Novel recombinant phenotypic assay for clonal analysis of reverse transcriptase mutations conferring drug resistance to HIV-1 variants

Stefania Paolucci1, Fausto Baldanti1,2, Maurizio Zavattoni1 and Giuseppe Gerna1,*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective: A novel rapid reverse transcriptase (RT) recombinant HIV-1 drug-susceptibility assay was developed to evaluate resistance to RT inhibitors.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human immunodeficiency virus type 1 (HIV-1) drug-susceptibility testing should be reliable and rapid enough to assist clinicians in selecting the best available therapeutic option. Several assays (both genotypic and phenotypic) for the evaluation of antiviral drug resistance have been developed. Although useful, genotypic assays provide only indirect evidence of drug resistance. This limitation has sometimes led to an underestimate of the clinical impact of unusual amino acid substitutions13 or combinations of mutations.4,5 In particular, consolidated assumptions associating specific reverse transcriptase (RT) mutations with resistance to specific classes of RT inhibitors (RTIs) have recently been revised following the discovery of single amino acid changes conferring cross-resistance to structurally unrelated drugs2,3 or simultaneous resistance to a wide spectrum of drugs.68 In addition, specific combinations of mutations appear selectively to decrease the susceptibility to different compounds belonging to the same class.4,8

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
HIV-1 isolates from five treatment-naive and 10 highly active antiretroviral therapy (HAART)-experienced HIV-1-infected patients were analysed. Virological failure of HAART regimens including RTIs was the criterion adopted for the selection of treatment-experienced patients.

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 pHXB2{Delta}2–261RT 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-{alpha} 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).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
At time of analysis, median HIV-RNA copies in plasma of treatment-naive and HAART-experienced patients was 42 259 (range 8702–125 539) and 19 693 (range 4922–193 953) copies/mL, respectively. Median CD4 cell counts were 320 (range 94–736) cells/mm3 and 161.5 (range 30–649) cells/mm3, respectively. HAART-experienced patients had been treated with 4–6 RTIs for a median time of 15 (range 1–84) months (Table 1), in combination with different protease inhibitors.


View this table:
[in this window]
[in a new window]
 
Table 1. Comparison of genotypes between virus recombinant strains and viral isolates of the 10 HAART-experienced patients
 
Sequencing of 10 RT gene clones from RNA of each HIV-1 viral isolate from the five treatment-naive patients showed no drug-resistance-associated mutation. In contrast, sequencing of 10 RT gene clones from each viral isolate recovered from 10 HAART-experienced patients showed a number of drug-resistance-associated mutations in all patients (Table 1). In detail, four HIV-1 isolates (from patients 1, 7, 8 and 9) showed a highly homogeneous viral population, with drug-resistance-associated mutations present in most or all clones analysed. In contrast, the remaining six isolates showed a more complex distribution of RT mutations within the viral population. In these patients, in addition to the most represented mutations, a minority of clones showed other mutations, suggesting the presence of additional viral variants at a very low rate in the viral isolate population (Table 1).

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.


View this table:
[in this window]
[in a new window]
 
Table 2. Comparisons of HIV-1 susceptibility assays to RTIs in 10 HAART-experienced patients
 
The presence of less abundantly represented HIV-1 variants with additional RT mutations in viral isolates from patients 2, 3, 4, 5, 6 and 10 could explain the higher resistance levels detected by the conventional isolate phenotypic assay (Table 1). These minor viral variants could expand during the multi-cycle replication required for assay completion and determine the final level of drug resistance. The same could be true for the HIV-1 isolate recovered from patient 9. In fact, although not detected by the clonal analysis, the presence of the mutation M184V was previously reported in a plasma sample from the same patient (data not shown), thus suggesting that viral variants carrying such a mutation could be present at a very low rate. On the other hand, the novel recombinant phenotypic assay (testing of homogeneous viral populations in a single-replication-cycle assay) could better define the level of clonal resistance associated with specific RT mutations, in the absence of multiple viral variants with different RT mutation profiles.

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).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The improved methodology has increased the reliability and efficacy of drug-resistance testing. Both genotypic and phenotypic systems are now available to clinicians. Although both systems are of clinical utility, phenotypic and genotypic assays show multiple advantages and disadvantages. Genotypic assays are rapid to perform but complex to interpret, mainly in patients extensively treated with antiretroviral drugs who carry a complex virus population showing multi-drug resistance. On the other hand, phenotypic drug-susceptibility testing provides direct evidence of drug resistance. For this reason, this approach is particularly helpful in the presence of HIV-1 strains with unusual mutations or combinations of mutations.14,68 However, phenotypic drug-susceptibility assays are difficult to perform and are time-consuming.

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.


    Acknowledgements
 
We thank Luca Dossena, Lucia Chezzi and Cinzia Zanello for excellent technical assistance, and Linda D’arrigo for revising the English.

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).


