Clinical: Original papers
Multiple dideoxynucleoside analogue‐resistant (MddNR) HIV‐1 strains isolated from patients from different European countries
Schmit, Jean-Claude1,2,8; Van Laethem, Kristel1; Ruiz, Lidia3; Hermans, Philippe4; Sprecher, Suzanne4; Sönnerborg, Anders5; Leal, Manuel6; Harrer, Thomas7; Clotet, Bonaventura3; Arendt, Vic2; Lissen, Eduardo6; Witvrouw, Myriam1; Desmyter, Jan1; Clercq, Erik De1; Vandamme, Anne-Mieke1
1Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium
2Laboratoire de Rétrovirologie, CRP-Santé, Luxembourg
3Retrovirology Laboratory IRSI-Caixa, Barcelona, Spain
4Centre Hospitalier Universitaire St Pierre and Institut Pasteur, Brussels, Belgium
5Division of Clinical Virology, Karolinska Institute, Stockholm, Sweden
6Viral Hepatitis and AIDS Study Group, Sevilla, Spain
7Department of Internal Medicine III, University of Erlangen-Nürnberg, Germany.
8Requests for reprints to: Jean-Claude Schmit, MD, PhD, Laboratoire de Rétrovirologie, Centre de Recherche Public-Santé, 4 rue Barblé, L-1210 Luxembourg.
Sponsorship: Supported by the Biomedical Research Programme of the European Union (EU Biomed 2 grant BMH4-CT-95–1634), the Belgian Geconcerteerde Onderzoeksacties (project GOA 95/5), the Belgian Nationaal Fonds voor Wetenschappelijk Onderzoek (NFWO) grant G.3304.96 and the Programa Nacional de Salud, Plan Nacional l+D, grant SAF 97-0219 (Spain). J.C. Schmit acknowledges a grant from the EU Biomed 1 Programme (grant BMH1-CT-94-5599) and from the Centre de Recherche Public-Santé, Luxembourg (grant 96/09). The Rega Institute for Medical Research, Leuven, Belgium, the Laboratoire de Rétrovirologie, Luxembourg-City, Luxembourg and the Retrovirology Laboratory IRSI-Caixa, Barcelona, Spain are members of the European Network for the Virological evaluation of international trials for new Anti-HIV therapies (ENVA).
Date of receipt: 29 April 1998; revised: 22 July 1998; accepted: 28 July 1998.
Objective: To study the prevalence of multiple dideoxynucleoside (ddN)-resistant (MddNR) HIV-1 in European patients under treatment with multiple ddN analogues, and to characterize MddNR strains genotypically and phenotypically.
Design and methods: Blood samples from patients after ≥ 6 months of treatment with multiple ddN were screened for the MddNR mutation Q 151M. After confirmation of MddNR in 15 patients from five European countries, genotypic resistance was evaluated by DNA sequencing of the reverse transcriptase (RT) gene. Phenotypic resistance was measured by the recombinant virus assay. Results were compared with the clinical evolution of the patients.
Results: The prevalence of MddNR strains in European patients treated with multiple ddN analogues was 3.5%. Viruses typically contained amino acid substitutions V75F, F77L, F116Y and Q151M in the RT gene. A new mutation, S68G, was frequently associated with MddNR. Phenotypically, viruses displayed high-level resistance to zidovudine (ZDV), didanosine (ddl), zalcitabine (ddC), stavudine (d4T) and partial resistance to lamivudine (3TC) once multiple mutations were present. Under in-vivo treatment pressure, some MddNR strains additionally developed resistance to protease inhibitors or non-nucleoside RT inhibitors (NNRTI). Clinically, most patients had advanced HIV disease with low CD4 cell counts, high viral loads and a rapid progression, but two patients harbouring MddNR virus responded well to dual protease inhibitor associations.
Conclusions: MddNR resistant HIV-1 can be found in European patients. MddNR is characterized by a specific set of drug resistance mutations, cross-resistance to most ddN analogues and a fast clinical progression. MddNR can be associated with protease inhibitor or NNRTI resistance.
