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.     

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.     

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.     

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.     

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.     

Resting CD4+ T lymphocytes but not thymocytes provide a latent viral reservoir in a simian immunodeficiency virus-Macaca nemestrina model of human immunodeficiency virus type 1-infected patients on highly active antiretroviral therapy

Despite suppression of viremia in patients on highly active antiretroviral therapy (HAART), human immunodeficiency virus type 1 persists in a latent reservoir in the resting memory CD4(+) T lymphocytes and possibly in other reservoirs. To better understand the mechanisms of viral persistence, we established a simian immunodeficiency virus (SIV)-macaque model to mimic the clinical situation of patients on suppressive HAART and developed assays to detect latently infected cells in the SIV-macaque system. In this model, treatment of SIV-infected pig-tailed macaques (Macaca nemestrina) with the combination of 9-R-(2-phosphonomethoxypropyl)adenine (PMPA; tenofovir) and beta-2',3'-dideoxy-3'-thia-5-fluorocytidine (FTC) suppressed the levels of plasma virus to below the limit of detection (100 copies of viral RNA per ml). In treated animals, levels of viremia remained close to or below the limit of detection for up to 6 months except for an isolated "blip" of detectable viremia in each animal. Latent virus was measured in blood, spleen, lymph nodes, and thymus by several different methods. Replication-competent virus was recovered after activation of a 99.5% pure population of resting CD4(+) T lymphocytes from a lymph node of a treated animal. Integrated SIV DNA was detected in resting CD4(+) T cells from spleen, peripheral blood, and various lymph nodes including those draining the gut, the head, and the limbs. In contrast to the wide distribution of latently infected cells in peripheral lymphoid tissues, neither replication-competent virus nor integrated SIV DNA was detected in thymocytes, suggesting that thymocytes are not a major reservoir for virus in pig-tailed macaques. The results provide the first evidence for a latent viral reservoir for SIV in macaques and the most extensive survey of the distribution of latently infected cells in the host.     

A stochastic model of latently infected cell reactivation and viral blip generation in treated HIV patients

Motivated by viral persistence in HIV+ patients on long-term anti-retroviral treatment (ART), we present a stochastic model of HIV viral dynamics in the blood stream. We consider the hypothesis that the residual viremia in patients on ART can be explained principally by the activation of cells latently infected by HIV before the initiation of ART and that viral blips (clinically-observed short periods of detectable viral load) represent large deviations from the mean. We model the system as a continuous-time, multi-type branching process. Deriving equations for the probability generating function we use a novel numerical approach to extract the probability distributions for latent reservoir sizes and viral loads. We find that latent reservoir extinction-time distributions underscore the importance of considering reservoir dynamics beyond simply the half-life. We calculate blip amplitudes and frequencies by computing complete viral load probability distributions, and study the duration of viral blips via direct numerical simulation. We find that our model qualitatively reproduces short small-amplitude blips detected in clinical studies of treated HIV infection. Stochastic models of this type provide insight into treatment-outcome variability that cannot be found from deterministic models.     

Significant reduction in HIV-1 plasma viral load but not in proviral infected cells during sub-optimal antiretroviral therapy

Attempts to eradicate HIV infection through highly active antiretroviral therapy (HAART) in the very early stages of the infection have failed due to the resumption of viral replication from unknown reservoirs. It has been postulated that antiretroviral therapy capable of suppressing viral replication, as shown by reduction of HIV-RNA copies in plasma and lymph nodes, should have less effect on the number of HIV-DNA carrying cells in the same districts. To test this hypothesis, plasma viremia and the proportion of provirally infected cells in peripheral blood and in lymph nodes were measured in patients at 3 and 6 months of treatment with zidovudine plus lamivudine. All patients showed a significant decrease in plasma viremia at 3 months that was maintained at 6 months (mean values of 1.6 +/- 0.6 log10 from baseline). Conversely the proportion of HIV-DNA carrying cells slightly declined at 3 months but remained substantially stable thereafter both in peripheral blood and in lymph nodes. Taken together these data suggest that this therapeutic regimen, although sub-optimal, is effective in significantly reducing the virus production by productively infected cells but does not seem to substantially affect the load of provirally infected cells.     

