Early antiretroviral therapy favors post-treatment SIV control associated with the expansion of enhanced memory CD8+ T-cells.

HIV remission can be achieved in some people, called post-treatment HIV controllers, after antiretroviral treatment discontinuation. Treatment initiation close to the time of infection was suggested to favor post-treatment control, but the circumstances and mechanisms leading to this outcome remain unclear. Here we evaluate the impact of early (week 4) vs. late (week 24 post-infection) treatment initiation in SIVmac251-infected male cynomolgus macaques receiving 2 years of therapy before analytical treatment interruption. We show that early treatment strongly promotes post-treatment control, which is not related to a lower frequency of infected cells at treatment interruption. Rather, early treatment favors the development of long-term memory CD8+ T cells with enhanced proliferative and SIV suppressive capacity that are able to mediate a robust secondary-like response upon viral rebound. Our model allows us to formally demonstrate a link between treatment initiation during primary infection and the promotion of post-treatment control and provides results that may guide the development of new immunotherapies for HIV remission.

In-Depth Characterization of Full-Length Archived Viral Genomes after Nine Years of Posttreatment HIV Control.

In the search for control of human immunodeficiency virus type 1 (HIV-1) infection without antiretroviral therapy, posttreatment controllers (PTCs) are models of HIV remission. To better understand their mechanisms of control, we characterized the HIV blood reservoirs of 8 PTCs (median of 9.4 years after treatment interruption) in comparison with those of 13 natural HIV infection controllers (HICs) (median of 18 years of infection) and with those of individuals receiving efficient antiretroviral therapy initiated during either primary HIV infection (PHIs; n = 8) or chronic HIV infection (CHIs; n = 6). This characterization was performed with single-genome amplification and deep sequencing. The proviral diversity, which reflects the history of past viral replication, was lower in the PTCs, PHIs, and aviremic HICs than in the blipper HICs and CHIs. The proportions of intact and defective proviruses among the proviral pool in PTCs were not significantly different from those of other groups. When looking at the quantities of proviruses per million peripheral blood mononuclear cells (PBMCs), they had similar amounts of intact proviruses as other groups but smaller amounts of defective proviruses than CHIs, suggesting a role of these forms in HIV pathogenesis. Two HICs but none of the PTCs harbored only proviruses with deletion in nef; these attenuated strains could contribute to viral control in these participants. We show, for the first time, the presence of intact proviruses and low viral diversity in PTCs long after treatment interruption, as well as the absence of evolution of the proviral quasispecies in subsequent samples. This reflects low residual replication over time. Further data are necessary to confirm these results. IMPORTANCE Most people living with HIV need antiretroviral therapy to control their infection and experience viral relapse in case of treatment interruption, because of viral reservoir (proviruses) persistence. Knowing that proviruses are very diverse and most of them are defective in treated individuals, we aimed to characterize the HIV blood reservoirs of posttreatment controllers (PTCs), rare models of drug-free remission, in comparison with spontaneous controllers and treated individuals. At a median time of 9 years after treatment interruption, which is unprecedented in the literature, we showed that the proportions and quantities of intact proviruses were similar between PTCs and other individuals. Unlike 2/7 spontaneous controllers who harbored only nef-deleted proviruses, which are attenuated strains, which could contribute to their control, no such case was observed in PTCs. Furthermore, PTCs displayed low viral genetic diversity and no evolution of their reservoirs, indicating very low residual replication, despite the presence of intact proviruses.

The build-up of stock of stable integrated proviruses overtime explains the difficulty in reducing HIV-1 DNA levels when treatment is initiated at the chronic stage of the infection.

Background: Understanding factors affecting the size and the evolution of the HIV reservoir is essential for the development of curative strategies. This study aimed to assess the impact of antiretroviral therapy (ART) initiated during primary infection (PHI) vs chronic infection (CHI) on the levels and dynamics of integrated HIV-1 DNA, a biomarker of viral persistence. Methods: Integrated and total HIV-1-DNA were measured in the blood of 92 patients treated during PHI (early group) and 41 during CHI (deferred group), at diagnosis, ART initiation, and 12-24 months on treatment. Results: On ART, detectable (>1.78 log10 copies/106 PBMCs) integrated HIV-1 DNA levels were significantly lower in the early vs deferred group (2.99 log10vs 3.29 log10,p = 0.005). The proportion of undetectable integrated HIV-1 DNA tended to be higher in the early group vs deferred group (61 % vs 46 %; p = 0.133). Conclusion: Treatment initiated at PHI limits the levels of integrated HIV-1 DNA in blood. However, initiating treatment at CHI does not allow reaching such low levels in most patients, probably because the stable proviruses at that stage are present in the less prone to elimination long-lived cells. Thus, early ART could provide an opportunity to preparing for functional cure and eradication strategies.

