Asymmetric division of activated latently infected cells may explain the decay kinetics of the HIV-1 latent reservoir and intermittent viral blips

Most HIV-infected patients when treated with combination antiretroviral therapy achieve viral loads that are below the current limit of detection of standard assays after a few months. Despite this, virus eradication from the host has not been achieved. Latent, replication-competent HIV-1 can generally be identified in resting memory CD4⁺ T cells in patients with “undetectable” viral loads. Turnover of these cells is extremely slow but virus can be released from the latent reservoir quickly upon cessation of therapy. In addition, a number of patients experience transient episodes of viremia, or HIV-1 blips, even with suppression of the viral load to below the limit of detection for many years. The mechanisms underlying the slow decay of the latent reservoir and the occurrence of intermittent viral blips have not been fully elucidated. In this study, we address these two issues by developing a mathematical model that explores a hypothesis about latently infected cell activation. We propose that asymmetric division of latently infected cells upon sporadic antigen encounter may both replenish the latent reservoir and generate intermittent viral blips. Interestingly, we show that occasional replenishment of the latent reservoir induced by reactivation of latently infected cells may reconcile the differences between the divergent estimates of the half-life of the latent reservoir in the literature.     

Cobas Ampliprep/Cobas TaqMan HIV-1 v2.0 Assay: Consequences at the Cohort Level

Background

High-sensitive real-time PCR assays are routinely used to monitor HIV-1 infected subjects. Inter-assay discrepancies have been described at the low viral load (VL) end, where clinical decisions regarding possible virological rebound are based.

Methods

A retrospective study was performed to analyze frequencies of viral blips after transition to the COBAS Ampliprep/COBAS TaqMan v2.0 HIV-1 assay (Taqman v2.0) in patients with prior undetectable VLs as measured with the Roche Cobas Ampliprep Amplicor HIV-1 Monitor Test, v1.5 (Amplicor) and was evaluated in comparison to a group of patients monitored with the Abbott Real-time HIV-1 assay (Abbott RT) during the same period of time.

Results

85 of 373 patients with VLs below the limit of quantification with Amplicor had VLs >50 copies/mL after transition to the TaqMan v2.0 assay. Among these 74.1% had VLs ranging from 50–499 copies/mL, 22.9% had VLs >500 copies/mL. From 22 patients with initial Taqman v2.0 based VLs exceeding 500 copies/mL, 6 patients had VLs <20 copies/mL after novel VL measurement on a next visit. In our control group with VL quantification using the Abbott RT assay, only 1 patient became detectable and showed a VL of <40 copies/mL after new measurement.

Conclusions

Transition to the Taqman v2.0 assay was accompanied by an increase of quantifiable HIV-1 VLs in patients with long term viral suppression under antiretroviral therapy that might be attributed to technical shortcomings of the Taqman v2.0 assay. A high test variability at the low VL end but also beyond was observed, making meaningful clinical interpretation of viral blips derived from different assays difficult.     

Dynamics of intermittent viremia during highly active antiretroviral therapy in patients who initiate therapy during chronic versus acute and early human immunodeficiency virus type 1 infection

The meaning of viral blips in human immunodeficiency virus type 1 (HIV-1)-infected patients treated with seemingly effective highly active antiretroviral therapy (HAART) is still controversial and under investigation. Blips might represent low-level ongoing viral replication in the presence of drug or simply release of virions from the latent reservoir. Patients treated early during HIV-1 infection are more likely to have a lower total body viral burden, a homogenous viral population, and preserved HIV-1-specific immune responses. Consequently, viral blips may be less frequent in them than in patients treated during chronic infection. To test this hypothesis, we compared the occurrence of viral blips in 76 acutely infected patients (primary HIV infection [PHI] group) who started therapy within 6 months of the onset of symptoms with that in 47 patients who started HAART therapy during chronic infection (chronic HIV infection [CHI] group). Viral blip frequency was approximately twofold higher in CHI patients (0.122 +/- 0.12/viral load [VL] sample, mean +/- standard deviation) than in PHI patients (0.066 +/- 0.09/VL sample). However, in both groups, viral blip frequency did not increase with longer periods of observation. Also, no difference in viral blip frequency was observed between treatment subgroups, and the occurrence of a blip was not associated with a recent change in CD4(+) T-cell count. Finally, in PHI patients the VL set point was a significant predictor of blip frequency during treatment.     

