Antiviral Therapy 6: 239-248
Genotypic correlates of resistance to HIV-1 protease inhibitors on longitudinal data: the role of secondary mutations Jean Servais1*, Jean-Marc Plesséria1, Christine Lambert1, Elodie Fontaine1, Isabelle Robert1, Vic Arendt1,2, Thérèse Staub1,2, François Schneider1,3, Robert Hemmer1,2 and Jean-Claude Schmit1,2 1
Laboratoire de Rétrovirologie, Centre de Recherche Public-Santé, Luxembourg Service National des Maladies Infectieuses, Centre Hospitalier de Luxembourg, Luxembourg 3 Laboratoire National de Santé, Luxembourg 2
*Corresponding author: Tel: +352 44 11 6105; Fax: +352 44 1279; E-mail:
[email protected]
Direct sequencing of the pol gene was assessed retrospectively with protease inhibitor susceptibility in a longitudinal study. A total of 134 samples from 26 patients were analysed at regular intervals up to 2 years. Patients were included in virological failure despite indinavir, ritonavir or saquinavir based triple-drug therapy. Both the type and number of certain secondary protease mutations modulated the effect of primary mutations on phenotypic resistance. This was notably applicable to L10I/V, and to lesser extents to A71V/T. However, combinations of primary mutations, including I54V could predict resistance to the drug used and nelfinavir in more than 80%. In contrast, in vitro cross-resistance to amprenavir was rarely encountered. In addition, there was a relationship between a higher number of key mutations
and poorer virological and clinical outcomes, respectively, from 6 and 3 months on. The key mutations were the protease mutations independently conferring phenotypic resistance and/or the reverse transcriptase mutations predicting treatment outcome. This relationship was independent from drug history, viral load and CD4 cell count measurements. In summary, even on a small sample size, sequence-based genotyping seems to be a good prognostic marker when performed longitudinally. In the context of primary resistance mutations, including additional secondary mutations, it may be useful in the prediction of phenotypic and clinical resistance. This should be assessed to optimize treatment monitoring before emergence of broadly cross-resistant virus.
Introduction Highly active antiretroviral therapy (HAART) based on protease inhibitors reduces HIV-related morbidity and mortality [1] by suppressing HIV RNA plasma viral load and restoring CD4 cell counts [2]. Viral drug resistance is a major cause of HAART failure. Mutations in the protease-coding regions of pol have been associated with a reduced susceptibility to different protease inhibitors both under in vitro and in vivo conditions, and broad cross-resistance between compounds is likely to emerge under continuing drug pressure [3]. Cross-resistance between first generation protease inhibitors is common especially between indinavir and ritonavir, and to a lesser extent between these drugs and saquinavir [3]. A stepwise accumulation of resistance mutations is required to reach a significant level of phenotypic resistance [4]. Interestingly, mutant viruses can maintain a good replication capacity, also referred to as virus fitness, through compensatory mutations [5]. The possible effects of multiple combined mutations and polymorphisms in the protease-coding regions of ©2002 International Medical Press 1359-6535/01/$17.00
pol on resistance and cross-resistance are incompletely understood. However, in second-line therapy, both sequence-based genotyping and recombinant virus phenotyping predict the virological outcome in both retrospective and prospective studies [6–9]. The relationship between sequence-based genotyping, phenotyping and virological and clinical outcomes was investigated in protease inhibitor treated patients experiencing HAART failure. Those patients were initially protease-inhibitor naive and followed longitudinally in an observational study. We further tried to understand how genotype could predict phenotype in that population, putting special emphasis on the possible role of so-called secondary mutations.
Materials and methods Patients Twenty-six HIV-1 infected patients, followed at the National Service of Infectious Diseases, Centre Hospitalier de Luxembourg, undergoing a subsequent 239
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virological failure after an initial response to HAART, were included in an observational study. Failure was defined as a decrease of less than 1 log10 copies/ml from baseline viral load. Baseline data were obtained at the time of the first protease inhibitor administration. The mean duration of follow-up was 2.6 ±0.6 years. Half of the patients had a prior exposure to nucleoside reverse transcriptase (RT) inhibitors and were advanced in disease. Baseline characteristics of the patients are presented in Table 1.