    Footnotes
 
* Corresponding author. Tel: +39-0382-502420; Fax: +39-0382-502599; E-mail: g.gerna{at}smatteo.pv.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Paolucci, S., Baldanti, F., Tinelli, M. et al. (2003). Detection of a new HIV-1 reverse transcriptase mutation (Q145M) conferring resistance to nucleoside and non-nucleoside inhibitors in a patient failing highly active antiretroviral therapy. AIDS 17, 924–7.[CrossRef][ISI][Medline]

2 . Baldanti, F., Paolucci, S., Maga, G. et al. (2003). Nevirapine-selected mutations Y181I/C of HIV-1 reverse transcriptase confer cross-resistance to stavudine. AIDS 17, 1568–70.[CrossRef][ISI][Medline]

3 . Blanca, G., Baldanti, F., Paolucci, S. et al. (2003). Nevirapine resistance mutation at codon 181 of the HIV-1 reverse transcriptase confers stavudine resistance by increasing nucleotide substrate discrimination and phosphorolytic activity. Journal of Biological Chemistry 278, 15469–72.[Abstract/Free Full Text]

4 . Ross, L., Scarsella, A., Raffanti, S. et al. (2001). Thymidine analog and multinucleoside resistance mutations are associated with decreased phenotypic susceptibility to stavudine in HIV type 1 isolated from zidovudine-naive patients experiencing viremia on stavudine-containing regimens. AIDS Research and Human Retroviruses 17, 1107–15.[CrossRef][ISI][Medline]

5 . Menzo, S., Monachetti, A., Balotta, C. et al. (2003). Processivity and drug-dependence of HIV-1 protease: determinants of viral fitness in variants resistant to protease inhibitors. AIDS 17, 663–71.[CrossRef][ISI][Medline]

6 . Iversen, A. K. N., Shafer, R. W., Wehrly, K. et al. (1996). Multidrug-resistant human immunodeficiency virus type 1 strains resulting from combination antiretroviral therapy. Journal of Virology 70, 1086–90.[Abstract]

7 . Larder, B. A., Bloor, S., Kemp, S. D. et al. (1999). A family of insertion mutations between codons 67 and 70 of human immunodeficiency virus type 1 reverse transcriptase confer multinucleoside analog resistance. Antimicrobial Agents and Chemotherapy 43, 1961–7.[Abstract/Free Full Text]

8 . Ross, L., Henry, K., Paar, D. et al. (2001). Thymidine-analog and multi-nucleoside resistance mutations are observed in both zidovudine-naive and zidovudine-experienced subjects with viremia after treatment with stavudine-containing regimens. Journal of Human Virology 4, 217–22.[ISI][Medline]

9 . Japour, A. J., Mayers, D. L., Johnson, V. A. et al. (1993). Standardized peripheral blood mononuclear cell culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type 1 isolates. Antimicrobial Agents and Chemotherapy 37, 1095–101.[Abstract]

10 . Boucher, C., Keulen, W., van Bommel, T. et al. (1996). Human immunodeficiency virus type 1 drug susceptibility determination by using recombinant viruses generated from patient sera tested in a cell-killing assay. Antimicrobial Agents and Chemotherapy 40, 2404–9.[Abstract]

11 . Hertogs, K., de Bethune, M. P., Miller, V. et al. (1998). A rapid method for simultaneous detection of phenotypic resistance to inhibitors of protease and reverse transcriptase in recombinant human immunodeficiency virus type 1 isolates from patients treated with antiretroviral drugs. Antimicrobial Agents and Chemotherapy 42, 269–76.[Abstract/Free Full Text]

12 . Petropoulos, C. J., Parkin, N. T., Limoli, K. L. et al. (2000). A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1. Antimicrobial Agents and Chemotherapy 44, 920–8.[Abstract/Free Full Text]

13 . Cattaneo, E., Zavattoni, M., Baldanti, F. et al. (1999). Diagnostic value of viral culture, polymerase chain reaction and Western blot for HIV-1 infection in 218 infants born to HIV-infected mothers and examined at different ages. New Microbiologica 22, 281–91.[Medline]

14 . The AIDS Clinical Trials Group Virology Technical Advisory Committee and the Division of AIDS, National Institutes of Allergy and Infectious Diseases. (1994). In HIV Drug Susceptibility Assay. ACTG Virology Manual for HIV Laboratories. pp. RES 1–12. Division of AIDS, National Institutes of Allergy and Infectious Diseases, Bethesda, MD, USA.

15 . Paolucci, S., Baldanti, F., Campanini, G. et al. (2001). Analysis of HIV drug-resistant quasispecies in plasma, peripheral blood mononuclear cells and viral isolates from treatment-naive and HAART patients. Journal of Medical Virology 65, 207–17.[CrossRef][ISI][Medline]

16 . Betts, B. J.& Shafer, R. W. (2003). Algorithm specification interface for human immunodeficiency virus type 1 genotypic interpretation. Journal of Clinical Microbiology 41, 2792–4.[Free Full Text]

17 . Kantor, R., Machekano, R., Gonzales, M. J. et al. (2001). Human immunodeficiency virus reverse transcriptase and protease sequence database: an expanded data model integrating natural language text and sequence analysis programs. Nucleic Acids Research 29, 296–9.[Abstract/Free Full Text]