Duration of an intermittent episode of viremia

HIV-1 infected patients after being treated with potent combinations of antiretroviral drugs for 2–6 months typically reach a state in which virus can no longer be detected within their blood. These patients with undetectable virus occasionally have viral load measurements that are above the limit of detection of current assays. Such measurements are called blips. Here we examine the possibility that such blips represent infrequent measurements taken during a period of time in which there is a transient elevation of virus in the patient’s blood, i.e., a so-called transient episode of viremia. By analyzing time series of blips from a large number of patients, we conclude that transient episodes of viremia exist and that on average they extend for a period of about 3 weeks.     

Opportunistic infection as a cause of transient viremia in chronically infected HIV patients under treatment with HAART

When highly active antiretroviral therapy is administered for long periods of time to HIV-1 infected patients, most patients achieve viral loads that are “undetectable” by standard assay (i.e., HIV-1 RNA < 50 copies/ml). Yet despite exhibiting sustained viral loads below the level of detection, a number of these patients experience unexplained episodes of transient viremia or viral “blips”. We propose here that transient activation of the immune system by opportunistic infection may explain these episodes of viremia. Indeed, immune activation by opportunistic infection may spur HIV replication, replenish viral reservoirs and contribute to accelerated disease progression. In order to investigate the effects of intercurrent infection on chronically infected HIV patients under treatment with highly active antiretroviral therapy (HAART), we extend a simple dynamic model of the effects of vaccination on HIV infection [Jones, L.E., Perelson, A.S., 2002. Modeling the effects of vaccination on chronically infected HIV-positive patients. JAIDS 31, 369–377] to include growing pathogens. We then propose a more realistic model for immune cell expansion in the presence of pathogen, and include this in a set of competing models that allow low baseline viral loads in the presence of drug treatment. Programmed expansion of immune cells upon exposure to antigen is a feature not previously included in HIV models, and one that is especially important to consider when simulating an immune response to opportunistic infection. Using these models we show that viral blips with realistic duration and amplitude can be generated by intercurrent infections in HAART treated patients.     

Viral blip dynamics during highly active antiretroviral therapy

Although intermittent episodes of low-level viremia are often observed in well-suppressed highly active antiretroviral therapy (HAART)-treated patients, the timing and amplitude of viral blips have never been examined in detail. We analyze here the dynamics of viral blips, i.e., plasma VL measurements of >50 copies/ml, in 123 HAART-treated patients monitored for a mean of 2.6 years (range, 5 months to 5.3 years). The mean (+/- the standard deviation) blip frequency was 0.09 +/- 0.11/sample, with about one-third of patients showing no viral blips. The mean viral blip amplitude was 158 +/- 132 human immunodeficiency virus type 1 (HIV-1) RNA copies/ml. Analysis of the blip frequency and amplitude distributions suggest that two blips less than 22 days apart have a significant chance of being part of the same episode of viremia. The data are consistent with a hypothetical model in which each episode of viremia consists of a phase of VL rise, followed by two-phase exponential decay. Thus, the term "viral blip" may be a misnomer, since viral replication appears to be occurring over an extended period. Neither the frequency nor the amplitude of viral blips increases with longer periods of observation, but the frequency is inversely correlated with the CD4(+)-T-cell count at the start of therapy, suggesting that host-specific factors but not treatment fatigue are determinants of blip frequency.     

Increased Risk of Virologic Rebound in Patients on Antiviral Therapy with a Detectable HIV Load <48 Copies/mL

We investigated the independent effects of HIV-1 ”target not detected” measurements versus those that were detectable but below the limit of quantification by Taqman RT-PCR assay on subsequent viral rebound as there are conflicting data regarding the clinical implications of arbitrary or isolated low-level viremia. Cox proportional hazard regression modeling was used to investigate the independent effects of the first HIV-1 load measurement after introduction of the Taqman RT-PCR assay (time-point 0 [T0]), pre-T0 viral loads, CD4 T cell count, race/ethnicity, gender, age and NNRTI use on risk of a confirmed VL >50, >200, >400 and >1000 copies/mL at 22 months follow-up in analyses of all patients and propensity-matched baseline cohorts. 778 patients had a viral load that was either not detected by RT-PCR (N = 596) or detectable, but below the limit of quantification (N = 182) at T0. Detectable viremia, lower T0 CD4 count, decreased age, and having detectable or unknown VL within a year prior to T0 were each associated with viral rebound to >50, >200 and >400 copies/mL. Overall failure rates were low and <5.5% of all patients had confirmed VL >1000 copies/mL. A majority of patients with rebound >200 copies/mL subsequently re-suppressed (28 of 53). A detectable VL <48 copies/mL was independently and significantly associated with subsequent viral rebound, and is cause for clinical concern.     

