JAIDS Journal of Acquired Immune Deficiency Syndromes:
Brief Report: Clinical Science
Viral Dynamics and In Vivo Fitness of HIV-1 in the Presence and Absence of Enfuvirtide
Marconi, Vincent MD*; Bonhoeffer, Sebastian PhD†; Paredes, Roger MD*; Lu, Jing MD*; Hoh, Rebecca RD‡; Martin, Jeffery N MPH§; Deeks, Steven G MD‡; Kuritzkes, Daniel R MD*
From the *Section of Retroviral Therapeutics, Brigham and Women's Hospital and Division of AIDS, Harvard Medical School, Boston, MA; †Theoretical Biology, ETH Zentrum, Zurich, Switzerland; and ‡Department of Medicine; §Epidemiology and Biostatistics, University of California, San Francisco, and San Francisco General Hospital, San Francisco, CA. Vincent Marconi is now with the HIV Unit, Wilford Hall United States Air Force Medical Center, 2200 Bergquist Drive, Suite 1, Lackland AFB, TX 78236.
Received for publication March 5, 2008; accepted March 6, 2008.
Presented in part at the 1st International Workshop on HIV Entry Inhibition, December, 2005, Bethesda, MD [abstract 10], and at the 13th Conference on Retroviruses and Opportunistic Infections, February 5-8, 2006, Denver, CO [abstract M-188].
Correspondence to: Dr Daniel R. Kuritzkes, MD, Section of Retroviral Therapeutics, Brigham and Women's Hospital, 65 Landsdowne Street, Room 449, Cambridge, MA 02139 (e-mail: firstname.lastname@example.org).
Objectives: To estimate the in vivo fitness cost of enfuvirtide (ENF) resistance, we analyzed dynamic shifts in the HIV-1 quasispecies under changing selective pressure in 3 subjects on failing ENF-based regimens who interrupted ENF while maintaining stable background regimens. Subsequently, ENF was readministered for 4 weeks as “pulse intensification.”
Methods: The proportion of plasma virus carrying the V38A mutation in gp41 was quantified by allele-specific real-time polymerase chain reaction in serial samples collected from 3 subjects at 1- to 4-week intervals. Fitness differences were calculated using a method that corrected for time dependence of the viral replication rate.
Results: The V38A mutant made up ≥85% of the quasispecies at baseline and decayed to <5% over 12-24 weeks; plasma HIV-1 RNA levels remained stable during this time. Fitness differences for mutant versus wild type ranged from −25% to −65%, providing in vivo evidence for the reduced fitness of ENF-resistant HIV-1. The V38A mutant virus reemerged rapidly during the ENF pulse.
Conclusions: These results demonstrate that the HIV-1 quasispecies undergoes dynamic changes in response to withdrawal and reinitiation of fusion inhibitor therapy. The relative stability of plasma HIV-1 titers during decay of V38A suggests that factors other than viral fitness likely define viral load set-point in patients with advanced disease.
The fusion inhibitor enfuvirtide (ENF) is a potent antiretroviral drug that is most effective when used in combination with 2 or more agents to which the virus remains at least partially susceptible.1,2 In the setting of partial viral suppression, ENF resistance develops rapidly due to emergence of mutations in the first heptad repeat (HR-1) of the gp41 ectodomain.3,4 The most commonly identified ENF resistance mutations include G36D or S, V38A, M or E, Q40H, and N43D.5
In vitro studies using growth competition assays have shown that ENF-resistant viruses are substantially less fit than wild type in the absence of drug.6 Our group conducted a prospective evaluation of the virologic and immunologic consequences of ENF interruption in subjects with incomplete viral suppression on an ENF-based regimen.7 Subjects discontinued ENF while remaining on a stable background regimen. ENF resistance waned over time in most subjects, suggesting that ENF resistance mutations reduce fitness of HIV-1 (as assessed in absence of drug). To determine more precisely the impact of these mutations on viral fitness in vivo, we developed a sensitive allele-specific real-time polymerase chain reaction (PCR) assay to quantify the relative proportion of mutant HIV-1 over time. This assay allowed us to study the dynamics of ENF-resistant and wild-type virus in 3 subjects under changing selective pressures as subjects discontinued and subsequently resumed ENF.
