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Intensification of Antiretroviral Therapy Accelerates the Decay of the HIV-1 Latent Reservoir and Decreases, But Does Not Eliminate, Ongoing Virus Replication

Ramratnam, Bharat, MD*; Ribeiro, Ruy, PhD; He, Tian*; Chung, Chris*; Simon, Viviana, MD*; Vanderhoeven, Jeroen*; Hurley, Arlene*; Zhang, Linqi, PhD*; Perelson, Alan S., PhD; Ho, David D., MD*; Markowitz, Martin, MD*

JAIDS Journal of Acquired Immune Deficiency Syndromes: January 1st, 2004 - Volume 35 - Issue 1 - p 33-37
Clinical Science
Free

This study evaluated whether intensification of standard antiretroviral therapy with abacavir, with or without efavirenz, leads to better viral suppression and acceleration of the rate of HIV-1 decay. Ten HIV-1–infected individuals were enrolled in a prospective, open-label study and received standard, combination antiretroviral therapy with either 3 or 4 agents. The rate of decay of the HIV-1 latent reservoir and the frequency of intermittent viremia were compared between 5 patients who underwent treatment intensification and 5 control subjects with comparable baseline characteristics. When compared with control patients, the median half-life (t1/2) of the latent reservoir decreased from 31 to 10 months (P = 0.016) in subjects who had treatment intensification. The frequency of intermittent viremia/year also decreased in 4 of 5 individuals following intensification (2.4/y vs. 0.8/y). These data suggest that ongoing virus replication during standard antiretroviral therapy is due, in part, to the inadequate antiviral potency of current regimens. Despite better viral suppression, treatment intensification did not completely block viral replication, as evidenced by continuing intermittent viremia in some individuals. Additional studies are needed to understand the host- and pathogen-related determinants of incomplete pharmacologic control of HIV-1 replication.

From *The Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY, and Los Alamos National Laboratory, Los Alamos, NM.

Received for publication April 10, 2003; accepted September 22, 2003.

Supported by National Institute of Health grants AI40387, RR06555, K23AI01780, the Columbia-Rockefeller Center for AIDS Research (AI42848), and the General Clinical Research Center of The Rockefeller University (MO1-RR00102). BR is a recipient of the Daland Fellowship in Clinical Investigation from The American Philosophical Society.

Reprints: Bharat Ramratnam, Laboratory of Retrovirology, 55 Claverick Street, 4th Floor, Providence, RI 02903 (e-mail: BRamratnam@Lifespan.org).

The goal of antiretroviral therapy is to achieve complete suppression of HIV-1 replication. During the first few months of treatment, virus burden decreases by several orders of magnitude, reflecting the rapid clearance of plasma virions and productively infected cells. 1,2 Thereafter, HIV-1 largely persists in a small reservoir of latently infected memory lymphocytes that are capable of releasing infectious virus upon withdrawal of therapy. 3 In the majority of patients, the latent reservoir remains stable despite prolonged treatment. 4 Although the decay kinetics of memory lymphocytes in humans and monkeys suggest that these cells have a half-life (t1/2) of approximately 6 months, the latent reservoir of HIV-1 has a t1/2 > 48 months in many patients. 4–6 We have previously demonstrated that this slow decay is largely a result of ongoing virus replication that serves to replenish the pool of infected cells. 5 Therefore, we hypothesized that the additional antiviral potency afforded by treatment intensification would decrease HIV-1 replication and thereby lead to an accelerated decay of viral burden. A prospective pilot study was performed to test this hypothesis.

