Evolution of HIV-1 multidrug-resistant genotypes during combination therapy and after the cessation of antiretroviral drugs

Yahi, Nouaraa; Tourrès, Christiana; Tivoli, Natachaa; Colson, Philippea; Dhiver, Catherineb; Quinson, Anne-Marieb; Tamalet, Catherinea

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aLaboratoire de Virologie, UF SIDA, CHRU La Timone, France; and bCISIH-Marseille, Marseille, France.

Received: 27 April 2000;

revised: 4 August 2000; accepted: 9 August 2000.

Article Outline

The current management of patients who have experienced extensive previous therapy, and especially those who have undergone ‘incremental therapy', is a major challenge for AIDS therapeutics. Indeed, the viral strains present in these patients may be resistant to most, if not all, currently available antiretroviral drugs. In this context, one should take into account the emergence of multiple nucleoside resistance involving specific mutational patterns of the HIV-1 pol gene that are independent of the classic mutations conferring resistance to individual dideoxynucleosides [1]. These include a cluster of five mutations in the reverse transcriptase (RT) coding region (A62V, V75I, F77L, F116Y and Q151M), generally referred to as multidrug resistance (MDR) mutations [2], and insertions of one or several amino acid residues between codons 67 and 70 of RT (β3–β4 insertions) [3]. Overall, these MDR genotypes were observed in 29 (4.6%) of the viruses sequenced from a cohort of 632 extensively treated patients followed in the Marseille area.

One therapeutic option in individuals in whom multiple regiments have previously failed is to interrupt therapy, which is expected to allow the outgrowth of a viral population with an increased sensitivity to antiretroviral drugs [4,5]. This approach (often referred to as washout strategy) may be particularly suitable for patients with multidrug-resistant HIV-1, because in the absence of drugs, viruses with MDR genotypes are far less fit than wild-type strains. Therefore, stopping therapy may allow the emergence of wild-type virus with increased fitness, and permit the use of recycled antiretroviral drugs to suppress viral replication. We investigated the evolution of multidrug resistant genotypes (MDR and insertion mutants of HIV-1 RT) during highly active antiretroviral therapy of selected patients, and we studied the effects of the interruption of therapy on the genotypic drug-resistance pattern of plasma HIV-1. The four patients in this study were selected for the following criteria: (i) virological failure after multiple regimens; and (ii) the presence of either a major MDR mutation or a β3–β4 insert in the RT gene. Genotypic HIV-1 drug resistance testing was performed on plasma samples by dye terminator cycle sequencing, as previously described [6]. The use of dye terminator cycle sequencing was effective in the detection of mixed viral populations representing at least 10–20% of the total genomes [6]. The accuracy of sequence data was assessed by phylogenetic analysis, which allowed us to rule out sample cross-contamination. In particular, patient signature patterns could be evidenced in longitudinal sequences from each patient.

The most potent response was obtained for patient A, who had a viraemia always less than 20 000 copies/ml over several years, and CD4 cell counts over 500 cells/mm3 at the time of treatment interruption (Table 1). Four months after stopping therapy, genotype analysis revealed important changes in the pol gene: all mutant amino acids in the RT gene were replaced by wild-type amino acids, whereas only one mutation (L63P) remained detectable in the protease gene. The washout also resulted in a burst of viraemia (a 24-fold increase), which is highly significant because the viraemia of this patient always remained almost constant (approximately 20 000 copies/ml) for several years on therapy. Overall, the success of the washout strategy in this particular case can be tentatively explained by the following reasons: (i) viraemia less than 20 000 copies/ml; (ii) CD4 cell counts greater than 500 cells/ml; (iii) genotype-based decision early after the detection of MDR mutations; (iv) switching to an entirely new regimen. In this respect, it is likely that the introduction of a new class of drug (i.e. efavirenz) may have significantly contributed to decrease the viral load. Neverthelessess, these data confirm that the withdrawal of antiretroviral drugs may allow the rebound of wild-type viruses that have a replicative advantage in the absence of drugs.

The three other patients (B, C, and D) in this study had been extensively treated with various combination regimens since 1991. Consequently, these patients were characterized by low CD4 cell counts (< 100 cells/ml), high viraemia (> 200 000 copies/ml), and high-level drug resistance because of a polymutated pattern of the RT and protease genes. In these cases, the interruption of therapy resulted in the re-emergence of viruses devoid of insertion mutations, but did not allow the rebound of wild-type viruses (Table 1). In only one case (patient B), was the polymutated pattern of the protease gene not affected by drug removal. Overall, the re-initiation of therapy in these three patients did not result in an effective decrease of viraemia. Moreover, it should be emphasized that stopping therapy in patients with very low CD4 cell counts carries important risks. The number of CD4 cells may further decrease, as observed in patients C and B, rendering them particularly exposed to opportunistic infections (a cerebral toxoplasmosis in the case of patient C, despite careful accompanying prophylaxis). Therefore, it should be questioned whether continuing the failing regimen until new drugs are available is the only therapeutic option for these patients.

In conclusion, despite the limited number of patients in this study, our data show that MDR genotypes, including both MDR mutations and insertions between RT codons 67 and 70, may disappear as a result of the cessation of therapy. Therefore, the presence of such mutations at the time of therapy interruption does not preclude the success of washout strategies.

Nouara Yahia

Christian Tourrèsa

Natacha Tivolia

Philippe Colsona

Catherine Dhiverb

Anne-Marie Quinsonb

Catherine Tamaleta

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© 2000 Lippincott Williams & Wilkins, Inc.