Subjects and Plasma Samples
Subjects were antiretroviral treatment-experienced HIV-1-infected patients enrolled in an ongoing prospective cohort Study of the Consequences of the Protease Inhibitor Era (SCOPE).8 This particular substudy evaluated the consequences of interrupting ENF while continuing to receive other drugs in the regimen [partial treatment interruption (PTI)]. All subjects provided written informed consent, and all aspects of this study were conducted according to institutional guidelines for experiments with human subjects. Plasma samples were obtained at baseline (before ENF interruption) and then weekly for the first 2 months and every other week for the subsequent 2 years after ENF interruption. Several months after the ENF interruption, a subset of subjects underwent pulse intensification of their antiretroviral regimen during which ENF was readministered for a 4-week period (the “background” regimen was not modified during this time). The purpose of this pulse intensification was to determine whether a brief resumption of ENF would have beneficial effects on CD4 count stability by transiently lowering plasma HIV-1 RNA levels or by reselecting potentially less pathogenic ENF-resistant viruses.9,10 Plasma samples were obtained weekly during and after the pulse therapy phase. Samples were stored as 1-mL aliquots at −80°C before analysis. Plasma HIV-1 RNA levels were determined using a PCR assay (Amplicor HIV Monitor version 1.5; Roche Molecular Systems, Branchburg, NJ; lower limit of quantification 50 copies RNA/mL).
Cloning and Sequencing of HIV-1 gp41
Viral RNA was extracted from plasma using the QIAamp viral RNA Kit (Qiagen, Valencia, CA). A 650-base pair fragment of gp41 that included the HR-1- and HR-2-coding region was amplified by a nested reverse transcriptase-coupled PCR, cloned, and sequenced as described.5
Quantification of Viral Populations Using Real-Time PCR
Viral RNA was extracted from plasma samples as above and subjected to reverse transcriptase-coupled PCR to obtain amplified complementary DNA products of the gp41-coding region of env. Reverse transcription was performed using a one-step process as described.11 To quantify the proportion of mutant sequences contained within each specimen, 5 μL of the reverse transcriptase-coupled PCR products were added to PCR reactions that permitted nonselective amplification of HR-1-coding sequences or selective amplification of sequences containing the V38A mutation. The limit of detection or sensitivity of the mutant-selective primer was 0.80%.11
Calculation of Relative Fitness
The relative fitness advantage or disadvantage was determined based on the growth-corrected method as described.12 (Fitness differences calculated without growth correction gave qualitatively similar results, but because the growth-corrected model gave the best fit, we show only those data.) We set the death rate of infected cells, δ, equal to 0.5 ± 0.1 as this value represents approximately the mean of many independent estimates.13 Furthermore, we assumed a coefficient of variation of 30% for the virus load measurements.14
Decay of V38A Mutant Populations During ENF Interruption
Samples from 3 ENF-treated subjects, in whom the gp41 V38A mutation was present as the predominant population in the quasispecies (based on clonal sequence analysis), were selected for analysis by allele-specific real-time PCR (Table 1). The duration of ENF therapy in these subjects before the PTI ranged from 27 to 39 weeks. The V38A mutation was present in 85% or more of the viral quasispecies at the start of the ENF interruption in all 3 subjects. After an initial lag of 6-12 weeks during which the viral population seemed to be unchanged, the V38A mutant population decayed at variable rates (Fig. 1). There was little or no change in plasma HIV-1 RNA levels during this period. The time to near-complete replacement of mutant virus by wild-type virus seemed to correlate with the duration of ENF treatment before the PTI: In subject 1, who received ENF for 27 weeks, the V38A mutants declined to less than 5% of the viral population by 12 weeks after PTI; in subject 2, who received 33 weeks of ENF, the V38A population declined to less than 5% by week 22; in subject 3, who received 39 weeks of ENF, the V38A population declined to less than 5% by week 24.