The dideoxynucleoside (ddN) analogues zidovudine (ZDV), didanosine (ddI), zalcitabine (ddC), stavudine (d4T) and lamivudine (3TC) are major components of current anti-HIV treatment regimens [1,2]. Viral resistance to each of these drugs separately, and patterns of cross-resistance or resistance reversal have been described under in-vitro and in-vivo drug pressure during the past few years . Since 1993, a small number of virus strains simultaneously resistant to multiple ddN analogues have been observed in HIV-1-infected patients from the United States [4,5], with a reported prevalence of 2–15% depending on the patient population investigated . To date, only one case of multiple ddN-resistant (MddNR) HIV-1 isolated from a European patient has been reported . From the limited data available it seems that MddNR emerges after combination therapy with multiple ddNs, mostly ZDV and ddI or ddC. In contrast to the classical ZDV resistance, related to amino acid substitutions M41L, D67N, K70R, L210W, T215Y/F and K219Q/E in HIV reverse transcriptase (RT), MddNR is genotypically characterized by mutations A62V, V75I, F77L, F116Y and Q151M. It results in high-level resistance to ZDV, ddI, ddC, d4T and partial cross-resistance to 3TC. The emergence of MddNR viruses under combination therapy is particularly worrying at a time when guidelines recommend two or three drug combinations as the standard protocol for HIV treatment [1,2].
The present study was designed to define the genotypic and phenotypic drug resistance patterns and the clinical course associated with MddNR, and to estimate its prevalence in European patients under treatment with multiple ddN analogues.
Materials and methods
Patient population and data collection
Blood samples from 174 HIV-1-seropositive patients from four European countries (Belgium, Spain, Luxembourg and Germany) were screened for the presence of mutation Q151M using a selective polymerase chain reaction (PCR) approach. A total of 116 patients had been treated with two or more ddN analogues, mostly ZDV-ddI or ZDV-ddC, for more than 6 months, 21 patients received ZDV, 12 patients ddI alone, and 25 patients were drug-naive. If mutation Q151M was found, other blood samples from the same patient were analysed, further characterization of the HIV strains was performed, and data on treatment, clinical evolution and biological parameters were collected from the patient's hospital files. In addition to the Q151M screening programme, we were able to identify, in different cohorts of patients, 11 more patients with MddNR strains during sequencing efforts of the HIV RT gene for other purposes. These strains were also further investigated and are included in the present report.
DNA/RNA extraction, cDNA synthesis, cloning of the RT gene, selective PCR for mutation Q151M and DNA sequencing of the RT gene
DNA extraction (QiaAmp blood kit; Qiagen, Hilden, Germany), respectively, RNA extraction (RNAzol B; Biotecx Laboratories, Houston, Texas, USA) and cDNA synthesis (RNA-PCR core kit; Perkin-Elmer, Brussels, Belgium) were performed as described previously . Selective PCR for the detection of mutation Q151M has been detailed elsewhere . Direct solidphase sequencing of codons 1 to 259 of the RT-PCR product was performed on a semi-automated sequencer (ALF, automated laser flurescence; Pharmacia, Uppsala, Sweden) . For cloning, the RT gene was amplified using the primers AV15-AV7  as outer primers and RT1-RT2 as inner primers. The 1755 bp PCR fragment (spanning the entire RT gene) was directly inserted into a plasmid vector (TOPO TA cloning kit; Invitrogen, Carlsbad, CA, USA). The clones were sequenced on an ABI Prism 310 (Perkin Elmer, Foster City, CA, USA) using the dye terminator cycle sequencing kit. Phylogenetic inferences were made by the neighbour-joining method  (software PHYLIP version 3.57c ), and were evaluated using 1000 bootstrap samples. Comparison between MddNR strains and reference HIV-1 strains were made by signature pattern analysis (software VESPA ).
