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Interruption of Enfuvirtide in HIV-1-Infected Adults with Incomplete Viral Suppression on an Enfuvirtide-Based Regimen
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The Journal of Infectious Diseases Feb 1, 2007;195:387-391
Steven G. Deeks,1 Jing Lu,3 Rebecca Hoh,1 Torsten B. Neilands,1 George Beatty,1 Wei Huang,2 Teri Liegler,1 Peter Hunt,1 Jeffrey N. Martin,1 and Daniel R. Kuritzkes3
1University of California, San Francisco, San Francisco, and 2Monogram Biosciences, Inc., South San Francisco, California; 3Section of Retroviral Therapeutics, Brigham and Women's Hospital and Division of AIDS, Harvard Medical School, Boston, Massachusetts
(See the editorial commentary by Morse and Maldarelli.)
"....In summary, interrupting enfuvirtide therapy in patients with enfuvirtide-resistant HIV-1 was associated with a small but measurable increase in viremia, which indicates persistent low-level activity of the drug. Enfuvirtide resistance waned rapidly in the absence of drug pressure. Although these mutations do not appear to limit the ability of HIV-1 to replicate in patients with advanced disease, they may alter the complex relationship that exists between viral replication and the preservation of target cells....The decision to continue or discontinue enfuvirtide in patients with incomplete viral suppression needs to be individualized and should be made on the basis of a number of factors. In our study, we found evidence that enfuvirtide had measurable but limited residual antiviral activity (particularly in those individuals with higher HIV-1 RNA levels), which suggests that the drug should be discontinued. Other factors that support the discontinuation of enfuvirtide in these patients include (1) the high cost of enfuvirtide, (2) the inconvenience of parental dosing, (3) the potential for significant adverse events (e.g., injection-site reactions), and (4) the risk of ongoing viral evolution and emergence of cross-resistance to second-generation enfuvirtide-like inhibitors. However, because enfuvirtide is often used in patients with very advanced disease, continuing any potentially beneficial drug may be reasonable, particularly if there is evidence of a CD4+ T cell benefit that cannot be fully explained by other factors. Along these lines, it has recently been reported that certain enfuvirtide-associated mutations may be associated with sustained CD4+ T cell counts (independent of HIV-1 RNA levels)..."
ABSTRACT
Many antiretroviral drugs continue to exert an anti-human immunodeficiency virus (HIV) benefit in the presence of drug resistance mutations. The degree to which enfuvirtide exerts continued antiviral activity in the presence of incomplete viral suppression has not been defined. To address this question, 25 subjects interrupted enfuvirtide while remaining on a stable background regimen. Enfuvirtide interruption was associated with an immediate but limited increase in plasma HIV-1 RNA levels. Enfuvirtide resistance waned rapidly in the absence of drug pressure and was no longer detectable by week 16 in most individuals. These data indicate that enfuvirtide has measurable antiviral activity in the setting of incomplete viral suppression. Although enfuvirtide resistance mutations are associated with significant fitness defects in vivo, the clinical significance of these mutations remains undefined.
Enfuvirtide is a 36-aa peptide that binds to the first heptad repeat of the transmembrane subunit of HIV-1 envelope glycoprotein (gp), thereby preventing cell fusion and viral entry. Although it is potent, enfuvirtide must be administered parenterally, and it is used primarily in heavily pretreated patients with limited therapeutic options. In 2 large phase III studies, the majority of patients treated with enfuvirtide and an optimized background regimen failed to achieve and maintain undetectable plasma HIV-1 RNA levels, in part because, for most patients, no other effective agents were available to combine with enfuvirtide [1, 2]. The optimal management of patients failing an enfuvirtide-based regimen is not known.
Antiretroviral therapy often has a persistent beneficial effect in the presence of incomplete viral suppression. Prior work from our group and others has indicated a persistent antiviral activity of nucleoside analogs against drug-resistant variants, whereas other drugs (including lamivudine and protease inhibitors) act to reduce replicative capacity [3-5]. On the basis of the assumption that knowledge regarding drug activity in the presence of drug resistance mutations can help define optimal long-term therapeutic strategies, we performed a prospective study in which patients with incomplete viral suppression but receiving an enfuvirtide-based regimen interrupted this drug while remaining on a stable background regimen.
Subjects, materials, and methods. This was a prospective, single-arm study. Eligibility criteria included (1) continuous therapy with an enfuvirtide-based regimen for at least 24 weeks, (2) persistent plasma HIV-1 RNA levels >400 copies/mL for the preceding 4 weeks, and (3) >90% self-reported adherence to therapy during the month preceding study entry.
Subjects were monitored in a manner similar to that of our prior "partial treatment interruption" study [5]. Briefly, eligible subjects were seen at least twice during a short lead-in period to estimate the steady-state level of viremia. During the subsequent interruption period, subjects were monitored weekly for 4 weeks, every 2 weeks for the next 8 weeks, and then every 4 weeks for the next 12 weeks. Subjects were encouraged to resume enfuvirtide (with or without modifying the background regimen) if plasma HIV-1 RNA levels increased by >0.5 log10 copies RNA/mL or if CD4+ T cell counts decreased by >50%.
