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Journal of Clinical Microbiology, February 2004, p. 862-866, Vol. 42, No. 2
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.2.862-866.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Different Viral Rebound following Discontinuation of Antiretroviral Therapy in Cases of Infection with Viruses Carrying L74V or Thymidine-Associated Mutations

Carmen de Mendoza,1 Ellen Paxinos,2 Pablo Barreiro,1 Nuria Camino,1 Marina Núñez,1 and Vincent Soriano1*

Service of Infectious Diseases, Hospital Carlos III, Madrid, Spain,1 ViroLogic, South San Francisco, California2

Received 29 April 2003/ Returned for modification 11 July 2003/ Accepted 29 October 2003


   ABSTRACT
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 Abstract
Introduction
 References
 
A total of 76 patients discontinued treatment with didanosine plus hydroxyurea after 1 year of maintenance therapy. The greatest human immunodeficiency virus (HIV)-RNA rebounds were seen in 10 patients harboring an L74V mutation, and the presence of viruses with this mutation rapidly waned. In contrast, viral rebounds were significantly less pronounced (P < 0.01) in 12 subjects harboring thymidine-associated mutations; these mutations persisted in all instances. Thus, selection of an L74V mutation during didanosine therapy may compromise HIV replication in vivo.


   INTRODUCTION
Top
 Abstract
 Introduction
References
 
Highly active antiretroviral therapy (HAART) has been shown to fully suppress human immunodeficiency virus (HIV) replication for long periods of time and dramatically improve the life expectancy of HIV-infected patients (13, 26, 28). However, complicated dosing schedules, side effects, and quality-of-life issues may make compliance with many antiretroviral regimens difficult for patients (21, 27). Simplification strategies or therapy interruptions have been explored in patients with good immunologic and virologic status while those patients were being treated with HAART. In many cases, unfortunately, both simpler maintenance therapies (10, 14, 20) and strategic treatment interruptions (12, 18) have failed to prolong the benefit of potent antiretroviral therapy.

Hydroxyurea (HU) has been found to be synergistic with didanosine (ddI) in blocking HIV replication (4, 11, 16). In the year 2000 we started a clinical trial in which patients treated with HAART who had undetectable levels of plasma viremia for long periods of time were randomized and were switched to ddI-HU maintenance therapy or continued on their triple-therapy regimen. Preliminary results were reported elsewhere (3). Overall, isolates from 50% of patients were found to contain less than 10,000 HIV-RNA copies/ml after 1 year of ddI-HU treatment. However, little is known about the genetic evolution of viruses under conditions of long-term exposure to ddI-HU or about its clinical implications. Herein, we analyzed the virologic and immunologic outcomes of a large group of HIV-infected patients who discontinued ddI-HU treatment after 1 year of treatment with this maintenance therapy.

HIV-infected individuals with CD4+ counts over 200 cells/µl and sustained plasma HIV-RNA suppression (<50 copies/ml) during at least 1 year on HAART were randomly assigned to switch to treatment with HU (500 mg administered twice a day) plus enterocoated ddI (400 mg administered once daily) or to continue with their standard triple-drug regimen. If plasma HIV-RNA levels rose above 10,000 copies/ml or the CD4 count dropped under 200 cells/µl at any time during ddI-HU treatment, patients resumed HAART (3).

Individuals who completed 1 year of ddI-HU treatment were advised to discontinue therapy for 2 months. The need to resume antiretroviral therapy was then reassessed. All participants signed an informed-consent form before entry into the study. Medical interviews and physical examinations were performed at the baseline time point and every 2 months thereafter. Specimens for the determination of plasma HIV-RNA, lymphocyte subset, and blood chemistry characteristics were obtained at each visit. Analyses of genotypes, phenotypes, and virus replicative capacity were performed with samples collected at 12 months of ddI-HU treatment and after 2 months off therapy. Nucleotide sequencing was performed using an ABI Prism 3100 automated sequencer (Applied Biosystems, Foster City, Calif.). Key drug resistance mutations at the reverse transcriptase (RT) gene were examined (taking as a reference a list of international guidelines recorded at a recent International AIDS Society meeting) (8, 15). The accuracy of the sequence data was assessed by phylogenetic analyses to rule out sample cross-contamination. Virus susceptibility to different antiretroviral drugs was examined using a PhenoSense HIV assay (ViroLogic, South San Francisco, Calif.) (19). Virus replicative capacity levels were measured using ViroLogic's replication capacity assay, which is based on the PhenoSense HIV drug susceptibility assay (2).