Phenotypic drug resistance
Phenotypic resistance to antiretroviral drugs was evaluated using the recombinant virus assay (RVA)  with modifications described previously . Briefly, starting from an outer PCR amplifying the RT and protease genes with primers AV150 and RT02, a second-nested PCR was performed to amplify either the RT gene with primers IN5 and IN3  or the protease gene with primers RVP5 and RVP3 . The inner PCR products were purified and mixed with linearized RT-deleted pHIVΔRT-BstEII plasmid (obtained from Dr Kellam and Larder, Glaxo Wellcome, UK via the National Institute for Biological Standards and Control (NIBSC) AIDS Reagent Project, UK) or protease-deleted pHIVΔPR-BstEII plasmid (courtesy of Dr E. Blair, Glaxo Wellcome, UK), and added to MT4 cells. After electrotransfection, MT4 cells were maintained in supplemented RPMI 1640 medium until the virus-induced cytopathogenic effect (CPE) was microscopically detected. Virus stocks were grown on MT4 cells and titrated for infectivity . A standardized virus input was used to infect MT4 cells in the presence of increasing concentrations of the drug to be tested. Inhibition of viral CPE detected by a colorimetric reaction  was used to calculate by the median effect equation  the 50% inhibition concentrations (IC50) for the ddN analogues ZDV, ddI, ddC, d4T and 3TC, the non-nucleoside RT inhibitor (NNRTI) delavirdine (DLV) and the protease inhibitors ritonavir (RTV), indinavir (IDV) and saquinavir (SQV). For this study ZDV, ddI, ddC and d4T were purchased from Sigma (Bornem, Belgium). 3TC and abacavir were a gift from Glaxo Wellcome, UK. Ritonavir was kindly provided by Abbott, US (Dr John Leonard), indinavir by Merck and Co., US (Dr Joel Hueff), saquinavir by Roche, UK, and delavirdine by Pharmacia and Upjohn, US. The IC50 values of the MddNR strains are expressed in μM and are compared with the mean IC50 values for a control wild-type strain (HIV-1 IIIB) tested in six to 10 independent runs.
Prevalence of patients with MddNR
Four patients with MddNR virus (patients LZ, SA, SJ and FO) were identified in the Q151M screening study, all in the group treated with multiple ddN analogues (n = 116). No MddNR virus was detected in patients under ZDV or ddI monotherapy, or without treatment. The prevalence of MddNR was thus 3.5% (four out of 116) among patients undergoing multiple ddN treatment.
Clinical description of patients with MddNR
Table 1 summarizes clinical and biological parameters for the 15 European MddNR patients. The detailed clinical evolution of patient L, the first European patient with MddNR, has been published previously . Most patients had an advanced clinical stage and multiple opportunistic infections at the time the samples were obtained. Consistently, when MddNR was present, plasma viral loads were high and CD4 cell counts low. There was no clear association with syncytium-inducing (SI) phenotype, although SI strains were frequently found, especially in late-stage patients. Six patients were treated with the ZDV-ddI combination at the time the Q151M mutation was first detected (patients AJ, SJ, SA, FO, S2, S3). MddNR virus emerged during ddI-d4T in three patients (FA and DE), ddI monotherapy in three patients (L, HA and NI), and, finally, under ddI-hydroxyurea (DI), ZDV-ddC (BI), and d4T-ddI-3TC (LZ) in one patient each (Table 1). For patient S1 only one blood sample was available, which to date did not allow the appearance of Q151M.
The remarkable clinical course of two patients is outlined in Figures 1 and 2. Patient DI (Fig. 1), who harboured drug-sensitive wild-type virus under ZDV followed by ddI monotherapy, showed mutation Q151M (as the only mutation) under ddI-hydroxyurea treatment, but continued to have low viral loads and stable CD4 cell counts. When this patient's treatment was changed to ZDV-ddI-ritonavir, the viral load decreased to undetectable levels (< 2.7 log RNA copies/ml), and CD4 cell counts started to rise. The patient continued to respond to a combination of indinavir with saquinavir, and a year later had high CD4 cell counts and still undetectable viral loads. The Q151M mutation was found to disappear under the dual protease inhibitor treatment indinavir and saquinavir. Similarly, patient HA (data not shown) had a long-lasting suppression of plasma viral load under the dual protease inhibitor treatment ritonavir and saquinavir, with a disappearance of the Q151M mutation.