Modeling HIV persistence, the latent reservoir, and viral blips

HIV-1 eradication from infected individuals has not been achieved with the prolonged use of highly active antiretroviral therapy (HAART). The cellular reservoir for HIV-1 in resting memory CD4⁺ T cells remains a major obstacle to viral elimination. The reservoir does not decay significantly over long periods of time but is able to release replication-competent HIV-1 upon cell activation. Residual ongoing viral replication may likely occur in many patients because low levels of virus can be detected in plasma by sensitive assays and transient episodes of viremia, or HIV-1 blips, are often observed in patients even with successful viral suppression for many years. Here we review our current knowledge of the factors contributing to viral persistence, the latent reservoir, and blips, and mathematical models developed to explore them and their relationships. We show how mathematical modeling has helped improve our understanding of HIV-1 dynamics in patients on HAART and of the quantitative events underlying HIV-1 latency, reservoir stability, low-level viremic persistence, and emergence of intermittent viral blips. We also discuss treatment implications related to these studies.     

CD4 dynamics over a 15 year-period among HIV controllers enrolled in the ANRS French observatory

Background

There are few large published studies of HIV controllers with long-term undetectable viral load (VL). We describe the characteristics and outcomes of 81 French HIV controllers.

Methods and Results

HIV controllers were defined as asymptomatic, antiretroviral-naïve persons infected ≥10 years previously, with HIV-RNA <400 copies/mL in >90% of plasma samples. All available CD4 and VL values were collected at enrolment. Mixed-effect linear models were used to analyze CD4 cell count slopes since diagnosis. HIV controllers represented 0.31% of all patients managed in French hospitals. Patients infected through intravenous drug use were overrepresented (31%) and homosexual men were underrepresented (26% of men) relative to the ANRS SEROCO cohort of subjects diagnosed during the same period. HIV controllers whose VL values were always below the detection limit of the assays were compared with those who had rare “blips” (<50% of VL values above the detection limit) or frequent blips (>50% of VL values above the detection limit). Estimated CD4 cell counts at HIV diagnosis were similar in the three groups. CD4 cell counts remained stable after HIV diagnosis in the “no blip” group, while they fell significantly in the two other groups (−0.26√CD4 and −0.28√CD4/mm³/year in the rare and frequent blip groups, respectively). No clinical, immunological or virological progression was observed in the no blip group, while 3 immunological and/or virological events and 4 cancers were observed in the blip subgroups.

Conclusions

Viral blips in HIV controllers are associated with a significant decline in CD4 T cells and may be associated with an increased risk of pathological events, possibly owing to chronic inflammation/immune activation.     

Plasma HIV-2 RNA According to CD4 Count Strata among HIV-2-Infected Adults in the IeDEA West Africa Collaboration

Background

Plasma HIV-1 RNA monitoring is one of the standard tests for the management of HIV-1 infection. While HIV-1 RNA can be quantified using several commercial tests, no test has been commercialized for HIV-2 RNA quantification. We studied the relationship between plasma HIV-2 viral load (VL) and CD4 count in West African patients who were either receiving antiretroviral therapy (ART) or treatment-naïve.

Method

A cross sectional survey was conducted among HIV-2-infected individuals followed in three countries in West Africa from March to December 2012. All HIV-2 infected-patients who attended one of the participating clinics were proposed a plasma HIV-2 viral load measurement. HIV-2 RNA was quantified using the new ultrasensitive in-house real-time PCR assay with a detection threshold of 10 copies/ mL (cps/mL).