Clonal analysis of HR-1 sequences from plasma HIV-1 RNA performed 16 weeks after the interruption showed persistence of the V38A mutation in none of 8 clones (0%) from subject 1, 2 of 8 clones (25%) from subject 2, and 5 of 9 clones (56%) from subject 3 (Table 1). The V38A mutation remained detectable by allele-specific PCR at low levels (approximately 1%) in the viral quasispecies of each subject for 9-19 months after ENF interruption but had fallen below the limit of detection by clonal analysis.
Reemergence of V38A During ENF Pulse Therapy
Each subject received a single 4-week “pulse” of ENF at various times after the ENF interruption. ENF was added to the regimen at weeks 76, 68, and 38 for subjects 1, 2, and 3, respectively. Pulse treatment with ENF resulted in only transient reductions in plasma HIV-1 RNA. Subjects 1 and 3 both had an initial 1.0-log10 decrease in plasma HIV-1 RNA, whereas subject 2 had a 0.5-log10 decrease. Subsequently, viral RNA increased by 0.7-log10 in subject 1 and by 1.4-log10 in subject 3. In the case of subject 2, viral RNA increases by 1.2-log10 above nadir then decreases again to 0.4-log10 above the nadir associated with the pulse. Increases in the proportion of virus carrying the V38A mutation were detectable within 1 week of the ENF pulse and were associated with the rebound in plasma HIV-1 RNA (Fig. 1). For example, in subject 3, the initial reduction in plasma HIV-1 RNA was accompanied by an increase in the proportion of V38A mutant from below the limit of detection to about 7%. As plasma HIV-1 RNA levels subsequently rebounded, the percent of V38A mutant virus increased in parallel, reaching approximately 52% before the end of the ENF pulse. Similar findings were observed in subject 2 (Fig. 1). Additional plasma samples available from subject 3 showed that during the second ENF interruption the V38A population decayed rapidly after a minimal lag time, declining to below the threshold of detection nearly 11 weeks after ENF was discontinued.
Determination of Viral Fitness In Vivo
The relative fitness difference between the V38A mutant and wild-type virus was calculated for each subject (Table 2). In the absence of ENF, the V38A mutant was approximately 25%-65% less fit than wild type. It is noteworthy that for subject 3 the V38A mutant seemed to have a greater fitness disadvantage relative to wild type during the second ENF interruption. By contrast, in the presence of ENF, the mutant viruses were substantially more fit than wild type (107%-498%). However, the limited number of data points available during the ENF pulse resulted in poor fits of the data to the model as indicated by the wide SDs. For this reason, estimates of the fitness differences during the ENF pulse phase are less reliable than those obtained during the interruption phase.
In this study, use of an allele-specific PCR assay allowed us to track the decay of HIV-1 variants carrying the V38A mutation for ENF resistance in gp41. Because we used a study design in which ENF was the only drug removed from or added to a stable background antiretroviral regimen, we were able to measure directly the impact of this ENF resistance-associated mutation on viral fitness in vivo in the presence and absence of drug. The V38A mutant population remained stable for 6-12 weeks in absence of ENF and then decayed rapidly to less than 5% of the virus population over an additional 6-12 weeks. Use of a growth-corrected model for estimating fitness difference found that the V38A mutant viruses were 25%-65% less fit than the wild type in the absence of ENF. These results provide in vivo confirmation of earlier work from our group that demonstrated reduced fitness of the V38A mutant compared with wild-type virus in vitro.6
Although marked differences in viral fitness led to turnover in the viral population in response to changing selection pressures, plasma HIV-1 RNA levels remained essentially unchanged during the PTI. This observation illustrates the difference between relative fitness and replication capacity-relative fitness determines the proportion of various members of the viral quasispecies, but their absolute titer is more closely related to replication capacity. An alternative explanation is that target cell availability, rather than viral fitness and replication capacity, is the limiting factor in determining viral load.15 If replacement of the V38A mutants by wild-type virus with a higher replication capacity results in greater depletion of available target cells, then it may be difficult to discern an impact of these mutations on viral load set-point.16 Detailed analysis on a larger number of subjects would be necessary to define more fully the relative impact of fitness and target cell availability on the level of viremia.