Patient BI (Fig. 2), with wild-type virus under ddI monotherapy, selected for MddNR virus when under ZDV-ddC treatment, concomitantly with a rise in viral load and a decrease in CD4 cell count. The patient had poor treatment compliance, failed to respond to ZDV-ddI, but showed a transient response to ZDV-ddC-ritonavir (June 1996). The addition of saquinavir again gave a transient effect and the patient finally acquired both MddNR (Tables 2 and 3) and protease inhibitor-resistant virus (protease mutations L101, M36V, G48V, 154V, A71V, V771, V82A, 184V).
Genomic features of MddNR
The nucleotide sequences of MddNR strains are available in the GenBank/EMBL/DDBJ databases under the following accession numbers (Patient L: AJ002370–AJ002375; other patients: AJ003201–AJ003213, AJ010410–AJ010422, and AJ010487–AJ010493). A phylogenetic analysis (results not shown) concluded that all MddNR strains, with the exception of DE-02.97, fall within subtype B. Table 2 summarizes nucleotide sequence-deduced amino acid substitutions in the RT protein (codon 1–259) for multiple MddNR isolates compared with the HIV-1 reference strain, HXB2 (GenBank/EMBL/DDBJ accession number: K03455).
Additional mutations known to correspond to normal polymorphism  are not mentioned here but can be deduced from the sequences in the database.
A signature pattern analysis was performed on amino acid residues 27 to 229 of the RT protein. Fifteen sequences derived from patients with MddNR strains were compared with 14 subgroup B wild-type HIV-1 reference strains (HXB2, LAI (accession number: K02013); SF2 (K02007); BCSG3C (L02317); CAM1 (D10112); D31 (U43096); HAN (U43141); JRCSF (M38429); LW123 (U12055); MN (M17449); NL43 (M19921); NY5CG (M38431); OYI (M26727) and YU2 (M93258)). The following mutations were characteristic of the MddNR strains: V751 (found in 60%), F77L (73%), F116Y (53%) and Q151M (100%). Mutation A62V, previously associated with MddNR, was found in only five out of 15 sequences (33%). A new mutation, S68G, was detected in seven out of 15 MddNR strains (47%). There was also a large polymorphism at positions T69N/I/D (47%) and K70R/S (47%). Drug resistance-related mutations were not found in the wild-type reference strains.
A second signature pattern analysis with 15 sequences containing the classical ddN resistance mutations (Genbank/EMBL/DDBJ accession numbers: Z99298-Z99313, previously published) as background revealed discordances at codons 41, 70, 75, 77, 116, 151, 210, 211 and 215. Mutations at codons 75, 77, 116 and 151 were found exclusively in the MddNR strains, which were always wild-type at codons 41, 210 and 215, although both types of resistant viruses were found to co-exist in the same patient (e.g. patients SA, SJ, HA). In contrast, background strains were always wild-type at codons 77, 116 and 151, and mostly at codons 75 (95%). As expected, they predominantly contained mutations at positions 41, 70, 210, and 215. Only one background sequence contained mutation S68T. Polymorphism at codon 69 was found in 20% of the background sequences. Mutation R211K, occasionally associated with multiple drug resistance, was found in both groups, but predominantly in the group with the standard ZDV mutations (50% versus 29% in the MddNR group).
The most striking genomic features of MddNR strains was that mutation Q151M appeared first in time for patients in whom an analysis of sequential samples was possible, and that the classical ZDV resistance mutations (M41L, L210W, T215Y/F) were never associated with MddNR strains. In rare cases, the ZDV resistance mutations, K70R (patients FA, AJ, DI, S1) and K219Q (FA) or K219G (S1), as well as the ddC-resistance mutations K65R (FA, S1), were found in association with MddNR mutations. In two patients (DI and HA) the MddNR mutation Q151M disappeared after prolonged treatment with the protease inhibitors indinavir-saquinavir and ritonavir-saquinavir, respectively. Mutation T215Y was detected as a mixture with the wild-type codon in patients SA, SJ and HA. Therefore, the RT gene was cloned and sequenced. Clones containing substitutions T215Y or Q151M were identified, but both mutations were never found in the same clone.