The distribution of viral blips observed in HIV-1 infected patients treated with combination antiretroviral therapy

Human immunodeficiency virus type 1 (HIV-1) infected patients treated with combination antiretroviral therapy frequently have the level of HIV-1 RNA detectable in plasma driven below the lower limit of detection of current assays, 50 copies ml⁻¹. Patients may continue to exhibit viral loads (VLs) below the assay limit for years, yet on some occasions the VL may be above the limit of detection. Whether these ‘blips’ in VL are simply assay errors or are indicative of intermittent episodes of increased viral replication is of great clinical concern. By analyzing the occurrence of viral blips in 123 treated HIV-infected patients, we show that patients do not share a common probability distribution of blip amplitude and thus reject the hypothesis that blips are solely due to assay variation. Work performed under the auspices of the U.S. Department of Energy.     

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.     

Limits on replenishment of the resting CD4+ T cell reservoir for HIV in patients on HAART

Whereas cells productively infected with human immunodeficiency virus type 1 (HIV-1) decay rapidly in the setting of highly active antiretroviral therapy (HAART), latently infected resting CD4(+) T cells decay very slowly, persisting for the lifetime of the patient and thus forming a stable reservoir for HIV-1. It has been suggested that the stability of the latent reservoir is due to low-level viral replication that continuously replenishes the reservoir despite HAART. Here, we offer the first quantitative study to our knowledge of inflow of newly infected cells into the latent reservoir due to viral replication in the setting of HAART. We make use of a previous observation that in some patients on HAART, the residual viremia is dominated by a predominant plasma clone (PPC) of HIV-1 not found in the latent reservoir. The unique sequence of the PPC serves as a functional label for new entries into the reservoir. We employ a simple mathematical model for the dynamics of the latent reservoir to constrain the inflow rate to between 0 and as few as 70 cells per day. The magnitude of the maximum daily inflow rate is small compared to the size of the latent reservoir, and therefore any inflow that occurs in patients on HAART is unlikely to significantly influence the decay rate of the reservoir. These results suggest that the stability of the latent reservoir is unlikely to arise from ongoing replication during HAART. Thus, intensification of standard HAART regimens should have minimal effects on the decay of the latent reservoir.     

Development of anti-retroviral resistance of HIV-1 infected individuals on therapy: is it inevitable?

Highly active antiretroviral therapy directed against HIV-1 has dramatically modified morbidity and mortality in infected individuals. Enthusiasm for the success of these medications have been tempered by an inability to clear virus from the infected host leaving a virus poised to leverage any advantage into one of productive survival. One mechanism used to accomplish escape from suppression secondary to antiretroviral therapy is by developing mutations. The goal of therapy has been to diminish viral replication, thereby effectively abrogating the development of these resistance-bearing mutations. This strategy has met with significant success but numerous host-viral factors impact on the ability of the clinician to persistently suppress viral load, thereby providing a window of opportunity for the virus to mutate. In particular we review evidence for ongoing viral replication in the face of suppressive antiretroviral therapy and viral replication in tissue compartments. We discuss whether viral resistance can develop during transient elevations in viral load (viral blips) or as a function of the rate of viral load decay while on therapy. Finally, we touch on the therapeutic strategy that diminished viral replication capacity of mutational species can maintain host immunity.     

Residual Viral Replication during Antiretroviral Therapy Boosts Human Immunodeficiency Virus Type 1-Specific CD8+ T-Cell Responses in Subjects Treated Early after Infection

Human immunodeficiency virus type 1 (HIV-1)-infected subjects treated early after infection have preserved HIV-1-specific CD4+ T-cell function. We studied the effect of highly active antiretroviral therapy (HAART) on the frequency of HIV-1-specific CD8+ T cells in patients treated during early (n = 31) or chronic (n = 23) infection. The degree of viral suppression and time of initiation of treatment influenced the magnitude of the CD8+ T-cell response. HIV-1-specific CD8+ T cells can increase in number after HAART in subjects treated early after infection who have episodes of transient viremia.     

