Longitudinal monitoring of viral load and resistance genotype was performed in plasma-HIV RNA and PBMC HIV DNA for at least 24 months in a subset of 10 patients. Phylogenetic analysis of HIV DNA protease gene clones was used to explore the diversity of quasi-species at baseline. RESULTS: Baseline resistance profile was identical in paired HIV RNA and PBMC HIV DNA for all 44 patients. All resistance-associated mutations persisted in plasma and PBMC over 2 years in the five untreated patients. Of the five patients started on empirical HAART, two achieved undetectable HIV RNA at month 6, with long-term persistence of archived drug-resistance mutations in PBMC HIV DNA. Virological failure was observed in the other three patients, resulting in the accumulation of additional drug-resistance mutations in HIV RNA and HIV DNA for two of them. Phylogenetic analysis of HIV DNA clones showed highly homogenous and exclusively resistant quasi-species in the cellular reservoir at baseline. CONCLUSION: HIV resistant strains acquired at the time of PHI massively fuel the cellular reservoir, and their prolonged persistence is supported by the early expansion of a dominant homogenous and resistant viral population. Results in treated patients showed that classical empirical triple-combination may be suboptimal.     

Rate of HIV-1 RNA rebound upon stopping antiretroviral therapy.

OBJECTIVE: To determine the rate of plasma HIV-1 RNA rebound in patients stopping highly active antiretroviral therapy (HAART) after achieving undetectable viral load. DESIGN: Sequential plasma HIV RNA levels were measured in six patients during the 21 days following withdrawal from HAART. METHODS: Plasma samples were obtained from six patients who chose to withdraw from HAART because of lipodystrophy, narcotic overdose, insomnia and/or high blood pressure. Longitudinal plasma viral load was determined in triplicate upon stopping therapy. RESULTS: All patients had plasma viral loads below 50 HIV RNA copies/ml at the time of stopping therapy and had had levels below 500 copies/ml for a median of 390 days (range 39-542 days). Plasma HIV rebound upon stopping therapy was rapid (median increase 0.2 log/day; range 0.15-0.42 log/day) and initially appeared to follow first-order kinetics. Plasma HIV RNA levels returned to greater than 500 copies/ml within 6 to 15 days (median 10 days) and approached or exceeded pre-therapy levels in all patients within 21 days of stopping therapy. Extrapolating backwards to the time at which individuals stopped therapy suggested that patients had tens of thousands of total body plasma HIV RNA copies despite having 'undetectable' plasma HIV RNA. CONCLUSIONS: HIV RNA in plasma rebounds within days of stopping antiretroviral therapy. A considerable burden of total body plasma HIV RNA likely remains even during effective HAART therapy.     

Effect of highly active antiretroviral therapy on cervicovaginal HIV-1 RNA

OBJECTIVES: To determine the frequency of cervicovaginal lavage and plasma HIV-1 RNA levels that are below detectable levels (< 400 copies/ml) among women on highly active antiretroviral therapy (HAART), non-HAART and on no therapy. To compare the effect of initiating HAART on the timing of HIV-1 RNA suppression in the blood plasma and genital tract among antiretroviral-na?ve women. METHODS: Data were obtained from 205 HIV-infected women with paired plasma and cervicovaginal lavage viral load measurements. Seven antiretroviral-na?ve women starting HAART had viral load measurements performed daily for one week, at 2 weeks and at 1 month after initiating therapy. Viral load quantification was carried out by nucleic acid sequence-based amplification assay. The lower limit of detection was 400 copies/ml. RESULTS: Plasma and cervicovaginal HIV-1 RNA was detectable in 71 and 26% of the women, respectively. Among women with plasma viral loads less than 400, 400-9999, and 10,000 copies/ml or over, genital tract HIV-1 RNA was detected in 3, 17 and 48%, respectively (P < 0.001). Fifty-one per cent of the women with CD4 cell counts of less than 200/mm3 had detectable cervicovaginal viral loads compared with 18% among women with CD4 cell counts of 200/mm3 or over (P < 0.001). Cervicovaginal HIV-1 RNA was less than 400 copies/ml in 85% of those on HAART, 69% of those on non-HAART and 69% of those on no therapy (P < 0.045). In seven antiretroviral-na?ve women initiating HAART, cervicovaginal HIV-1 RNA decreased by 0.7-2.1 log10 within 1-14 days of starting therapy. CONCLUSION: The cervicovaginal HIV-1 RNA level was positively correlated with plasma HIV-1 RNA and negatively with the CD4 cell count. The use of HAART was significantly associated with below-detectable levels of HIV-1 RNA in both plasma and the genital tract. HIV-1 RNA suppression in the genital tract may occur rapidly after initiating therapy.     

Human immunodeficiency virus type 1-infected persons with residual disease and virus reservoirs on suppressive highly active antiretroviral therapy can be stratified into relevant virologic and immunologic subgroups

A significant percentage of human immunodeficiency virus type 1 (HIV-1)-infected persons treated with highly active antiretroviral therapy (HAART) will develop plasma HIV-1-specific virion RNA levels <50 copies/mL. HIV-1-infected persons receiving virally suppressive HAART were studied with a viral outgrowth assay of the patients' peripheral blood mononuclear cells (PBMC), and a quantitative polymerase chain reaction assay was used to analyze HIV-1 2-long terminal repeat (2-LTR) circular DNA in PBMC, which indicates new HIV-1 infections of cells in vivo. Viral outgrowth in vitro correlated inversely with the level of peripheral blood CD4(+) T lymphocytes. Detection and quantitation of 2-LTR circular DNA correlated strongly with viral outgrowth patterns and inversely with CD4(+) T lymphocyte counts. Relevant subgroups of HIV-1-infected subjects on suppressive HAART with residual viral disease and reservoirs can now be stratified.