Results

A total of 351 HIV-2-infected individuals participated in this study, of whom 131 (37.3%) were treatment naïve and 220 (62.7%) had initiated ART. Among treatment-naïve patients, 60 (46.5%) had undetectable plasma HIV-2 viral load (<10 cps/mL), it was detectable between 10-100 cps/mL in 35.8%, between 100-1000 cps/mL in 11.7% and >1000 cps/mL in 6.0% of the patients. Most of the treatment-naïve patients (70.2%) had CD4-T cell count ≥500 cells/mm³ and 43 (46.7%) of these patients had a detectable VL (≥10 cps/mL). Among the 220 patients receiving ART, the median CD4-T cell count rose from 231 to 393 cells/mm³ (IQR [259-561]) after a median follow-up duration of 38 months and 145 (66.0%) patients had CD4-T cell count ≤ 500 cells/mm³ with a median viral load of 10 cps/mL (IQR [10-33]). Seventy five (34.0%) patients had CD4-T cell count ≥ 500 cells/mm³, among them 14 (18.7%) had a VL between 10-100 cps/mL and 2 (2.6%) had VL >100 cps/mL.

Conclusion

This study suggests that the combination of CD4-T cell count and ultrasensitive HIV-2 viral load quantification with a threshold of 10 cps/mL, could improve ART initiation among treatment naïve HIV-2-infected patients and the monitoring of ART response among patients receiving treatment.     

Residual Viremia Is Preceding Viral Blips and Persistent Low-Level Viremia in Treated HIV-1 Patients

Background

It has been suggested that low-level viremia or blips in HIV-infected patients on antiretroviral treatment are related to assay variation and/or increased sensitivity of new commercial assays. The 50-copy cut-off for virologic failure is, therefore, under debate.

Methods

Treated patients with low-level viremia (persistent viral loads (VL) of 50–1000 copies/mL, group A, N = 16) or a blip (single detectable VL, group B, N = 77) were compared to a control group (consistently suppressed viremia since start therapy (<50 copies/mL), N = 79). Residual viremia (detectable viral RNA <50 copies/ml) in the year preceding the first VL above 50 copies/mL (T0) was determined using Roche Cobas-Amplicor v1.5 or CAP-CTM v2.0. Subsequent virologic failure (2 consecutive VLs>500 or 1 VL>1000 copies/mL that was not followed by a VL<50 copies/mL; median follow up 34 months) was assessed.

Results

Significantly more patients in groups A and B had residual viremia in the year preceding T0 compared to controls (50% and 19% vs 3% respectively; p<0.001). Residual viremia was associated with development of low-level viremia or blips (OR 10.9 (95% CI 2.9–40.6)). Subsequent virologic failure was seen more often in group A (3/16) and B (2/77) than in the control group (0/79).

Conclusion

Residual viremia is associated with development of blips and low-level viremia. Virologic failure occurred more often in patients with low-level viremia. These results suggest that low-level viremia results from viral production/replication rather than only assay variation.     

The amplitudes of viral blips in HIV-1 infected patients treated with antiretroviral therapy are power-law distributed

We previously reported that in patients treated with highly active antiretroviral therapy (HAART) who achieve viral load (VL) suppression, low fluctuations of viral load over the threshold of detection (viral blips) more than 4 weeks apart occur at random, with a frequency that does not change with longer times of observation. The etiology of viral blips is currently unknown, but viral blip frequency inversely correlates with the decay of the latent reservoir, whose stability has been proposed as the major hurdle to HIV eradication. We show here that the distribution of viral blip amplitudes observed in a group of 272 patients successfully treated with highly active antiretroviral therapy appears to be power-law distributed. Such a distribution can be theoretically generated by randomly sampling the arrival of asynchronous and overlapping elementary pulses of viremia, with asymptotic exponential decay of kinetics, thus suggesting that the low fluctuations of viremia observed in patients during HAART treatment is, in part, a discrete phenomenon consistent with random activation of latently infected cells or release of virus and infected cells into the blood compartment from unknown sites of active viral replication.     

