Antiretroviral therapy with the integrase inhibitor raltegravir alters decay kinetics of HIV, significantly reducing the second phase
Murray, John Ma,b; Emery, Seana; Kelleher, Anthony Da; Law, Matthewa; Chen, Joshuac; Hazuda, Daria Jc; Nguyen, Bach-Yen Tc; Teppler, Hedyc; Cooper, David Aa
From the aNational Centre in HIV Epidemiology and Clinical Research, Australia
bSchool of Mathematics and Statistics, University of New South Wales, Sydney, Australia
cMerck Research Laboratories, North Wales, Pennsylvania, USA.
Received 14 June, 2007
Revised 10 August, 2007
Accepted 13 August, 2007
Correspondence to Dr J. Murray, School of Mathematics and Statistics, University of New South Wales, Sydney, NSW 2052, Australia. E-mail: J.Murray@unsw.edu.au
Objective: Raltegravir (MK-0518) belongs to the new class of HIV integrase inhibitors. To date, there have been no reports investigating the potential for differential effects on viral dynamics with integrase inhibitors relative to current antiretroviral drugs.
Methods: Patients in this phase II study (P004) were antiretroviral treatment naive. Part 1 of this study compared monotherapy with raltegravir (100 mg, 200 mg, 400 mg, or 600 mg twice daily) with placebo over 10 days. In part 2, patients were enrolled for 48 weeks of combination therapy, with randomization to one of the four dosages of raltegravir or to efavirenz, in addition to tenofovir and lamivudine. Mathematical models were used to investigate processes underlying viral dynamics.
Results: From day 15 through to day 57, individuals in the raltegravir arm were significantly more likely to have HIV RNA < 50 copies/ml (P ≤ 0.047). Plasma viral loads were 70% lower at initiation of second-phase decay for individuals taking raltegravir than for those taking efavirenz (P < 0.0001). This challenges the current hypothesis that second-phase virus originates from infected long-lived cells, as an integrase inhibitor should not impact on viral production from this cell population. Mathematical modeling supported two hypotheses as consistent with these observations: (i) that second-phase virus arises from cells newly infected by long-lived infected cells and (2) that it arises from activation of latently infected cells with full-length unintegrated HIV DNA.
Conclusions: These observations challenge the current understanding of HIV-1 turnover and compartmentalization. They also indicate the promise of this new integrase inhibitor raltegravir.
HIV integrase inhibitors are a new class of antiretroviral drugs that target a crucial process in the life cycle of HIV [1–3]. They act by blocking incorporation of the proviral HIV DNA into the host cell DNA. As productive infection requires integration of HIV DNA, an integrase inhibitor prevents an infectious virion that has progressed through reverse transcription from successfully infecting the cell. This class of drugs has a mode of action that is independent of reverse transcriptase and protease inhibitors and has the capacity to assist in control of HIV replication.
Ten-day monotherapy trials investigating dosing levels and tolerability of integrase inhibitors have been reported [1,3], and preliminary results from phase II studies suggest that this new class of drugs can be effective in controlling HIV replication even in individuals infected with multiclass drug-resistant virus . To date, however, there have been no reports investigating the potential for differential effects on viral dynamics with integrase inhibitors relative to current antiretroviral drugs.
Classically, the reduction in HIV replication under antiretroviral therapy (ART) is characterized by a biphasic decay pattern of HIV RNA in blood. The first phase exhibits a mean half-life of between 0.9 and 1.6 days and occurs over the first 7 to 10 days [5–7]. The second phase has a mean half-life of 14 days and successful therapy reduces plasma HIV RNA below standard limits of detection . Analysis of HIV RNA < 50 copies/ml suggests that HIV RNA stabilizes at low but detectable levels in plasma, with 50% of patients who have been taking ART for at least 6 months having HIV RNA > 2.5 copies/ml .
It is generally believed that virus observed during the first-phase decay arises from productively infected CD4 T cells, and that this phase of viral decay tracks their death [9,10]. The origins of HIV RNA during the second phase are not clearly defined. It has been hypothesized the virus may arise from long-lived infected cells , from dissociation of virus from follicular dendritic cells  or from productively infected CD4 T cells that are not subject to clearance by the host immune responses given the waning levels of HIV antigen .
The addition of an integrase inhibitor such as raltegravir (formerly known as MK-0518) to antiretroviral drugs from other classes may be expected to provide at least an additive effect on inhibition of HIV replication. However, it would not be expected to interfere with production of progeny virus from the above sources since the cells that are hypothesized to be responsible for the observed second-phase virus are already infected or, in the case of dissociation from dendritic cells, these virions have already been produced.