A total of 76 patients completed 1 year of ddI-HU treatment; of those patients, 47 (62%) had plasma HIV-RNA levels below 200 copies/ml (group A) and 29 had levels between 200 and 10,000 copies/ml (group B). Baseline characteristics of the study population are recorded in Table 1.


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  		TABLE 1. Baseline characteristics of the study population

 
Median decreases in viral loads and CD4 counts were calculated as medians of the differences recorded for each given patient. After patients were off ddI-HU maintenance therapy for 2 months, the median plasma HIV-RNA in patients belonging to group A rose from <50 (interquartile range [IQR], <50 to 87) to 5,150 (IQR, 2,219 to 10,927) copies/ml. For patients from group B, the median plasma HIV-RNA level increased from 1,432 (IQR, 493 to 3,767) to 16,872 (IQR, 9,855 to 29,914) copies/ml. The two groups differed significantly in the magnitude of the viral rebound (P = 0.02) as well as in the rate of rebounds above 10,000 HIV-RNA copies/ml. Rebounds to above 10,000 HIV-RNA copies/ml were seen for only 15 (32%) of 47 patients from group A, while rebounding occurred for 22 (76%) of 30 patients from group B (P < 0.01).

At the time of discontinuation of ddI-HU treatment, the median CD4 count was higher for subjects belonging to group A than for those from group B (608 versus 455; P = 0.023). After 2 months off therapy, the median fall in the CD4 count was 90 cells/µl (IQR, -227 to -3) for subjects from group A and 30 cells/µl (IQR, -92 to +16) for those from group B. Although this difference did not reach statistical significance, the declines in the CD4 counts were significantly different for the members of groups A and B when the percentages of these cells are considered (-7.1% versus -3.9%, respectively; P = 0.028), which (given that HU may cause leukopenia) most likely was a more appropriate analysis method.

Genotypes were determined for patients with viral loads above 200 copies/ml at two time points: (i) upon completion of 12 months of ddI-HU maintenance therapy and (ii) at 2 months after cessation of this therapy. These data were not available for group A patients at the first time point, as viral loads then were below 200 copies/ml.

Genotypic results were obtained from 25 of 29 group B subjects at the end of 1 year of ddI-HU treatment. For the remaining four patients, relatively low viral loads compromised the performance of the technique. A total of 4 patients had wild-type (WT) viruses, 7 had viruses with an L74V mutation, 11 had viruses with thymidine-associated mutations (TAMs), and 3 had viruses with both TAMs and an L74V mutation.

After ddI-HU treatment was stopped, the greatest HIV-RNA rebounds were seen for subjects from group B carrying an L74V mutation (median variation, +4.53 log copies/ml). In all 10 of these patients, the L74V mutation disappeared after 2 months off therapy. In contrast, viral rebounds were significantly less pronounced (P < 0.01) for 10 subjects harboring TAMs (median variation, +3.68 log copies/ml); these mutations persisted in all instances (Table 2). We excluded from the analysis patient no. 57, since he had been on prolonged ddI monotherapy before beginning HAART and we suspected that L74V mutation viruses were present as minority quasispecies in the patient's virus population at the time of discontinuation of ddI-HU treatment. Further cloning analyses are under way to test this hypothesis.


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  		TABLE 2. Virologic and immunologic outcomes for patients from group B at month 12 of ddI-HU maintenance therapy and after 2 months off therapy

 
Up to 39 (83%) of the 47 patients in group A harbored WT viruses after 2 months off ddI-HU treatment. Their median viral load was 6,074 copies/ml. For seven subjects in group A who were carrying TAMs, the median load was 4,624 copies/ml. Isolates from the only subject from group A with viruses harboring both TAMs and an L74V mutation had 3,551 HIV-RNA copies/ml.

