Interruption of reverse transcriptase inhibitors or a switch from reverse transcriptase to protease inhibitors resulted in a fast reappearance of virus strains with a reverse transcriptase inhibitor-sensitive genotype

Objectives: To study the effect of the interruption of reverse transriptase inhibitor (RTI) therapy or a switch from RTI to protease inhibitors, on the genotypic drug-resistance pattern of plasma HIV-1.

Methods: Nine patients who completely stopped all medication, and five patients who switched from a treatment with RTI to a regimen containing protease inhibitors only, were studied. Direct sequencing of the plasma HIV-1 RT and protease gene was performed on follow-up samples taken before and after the interruption of treatment.

Results: Comparison of the amino acid sequence of the RT and protease genes in successive samples showed the rapid reappearance of wild-type viral variants in 12 of the 14 patients studied. Wild-type virus replaced the mutant strains 14 days to 2 months after the interruption of therapy, even in patients with a long treatment history.

Conclusion: The results of this study indicate the sustained lower fitness of mutant strains in vivo. As a result, wild-type virus remains capable of outcompeting the RT or protease mutant strains very fast after removal of the drug. These findings highlight the importance of ‚treatment history‚ in addition to genotypic and phenotypic markers determined at one time- point, when making therapeutic decisions for patients.

Introduction

Viral resistance to drugs is an important cause of treatment failure in the HIV-1-infected patient. Since the first discovery of specific nucleotide changes in the coding region of reverse transcriptase (RT) associated with zidovudine (ZDV) resistance[¹], genotypic changes conferring resistance to antiviral agents have been described for almost all drugs currently available[²]. Subsequently, retrospective studies [^3,4] have shown that the viral genotype and phenotype can predict the response to drugs. The discovery of a genetic base for resistance led to the development of rapid and simple genotyping methods for determining drug susceptibility[^5,6]. In addition, a modified phenotyping assay, the recombinant virus assay, became available[⁷]. It is likely that in the near future the screening of resistance will be performed on a routine basis to guide therapeutic decisions, both at the start of therapy and after failure. Continuous efforts to improve our understanding of how resistance arises and evolves during subsequent treatment regimens is therefore necessary.

Mutants resistant to antiretroviral agents are rarely seen before the initiation of therapy, so it is assumed that mutant strains have a reduced fitness compared with wild-type virus. Primary mutations appear early during the development of resistance and are usually drug specific. Secondary mutations, which accumulate over time, are not drug specific and are seen as compensatory changes leading to the improvement of viral fitness[⁸]. If resistant viral variants finally acquire a replication competence approaching that of wild-type virus then one would expect a long preservation of the mutant virus after removal of the drug pressure. In this study, we analysed the evolution of mutant virus after the cessation or switch of antiretroviral drugs in patients treated for a long time with the reverse transcriptase inhibitors (RTI) ZDV or lamivudine (3TC), or both, and found a quick reappearance of wild-type virus after the cessation of therapy.

Methods

Study population

Fourteen patients with different treatment histories were selected prospectively from the patients visiting the AIDS Reference Centre of our hospital. Only patients with genotypic resistance before cessation or switch of drugs were included. In nine patients, antiretroviral therapy was completely interrupted because of drug toxicity or because of therapy failure, as assessed by viral load determination. In five patients a regimen of RTI was stopped and replaced by a regimen of two protease inhibitors: ritonavir (RTV) and saquinavir (SQV). The latter five patients participated in the Dutch ‚Prometheus‚ study conducted during 1997.

Tables 1 and 2 summarize the treatment history. All patients were treated for more than 1 year. With the exception of patient 8 they all received ZDV, which was initiated between 1992 and 1996. ZDV remained a component of the combination drug regimen until the stop or switch in eight of the 14 patients. In the remaining six, ZDV was replaced by stavudine (d4T) during 1997; 3TC was added between 1995 and 1997 in six patients. Mutations associated with ZDV resistance had already been seen in viral RNA from plasma samples taken 4 years (patient 3) or 3 years (patients 2, 4 and 5) before the interruption of therapy (data not shown). For the other patients, the necessary samples to date genotypic resistance were not available.