To investigate these matters, viral dynamics were analyzed from treatment-naive patients enrolled in a phase II dose-ranging study of raltegravir over 10 days of monotherapy and then in comparison over 48 weeks with efavirenz, each in combination with two other antiretroviral drugs.
Patients were naive to ART or had received less than 7 days total ART. Patients were at least 18 years of age with a positive HIV enzyme-linked immunosorbent test, had HIV RNA of ≥ 5000 copies/ml and a CD4 cell count of ≥ 100 cells/μl. HIV RNA was assessed by the AMPLICOR HIV-1 Monitor Standard Assay version 1.5 (limit of detection, 400 copies/ml; Roche Molecular Systems, Branchburg, New Jersey, USA); those samples below the limit of detection were reassessed by the AMPLICOR HIV-1 Monitor UltraSensitive Assay version 1.5 (limit of detection, 50 copies/ml).
The mathematical models are described in the supplementary text. Statistical comparisons for continuously distributed variables between groups were performed with the Wilcoxon rank sum test, while comparisons for proportions of patients above or below detection limits were performed using Fisher's exact test. Nominal P values < 0.05 were considered significant and all tests were two sided. In this exploratory analysis, no adjustments were made to P values for multiple testing. Mathematical modeling and statistical analyses were conducted within Matlab, version 7, The MathWorks.
Patients in this study were naive for ART, had baseline plasma HIV RNA of ≥ 5000 copies/ml and CD4 T cell counts ≥ 100 cells/μl. This phase II dose ranging study of raltegravir was conducted in two parts. Part 1 compared monotherapy with raltegravir against placebo over 10 days . Patients entering part 1 were randomized to one of four different dosages of raltegravir (100, 200, 400 and 600 mg), each taken twice daily, or matched placebo. In Part 1 there were approximately eight patients in each of the five groups. At the end of Part 1 therapy was stopped before commencement of Part 2. In Part 2, an additional 150 patients were enrolled for 48 weeks of combination therapy, with randomization to one of the four dosages of raltegravir or to efavirenz (600 mg every night), in addition to tenofovir (300 mg once a day) and lamivudine (300 mg once a day) for all individuals.
First-phase decay with raltegravir monotherapy
Monotherapy with raltegravir over 10 days produced extensive monophasic decay for all dosage groups, with a median 2.2 log10 copies/ml decrease. Linear regression analysis determined no significant differences in the first-phase half-lives between the dosage groups (P ≥ 0.18). Mean half-lives were 1.3, 1.1, 1.3, and 1.2 days for the 100, 200, 400 and 600 mg raltegravir groups, respectively, with an overall mean of 1.2 days (SD, 0.26). The first-phase half-lives were consistent with the ranges previously observed [5–7].
Combination therapy with raltegravir
Under combination therapy, each of the raltegravir treatment groups achieved undetectable HIV RNA (<50 copies/ml), faster than the efavirenz group. From the second (day 15 of therapy, on average) to the fourth time point (day 57), patients in the raltegravir groups were more likely to be below the level of HIV detection (Table 1). Since first-phase monotherapy decay rates were indistinguishable for each of the dosage groups, and all raltegravir dosages achieved undetectable RNA at similar rates with combination therapy, subsequent analysis was performed on the combined raltegravir patients. Plasma HIV RNA was significantly lower for the combined raltegravir group until day 168 (Fig. 1). From day 1 to day 15, median HIV RNA fell from 58 350 to 98 copies/ml for the combined raltegravir patients, while those in the efavirenz group decreased from 70 200 to 537 copies/ml.
Second-phase half-lives were calculated using linear regression on HIV RNA log10 copies/ml from the second time point (day 15 on average), until the first point where HIV RNA fell below the 50 copies/ml limit. The median second-phase half-life for raltegravir individuals was 15.5 days [interquartile range (IQR), 14], whereas it was 18.3 days (IQR, 10) for those on efavirenz, but these half-lives were not significantly different (P = 0.2). The slightly numerically faster second-phase dynamics for the raltegravir group may be because some of these individuals had a first-phase decline that was prolonged beyond day 15. This appears likely given the extended first-phase decline exhibited under monotherapy. Including only those individuals on raltegravir who had detectable HIV RNA at days 15 and 29 (n = 42), gave a median second-phase half-life of 19.6 days, which also was not significantly different to the efavirenz group (P = 0.6).
