1 Servizio di Virologia and 2 Laboratori Sperimentali di Ricerca, IRCCS Policlinico San Matteo, 27100 Pavia, Italy
Received 26 September 2003; returned 8 December 2003; revised 28 January 2004; accepted 3 February 2004
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Abstract |
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Material and methods: HIV-1 RTs from five treatment-naive and 10 highly active antiretroviral therapy-experienced patients were evaluated. HIV-1 isolates recovered by culturing peripheral blood mononuclear cells from patients were used in the conventional isolate phenotype analysis. Recombinant HIV-1 strains were obtained by cloning the RT gene amplified from the supernatant of HIV-1 cultures in a plasmid carrying the HIV-1 strain HXB2 backbone, and the most represented clone for each virus isolate was then tested for antiviral drug susceptibility in parallel with HIV-1 isolates.
Results: Comparison of conventional virus isolate and the novel recombinant virus phenotypic assays showed a large concordance of results. However, some discrepant results were observed, in that higher drug-resistance levels were detected by the conventional isolate phenotypic assay in HIV-1 isolates showing the presence of a mixture of HIV-1 variants, whereas the novel recombinant phenotypic assay could more precisely detect the level of drug resistance of the single viral clones selected for the analysis.
Discussion: The novel recombinant phenotype assay, compared with the conventional virus isolate phenotype assay, showed widely overlapping results. The comparison of the two assays show that the conventional phenotypic assay is able to identify more efficiently the combined effect of drug-resistant viral variants, whereas the novel recombinant phenotypic assay is better able to define the level of drug resistance of the single viral variants. In addition, rapidity (2 weeks versus 4 weeks required by the reference recombinant assay and 6 weeks required by the conventional virus isolate phenotypic assay) is a major advantage of the novel assay.
Keywords: NRTIs, NNRTIs, IC50, HIV-1 isolates, viral population
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Introduction |
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For several years, different phenotypic assays aimed at determining the 50% RT inhibitory concentration (IC50) fold-increase of HIV-1 clinical strains, with respect to reference HIV-1 strains, have been available. In particular, methods based upon the evaluation of drug susceptibility of viral isolates9 and recombinant viruses1012 have been developed. However, the information obtained with the two types of assays is not overlapping, since both methods bear intrinsic advantages and disadvantages.9,11 In addition, both methods for phenotypic drug-resistance testing of either viral isolates or recombinant viruses are time-consuming, requiring culturing and titration of HIV-1 progeny.
In this study, a novel recombinant drug-susceptibility assay for determination of resistance to HIV-1 RTIs was developed and evaluated in comparison with the conventional phenotypic assay for drug-susceptibility testing of HIV-1 isolates. The novel assay does not require either virus culturing or titration, unlike the conventional drug-susceptibility assay of viral isolates and the reference recombinant assay.
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Materials and methods |
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HIV-1 isolates were obtained by co-culturing peripheral blood mononuclear cells (PBMCs) from HIV-1 patients with PBMCs from healthy donors, as reported previously.13 Following determination of the infectivity titre of each HIV-1 isolate, the in vitro drug-susceptibility assay was performed following a modified consensus method.14 The degree of inhibition of viral replication was measured by determination of the HIV-1 p24 antigen level (NEN Research Products, Boston, MA, USA) in the supernatant of cell cultures and was expressed as a fold-change in IC50 values for each drug. This value was obtained by dividing the IC50 value of viral isolates from each of the 10 HAART-experienced patients by the mean IC50 value of viral isolates from the five treatment-naive patients. Each test was performed in triplicate. Drug-resistance values were then arbitrarily grouped into one of the following three categories: susceptible, low-resistance and high-resistance, representing IC50 fold-increase values of 3, between 3.0 and 10, and >10, respectively. The following RTIs were assayed: zidovudine, didanosine, zalcitabine, lamivudine, stavudine, efavirenz, nevirapine and delavirdine.