Table 1

Table 2

In most patients, viral load remained detectable during the whole treatment period. Only in two patients (patients 2 and 3) was the viral load reduced to below the detection limit of 50 copies/ml plasma for 5 and 9 months, respectively, before an increase in viral load and subsequent therapy interruption.

HIV RNA quantification

Plasma samples were obtained at each visit and stored at -70°C. HIV RNA quantification was performed using the Ultrasensitive Amplicor HIV-1 Monitor Test (Roche Molecular Systems, Branchburg, NJ, USA). The lower detection limit of quantification for this assay is 50 copies/ml.

Genotypic analysis

Reverse transcription-polymerase chain reaction (RT-PCR) of the RT and protease genes was performed on plasma viral RNA using the Titan One Tube RT-PCR System (Boehringer Mannheim, Germany). Direct sequencing of the PCR product was performed using the dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, CA, USA). Sequencing reaction products were analysed on an ABI 310 Genetic Analyser (Applied Biosystems). Amino acid substitutions were identified by comparison of the plasma RNA sequences with a consensus sequence.

Results

For the nine patients who stopped all antiretroviral drugs, the sequencing results for the RT and the protease gene are shown in Table 1. With the exception of a T/D mix at position 69, associated with zalcitabine (ddC) resistance, the observed mutations in the RT gene correlated with ZDV resistance (41L, 67N, 70R, 210W, 215Y/F and 219Q/N) or 3TC resistance (184V)[²].

Interruption of the antiretroviral medication resulted in a fast rebound of the viral load in seven of the nine patients. Concurrently, therapy interruption resulted in the disappearance of resistance mutations in the RT gene in eight patients. None of the mutations associated with ZDV or 3TC resistance were systematically preserved. 41L was replaced by 41M in four of six patients; 67N by 67D in five of six patients; 70R by 70K in one of four patients; 184V by 184M in all four patients; 210W by 210L in three of four patients; 215Y/F by 215T in four of seven patients and 219Q/N by 219K in both patients with Q or N at position 219. In patient 2, replacement of the mutant virus by the wild type had already been seen on the first follow-up sample, 2 weeks after the cessation of therapy.

Reappearance of the wild-type amino acids was also seen in the protease gene, with changes at codon positions 10, 20, 36, 46, 54, 82, 84 and 90. The 63P was conserved, and a reversal of T/V to the wild-type 71A was also not observed.

Table 2 summarizes the results for five patients who switched therapy from RTI to protease inhibitors. In three patients all mutant amino acids in the RT gene were replaced by the wild-type amino acids, in one patient only the 219Q was replaced whereas the 69N and 70R remained unchanged. Reappearance of the wild-type amino acids occurred very rapidly: within 1-2 months after the switch.

In patient 14, no change in the RT mutations was observed. This patient subsequently stopped all medication after 137 days of treatment with protease inhibitors. This complete therapy interruption also had only a minor effect on the RT gene, but the protease gene returned to wild type. In four of five patients, naturally occurring polymorphisms were seen in the protease gene at resistance-related codon positions (63P, 20I and 36I). Three of the five patients showed upcoming resistance against the protease inhibitors during follow-up: 36V (one patient), 48V (one patient), 82A (one patient) and 90M (two patients).

Discussion

The use of novel means for evaluating resistance may prove helpful to guide clinicians in choosing agents to which the patient‚s dominant viral quasispecies remain sensitive, thereby potentially increasing the chances of therapeutic success. At present, however, a resistance-driven treatment algorithm is not available. The design of such an algorithm not only requires a thorough knowledge of the association between viral resistance and therapy outcome, but also an insight into the viral dynamics of mutant strains in complex treatment regimens.