Initiating Antiretroviral Treatment Early in Infancy Has Long-term Benefits on the Human Immunodeficiency Virus Reservoir in Late Childhood and Adolescence.

BACKGROUND: Early combined antiretroviral therapy (cART) limits the total HIV-DNA load in children. However, data on its impact in older children and adolescents remain scarce. This study compares HIV reservoirs in children (5-12 years) and adolescents (13-17 years) who started cART <6 months (early [E-] group) or >2 years (late [L-] group). METHODS: The ANRS-EP59-CLEAC study prospectively enrolled 76 patients perinatally infected with HIV-1 who reached HIV-RNA <400 copies/mL <24 months after cART initiation, regardless of subsequent viral suppression (E-group: 27 children, 9 adolescents; L-group: 19 children, 21 adolescents). Total and integrated HIV-DNA were quantified in blood and in CD4+ T-cell subsets. A substudy assessed HIV reservoir inducibility after ex vivo peripheral blood mononuclear cell (PBMC) stimulation. RESULTS: Total HIV-DNA levels were lower in early- versus late-treated patients (children: 2.14 vs 2.87 log copies/million PBMCs; adolescents: 2.25 vs 2.74 log; P < .0001 for both). Low reservoir was independently associated with treatment precocity, protective HLA, and low cumulative viremia since cART initiation. The 60 participants with undetectable integrated HIV-DNA started cART earlier than other patients (4 vs 54 months; P = .03). In those with sustained virological control, transitional and effector memory CD4+ T cells were less infected in the E-group than in the L-group (P = .03 and .02, respectively). Viral inducibility of reservoir cells after normalization to HIV-DNA levels was similar between groups. CONCLUSIONS: Early cART results in a smaller blood HIV reservoir until adolescence, but all tested participants had an inducible reservoir. This deserves cautious consideration for HIV remission strategies.

False-negative Results of Human Immunodeficiency Virus (HIV) Rapid Testing in HIV Controllers.

Serological assays were performed on 85 human immunodeficiency virus-controller samples . 6% presented a negative rapid screening test 7% presented an indeterminate Western blot. The enzyme immunoassay ratio decreased in controllers who had continual negative ultrasensitive HIV RNA results since inclusion.

Increasing contribution of integrated forms to total HIV DNA in blood during HIV disease progression from primary infection.

BACKGROUND: In the current context of research on HIV reservoirs, offering new insights into the persistence of HIV DNA in infected cells, which prevents viral eradication, may aid in identifying cure strategies. This study aimed to describe the establishment of stable integrated forms among total HIV DNA during primary infection (PHI) and their dynamics during the natural history of infection. METHODS: Total and integrated HIV DNA were quantified in blood from 74 PHI patients and 97 recent seroconverters (<12 months following infection, "progression cohort"). The evolution of both markers over six years was modelled (mixed-effect linear models). Their predictive values for disease progression were studied (Cox models). FINDINGS: For most patients during PHI, stable integrated forms were a minority among total HIV DNA (median: 12%) and became predominant thereafter (median at AIDS stage: 100%). Both total and integrated HIV DNA increased over a six-year period. Patients from the progression cohort who reached clinical AIDS during follow-up (n = 34) exhibited higher total and integrated HIV DNA levels at seroconversion and a higher percentage of integrated forms than did slower progressors (n = 63) (median: 100% vs 44%). The integrated HIV DNA load was strongly associated with the risk of developing AIDS (aRR = 2.63, p = 0.002). INTERPRETATION: The profile of "rapid" or "slower" progression in the natural history of HIV infection appears to be determined early in the course of HIV infection. The strong predominance of unstable unintegrated forms in PHI may explain the great benefit of this early treatment, which induces a sharp decrease in total HIV DNA. FUND: French National Agency for Research on AIDS and Viral Hepatitis.