Phenotypic drug resistance
Table 3 summarizes the results of the recombinant virus assay for multiple virus isolates from nine patients for whom recombinant virus was obtained. In samples with several MddNR mutations high-level resistance to ZDV, ddI, ddC and d4T was found (patients AJ, NI, FO and BI). High-level resistance to 3TC was only present when the virus strain also contained the M1841/V mutation (e.g. patient AJ). Q151M, as a single substitution, was not sufficient for high-level ddN resistance (patient DI or LZ). Two MddNR strains were tested against abacavir and displayed a seven-fold increase of IC50 compared with wild-type (data not shown). MddNR concomitantly with multiple protease inhibitor resistance was found in one patient (BI), whose last virus isolate displayed an IC50 of >5 μM for RTV, 2.41 μM for IDV and > 10 μM for SQV.
We report here the presence of MddNR HIV-1 in patients from different risk groups (homosexuals, heterosexuals, intravenous drug users) coming from five European countries. The absence of any direct relationship between the patients and the presence of drugsensitive virus without resistance mutations, in all patients from whom pretreatment blood samples were available, argue against patient-to-patient transmission of MddNR strains and in favour of a drug-induced resistance selection. Interestingly, when including the previously described patient, L , 14 out of 15 patients were under ddI-containing treatment when MddNR emerged. The 15th patient had received ddI just before the emergence of MddNR. Additionally, all patients were receiving or had been receiving ZDV as single drug or in combination with other drugs. Most American MddNR isolates were also found under ddI-containing treatments, with previous ZDV experience [18–20], rising the hypothesis that ZDV and ddI in combination or sequentially might play a crucial role in the selection of MddNR. As a result of the large-scale prescription of ZDV or ddI, or both, in HIV-infected people and the limited number of MddNR strains investigated to date, it is too early to draw conclusions on this important issue, especially as the majority of patients treated with ZDV and ddI do not develop MddNR. In our population with over 6 months' experience with multiple ddN analogues, the prevalence of MddNR was 3.5%. Studies in settings with much longer treatment periods would help to determine whether the prevalence of MddNR is dependent upon the duration of ddN analogue exposure. On the basis of the frequency of MddNR, we believe that patients failing nucleoside combinations should not only be screened for the classical drug resistance mutations, but also for Q151M. There are still controversial opinions on when and how to initiate antiretroviral drugs in naive patients with a preserved immune function and low viral load [1,2]. Prospective studies in these patients with ddN analogues associated or not with protease inhibitors, would allow a better definition of the rate of occurrence of MddNR in both treatment strategies, and the potential benefit of adding a protease inhibitor early in the course of infection.
This study confirms the pattern of MddNR mutations described previously in smaller patient series [4,5,7]. Signature pattern analysis revealed that MddNR strains do not harbour the major classical ddN resistance mutations. This suggests that there are two distinct resistance pathways in HIV-1. The first is the classical ZDV resistance pathway with amino acid substitution T215Y/F as a key marker, followed by a successive accumulation of ZDV-or other ddN-related mutations resulting in high-level phenotypic resistance to ZDV. A hallmark of this pathway is the antagonism between some of these ddN analogues, resulting in phenotypic resistance reversal, the best known example being the reversal of ZDV resistance by the selection of the 3TC resistance mutation, M184V , or the ddI resistance mutation L74V . The second resistance pathway is characterized by the Q151M mutation, which together with additional substitutions gives rise to high-level resistance to most known ddN analogues. Reversal of ddN resistance caused by the M1841/V mutation is not observed in the MddNR strains (e.g. in patients L, LZ, DE, AJ, BI, HA). Although in rare cases, both mutations T215Y and Q151M can be found in the same patient, they are never found in the same HIV-1 strain. Therefore, both drug resistance pathways seem to be mutually exclusive.