Although the rate of decline during the second phase did not differ between the raltegravir and efavirenz regimens, the point at which this decline initiated did differ. The significantly lower viral levels in plasma from day 15 onwards for the raltegravir group meant that the first phase of viral decay was more extensive, thereby reducing the viral level at which the second phase commenced (Fig. 1). Estimation of the size of the viral compartment contributing to second-phase viral decay was calculated by extrapolating the regression line of this second phase back to baseline. The ratio of this baseline second-phase value to baseline HIV RNA (‘M ratio’), has been listed for six individuals in Perelson et al.  (Table 1, pM*0/cV0). In that study combination therapy with nelfinavir, zidovudine and lamividine resulted in M ratios with a mean of 0.04 and median of 0.03. The lower the M ratio the more extensive was first phase decline. In the present study, M ratios for the efavirenz group were lower but not statistically different to values reported in the literature, with the higher literature values perhaps arising from a slightly different method where a viral load curve was fitted to the data in that study  (P = 0.08). The M ratio was 70% lower for individuals taking raltegravir than efavirenz (median 0.0042 versus 0.014; P < 0.0001), implying that raltegravir reduces viral production from the second-phase source by this amount over and above standard regimens.
The decrease in the second-phase viral production with the addition of raltegravir necessitates rethinking current theories of the source of virus responsible for the second phase. Current hypotheses are that second-phase viral load arises through production by long-lived infected cells , release from follicular dendritic cells  or decreased cytotoxic lymphocyte response . None of these theories can explain the effects produced by raltegravir, if it is acting as a pure integrase inhibitor, since they suggest that second-phase viral load originates from sources that an integrase inhibitor would not affect: previously infected cells or virus already produced but bound to dendritic cells.
Hypotheses for second-phase virus
If an integrase inhibitor is to influence viral levels in the second phase, the virus at that stage must arise from (i) cells that are either newly infected and where the integrase inhibitor provides an additional benefit above that of reverse transcriptase and protease inhibitors, (ii) cells that have been previously infected but where the proviral DNA has yet to be integrated into the host cell genome, or (iii) a sanctuary site where the integrase inhibitor has improved penetration. To investigate the viability of these hypotheses as explanations of the reduced second phase under an integrase inhibitor, three mathematical models were developed and applied to the median data derived from the monotherapy and combination trials with raltegravir and efavirenz. Model 1 simulated the first hypothesis above, model 2 simulated the second, and model 3 simulated the third (supplementary text). A cartoon of the effects of raltegravir in these models is displayed in Fig. 2.
In model 1, new productive infection (I), which generates virus (V) in the second phase, originates from long-lived infected cells (M). These latter cells decay at the slow rate observed in this phase, thereby providing this same dynamic to newly infected cells and second-phase virus (Fig. 3a,b). The integrase inhibitor provides an additional effect to reverse transcriptase and protease inhibitors in their inhibition of new productive infection from long-lived infected cells, decreasing second-phase viral levels more (solid lines Fig. 3a,b) than with efavirenz (dashed lines).
The results achieved with the integrase inhibitor in model 1 could theoretically be achieved through more potent reverse transcriptase or protease inhibitors. By comparison, model 2 simulates a process of infection that is not susceptible to traditional drugs, where virus from the second phase arises from latently infected cells that contain proviral DNA in a full-length unintegrated form, which has been estimated to form the vast majority of latent infection . In this model, second-phase virus originates from latently infected cells with unintegrated viral DNA (D) that are activated and converted to productively infected cells (I). Increased potency of traditional drugs will reduce the replenishment of the latent pool D but will not stop its conversion to productive infection. Model 2 also provides a consistent explanation for the effect achieved by the integrase inhibitor (Fig. 3c,d).
The third model, where the differential effects of the integrase inhibitor are achieved through better penetration of a sanctuary site, was less successful in duplicating the data (Fig. 3e,f). Although it achieved lower second-phase viral load, the slope of this second phase was significantly faster for the integrase inhibitor (solid line Fig. 3e) than for the efavirenz arm (dashed line Fig. 3e). This was at odds with the results described above, where there was no significant difference. The second-phase virus in this model traffics from the sanctuary site, where it is produced by productively infected cells. In order to achieve this differential effect for the integrase inhibitor, the efavirenz arm must have extremely low efficacy, 6%, in order for the additional efficacy provided by the integrase inhibitor to significantly perturb the decay rate of productively infected cells in the sanctuary site (green solid line versus green dashed line Fig. 3f).
Raltegravir significantly extended the first phase of viral decay and reduced the plasma HIV RNA level at which the second phase commenced (Fig. 1). It enabled individuals to suppress HIV RNA to below detection limits significantly faster than current regimens (Table 1). The 50 copies/ml assay limit did not allow investigation of any effects this may have on subsequent viral levels. By comparing M ratios for individuals enrolled on the raltegravir arm with those on the efavirenz arm, raltegravir achieved a 70% reduction of second-phase viral levels.
The effect of raltegravir on the second phase of viral decay was unexpected given current hypotheses for the processes underlying this phase [6,11,12] and the mechanism by which the drug is thought to work. We proposed three hypotheses to explain the observed effects and compared simulations of mathematical models for these hypotheses to median data for ART with or without an integrase inhibitor. Models 1 and 2 successfully reproduced the clinical data (Fig. 3).