New Highly Sensitive Real-Time PCR Assay for HIV-2 Group A and Group B DNA Quantification.

HIV-2 infection is characterized by a very low replication rate in most cases and low progression. This necessitates an approach to patient monitoring that differs from that for HIV-1 infection. Here, a new highly specific and sensitive method for HIV-2 DNA quantification was developed. The new test is based on quantitative real-time PCR targeting the long terminal repeat (LTR) and gag regions and using an internal control. Analytical performance was determined in three laboratories, and clinical performance was determined on blood samples from 63 patients infected with HIV-2 group A (n = 35) or group B (n = 28). The specificity was 100%. The 95% limit of detection was three copies/PCR and the limit of quantification was six copies/PCR. The within-run coefficients of variation were between 1.03% at 3.78 log10 copies/PCR and 27.02% at 0.78 log10 copies/PCR. The between-run coefficient of variation was 5.10%. Both manual and automated nucleic acid extraction methods were validated. HIV-2 DNA loads were detectable in blood cells from all 63 patients. When HIV-2 DNA was quantifiable, median loads were significantly higher in antiretroviral-treated than in naive patients and were similar for groups A and B. HIV-2 DNA load was correlated with HIV-2 RNA load (r = 0.68; 95% confidence interval [CI], 0.4 to 0.8; P < 0.0001). Our data show that this new assay is highly sensitive and quantifies the two main HIV-2 groups, making it useful for the diagnosis of HIV-2 infection and for pathogenesis studies on HIV-2 reservoirs.

Impact of early cART on HIV blood and semen compartments at the time of primary infection.

BACKGROUND: HIV-infected cells in semen facilitate viral transmission. We studied the establishment of HIV reservoirs in semen and blood during PHI, along with systemic immune activation and the impact of early cART. METHODS: Patients in the ANRS-147-OPTIPRIM trial received two years of early cART. Nineteen patients of the trial were analyzed, out of which 8 had acute PHI (WB ≤1 Ab). We quantified total cell-associated (ca) HIV-DNA in blood and semen and HIV-RNA in blood and semen plasma samples, collected during PHI and at 24 months of treatment. RESULTS: At enrollment, HIV-RNA load was higher in blood than in semen (median 5.66 vs 4.22 log10 cp/mL, p<0.0001). Semen HIV-RNA load correlated strongly with blood HIV-RNA load (r = 0.81, p = 0.02, the CD4 cell count (r = -0.98, p<0.0001), and the CD4/CD8 ratio (r = -0.85, p<0.01) in acute infection but not in later stages of PHI. Median blood and seminal cellular HIV-DNA levels were 3.59 and 0.31 log10cp/106 cells, respectively. HIV-DNA load peaked in semen later than in blood and then correlated with blood IP10 level (r = 0.62, p = 0.04). HIV-RNA was undetectable in blood and semen after two years of effective cART. Semen HIV-DNA load declined similarly, except in one patient who had persistently high IP-10 and IL-6 levels and used recreational drugs. CONCLUSIONS: HIV reservoir cells are found in semen during PHI, with gradual compartmentalization. Its size was linked to the plasma IP-10 level. Early treatment purges both the virus and infected cells, reducing the high risk of transmission during PHI. CLINICAL TRIALS REGISTRATION: NCT01033760.

Interlaboratory quality control of total HIV-1 DNA load measurement for multicenter reservoir studies.

BACKGROUND: Viral reservoirs represent an important barrier to HIV cure. Accurate markers of HIV reservoirs are needed to develop multicenter studies. The aim of this multicenter quality control (QC) was to evaluate the inter-laboratory reproducibility of total HIV-1-DNA quantification. METHODS: Ten laboratories of the ANRS-AC11 working group participated by quantifying HIV-DNA with a real-time qPCR assay (Biocentric) in four samples (QCMD). RESULTS: Good reproducibility was found between laboratories (standard deviation ≤ 0.2 log10 copies/106 PBMC) for the three positive QC that were correctly classified by each laboratory (QC1

Total HIV-1 DNA, a Marker of Viral Reservoir Dynamics with Clinical Implications.