Treatment First-line treatment combined two nucleoside analogues and one protease inhibitor at recommended dosages. The mean duration of the initial protease inhibitor was 1.3 ±0.7 years for indinavir (n=6), 1.2 ±0.8 years for ritonavir (n=10) and 0.9 ±0.6 years saquinavir hard gel (n=10). Initially, in addition to protease inhibitors, 13 patients were treated with zidovudine and zalcitabine, six had zidovudine and lamivudine, four had zidovudine and didanosine, two had lamivudine and zalcitabine, and one had lamivudine and didanosine therapy. During follow-up, treatment was changed because of viral load rebound. Thus treatment changes were made over the study period. Five patients ceased protease inhibitor therapy. Two of them, treated by saquinavir and ritonavir, stopped treatment because of associated toxicity without salvage therapy. For three patients, treatment changed from indinavir (n=2) or saquinavir (n=1) to nevirapine plus stavudine/lamivudine, zidovudine/lamivudine or stavudine/didanosine. Two patients stayed on initial indinavir therapy; 11 were switched to indinavir (three from saquinavir and eight from ritonavir). Six were switched to ritonavir therapy (one from indinavir and five from saquinavir). Two were changed to nelfinavir (one from saquinavir and one from indinavir). Second-line therapy based on protease inhibitor (n=21) combined two nucleoside analogues as follows: 13 patients had stavudine/lamivudine, three had zidovudine/lamivudine, three had stavudine/didanosine, and two had zidovudine/zalcitabine. Resistance testing was not used in a prospective way during the study period.
Samples Samples, taken between April 1995 and February 1999, were retrospectively screened for drug resistance at 3–6 month intervals for up to 2 years, and in the case of clinical event. Direct sequencing of the protease- and RT-coding regions of pol gave interpretable sequences for 134 strains. They were submitted to phenotypic protease inhibitor resistance testing for indinavir, ritonavir, saquinavir and nelfinavir giving 87 interpretable phenotypic results 240
Table 1. Baseline characteristics Patients
n=26
Age (years)* Gender, n (%) Men Ethnicity, n (%) Caucasian Risk factor for infection, n (%) Homosexual Heterosexual Transfusion Drug user Viral subtype, n (%) Subtype B CDC clinical stage, n (%) CDC stage C HIV RNA level (log10 copies/ml)* CD4 cell count (cells/mm3)†
37.1 ±9.98 22 (85) 23 (88) 15 (58) 9 (35) 2 (8) 0 22 (85) 14 (54) 4.41 ±0.90 70 (38–164)
CDC CD4 stage, n (%) Stage 3 History of nucleoside analogue treatment Proportion of experienced patients, n (%) Number of drugs† Months of treatment†
21 (81) 13 (50) 2 (0–4) 6 (0–12)
CDC, Centers for Disease Control and Prevention. *Mean ±SD. †Median and quartiles.
(65%, a median of three per patient, quartiles 2–6.5). Baseline samples could be phenotyped for 11 out of 26 patients. In a further step, assessment of in vitro crossresistance to amprenavir was performed only for strains with at least fourfold resistance to indinavir, ritonavir and saquinavir (n=32) as the mutational resistance pathway for amprenavir is different from other protease inhibitors [10]. Amprenavir was not used clinically during the study period.
Nucleic acid extraction Viral RNA was extracted from 140 µl EDTA-plasma using the QIAamp viral RNA kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. RNA was eluted in 50 µl of RNAse-free water, stored at –80°C and used for the different assays.
Sequencing of the protease- and RT-coding regions of pol Complete protease-respective nucleotides 111–741 of RT sequencing was performed by the TruGene HIV-1 Assay (Visible Genetics, Toronto, Canada). Secondgeneration primers from the manufacturer’s new commercial kit were used in case of failures of protease sequencing. All sequences were compared with HIV1LAV 1 (GenBank/EMBL accession number K02013), submitted to the GenBank/EMBL databases and are ©2002 International Medical Press
Secondary resistance mutations to protease inhibitors
currently available under accession numbers AJ10415–17, 10488–90, 11401–07 and 401723–1977 [11].
The recombinant virus assay The recombinant virus assay (RVA) has been previously described [12]. A replicating virus was obtained through homologous recombination of a proteasedeleted, HIV-1HXB2-derived provirus carried by a plasmid, with the PCR amplification product of the protease-coding regions of pol from a clinical isolate. Briefly, a nested PCR produced a fragment (643 bp) containing the protease-coding region of pol. This product was transfected with 10 µg of the linearized protease-deleted pHxb∆Pr plasmid (Glaxo Wellcome Research and Development, Hertfordshire, UK) in MT4 cells. Stocks of chimeric viruses were harvested from the culture supernatant while a cytopathic effect (CPE) appeared. A MT-4/MTT assay was used for titration and drug susceptibility testing. The inhibition of viral CPE by a protease inhibitor was determined, measuring the absorbance (OD540) of the infected sample after transformation of MTT to blue formazan. The 50% inhibitory concentration (IC50) was calculated using the median effect equation [13]. For each strain, seven threefold dilutions of drugs were carried out in triplicate. The wild-type reference strain HIV-1IIIB was tested during the same run, serving as control. Results were expressed as fold-resistance, that is, relative-fold increase in IC50, as compared with the wild-type reference strain.
(four- to eightfold) and susceptible (
