This study established the prevalence of preexisting bevirimat resistance in the QVT motif and its biological variability over the course of standard HAART regimens which do not include bevirimat. It also demonstrated the feasibility of using mixed populations of recombinant virus coupled with deep sequencing in order to screen for clinically relevant drug resistance mutations. Finally, this study showed the effect of bevirimat selection on viral diversity in vitro.
The data from our large treatment-naïve cohort showed a nearly 50% prevalence of preexisting bevirimat resistance in the form of QVT motif polymorphisms, as reported for other studies (20
). The prevalence of naturally occurring polymorphisms in the QVT motif was not substantially affected by exposure to long-term PI- and/or nonnucleoside reverse transcriptase inhibitor (NNRTI)-based HAART (Table ). These results appear to agree with the relatively inconsequential differences in bevirimat resistance observed by both Margot et al. and Seclén et al. (17
) between treatment-experienced and treatment-naïve patients (17
) and with the high degree of sequence conservation in the CA-SP1 cleavage site found by Malet et al. in PI-experienced patients (16
) but appear to contradict the increased prevalence of bevirimat resistance in patients with PI resistance found by Verheyen et al. (27
). While the vast majority of patients within the group observed in this study were exposed to PIs at some point during treatment, PI resistance was rare. As such, the findings of this study do not necessarily contradict those of Verheyen et al. but instead represent a more general look at the relationship between HAART exposure and naturally occurring genetic variations in gag
associated with decreased susceptibility to bevirimat rather than the relationship specifically seen in the PI-resistant population.
The use of mixed recombinant virus derived from patient samples allowed a range of drug concentrations to be queried against a panel of clinically derived isolates from the target population to screen for clinically relevant resistance. It also allowed the testing of 30 different clinically derived recombinant viruses in six different drug concentrations, while requiring maintenance of only three 6-well culture plates, and was capable of yielding results in less than 3 months. The ability to rapidly detect preexisting drug resistance present in the population could allow modifications to be made to drug development procedures and thus reduce or avoid the problems that have haunted the clinical development of bevirimat. To address this issue, we tested a mixed culture system coupled with deep sequencing. As it is likely that not all drugs would have levels of preexisting mutations as high as were encountered with bevirimat, bulk sequencing was used to screen out obvious cases of preexisting resistance in this “proof of principle” study. Despite this screening, the approach was still capable of detecting commonly occurring preexisting resistance in all test groups. Had this method been used for bevirimat, it would have been obvious to the developers that, if implementation of the drug in therapy were to proceed, patients infected with virus that had the key mutations would have to be screened out. With this insight, the efforts of the developers, who spent tens of millions of dollars on the clinical development of bevirimat, would have yielded very different results.
Where V370A took over the population, it was consistently observed to arise alongside other bevirimat-associated mutations. This could occur when these other mutations are not sufficient on their own to completely overcome the effects of the drug and take over the population but allow enough replication to facilitate the development of V370A. It thus may be possible that two bevirimat resistance mutations are required to provide sufficient selective advantage for a single virus to effectively take over the population.
The emergence of a single dominant resistance profile was associated with decreased viral diversity, which is consistent with the observation that resistance arose from a single patient strain rather than from recombination. This would be as expected from a particular virus subpopulation gaining a mutation and arising to take over the population, as was suggested by the finding of a single patient of origin of each dominant resistance profile. The lack of an apparently dominant virus under conditions in which a low concentration of or no drug was present is consistent with the comparable replication capacities of the input viruses and suggests that the competitive advantage displayed by patient viruses that are dominant at higher concentrations is dependent on the drug rather than an intrinsic property of the viruses themselves.
Mixtures of gag-protease
recombinant viruses derived from clinical samples gave a closer approximation of the development of resistance seen in vivo
than have previous in vitro
resistance assays that failed to detect mutations in the clinically important QVT motif (1
). The use of deep sequencing allowed these mutations to be traced back to the individual mutational background and to the specific patient of origin, allowing a better view of the interactions between different mutations as well as identifying specific patients likely to develop resistance when receiving bevirimat therapy. The benefits of this methodology are likely due to a combination of mixtures of virus genotypes directly providing resistance and/or a background of greater diversity in which resistance can emerge. The inability of the RDP algorithm to detect recombination could have been due to a lack of power resulting from the relatively small region of gag
that was analyzed rather than to a true absence of recombination, particularly because some recombination was predicted by the population genetics model. If recombination were occurring at a high frequency, however, it would likely have been detected.
The use of several different constant drug concentrations rather than a single concentration as used by Adamson et al. (1
) or gradually increasing concentrations as used by Li et al. and Zhou et al. (14
) may have also contributed the ability of the present method to select V370A despite the failure of previous methods to do so (1
). In the study by Adamson et al., selection was performed using 0.0766 μM bevirimat (1
), a concentration below the NL4-3-specific IC50
(0.204 μM) of bevirimat (8
); thus, that experiment may not have provided sufficient drug pressure to select for resistant variants in the QVT motif. In studies by Li et al. and Zhou et al., initial selection for resistance was performed at a set concentration that was maintained until evidence of resistance was observed, at which time the drug concentrations were gradually increased (14
). As was seen in this study, however, V370A on its own may not provide a sufficient replication advantage to emerge at the concentrations of 0.12 μM (28
) and 2.45 μM (14
) that were used for initial selection in the studies by Li et al. and Zhou et al. or at the concentration of 0.0766 μM that was used by Adamson et al. (1
). The genetic barrier to generation of V370A mutations in a background without other resistance mutations may thus be higher than the genetic barrier to generation of mutations in the CA/SP1 site, as it would require the mutation of two nucleotides (one at 370 and one at another resistance site) rather than one, thus making the generation of mutations in the CA/SP1 sites more likely to occur. This would be consistent with the resistance patterns observed in those previous studies (1
While the method used in this study allowed the development of resistance similar to that seen in vivo, it had limitations in that it did not control for drug-independent differences in replication capacity among recombinant viruses combined in the initial inoculum. It should be noted that reproducibility was not perfect when groups of 10 samples were used. The number of samples per group or the total number of groups could be increased to provide a more reproducible estimate of even rare clinically relevant mutations. Also, the use of recombinant viruses and deep sequencing restricts analysis to a specific region of interest, thus causing any mutations outside the analyzed area to be missed. This could have affected the fitness of specific variants if additional compensatory mutations were present outside the analyzed area.
This study demonstrated that the prevalence of polymorphisms in the QVT motif conferring bevirimat resistance was not strongly affected by long-term PI- and/or NNRTI-based HAART and showed little variability over several years of observation, suggesting that genotypic screening is feasible, though it may require greater sensitivity than current population-based sequencing methods are capable of providing. In addition to this, a minimum concentration of 0.1 μM bevirimat was required for the selection of resistance mutations in vitro, a concentration below which no major selective forces were observed. Finally, even at relatively low levels of baseline prevalence, viruses containing polymorphisms in the QVT motif were rapidly selected. This confirms that preexisting resistance mutations at low levels in a population would be rapidly selected in mixed cultures. These results further confirm the applicability of the use of mixtures of recombinant viruses derived from clinical isolates coupled with deep sequencing-based observation as an efficient in vitro method for screening preexisting resistance mutations for bevirimat and other emerging drugs.