HIV-1 DNA persists in infected cells despite combined antiretroviral therapy (cART), forming viral reservoirs. Recent trials of strategies targeting latent HIV reservoirs have rekindled hopes of curing HIV infection, and reliable markers are thus needed to evaluate viral reservoirs. Total HIV DNA quantification is simple, standardized, sensitive, and reproducible. Total HIV DNA load influences the course of the infection and is therefore clinically relevant. In particular, it is predictive of progression to AIDS and death, independently of HIV RNA load and the CD4 cell count. Baseline total HIV DNA load is predictive of the response to cART. It declines during cART but remains quantifiable, at a level that reflects both the history of infection (HIV RNA zenith, CD4 cell count nadir) and treatment efficacy (residual viremia, cumulative viremia, immune restoration, immune cell activation). Total HIV DNA load in blood is also predictive of the presence and severity of some HIV-1-associated end-organ disorders. It can be useful to guide individual treatment, notably, therapeutic de-escalation. Although it does not distinguish between replication-competent and -defective latent viruses, the total HIV DNA load in blood, tissues, and cells provides insights into HIV pathogenesis, probably because all viral forms participate in host cell activation and HIV pathogenesis. Total HIV DNA is thus a biomarker of HIV reservoirs, which can be defined as all infected cells and tissues containing all forms of HIV persistence that participate in pathogenesis. This participation may occur through the production of new virions, creating new cycles of infection and disseminating infected cells; maintenance or amplification of reservoirs by homeostatic cell proliferation; and viral transcription and synthesis of viral proteins without new virion production. These proteins can induce immune activation, thus participating in the vicious circle of HIV pathogenesis.

Sequential treatment with 5-aza-2'-deoxycytidine and deacetylase inhibitors reactivates HIV-1.

Reactivation of HIV gene expression in latently infected cells together with an efficient cART has been proposed as an adjuvant therapy aimed at eliminating/decreasing the reservoir size. Results from HIV clinical trials using deacetylase inhibitors (HDACIs) question the efficiency of these latency-reversing agents (LRAs) used alone and underline the need to evaluate other LRAs in combination with HDACIs. Here, we evaluated the therapeutic potential of a demethylating agent (5-AzadC) in combination with clinically tolerable HDACIs in reactivating HIV-1 from latency first in vitro and next ex vivo. We showed that a sequential treatment with 5-AzadC and HDACIs was more effective than the corresponding simultaneous treatment both in vitro and ex vivo. Interestingly, only two of the sequential LRA combinatory treatments tested induced HIV-1 particle recovery in a higher manner than the drugs alone ex vivo and at concentrations lower than the human tolerable plasmatic concentrations. Taken together, our data reveal the benefit of using combinations of 5-AzadC with an HDACI and, for the first time, the importance of treatment time schedule for LRA combinations in order to reactivate HIV.

A low HIV-DNA level in peripheral blood mononuclear cells at antiretroviral treatment interruption predicts a higher probability of maintaining viral control.

OBJECTIVE: The main aim of this study was to determine whether HIV replication can be controlled following interruption of treatment started early in the course of infection (CD4 >350 cells/µl and viral load <50 000 copies/ml), but not during the primary infection. METHODS: Patients enrolled in a multicenter trial of treatment interruption (ANRS 116 SALTO) with CD4 above 450 cells/µl and viral load below 400 copies/ml at treatment interruption were selected for this second analysis. We determined the proportion of patients whose plasma HIV-RNA load remained below 400 copies/ml during the first 12 months of treatment interruption, and baseline factors predictive of time to loss of viral control. Viral load rebound was defined as two successive values above 400 copies/ml, or as one value above 400 copies/ml, followed by treatment resumption. RESULTS: We studied 95 patients with a median CD4 nadir of 382 cells/µl (340-492). At treatment interruption, the median CD4 cell count and HIV-DNA load were 813/µl (695-988) and 206 copies/10 peripheral blood mononuclear cells (PBMCs) (53-556). Twelve months after treatment interruption, seven patients still had viral load below 400 copies/ml (Kaplan-Meier estimate 7.5%, 95% confidence interval 3.7-14.6), and four of them still had viral load below 400 copies/ml at 36 months. A multivariable Cox proportional-hazards model showed that time to loss of viral control was more shorter in patients with HIV-DNA at least 150 copies/10 PBMCs at treatment interruption (hazard ratio 2.1, 95% confidence interval 1.3-3.3, P = 0.002) than in those with HIV-DNA below 150 copies/10 PBMCs. CONCLUSION: Patients who have low HIV-DNA levels at antiretroviral treatment interruption are more likely to maintain viral control for long periods.