We analysed the evolution of mutant virus after the cessation or switch of antiretroviral drugs in patients with a long treatment history of ZDV or 3TC, or both. Some of the patients studied also received other RTI and protease inhibitors. The results revealed the disappearance of drug-associated mutations in the RT gene, after the complete interruption of therapy, or after a therapy switch from RTI to protease inhibitors. Reappearance of the wild-type amino acid was seen at all codon positions studied, most frequently at positions 184 and 219, and least often at position 70. Reversal to the wild-type amino acid was also observed for mutant codons in the protease gene. These observations confirm in-vitro studies[^9-13], showing the impaired viral fitness of mutant virus. However, they are in discordance with reports of the long-term persistence of ZDV mutations after the discontinuation of ZDV therapy[^14,15]. Considerable individual variations between the period of persistence of the mutant virus were seen in the patients presented here. Although we were not able to assess a correlation between the period of reversal from mutant to wild-type virus and the estimated drug adherence of the patients, the last parameters based exclusively on the clinician‚s impression, we cannot exclude the possibility that differences in adherence account for some of the individual variations observed.

Reappearance of virus with a wild-type RT gene occurred within 2-4 weeks after a change in or interruption of therapy, despite a sustained previous exposure to ZDV for several years and a predominance of mutant virus in the plasma for more than 2 years. In the majority of patients, the reversal results in a total replacement of the mutant by the wild-type virus, rather than a gradual reversion of individual mutations.

In two of the 14 patients we were not able to document a reappearance of wild-type RT. In patient 3 this can be explained by the very short follow-up period (17 days). No direct explanation is available for patient 14. In this patient only the mutations in the protease gene disappeared. It is possible that other adaptations have improved the fitness of this patient‚s RT resistant strain.

The observed switch from mutant to wild-type amino acids can be the result of a reversal of the mutant codons, or can be due to a rebound of the original wild-type strains. Yerly et al. [¹⁶] studied successive samples from seroconverting patients infected with virus bearing the T215Y/F amino acid substitution, and found that a reversal to the original wild-type amino acid occurred very infrequently in these patients. Instead, unusual amino acids such as aspartatic acid (D) and cysteine (C) appeared at position 215 in half of the patients, as an attempt of the virus to improve its fitness. The appearance of these unusual amino acids was never observed in our patients. This is an argument against the occurrence of reversal mutations, and an indication in favour of a rebound of the existing wild type.

The quick rebound of existing wild-type virus was still unexpected, however, because one would assume that after several years of drug pressure and adaptation of the virus to the drug, a virus with a replication capacity as high as that of the wild-type variant would have evolved. Moreover, the continuous presence of the drugs for a longer period of time is supposed to lead to an elimination or at least an important reduction of the population of drug-sensitive virus through a natural process of cell death, whereby cells infected with the wild-type strain are lost preferentially. Two possible explanations exist: either a latent cellular reservoir of wild-type HIV, capable of re-inducing highly active viral replication after removing the drug pressure, is preserved for a long period of time. Alternatively, the wild-type virus continues to replicate, despite the presence of drugs, and is ready to take over as soon as the pressure is interrupted. More detailed studies are necessary to address these assumptions. If the first hypothesis proves to be true, then it is clear that mutant viral strains will take over again as soon as the drug is re-introduced, and that the recycling of drugs, even after a very long interval, will remain impossible.

In seven of the patients studied, ZDV was replaced by d4T several months before cessation of the therapy. In none of these patients did this result in a reversal of the ZDV-resistant mutations as does a complete withdrawal of RTI. This finding indicates that the ZDV mutant virus has a higher replicative competence in the presence of d4T than the wild-type virus. This observation is in accordance with results of in-vivo studies[¹⁷], showing a reduced response to d4T in patients with genotypic ZDV-resistant virus compared with patients who lack ZDV resistance.

The fact that mutant viral strains have a lower replicative capacity than wild-type virus can be an argument in favour of continuing certain drugs despite viral resistance. A reduced viral fitness not only results in lower steady-state levels, but will also lower the mutation rate of the virus. More data on the fitness of resistant virus in vivo are needed. This information might help in the design of drug combinations that are not only effective in reducing the viral load to below detection limits, but that additionally result in a maximum decrease of viral fitness in case resistance occurs.

Although this study is limited in size, the findings presented illustrate the complexity of the interpretation of genotypic data. Looking at ‚treatment history‚ in addition to genotypic and phenotypic markers determined at one time-point is indispensable when making therapeutic decisions for patients. A longitudinal recording of resistance test results, from the time of initial presentation on and through the whole treatment period, will be necessary to guide therapy in the heavily pre-treated individual.