The effect of adding an integrase inhibitor to model 1 is to provide an additional effect to that of the reverse transcriptase inhibitors (and any protease inhibitors that act in the model through decreasing infectivity), thereby decreasing new infection from virus associated with long-lived infected cells. Hence under these assumptions, the integrase inhibitor does not target new infection mechanisms but reduces the success of transmission from long-lived infected cells to productively infected cells. Model 2, where the second phase is assumed to arise from activation of latently infected cells that have HIV DNA in a predominantly unintegrated form, provides a mechanism that is completely resistant to any additional benefit from improvements in reverse transcriptase inhibition. For this second model, the integrase inhibitor provides a protective mechanism that cannot be replaced by reverse transcriptase or protease inhibitors, no matter their potency, since for reverse transcriptase and protease inhibitors new virions will be produced, although these will be mostly noninfectious in the presence of protease inhibitors . Under each of these models, there may be an increase in aborted HIV DNA integration and a resulting rise in extrachromosomal viral DNA in circular forms containing one or two long terminal repeats (2LTR circles), as observed in vitro . In those experiments, cell viability was not affected, and clinical trials with raltegravir have not shown toxicities higher than with current antiretroviral drugs [3,4]. Therefore, any resulting increases in unintegrated HIV DNA would not be expected to be detrimental to these cells. Although not conclusive, the simulations of model 3 suggest that second-phase virus does not originate from a sanctuary site where raltegravir is more successful in penetrating than standard antiretroviral drugs.
Availability of protease inhibitors in the 1990s and their combination with reverse transcriptase inhibitors evoked hopes that this new potent ART would at last allow clearance of HIV infection. Estimates were made on the length of treatment needed before this could be achieved . Although plasma levels of HIV RNA fall below detection limits within several months of ART, it was soon realized that latent infection provided a lower bound on the rate at which HIV infection could be removed from the body. This component is established early in infection  and consists of both unintegrated and integrated HIV DNA even under ART . The high levels under ART of unintegrated HIV DNA in resting CD4 T cells , and proviral sequence evolution in individuals with undetectable HIV RNA , suggest that there is persistent viral replication even in the presence of seemingly successful ART. Regardless of whether ongoing infection or the slow turnover of resting CD4 T cells is primarily responsible, the latent pool decreases slowly under standard ART, with a half-life of anywhere between 6 months [16,17] and 44 months . This leads to estimates of between 8 years [16,18] and more than 60 years for clearance of infection. The rate of decrease is dependent on residual viral replication , and hence drugs like raltegravir that increase the effectiveness of ART, especially for viral components such as unintegrated HIV DNA that are immune to current drugs, may improve this rate of decay.
There is considerable uncertainty as to the prevalence of unintegrated HIV DNA in resting CD4 T cells during ART, and the impact this component makes to renewing the integrated pool. In-vitro experiments have established that HIV can enter resting CD4 T cells with high efficiency, but that productive infection does not ensue. The cause of this failure seems dependent on the cell culture conditions, as infection of resting cells can fail during reverse transcription [19,20] or through the lability of the full-length unintegrated HIV DNA transcript , or it can lead to stable infection that is transcriptionally inactive without cellular activation . The in-vivo situation under ART is equally unclear. Total HIV DNA exceeds integrated HIV DNA in resting CD4 T cells by 100-fold, yet does not seem to contribute significantly to replication competent virus . The presence of unintegrated HIV DNA under ART, however, indicates either that there is substantial ongoing infection, if this molecule is labile, or that it is long lived. If it is the latter, then integrase inhibitors can provide an effective block in new productive infection upon activation of this latent component, as hypothesized in model 2. Regardless, integrated HIV DNA must undergo some replenishment since it decays slowly under ART but rebounds within weeks upon cessation of therapy . It is difficult to reconcile these very different dynamics if there is no compensatory loss in this compartment, and hence continual replacement. Detailed analysis of integrated and unintegrated HIV DNA in resting CD4 T cells, in the presence and absence of an integrase inhibitor, would provide valuable information on this matter.
We thank the patients and investigators who participated in this study.
Sponsorship: The National Centre in HIV Epidemiology and Clinical Research is funded by the Australian Commonwealth Department of Health and Ageing and is affiliated with the Faculty of Medicine at the University of New South Wales. AK is supported by a Practitioner Fellowship from the NHMRC.
Note: The P004 study is registered at ClinicalTrials.gov under identifier NCT00100048.
Note: Supplementary material can be accessed online.
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antiviral therapy; HIV-1; integrase inhibitors; mathematical models; raltegravir; viral load
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