Adipose Tissue Is a Neglected Viral Reservoir and an Inflammatory Site during Chronic HIV and SIV Infection.

Two of the crucial aspects of human immunodeficiency virus (HIV) infection are (i) viral persistence in reservoirs (precluding viral eradication) and (ii) chronic inflammation (directly associated with all-cause morbidities in antiretroviral therapy (ART)-controlled HIV-infected patients). The objective of the present study was to assess the potential involvement of adipose tissue in these two aspects. Adipose tissue is composed of adipocytes and the stromal vascular fraction (SVF); the latter comprises immune cells such as CD4+ T cells and macrophages (both of which are important target cells for HIV). The inflammatory potential of adipose tissue has been extensively described in the context of obesity. During HIV infection, the inflammatory profile of adipose tissue has been revealed by the occurrence of lipodystrophies (primarily related to ART). Data on the impact of HIV on the SVF (especially in individuals not receiving ART) are scarce. We first analyzed the impact of simian immunodeficiency virus (SIV) infection on abdominal subcutaneous and visceral adipose tissues in SIVmac251 infected macaques and found that both adipocytes and adipose tissue immune cells were affected. The adipocyte density was elevated, and adipose tissue immune cells presented enhanced immune activation and/or inflammatory profiles. We detected cell-associated SIV DNA and RNA in the SVF and in sorted CD4+ T cells and macrophages from adipose tissue. We demonstrated that SVF cells (including CD4+ T cells) are infected in ART-controlled HIV-infected patients. Importantly, the production of HIV RNA was detected by in situ hybridization, and after the in vitro reactivation of sorted CD4+ T cells from adipose tissue. We thus identified adipose tissue as a crucial cofactor in both viral persistence and chronic immune activation/inflammation during HIV infection. These observations open up new therapeutic strategies for limiting the size of the viral reservoir and decreasing low-grade chronic inflammation via the modulation of adipose tissue-related pathways.

An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression.

The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

Combined ART started during acute HIV infection protects central memory CD4+ T cells and can induce remission.

BACKGROUND: Therapeutic control of HIV replication reduces the size of the viral reservoir, particularly among central memory CD4+ T cells, and this effect might be accentuated by early treatment. METHODS: We examined the effect of ART initiated at the time of the primary HIV infection (early ART), lasting 2 and 6 years in 11 and 10 patients, respectively, on the HIV reservoir in peripheral resting CD4+ T cells, sorted into naive (TN), central memory (TCM), transitional memory (TTM) and effector memory (TEM) cells, by comparison with 11 post-treatment controllers (PTCs). RESULTS: Between baseline and 2 years, CD4+ T cell subset numbers increased markedly (P < 0.004) and HIV DNA levels decreased in all subsets (P < 0.009). TTM cells represented the majority of reservoir cells at both timepoints, T cell activation status normalized and viral diversity remained stable over time. The HIV reservoir was smaller after 6 years of early ART than after 2 years (P < 0.019), and did not differ between PTCs and patients treated for 6 years. One patient, who had low reservoir levels in all T cell subsets after 2 years of treatment similar to the levels in PTCs, spontaneously controlled viral replication during 18 months off treatment. CONCLUSIONS: Early prolonged ART thus limits the size of the HIV reservoir, protects long-lived cells from persistent infection and may enhance post-treatment control.

Impact of the Timing of Initiation of Antiretroviral Therapy During Primary HIV-1 Infection on the Decay of Cell-Associated HIV-DNA.

