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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Keywords:© 2008 Lippincott Williams & Wilkins, Inc.
HIV-1; enfuvirtide; treatment interruption; viral dynamics; viral fitness; allele-specific PCR