Lymphocyte subsets and viral load in patients with HIV-associated non-Hodgkin's lymphoma treated with anti-CD20 monoclonal antibody and chemotherapy

The anti-CD20 monoclonal antibody Rituximab is a novel antitumor agent used in association with chemotherapy (CT) for the treatment of high-grade/intermediate non-Hodgkin's lymphomas (NHL) in HIV-negative populations. This therapeutic combination is currently also being explored in HIV-positive patients with NHL (HIV-NHL). The objective of our study was to determine CD4 and CD8T cell counts, HIV plasma viremia and proviral load in patients with CD20-positive HIV-NHL treated with Rituximab plus CT and highly active antiretroviral therapy (HAART). We studied eight patients with HIV-NHL treated by anti-CD20 and CT before, after three, and after six cycles of therapy; CD4, CD8 and CD19 lymphocyte subsets were measured by monoclonal antibodies and flow cytometry. HIV plasma viremia was determined by the b-DNA assay, and proviral load by a quantitative competitive PCR. CD4T cell counts remained stable after three cycles of therapy, while a significant reduction of this subset was present at the end of therapy. HIV plasma viremia was significantly reduced after the third cycle, but returned to pretreatment levels at the end of therapy; we also observed individual fluctuations of proviral load during therapy, this marker being increased in two out of three patients at the end of therapy. These observations suggest that Rituximab plus CT accelerated the rate of CD4 depletion and of HIV replication in the peripheral blood of HIV-NHL patients and that HAART may be able to delay these effects. Received: 1 December 2000 / Accepted: 8 February 2001     

Replication-competent simian immunodeficiency virus (SIV) Gag escape mutations archived in latent reservoirs during antiretroviral treatment of SIV-infected macaques

In response to pressure exerted by major histocompatibility complex (MHC) class I-mediated CD8(+) T cell control, human immunodeficiency virus (HIV) escape mutations often arise in immunodominant epitopes recognized by MHC class I alleles. While the current standard of care for HIV-infected patients is treatment with highly active antiretroviral therapy (HAART), suppression of viral replication in these patients is not absolute and latently infected cells persist as lifelong reservoirs. To determine whether HIV escape from MHC class I-restricted CD8(+) T cell control develops during HAART treatment and then enters latent reservoirs in the periphery and central nervous system (CNS), with the potential to emerge as replication-competent virus, we tracked the longitudinal development of the simian immunodeficiency virus (SIV) Gag escape mutation K165R in HAART-treated SIV-infected pigtailed macaques. Key findings of these studies included: (i) SIV Gag K165R escape mutations emerged in both plasma and cerebrospinal fluid (CSF) during the decaying phase of viremia after HAART initiation before suppression of viral replication, (ii) SIV K165R Gag escape mutations were archived in latent proviral DNA reservoirs, including the brain in animals receiving HAART that suppressed viral replication, and (iii) replication-competent SIV Gag K165R escape mutations were present in the resting CD4(+) T cell reservoir in HAART-treated SIV-infected macaques. Despite early administration of aggressive antiretroviral treatment, HIV immune escape from CD8(+) T cell control can still develop during the decaying phases of viremia and then persist in latent reservoirs, including the brain, with the potential to emerge if HAART therapy is interrupted.     