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METHODS

Subjects

To evaluate the antiviral efficacy of treatment intensification, we approached individuals who were participating in antiretroviral drug therapy trials at the Aaron Diamond AIDS Research Center with the aim of recruiting individuals in the treatment and control arms of this prospective nonrandomized pilot study. We limited inclusion to those individuals treated for a minimum of 30 months with at least 1 protease inhibitor and 2 reverse transcriptase inhibitors. Ten individuals receiving prolonged antiretroviral therapy with zidovudine, lamivudine, and nelfinavir or ritonavir/saquinavir were enrolled. Subjects were offered treatment intensification with abacavir, with or without efavirenz, in an open-label, prospective trial. All individuals were chronically infected with HIV-1 and harbored drug-sensitive virus as determined by genotypic analysis of either proviral DNA or virus rescued from culture supernatants. There was no difference in baseline CD4 count (P = 0.296) or levels of plasma HIV-1 RNA (P = 0.834) between the intensification and control groups (Table 1). The frequency of intermittent viremia and the absolute size and decay rate of the latent reservoir of HIV-1 were then compared in 5 individuals who underwent treatment intensification and 5 control subjects. Individuals were initially treated with standard combination antiretroviral therapy (Table 1).

TABLE 1

TABLE 1

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Plasma HIV-1 RNA

The levels of HIV-1 RNA in plasma were determined at monthly intervals using the Roche Ultra-Sensitive HIV-1 Monitor assay with a limit of detection of 50 HIV-1 RNA copies/mL (Roche Molecular Diagnostic Systems, Branchburg, NJ). Intermittent viremia was defined as an episode of plasma HIV-1 RNA > 50 copies/mL after attaining an undetectable level. The frequency of determinations did not vary over the study period or among different patients.

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Estimation of the HIV-1 Latent Reservoir

The absolute size of the latent reservoir of HIV-1 was determined by analyzing the results of serial limiting-dilution microcultures of highly enriched preparations of CD4+ T lymphocytes, as described previously. 5 Briefly, peripheral blood mononuclear cells (PBMCs) were depleted of CD8+ T lymphocytes using magnetic beads (Dynal, Lake Success, NY) and plated in 1:2 dilutions with HIV-1-seronegative donor phytohemagglutinin (PHA)-stimulated CD4+ T-lymphoblasts and irradiated PBMCs. The cultures were maintained over 28 days with regular change of media every 3 days and the weekly addition of PHA-stimulated CD4+ T-lymphoblasts. At day 28, HIV-1 p24 antigen was determined by immunoassay (Abbott Laboratories, Abbott Park, IL). Previous work has demonstrated the validity of this assay for the quantification of the decay kinetics of the latent reservoir of HIV-1 in CD4+ T lymphocytes. Co-cultures were planned so as to occur at even intervals (average ∼8 mo, range 6.5–9 mo) during the preintensification/intensification study period such that individuals were studied a total of 5–7 times. Co-culture results were expressed as infectious units per million (IUPM) PBMCs. Linear regression was used to generate a best-fit line through a plot of ln(IUPM) vs. time. The slope of this line was used to calculate the latent reservoir t1/2 assuming first-order decay kinetics.

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RESULTS

During the first 3 years of therapy, both the intensification and control groups had comparable levels of ongoing HIV-1 replication as suggested by similar frequencies of intermittent viremia (median 3.3/y. vs. 2.8/y, P = 0.531) detected during monthly examinations of plasma HIV-1 RNA level. Furthermore, the kinetics of latent reservoir decay were similar in both groups, with a t1/2 > 29 months for both groups during standard antiretroviral therapy. Thus, by 2 independent measures, viral burden and degree of viral control were similar in the 2 groups. Abacavir alone (n = 4) or with efavirenz (n = 1) was added after individuals had received a mean of 34 months (range: 31–37 months) of treatment. All individuals had relatively well controlled viral replication during the first 3 years of standard therapy with approximately 3–4 episodes of intermittent viremia per year during monthly plasma HIV-1 RNA determinations. The duration of antiretroviral intensification ranged from 5–18 months. When compared with control patients, the median t1/2 of the HIV-1 latent reservoir decreased from 31 to 10 months (P = 0.016) in patients with treatment intensification. Among individual patients, 3 of 5 individuals (#33, 1002, 1013) had a dramatic reduction in latent reservoir size, but 2 patients (#37,39) had no significant change. The 2 individuals with no change in decay pattern were extremely well controlled in terms of virus replication, experiencing 1–2 episodes of intermittent viremia per year. When all the culture data were pooled, the slope of decay of the HIV-1 latent reservoir increased by more than 4-fold and became significantly different from zero (−0.008/mo vs. −0.034/mo, P = 0.007) (Fig. 1). The frequency of intermittent viremia also decreased in 4 of 5 individuals following intensification (median 3.3/y vs. 0.7/y) (Table 1). In contrast, control subjects continued to experience intermittent viremia at the same frequency as during the first 30 months of treatment (median 2.8/y vs. 1.9/y, P = 0.79). Consequently, 4 of 5 of the control patients had no appreciable decay of the latent reservoir. A strong inverse correlation (r = 0.64, P = 0.01) was found between the rate of decay of the HIV-1 latent reservoir and the frequency of intermittent viremia, in agreement with previous findings. 5 CD4+ T-lymphocyte counts increased in all patients and no significant difference was observed among the intensification and control groups at the conclusion of the study (Table 2).