Phenotypic analyses and replicative capacity assays were performed with a subgroup of 14 patients (9 from group A and 5 from group B) from whom further samples were available. The results are recorded in Table 3. At 2 months after discontinuation of therapy, all subjects from group A carried WT viruses and none showed phenotypic resistance to ddI. The virus replicative capacity after 2 months off therapy remained unchanged for 5 of these patients, whose viral load levels rose only slightly (median, 6,990 copies/ml). In contrast, another 2 patients from group A (no. 5 and 8) had viruses with an increased replicative capacity after ddI-HU treatment was discontinued; for these patients, the viral load rose to 16,556 and 81,163 copies/ml, respectively. Finally, isolates from one patient (no. 7) showed a significant decline in virus replicative capacity (accompanied by a twofold reduction in viral load) after discontinuation of ddI-HU treatment (Table 3).


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  		TABLE 3. Evolution of virologic parameters with ddI-HU maintenance therapy and 2 months after drug removal.

 
In group B, three patients carrying an L74V mutation subsequently lost this mutation upon discontinuation of therapy. After 1 year of ddI-HU treatment, the virus replicative capacity was at a low level in all cases. However, it returned to baseline values once ddI-HU treatment was discontinued. Only one patient (no. 50), in whom a virus harboring a Q151M mutation outgrew an L74V mutation virus just after the patient completed 1 year of ddI-HU treatment, showed a significant reduction in ddI susceptibility (an increase of 3.1-fold in the 50% inhibitory concentration), which disappeared (0.8-fold reduction) after the discontinuation of ddI-HU treatment.

Shortly after the introduction of HAART, initial enthusiasm was replaced by growing concern respecting the long-term side effects of antiretroviral therapy. Accordingly, most recent guidelines recommend deferring antiretroviral treatment until CD4 counts fall below 350 cells/µl and plasma HIV-RNA levels exceed 50,000 copies/ml (28). For many patients currently showing complete virus suppression and high CD4 counts, attempts to either simplify or discontinue therapy have been explored. However, maintenance-induction studies in most instances have failed to show beneficial results after treatment designed to control HIV replication with standard triple-drug combinations was instituted (10, 14, 20). Structured treatment interruptions are now viewed with concern, on the other hand, as they may lead to rapid loss of CD4+ T cells and drug resistance without improvement of HIV-specific immunity (1, 12, 17, 18, 22).

In our institution, patients treated with triple-drug combinations and having long-term virus suppression were allowed to participate in a trial of maintenance therapy with ddI-HU in 2000 (3, 5). After 1 year of treatment with this regimen, 76 patients with plasma HIV-RNA values below 10,000 copies/ml were invited to discontinue therapy for 2 months. For most of the patients there was an overall increase in plasma HIV-RNA levels, a decline in CD4+ lymphocyte counts, and a disappearance of drug resistance mutations, which is somewhat in agreement with previous reports (9). However, viral rebounds were much more pronounced for subjects carrying viruses with an L74V mutation than in those harboring no mutations or TAMs alone. Whereas L74V mutation viruses disappeared after ddI-HU treatment was discontinued, moreover, viruses harboring TAMs did not. Not surprisingly, the replicative capacity of viruses carrying an L74V mutation was markedly diminished with respect to that of WT viruses or viruses with TAMs. The virus replicative capacity returned to baseline in subjects with L74V mutation viruses after ddI-HU treatment discontinuation and the disappearance of the L74V mutation.

In vitro studies have shown that zidovudine-resistant strains have a replication capacity similar to WT viruses (6, 7, 25) and that, conversely, L74V mutation viruses show reduced RT processivity and low-level viral fitness (23, 24). To our knowledge, however, this is the first report to prove that the selection of L74V mutation viruses during ddI therapy can compromise HIV replication in vivo.


   ACKNOWLEDGMENTS
 
We thank Angelica Corral for her excellent technical assistance, Franco Lori for his helpful comments, Eulalia Valencia for her clinical contribution, and Juan Gonzalez-Lahoz for his continuous support.

This work was funded in part by grants from AIES, RIS (project 173), CAM, and FIPSE.


   FOOTNOTES
 
* Corresponding author. Mailing address: Service of Infectious Diseases, Hospital Carlos III, Calle Nueva Zelanda 54, 4 B, 28035 Madrid, Spain. Phone: 34 91 453 26 61. Fax: 34 91 733 66 14. E-mail: vsoriano{at}dragonet.es. Back


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Journal of Clinical Microbiology, February 2004, p. 862-866, Vol. 42, No. 2
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.2.862-866.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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