The V38A mutant reemerged rapidly during the pulse intensification phase of this study and was accompanied by rapid rebound in plasma HIV-1 RNA levels. The more rapid turnover of the virus population during the ENF pulse suggests that ENF imposes stronger selective pressure on the virus population than does the PTI and that the advantage of the V38A mutant over wild type in the presence of drug is greater than the disadvantage of the mutant compared with wild type in the absence of drug. The rapid reemergence of the V38A mutant population could also be explained by a persistent reservoir of actively replicating mutant virus.17,18 Indirect evidence for this possibility is provided by the finding that in our study the level of V38A mutants detectable by allele-specific PCR never dropped below 1% of the population. This finding is consistent with previous studies in which persistence of drug-resistant virus as minority variants after treatment interruption was associated with virologic failure when antiretroviral therapy with the same drugs was resumed.19,20
This study has a number of limitations. The detailed analysis performed allowed only a relatively small number of subjects to be studied. Consequently, the results presented here may not be representative of all patients with ENF failure associated with a V38A mutation. In addition, the small number of samples available during the pulse intensification phase resulted in a relatively poor fit of the data to the model used for determining fitness differences in the presence of ENF; hence, those estimates must be considered provisional. Although our analysis focused on the dynamics of the V38A mutation in the HR-1 domain of gp41, other mutations contributing to ENF resistance may have been present and could have contributed to overall viral fitness. For this reason, the presence or absence of V38A served only as a marker of viral turnover in response to ENF withdrawal or administration, and the fitness differences we observed cannot be ascribed solely to the presence of a valine or an alanine at amino acid 38. In addition, given the heterogeneity of HIV-1 env, it is possible that the magnitude of the fitness differences we observed could vary depending on the specific envelope backbone in which the V38A mutation is found.
Work by others has shown that changes in virus populations accompanying the development of ENF resistance involve simultaneous or sequential emergence of viral variants that may carry similar mutations in HR-1 but may differ markedly in other regions of envelope. These subpopulations may differ in replicative capacity and fitness in the absence of ENF and could exhibit different decay rates when ENF is interrupted. Our results provide an estimate of the average fitness of the viral population.
In conclusion, the HIV-1 quasispecies undergoes dynamic changes in response to varying conditions imposed by the withdrawal and reinitiation of fusion inhibitor therapy. Differences in the kinetics of the decay and reemergence of V38A mutant virus reflect differences in the strength of the selection pressures applied in this study. The rapid reemergence of ENF resistance in association with virologic failure and a return to baseline in plasma HIV-1 RNA levels after the ENF pulse suggest that retreatment with ENF after previous failure of this drug is unlikely to produce a durable virologic response. Further studies are needed to identify the factors that determine the lag period between treatment interruption and reemergence of wild-type virus and to understand the relationship between treatment duration and size of the reservoir of drug-resistant virus.
Supported in part by Public Health Service grants from the National Institutes of Health (K24 RR016482, R01 AI052745, R01 AI055273, R01 AI055357), the Harvard AIDS Clinical Trials Group Virology Support Laboratory (U01 AI38858), the California AIDS Research Center (ID01-SF-049), the UCSF/Gladstone Institute of Virology and Immunology Center for AIDS Research (P30 MH59037), the Harvard Medical School Center for AIDS Research (AI-060354), the General Clinical Research Center at San Francisco General Hospital (M01 RR00083), the Swiss National Science Foundation (PP00A-106751), “La Caixa” Fellowship Grant to R.P. provided by Caixa d'Estalvis i Pensions de Barcelona, Spain, and by a research grant to S.G.D. provided by Roche and Trimeris.
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