The YMDD motive at codons 183–186 and the amino acid sequence PQG at codons 150–152 are the only parts of the RT gene that are highly conserved among retroviruses . It is therefore surprising that HIV can tolerate the MddNR-associated Q151M mutation. Moreover, although substitutions at codons 75, 77 and 116 occur spontaneously in some animal retroviruses (e.g. simian immunodeficiency viruses; SIV), these regions are generally highly conserved. In contrast, positions 41 and 215, significant locations for classical ZDV resistance, show natural polymorphism among retroviruses. The high evolutionary conservation of regions involved in MddNR would suggest a reduced fitness of viruses carrying mutations in these regions. Yet, we and authors [7,19,24] found that MddNR viruses have a conserved replication rate in vitro, and in vivo, as deducible from the high plasma viral loads in MddNR patients (Table 1), and the stability of the MddNR genotype in competition experiments in vitro . Nevertheless, some data argue in favour of a reduced fitness for HIV-1 carrying the Q151M mutation alone. This single mutant has been shown to have reduced growth kinetics in vitro. Mutations A62V, V751 and others presumably act as compensatory mutations in this regard . The lack of these compensatory mutations in patient strains is consistently clinically associated with declining viral loads (e.g. patient DI, Fig. 1). In a similar way, mutation Q151M was recently found to be induced by ZDV treatment in SIVmac-infected macaques, which already presented a mutant V751 before drug treatment, again without any deleterious effect on viral replication and with a fast progression to simian AIDS .
The clinical course of the MddNR patients was heterogeneous. Still, most patients had high viral loads reflecting a sustained viral replication. As a rule, patients failed to respond to ddN treatments and had a rather fast decline of their CD4 cell counts and, as a consequence, a high frequency of opportunistic infections.
MddNR greatly reduces treatment options. In these circumstances ZDV, ddI, ddC and d4T are inefficient and 3TC is a poor option because of partial cross-resistance and rapid selection of M1841/V variants (e.g. in patients L, LZ, DE, AJ, BI and HA). In addition to MddNR, virus strains are also able to acquire in vivo genotypic and phenotypic resistance to NNRTIs  (patient L) or to multiple protease inhibitors (patient BI), increasing the possibility of the emergence of virus strains resistant to all currently approved antiretroviral drugs. Dual protease inhibitor combinations seem to be an attractive treatment alternative, and have resulted in undetectable viral loads for a prolonged time period, and the disappearance of Q151M in two patients. Also of interest is a preliminary report , suggesting that the second-generation NNRTI quinoxaline compound, HBY097, has the property of inducing a partial reversal of the MddNR phenotype in vitro. The relevance of this observation for in-vivo treatment is still unknown. Previous work from our group  suggests that treatment options for MddNR virus could include acyclic nucleoside phosphonates (e.g. adefovir dipivoxil, formerly bis-POM-PMEA), all types of NNRTI and investigational drugs such as the bicyclam derivative, JM3100, which has now entered clinical evaluation. It may be speculated that hydroxyurea, being a cell-specific inhibitor, could suppress the consequences of Q151M resistance. Patient DI developed mutation Q151M under ddI-hydroxyurea, but had a favourable clinical course and declining viral loads. This observation deserves further investigation in order to understand the role of hydroxyurea. Finally, the presence of MddNR strains is also likely to affect treatment choices for primary HIV infection, the prevention of mother-to-child transmission and post-exposure prophylaxis.
In conclusion, this data, although limited to a rather small number of patients, suggest that multiple drug resistance is an emerging problem in the treatment of HIV-infected individuals. Currently still low, the number of MddNR cases is likely to increase in the near future as more patients will be treated with multiple ddNs and for longer time periods. We believe that the occurrence of MddNR under multiple drug regimens, however, does not argue against combination therapy in general, but instead emphasizes the need for more efficient drug associations that are able rapidly and completely to suppress viral replication, thus avoiding escape mutants .
We thank Jean-Marc Plesséria, Elodie Fontaine and Christine Lambert for excellent technical assistance.
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Dideoxynucleoside analogues; HIV-1; multiple drug resistance
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