Using Exclusion-Based Sample Preparation (ESP) to Reduce Viral Load Assay Cost

Viral load (VL) measurements are critical to the proper management of HIV in developing countries. However, access to VL assays is limited by the high cost and complexity of existing assays. While there is a need for low cost VL assays, performance must not be compromised. Thus, new assays must be validated on metrics of limit of detection (LOD), accuracy, and dynamic range. Patient plasma samples from the Joint Clinical Research Centre in Uganda were de-identified and measured using both an existing VL assay (Abbott RealTime HIV-1) and our assay, which combines low cost reagents with a simplified method of RNA isolation termed Exclusion-Based Sample Preparation (ESP).71 patient samples with VLs ranging from <40 to >3,000,000 copies/mL were used to compare the two methods. We demonstrated equivalent LOD (~50 copies/mL) and high accuracy (average difference between methods of 0.08 log, R² = 0.97). Using expenditures from this trial, we estimate that the cost of the reagents and consumables for this assay to be approximately $5 USD. As cost is a significant barrier to implementation of VL testing, we anticipate that our assay will enhance access to this critical monitoring test in developing countries.     

Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling

To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10^-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10^-18 moles of the p24/assay corresponds to ca. 10³ copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (10² copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.     

Preservation of FoxP3+ regulatory T cells in the peripheral blood of human immunodeficiency virus type 1-infected elite suppressors correlates with low CD4+ T-cell activation

Elite suppressors (ES) are untreated human immunodeficiency virus type 1 (HIV-1)-infected individuals who maintain normal CD4⁺ T-cell counts and control viremia to levels that are below the limit of detection of current assays. The mechanisms involved in long-term control of viremia have not been fully elucidated. CD4⁺ CD25⁺ regulatory T cells (Tregs) downmodulate chronic inflammation by suppressing the activation and proliferation of effector lymphocytes. We found that while Tregs were functional in ES and patients on highly active antiretroviral therapy (HAART), ES maintained high levels of Tregs in peripheral blood mononuclear cells whereas patients on HAART had evidence of Treg depletion. We also demonstrated that Tregs can serve as reservoirs for HIV-1 in vivo. These data suggest that both direct infection by HIV-1 and tissue redistribution are possible explanations for declining FoxP3⁺ Tregs in progressive HIV-1 infection. Furthermore, the maintenance of Tregs may be one mechanism associated with the nonprogressive nature of HIV-1 infection in ES.     

A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml.

The branched DNA hybridization assay has been improved by the inclusion of the novel nucleotides, isoC and isoG, in the amplification sequences to prevent non-specific hybridization. The novel isoC, isoG-containing amplification sequences have no detectable interaction with any natural DNA sequence. The control of non-specific hybridization in turn permits increased signal amplification. Addition of a 14 site preamplifier was found to increase the signal/noise ratio 8-fold. A set of 74 oligonucleotide probes was designed to the consensus HIV POL sequence. The detection limit of this new HIV branched DNA amplifier assay was approximately 50 molecules/ml. The assay was used to measure viral load in 87 plasma samples of HIV- infected patients on triple drug therapy whose RNA titers were <500 molecules/ml. In all 11 patients viral load eventually declined to below the detection limit with the new assay.     

Induction of plasma (TRAIL), TNFR-2, Fas ligand, and plasma microparticles after human immunodeficiency virus type 1 (HIV-1) transmission: implications for HIV-1 vaccine design

The death of CD4⁺ CCR5⁺ T cells is a hallmark of human immunodeficiency virus (HIV) infection. We studied the plasma levels of cell death mediators and products—tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), Fas ligand, TNF receptor type 2 (TNFR-2), and plasma microparticles—during the earliest stages of infection following HIV type 1 (HIV-1) transmission in plasma samples from U.S. plasma donors. Significant plasma TRAIL level elevations occurred a mean of 7.2 days before the peak of plasma viral load (VL), while TNFR-2, Fas ligand, and microparticle level elevations occurred concurrently with maximum VL. Microparticles had been previously shown to mediate immunosuppressive effects on T cells and macrophages. We found that T-cell apoptotic microparticles also potently suppressed in vitro immunoglobulin G (IgG) and IgA antibody production by memory B cells. Thus, release of TRAIL during the onset of plasma viremia (i.e., the eclipse phase) in HIV-1 transmission may initiate or amplify early HIV-1-induced cell death. The window of opportunity for a HIV-1 vaccine is from the time of HIV-1 transmission until establishment of the latently infected CD4⁺ T cells. Release of products of cell death and subsequent immunosuppression following HIV-1 transmission could potentially narrow the window of opportunity during which a vaccine is able to extinguish HIV-1 infection and could place severe constraints on the amount of time available for the immune system to respond to the transmitted virus.