For construction of recombinant HIV-1 strains, viral RNA from HIV-1 isolates was extracted from cell-culture supernatants using a commercial kit (QIAamp Viral RNA, Qiagen Inc., Valencia, CA, USA). HIV-1 populations were analysed by amplifying HIV-1 RT sequences from isolate supernatants according to a previously reported RT-PCR method.15 Subsequently, PCR products were cloned in pCR 2.1 vector (TA Cloning Kit, Invitrogen, Groningen, The Netherlands) and single RT gene clones were directly sequenced (ABI PRISM 377XL DNA Sequencer; Applied Biosystems, Foster City, CA, USA). The distribution of mutations associated with resistance to RTIs was evaluated in 10 RT gene clones for each viral isolate from the five treatment-naive patients and the 10 HAART-experienced patients.15
To obtain recombinant HIV-1 strains, blunt-end PCR products of RT genes from culture supernatants of HIV-1 isolates were cloned into pHXB22261RT plasmid carrying the HIV-1 strain HXB2 genome, deleted of the RT gene (kindly provided by C. Boucher, Utrecht, The Netherlands). Following propagation of plasmid DNA in Inv-
competent cells (Invitrogen, San Diego, CA, USA), recombinant plasmid clones were analysed by direct sequencing to verify the presence of the correct insert. The single plasmid clone representative of the most abundant viral variant present in the HIV-1 isolate from each patient was selected for drug-susceptibility analysis. Finally, viable virus strains were reconstituted by transfecting 125 ng of plasmid DNA into 30% confluent HeLa CD4 cells using lipofectin (Life Technologies Ltd, Paisley, UK). The evaluation of drug susceptibility was coincident with virus reconstitution. In fact, after 6 h of incubation at 37°C following transfection, cell-culture supernatant was removed and replaced with four-fold dilutions of the following antiretroviral drugs: zidovudine, didanosine, zalcitabine, lamivudine, stavudine, efavirenz, nevirapine or delavirdine. Thus, HIV-1 culturing, titration and infection of the cell line with a standardized viral inoculum, as routinely performed in the recombinant assay developed by Boucher et al.10 (here referred to as the reference recombinant assay) were skipped, hence shortening remarkably (by about 2 weeks) the test duration. Drug-free controls for each drug dilution were included in each assay. After 72 h of incubation (the time necessary to perform a single replication cycle in the newly infected HeLa CD4 cells), HIV-1 p24 antigen was quantified in cell-culture supernatant. The degree of antiretroviral drug resistance was determined as an IC50 fold-increase, as described above, for the conventional drug-susceptibility assay. Each test was performed in triplicate.
Genotypic analysis of antiretroviral drug resistance of HIV-1 isolates and recombinant strains from each patient was performed by feeding the relevant RT sequences into the Stanford software system (http://hivdb.stanford.edu/), which interprets the genotype sequences by assigning a score to each mutation associated with resistance to RTIs.16,17 The genotypic interpretation method takes into account four levels of drug resistance (susceptible, low, intermediate and high) instead of three. However, for clinical purposes, low and intermediate drug resistance were cumulated into a single category (low).
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Results |
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In the novel recombinant assay, the most abundantly represented viral variant was selected for analysis of each virus isolate, to reproduce the situation that most frequently occurs when performing the assay on clinical isolates.
Mean IC50 values for each RTI of five HIV-1 control isolates from treatment-naive patients, as determined by the conventional drug susceptibility assay, were as follows: zidovudine, 0.01 ± 0.007 µM; didanosine, 1.59 ± 0.84 µM; zalcitabine, 0.12 ± 0.13 µM; stavudine, 0.10 ± 0.07 µM; lamivudine, 0.06 ± 0.03 µM; efavirenz, 0.0004 ± 0.0002 µM; nevirapine, 0.08 ± 0.05 µM; delavirdine, 0.02 ± 0.02 µM.
In parallel, the relevant mean IC50 values, as determined by the novel recombinant phenotypic assays, were as follows: zidovudine, 0.01 ± 0.009 µM; didanosine, 1.46 ± 0.55 µM; zalcitabine, 0.43 ± 0.32 µM; stavudine, 0.64 ± 0.24 µM; lamivudine, 0.53 ± 0.29 µM; efavirenz, 0.0008 ± 0.0009 µM; nevirapine, 0.17 ± 0.07 µM; delavirdine, 0.27 ± 0.20 µM.
The above reported results show that the two assays have comparable variability. However, the two assays provide different IC50 values for each drug. Thus, drug-resistance levels of HIV-1 strains from HAART-experienced patients were normalized by expressing results as a fold-increase over the mean value of resistance levels of HIV-1 strains from treatment-naive patients, as obtained by each assay.