HIV-1 DNA and RNA kinetics in primary HIV infection

BACKGROUND: HIV-1 reservoir is early established during PHI. It is reduced, but not extinguished by early therapy: DNA containing cells are still detectable after months of successful viremia suppression. To define the best method to measure low level viral replication, we determined the extent of HIV reservoir in 11 acutely infected patients and evaluated how it is renewed even during successful treatment. METHODS: Eleven acutely infected HIV patients were included in the study. Three where not treated with antiretroviral drugs while 8 underwent early aggressive antiretroviral treatment (HAART) which, in 3 cases, was associated to cyclosporin A (CsA) administration. HIV viremia was monitored by commercially available methods while HIV-DNA and cellular RNA quantitation were obtained by in house PCR and RT-PCR respectively, in the gag region. RESULTS: Significant CD4 recover and HIV viremia suppression were reached in a mean period of three to six months in all treated patients. The course of the HIV-DNA and of cellular HIV RNA reduction showed a similar trend. This variation was slower, if compared to plasma viremia and never reached undetectable levels, justifying the rebound of viremia observed at therapy interruption. CONCLUSIONS: These data suggest and confirm that complete abolition of viral replication is not achieved and viral reservoir may be re-expanded even after short term rebound of viremia. Scheduling of possible structured therapy interruption should be designed based on multiple virological parameters and on the individual characteristics of the patients.     

Immunologic pressure within class I-restricted cognate human immunodeficiency virus epitopes during highly active antiretroviral therapy

Cytotoxic T lymphocytes (CTL) and highly active antiretroviral therapy (HAART) are known to exert strong evolutionary pressures on the virus population during human immunodeficiency virus (HIV) infection. However, it is not known whether CTL responses continue to substantially affect viral evolution during treatment. To study the effect of immunologic pressure on viral sequences during HAART, we identified 10 targeted HIV-specific CD8+-T-cell epitopes in five treatment-naive patients, sequenced each epitope in plasma-derived viruses, and then identified evidence of immunologic pressure at these epitopes by comparing the frequency of viral variants in plasma to the frequency of the CD8+-T-cell response for each variant identified. For one of the five patients, evidence of viral evolution was found during therapy. The sequence of the CTL-targeted epitope changed from an apparent escape variant prior to the initiation of therapy, to the sequence that is best recognized by the CTL response after the initiation of therapy, and then finally to a new escape variant during continued therapy. These data show that CTL-mediated pressure can continue to affect viral evolution after the initiation of HAART, even when treatment drives the viral load below detectable levels, and suggest that antiretroviral therapy may preferentially inhibit those virus variants that escape the CTL response.     

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.     

Evidence for Human Immunodeficiency Virus Type 1 Replication In Vivo in CD14+ Monocytes and Its Potential Role as a Source of Virus in Patients on Highly Active Antiretroviral Therapy

In vitro studies show that human immunodeficiency virus type 1 (HIV-1) does not replicate in freshly isolated monocytes unless monocytes differentiate to monocyte-derived macrophages. Similarly, HIV-1 may replicate in macrophages in vivo, whereas it is unclear whether blood monocytes are permissive to productive infection with HIV-1. We investigated HIV-1 replication in CD14(+) monocytes and resting and activated CD4(+) T cells by measuring the levels of cell-associated viral DNA and mRNA and the genetic evolution of HIV-1 in seven acutely infected patients whose plasma viremia had been <100 copies/ml for 803 to 1,544 days during highly active antiretroviral therapy (HAART). HIV-1 DNA was detected in CD14(+) monocytes as well as in activated and resting CD4(+) T cells throughout the course of study. While significant variation in the decay slopes of HIV-1 DNA was seen among individual patients, viral decay in CD14(+) monocytes was on average slower than that in activated and resting CD4(+) T cells. Measurements of HIV-1 sequence evolution and the concentrations of unspliced and multiply spliced mRNA provided evidence of ongoing HIV-1 replication, more pronounced in CD14(+) monocytes than in resting CD4(+) T cells. Phylogenetic analyses of HIV-1 sequences indicated that after prolonged HAART, viral populations related or identical to those found only in CD14(+) monocytes were seen in plasma from three of the seven patients. In the other four patients, HIV-1 sequences in plasma and the three cell populations were identical. CD14(+) monocytes appear to be one of the potential in vivo sources of HIV-1 in patients receiving HAART.