The study was approved by institutional review boards at the University of California, San Francisco, and Brigham and Women's Hospital. All subjects provided written, informed consent.
Clonal analysis was performed on samples obtained at baseline and during follow-up. Viral RNA was extracted from plasma using the QIAamp viral RNA Kit (Qiagen). A 650-bp fragment of gp41 that included the heptad-repeat (HR)-1 and -2 coding region was amplified by nested reverse transcription-polymerase chain reaction (PCR). Bidirectional DNA sequencing of the purified PCR products was performed using Taq DyeDeoxy Terminator cycle-sequencing kits (Applied Biosystems). The DNA sequences were edited and translated using BioEdit software (version 7.0.1; available at: http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The phenotypic susceptibility of enfuvirtide was measured using a single-cycle viral entry assay in which patient-derived env genes were cotransfected with a luciferase-containing env-defective HIV-1 genomic vector (Monogram Biosciences). This assay also provided insight into the infectivity of HIV-1, as assessed by measuring the production of relative light units (RLUs) in cell lines expressing CCR5 or CXCR4 [6]. The variability of the assay was <3-fold (99% of all paired comparisons were within 3-fold).
Repeated-measures analyses were performed using mixed-effects linear models, implemented in the PROC MIXED function of SAS software (version 9.1.3; SAS). Two models of change after the interruption of therapy were evaluated. The first assumed a rapid change in plasma HIV-1 RNA level followed by a plateau, whereas the second assumed a monotonic increase over time. The final model was chosen by means of comparing Akaike information criteria. All other analyses were performed using standard nonparametric techniques.
Results.
Twenty-five subjects were studied. At the time when enfuvirtide was interrupted (baseline for the study), subjects had been receiving an enfuvirtide-based regimen for a median of 34 weeks (interquartile range [IQR], 28-50). The median baseline plasma HIV-1 RNA level was 4.78 log10 copies/mL (IQR, 4.33-5.43), and the median CD4+ T cell count was 95 cells/mm3 (IQR, 46-124). The change in HIV-1 RNA levels and CD4+ T cell counts during enfuvirtide treatment was +0.03 log10 copies RNA/mL (IQR, -0.36 to +0.46) and +35 cells/mm3 (IQR, 5-67 cells/mm3), respectively. The median number of missed enfuvirtide doses during the 30 days preceding study entry was 0 (IQR, 0-1).
Of the 25 subjects studied, 3 modified therapy before week 16 (2 because of toxicity; 1 enrolled in a clinical trial). Four subjects modified therapy between weeks 16 and 24 (2 because of toxicity and because of decreasing CD4+ T cell count). Two subjects had an adverse event during the 24-week study period (pneumonia and pneumothorax).
The baseline viral load and CD4+ T cell counts were based on repeated measurements performed over the course of a 4-week screening period (median number of visits, 6; range, 1-11). During this screening period, plasma HIV-1 RNA levels were stable (mean change, +0.002 log10 copies/mL/week; P = .83). CD4+ T cell counts were also stable during this time (mean change, -0.13 cells/mm3/week, P = .53).
At weeks 2 and 4 off enfuvirtide, the median change in viral load relative to the mean value before discontinuation was +0.22 log10 RNA copies/mL (IQR, -0.04 to 0.33 log10 RNA copies/mL) (P = .046) and +0.11 RNA copies/mL (IQR, -0.03 to +0.37 RNA copies/mL) (P = .006), respectively (figure 1). The mean plasma HIV-1 RNA slope increased by 0.17 log10 copies RNA/mL per week between the period before discontinuation and the period immediately after treatment interruption (P = .07). When all of the data through 24 weeks after the enfuvirtide interruption were examined, there was evidence of an immediate increase in mean plasma HIV-1 RNA levels of 0.19 log10 copies/mL (95% confidence interval, 0.06-0.33; P = .004) followed by a plateau without a subsequent increase. These models also demonstrated a small decrease in mean CD4+ T cell counts over time (-0.6 CD4+ T cells/mm3/week, P = .02). The slope of the CD4+ T cell count decrease was also greater during the period after interruption than during the period before interruption (change in slope, -0.64 cells/mm3/week), although this difference was not significant (P = .55).
We next assessed predictors of outcome, using the change in HIV-1 RNA levels at week 4 as the primary outcome (week 4 was chosen in this post hoc analysis because most subjects appeared to have achieved a new steady state by that time). Both a lower baseline plasma HIV-1 RNA level (P = -0.59; P = .002) and higher baseline CD4+ T cell count (P = 0.45; P = .024) were predictive of change, whereas the duration of enfuvirtide therapy, chemokine receptor tropism, and enfuvirtide IC50 were not predictive (P > .1 for all).