Comparison between the conventional isolate phenotypic assay and the novel recombinant phenotypic assay in HAART-experienced patients showed concordant results in 54/80 (67.5%) determinations; 26 discrepancies (32.5%) were observed (Table 2). Of these, 15 (57.7%) consisted of higher resistance levels detected by the conventional isolate phenotypic assay in virus isolates from all patients except patient 1. The remaining 11 discrepant results (42.3%) were due to a higher score determined by the novel recombinant assay, and were detected in all patients except patients 3 and 5.
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The analysis of the amplitude of the divergence of discrepant results showed the following: (i) the good agreement between the two assays was documented by the fact that discrepant results were mostly confined within the next superior or inferior resistance class; (ii) the widest divergence of results was relevant to lamivudine and was mostly related to the higher resistance score determined by the conventional phenotypic assay; (iii) the largest number of discrepancies was relevant to stavudine and was mostly related to the higher resistance score detected by the novel recombinant phenotypic assay.
Finally, the comparison between the novel recombinant assay and the genotypic interpretation method showed 66/80 (82.5%) concordant results and 14 discrepancies (17.5%). All discrepancies were relevant to the shift from one to the next inferior or superior class of resistance (Table 2).
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Discussion |
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In this respect, we set up a novel recombinant drug-susceptibility assay, which was faster to perform than the previously developed reference recombinant phenotypic assay 10 (2 versus 4 weeks) since it did not require virus culturing and titration prior to drug-susceptibility testing. In order to validate the novel assay it had to be compared with a reference assay on the same biological material. Thus, we compared the drug-resistance level obtained by the conventional phenotypic assay performed on HIV-1 isolates from 10 HAART-experienced patients and that determined by the novel recombinant phenotypic assay performed on the recombinant HIV-1 strains obtained from the RT clones mostly represented in cell culture supernatants of the same 10 HIV-1 isolates. We decided to test the most represented viral variant instead of testing the most mutated one by the novel assay, because the former should play a major role in determining antiviral drug resistance when applying recombinant phenotypic drug-resistance assays to mixed virus populations in the absence of clonal analysis of the viral mixture.
The novel assay proved to be reliable in detecting drug resistance, since it generated results that largely overlapped with those of the reference conventional phenotypic assay. On the other hand, some discrepancies between the two assays were observed, which appear to be related to the specific design of each method rather than to the poorer performance of the novel assay with respect to the conventional assay. In fact: (i) discrepant results mostly consisted of a shift to the next superior or inferior drug-resistance class; (ii) in most cases, the discrepancies could be explained by the presence of less represented drug-resistant variants in the viral isolate utilized to perform the conventional phenotypic assay. In particular, the widest divergence was observed with lamivudine resistance levels, which was supported by viral variants not dominantly present in the isolate population and therefore not analysed in the novel recombinant assay; (iii) testing of homogeneous viral populations in the novel recombinant phenotypic assay enhanced the detection of drug resistance associated with specific HIV-1 clones, without the interference of multiple viral variants with different RT mutation profiles. This mechanism could explain the higher levels of stavudine resistance (which is affected by complex patterns of RT mutations, still largely obscure) scored by the novel recombinat assay.
From the clinical standpoint, it would appear that the conventional drug-susceptibility assay, which takes into account the presence of minor resistant viral variants, could provide more information than the novel recombinant phenotypic assay. However, the former assay is cumbersome and slow to perform. On the other hand, recombinant phenotypic assays are indispensable for a precise definition of drug-resistance levels associated with single or multiple RT mutations. Once obtained, the information can be utilized to improve the available genotypic assays. Thus, recombinant phenotypic assays such as the one described here, which are characterized by high reproducibility and reduced reporting time, could help in the near future in providing tools for rapid diagnosis of HIV-1 drug resistance.
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Acknowledgements |
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This work was partially supported by Ministero della Salute, Ricerca Finalizzata (grant no. 126), Ricerca Corrente (grant no. 80207) and by Istituto Superiore di Sanità, Progetto Nazionale AIDS (grant no. 30D.36).
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Footnotes |
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References |
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