TABLE 2

TABLE 2

FIGURE 1.

FIGURE 1.

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DISCUSSION

The etiology of ongoing virus replication during highly active antiretroviral therapy is unclear. Recent studies estimate that a significant number of individuals (up to 40%) harbor low-level virus replication during treatment. 7–9 Though intermittent viremia has not been associated with overall virologic failure during 1 year of follow-up, recent reports have demonstrated their association with the selection of drug-resistant HIV. 10 Given the life-long nature of HIV treatment, completely suppressive regimens will be needed to minimize the risk of developing drug resistance.

Although treatment intensification in our study yielded detectable improvements in antiviral potency, HIV-1 replication was not completely suppressed. Three of 5 patients continued to experience intermittent viremia, albeit at lower frequencies. The limitations of this study are perhaps best illustrated by Patient #33, who had no appreciable decrease in intermittent viremia but a rapid decay of the latent reservoir of HIV-1. Upon further analysis, the patient's episodes of intermittent viremia were closely clustered during intensification. Of a total of 20 plasma HIV-1 RNA determinations over the 17-month intensification period, 5 were detectable. Of these 5 episodes, 4 occurred during the first month of treatment intensification and 15 of the 16 remaining determinations were undetectable. Thus, Patient #33 had a prolonged (>1 year) period of optimum viral suppression that translated into a more rapid decay of the latent reservoir. In contrast, Patients #37 and #39 had no accelerated decay of the reservoir despite a decrease in the frequency of intermittent viremia. For example, Patient #37 experienced a total of 6 episodes of intermittent viremia during 3 years of monthly determinations and no episodes during the 17 months of intensification. Five of 6 episodes were below 150 HIV-1 RNA copies/mL plasma. We hypothesize that the co-culture assay may not be sensitive enough to detect differences in viral burden in such individuals who began our pilot study with relatively well suppressed levels of viral replication.

Our findings are in contrast to a recent report in which a combination of treatment intensification (didanosine and hydroxyurea) and immune stimulation (OKT3 followed by interleukin-2) reduced viral burden to undetectable levels in 3 patients. 11 A limit to drug efficacy may be operational for HIV-1. A threshold of potency may exist based upon host factors such as drug efflux proteins with their generic affinity to certain classes of antiviral drugs (e.g., p-glycoprotein in the case of protease inhibitors and multidrug-resistant proteins [4/5] for reverse transcriptase inhibitors). Alternatively, current drugs may not sufficiently permeate sanctuary sites for the complete elimination of all viral replication. Recent work has demonstrated that standard prolonged treatment does not lead to the decay of the latent reservoir and indeed 4 of 5 patients in our control group had slow or no decay. 12

Based on these data, the antiviral potency of current regimens emerges as an important, but not the sole, determinant of complete viral control. In certain patients, latent reservoir decay can be hastened with treatment intensification, even when intensification occurs after 3 years of standard treatment. Future investigations must address host factors, such as cellular drug efflux proteins and differential drug metabolism, that may limit the antiviral efficacy of HIV-1 therapy. Such studies may allow the design of agents and regimens that are more effective in reducing total HIV-1 burden and controlling virus replication.

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REFERENCES

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Keywords:

antiretroviral therapy; HIV-1 latency; treatment intensification; virus replication

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