Nucleotide sequencing of multiple clones of the gp41 ectodomain obtained at baseline and during follow-up was performed on samples from the first 11 subjects studied (table 1). In 8 of 11 subjects, enfuvirtide resistance mutations were present at baseline in all clones, whereas a mixture of mutant and wild-type clones was detected in samples from 3 subjects. After the enfuvirtide interruption, the enfuvirtide-resistant population was replaced by wild-type virus in 10 of 11 subjects. All clones were wild-type virus at weeks 16-24 in 5 subjects, whereas samples from the remaining subjects exhibited a mixture of wild-type and mutant clones.
Phenotypic resistance to enfuvirtide generally remained stable for 4 weeks and then decreased, with all but 2 subjects having no evidence of phenotypic resistance at week 16 (figure 1). The relative capacity of HIV-1 to enter target cells was estimated before and after the interruption by measuring luciferase activity in U89 cell lines expressing CCR5 or CXCR4. Entry assays were performed in the absence of any inhibitor and therefore provided some insights into the impact of enfuvirtide resistance-associated mutations on replication capacity. The luciferase activity increased from baseline to week 16 by a median 0.26 log10 RLU (P = .01).
Discussion.
Continued antiretroviral therapy in patients with ongoing viral replication is commonly associated with a durable (but not permanent) virologic, immunologic, and clinical benefit [7, 8]. This benefit occurs despite the presence of high-level genotypic and phenotypic resistance in the dominant virus population. Several factors contribute to this benefit, including the persistent antiviral activity of therapy against the drug-resistant variant, the selective maintenance of a virus population with reduced replicative capacity, and perhaps the selective maintenance of a virus population with reduced capacity to cause disease ("virulence") [5, 9]. In the present study, we sought to determine whether enfuvirtide has continued activity in patients with incomplete viral suppression and to define the mechanism of any such benefit. Among a cohort of individuals with relatively high plasma HIV-1 RNA levels while receiving enfuvirtide, interruption of enfuvirtide was associated with a small but statistically significant increase in plasma HIV-1 RNA levels. This increase occurred immediately after interruption of the drug and before any measurable change in genotypic or phenotypic resistance, which suggests that the drug had some residual activity, at least in some individuals.
Viral fitness generally refers to the ability of one virus to out-compete a second virus in a defined environment [10]. The present study was designed specifically to determine the in vivo significance of enfuvirtide-selected mutations on viral fitness by competing the drug-resistant variant with the wild-type variant in the absence of enfuvirtide. The continued use of a stable background regimen acted to prevent the emergence of an archived "wild-type" virus, which thus allowed more precise measurements of the impact of drug resistance mutations on envelope function. Virus from the majority of subjects exhibited a loss of genotypic and phenotypic drug resistance by weeks 8-16. This shift in the virus population was associated with the emergence of a virus that more efficiently entered target cells expressing CD4 and either CCR5 or CXCR4. Collectively, these observations are consistent with prior observations regarding the impact of enfuvirtide resistance-conferring mutations on replicative capacity [11-13] and provide in vivo evidence that enfuvirtide-mediated changes in gp41 reduce the ability of that protein to support viral replication.
The decision to continue or discontinue enfuvirtide in patients with incomplete viral suppression needs to be individualized and should be made on the basis of a number of factors. In our study, we found evidence that enfuvirtide had measurable but limited residual antiviral activity (particularly in those individuals with higher HIV-1 RNA levels), which suggests that the drug should be discontinued. Other factors that support the discontinuation of enfuvirtide in these patients include (1) the high cost of enfuvirtide, (2) the inconvenience of parental dosing, (3) the potential for significant adverse events (e.g., injection-site reactions), and (4) the risk of ongoing viral evolution and emergence of cross-resistance to second-generation enfuvirtide-like inhibitors. However, because enfuvirtide is often used in patients with very advanced disease, continuing any potentially beneficial drug may be reasonable, particularly if there is evidence of a CD4+ T cell benefit that cannot be fully explained by other factors. Along these lines, it has recently been reported that certain enfuvirtide-associated mutations may be associated with sustained CD4+ T cell counts (independent of HIV-1 RNA levels) [14].
Certain limitations of the present study deserve comment. First, because this was an uncontrolled study, it is possible that the increased HIV-1 RNA levels detected during the early interruption period may reflect the natural history of plasma viremia. This seems unlikely, given the biphasic nature of the postinterruption change in viremia (i.e., an immediate increase followed by a plateau). Second, it is not possible to determine whether the interruption accelerated the loss of CD4+ T cells. A large, randomized study of continued versus interrupted therapy would be needed to address these issues, but such a study might be impractical.
In summary, interrupting enfuvirtide therapy in patients with enfuvirtide-resistant HIV-1 was associated with a small but measurable increase in viremia, which indicates persistent low-level activity of the drug. Enfuvirtide resistance waned rapidly in the absence of drug pressure. Although these mutations do not appear to limit the ability of HIV-1 to replicate in patients with advanced disease, they may alter the complex relationship that exists between viral replication and the preservation of target cells [15].
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