BACKGROUND: Combined antiretroviral therapy (cART) initiation during primary human immunodeficiency virus (HIV) infection (PHI) yields a larger decrease in cell-associated HIV-DNA (CA-HIV-DNA) than initiation during the chronic phase. Our objective was to model the short and long-term decay of CA-HIV-DNA blood reservoir in patients initiating cART during PHI and to assess the impact of the timing of cART initiation on CA-HIV-DNA decay. METHODS: We included patients enrolled during PHI in the Agence Nationale de Recherche sur le Sida PRIMO cohort, treated within the month following enrollment and achieving sustained virologic response. The decay of CA-HIV-DNA over time while on successful cART was modeled with a 3-slope linear mixed-effects model according to the delay between estimated date of infection and cART initiation. RESULTS: Three hundred twenty-seven patients were included, accounting for 1305 CA-HIV-DNA quantifications. Median time between infection and cART initiation was 41 days (interquartile range, 33-54 days). Median follow-up under cART was 2.3 years (range, 0.4-16.6 years). The timing of cART initiation had significant impact on the first slope of decrease: The earlier cART was initiated after HIV infection, the faster CA-HIV-DNA level decreased during the first 8 months of cART: -0.171, -0.131, and -0.068 log10 copies/10(6) peripheral blood mononuclear cells (PBMCs) per month when cART was initiated 15 days, 1 month, and 3 months after infection, respectively (P < .0001). The predicted mean CA-HIV-DNA level achieved after 5 years of successful cART was 1.62 and 2.24 log10 copies/10(6) PBMCs when cART was initiated 15 days and 3 months after infection, respectively (P = .0006). CONCLUSIONS: This study provides strong arguments in favor of cART initiation at the earliest possible time point after HIV infection.

Intensive five-drug antiretroviral therapy regimen versus standard triple-drug therapy during primary HIV-1 infection (OPTIPRIM-ANRS 147): a randomised, open-label, phase 3 trial.

BACKGROUND: Early combination antiretroviral therapy (cART) initiation at the time of primary HIV-1 infection could restrict the establishment of HIV reservoirs. We aimed to assess the effect of a cART regimen intensified with raltegravir and maraviroc, compared with standard triple-drug cART, on HIV-DNA load. METHODS: In this randomised, open-label, phase 3 trial, we recruited patients from hospitals across France. Inclusion criteria were primary HIV-1 infection (an incomplete HIV-1 western blot and detectable plasma HIV-RNA), with either symptoms or a CD4+ cell count below 500 cells per µL. Patients were randomly assigned (1:1) to an intensive, five-drug cART regimen (raltegravir 400 mg and maraviroc 150 mg twice daily, and a fixed-dose combination of tenofovir disoproxil fumarate 300 g plus emtricitabine 200 g, darunavir 800 g, and ritonavir 100 g once daily) or a standard triple-drug cART regimen (tenofovir disoproxil fumarate 300 g plus emtricitabine 200 g, darunavir 800 g, and ritonavir 100 g once daily) using a predefined randomised list generated by randomly selected variable block sizes. The primary endpoint was the median number of HIV-DNA copies per 10(6) peripheral blood mononuclear cells (PBMC) at month 24, analysed in the modified intention-to-treat population, defined as all patients who started their assigned treatment. This study is registered with ClinicalTrials.gov, number NCT01033760. FINDINGS: Between April 26, 2010, and July 13, 2011, 110 patients were enrolled, of whom 92 were randomly assigned and 90 started treatment (45 in each treatment group). Six (13%) patients in the intensive cART group and two (4%) in the standard cART group discontinued before month 24. At month 24, HIV-DNA loads were similar between groups (2·35 [IQR 2·05-2·50] log10 per 10(6) PBMC in the intensive cART group vs 2·25 [1·71-2·55] in the standard cART group; p=0·21). Eight grade 3-4 clinical adverse events were reported in seven patients in the intensive cART group and seven grade 3-4 clinical adverse events were reported in seven patients in the standard cART group. Three serious clinical adverse events occurred: two (pancreatitis and lipodystrophy) in the standard cART group, which were regarded as treatment related, and one event (suicide attempt) in the intensive cART group that was unrelated to treatment. INTERPRETATION: After 24 months, cART intensified with raltegravir and maraviroc did not have a greater effect on HIV blood reservoirs than did standard cART. These results should help to design future trials of treatments aiming to decrease the HIV reservoir in patients with primary HIV-1 infection. FUNDING: Inserm-ANRS, Gilead Sciences, Janssen Pharmaceuticals, Merck, and ViiV Laboratories.

New sensitive one-step real-time duplex PCR method for group A and B HIV-2 RNA load.

The Agence Nationale de Recherche sur le Sida et les hépatites virales (ANRS) previously developed a widely used method for HIV-1 RNA quantification (Biocentric). Here, we report the development of a new specific and sensitive method for HIV-2 RNA quantification, based on an adaptation of the existing HIV-1 protocol. The new test is based on TaqMan one-step reverse transcription-quantitative PCR (qRT-PCR) targeting two conserved consensus regions of HIV-2 (long terminal repeat [LTR] and gag). Analytic performances were determined in three laboratories. Clinical performances were evaluated on 100 plasma samples from HIV-2-infected patients (groups A, B, and H) by comparison with the assay currently used for the ANRS HIV-2 cohort. The specificity was 100%. Sensitivity was 50 copies/ml (cp/ml) and was optimized to 10 cp/ml. The within-run coefficients of variation in the three laboratories varied from 0.54% to 1.61% at 4 log10 copies/ml and from 7.24% to 14.32% at 2 log10 cp/ml. The between-run coefficients of variation varied from 2.28% to 6.43%. Of the 39 clinical samples below 2 log10 in the current assay, the new test improved the detection or quantification of 17 samples, including eight group B samples. For quantifiable samples, similar loads were obtained with the two assays for group A samples. The median difference between the two assays for group B samples was +0.18 but with greater heterogeneity than for group A. The HIV-2 group H sample had similar results with the two assays. This new assay is highly sensitive and accurately quantifies the most prevalent HIV-2 groups. This test will be useful for monitoring low viral loads in HIV-2-infected patients.

Quantification of total HIV1-DNA in peripheral blood mononuclear cells.

HIV reservoir measurement in patients is one of the challenges at the time of testing new treatment approaches aiming at eradicating HIV infection. HIV reservoirs are complex and disseminated in a large number of organs and lymphoid tissues. We chose to quantify total cell-associated HIV-DNA in PBMC as a marker of HIV reservoirs and described the method we developed. The marker was used in large cohort studies at different stages of HIV disease and in therapeutical trials. Our results show how informative is this marker, as well as that plasma HIV-RNA and CD4 T cell count are representative of each patient when measured in blood. Such a series of results might help to adapt simplification or structured interruption strategies, design new clinical trials targeting HIV reservoirs, and select populations that could benefit of such new treatments.

Naive T lymphocytes and recent thymic emigrants are associated with HIV-1 disease history in french adolescents and young adults infected in the perinatal period: the ANRS-EP38-IMMIP study.

BACKGROUND: Children born at the start of the human immunodeficiency virus (HIV) epidemic and infected during the perinatal period are now young adults living with the virus. Naive T-lymphocyte restoration is essential for the maintenance of a diverse T-cell receptor repertoire and for immunity to pathogens. METHODS: The ANRS-EP38-IMMIP study included 93 patients infected with HIV type 1 (HIV-1) during the perinatal period. Naive CD4 (CD4N) and CD8 (CD8N) T lymphocytes and CD4 recent thymic emigrants (CD4RTE) were quantified in the peripheral blood by flow cytometry. Wilcoxon tests, Pearson correlation coefficients, and linear regressions were used to study their associations with HIV disease parameters. RESULTS: Median CD4N, CD8N, and CD4RTE percentages were 56% (interquartile range [IQR], 44-64), 31% (IQR, 22-44), and 79% (IQR, 74-83), respectively. The three T-lymphocyte subsets were positively correlated with CD4 T-cell count. Patients aviremic at the time of the study tended to have a lower CD4N percentage (55% vs 58%; P = .10), a significantly higher CD8N percentage (39% vs 22%; P < .0001), and a significantly lower CD4RTE percentage (77% vs 81%; P = .003) than viremic patients. In aviremic patients, CD4N percentages were positively associated with cumulative viremia over the last 10 years (r = 0.335; P = .01) and were significantly higher in patients harboring X4R5 viruses than in those harboring R5 viruses (61% vs 44%; P = .001). CONCLUSIONS: After at least 15 years of HIV infection, perinatally infected youths had preserved CD4N and CD4RTE levels. This persistence of high levels of thymic activity potentially compensating for the deleterious effects of current and past HIV replication is remarkable.