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A novel hepatitis B virus mutation with resistance to adefovir but not to tenofovir in an HIV-hepatitis B virus-co-infected patient
  [Research Letters]
AIDS: Volume 20(17) 14 November 2006 p 2229-2231
Lacombe, Karinea,b,c; Ollivet, Aurelied; Gozlan, Joelc,e; Durantel, Sandrad; Tran, Nathalief; Girard, Pierre-Marieb,c; Zoulim, Fabiend,g aInserm, U707, Epidemiologie, Systemes d'informations, Modelisation, Paris, F-75012 France bUniversite Pierre et Marie Curie, Paris, F-75012 France cAP HP, Hopital Saint-Antoine, Service de Maladies Infectieuses et Tropicales, Paris, F-75012 France dInserm, U271, Laboratoire des virus hepatiques et pathologies associees, Lyon, F-69000 France eUMR 7079, Universite Pierre et Marie Curie, Centre de Recherche des Cordeliers, F-75005 Paris, France fLaboratoire Biomerieux, Marcy-L'Etoile, France gHospices Civils de Lyon, Hopital de l'Hotel-Dieu, Service d'Hepatologie, Lyon, F-69288 France.
A molecular virology analysis performed in an HIV-hepatitis B virus (HBV) co-infected patient showed the emergence of an unusual HBV polymerase gene mutation (rt A181T) under adefovir therapy, conferring resistance to adefovir but not to tenofovir, as proved by in-vitro phenotypic analysis. This observation suggests that careful monitoring of co-infected patients is required to diagnose HBV resistance to nucleos(t)ide analogues, and that tenofovir may be active at least against some of the adefovir-resistant strains.
Although lamivudine, adefovir dipivoxil and entecavir are the only reverse transcriptase nucleos(t)ide analogues legally licensed to treat chronic hepatitis B virus (HBV), the use of tenofovir disoproxil fumarate is widely expanding in HIV-HBV-co-infected patients because of its dual activity against HIV and HBV. However, although the mechanism and frequency of HBV resistance to lamivudine is well characterized, resistance to adefovir and tenofovir still needs to be characterized in such patients. In this report, we describe the genotypic and phenotypic characteristics of an unusual adefovir-resistant HBV strain identified in an HIV-HBV-co-infected patient included in a multicentric HIV-HBV Cohort Study [1].
Case report
M.J. is a 42-year-old patient dually infected with HIV and positive HBe-antigen hepatitis B with neither a history of opportunistic infection nor hepatitis C virus/hepatitic D virus co-infection. Antiretroviral treatment was first introduced in February 1997 because of a low CD4 cell count (230 cells/μl, 18%). Because of its dual activity on HIV and HBV, lamivudine 150 mg twice a day (dosage recommended for treating HIV) was associated with zidovudine 300 mg twice a day. In August 1998, a hepatic flare (aspartate aminotransferase 310 UI/ml and alanine aminotransferase 747 UI/ml) was attributed to a switch of lamivudine to stavudine and indinavir. It resolved totally after the re-introduction of lamivudine (Fig. 1a). The antiretroviral treatment was changed twice during follow-up because of HIV breakthrough (lamivudine, stavudine, indinavir, then lamivudine, stavudine, efavirenz). A liver biopsy performed in September 2001 showed F2 and A2 levels of fibrosis and activity graded with METAVIR, with serum transaminase levels within the normal range. Because the HBV load was not controlled with lamivudine, the patient was switched in January 2002 to adefovir dipivoxil 10 mg a day, a new reverse transcriptase nucleotide analogue recently released at that time (concomitantly with indinavir/ritonavir, stavudine and didanosine). During adefovir therapy, serum HBV-DNA levels dropped from 9.26 to 5.34 log in 23 months, then rose again above 8 log in December 2003. Transaminases also increased steadily. The numerous drug monitorings performed to assess the compliance to treatment revealed an adefovir concentration within the therapeutic range. In January 2005, a new regimen was initiated because of a CD4 cell decrease (306 cells/μl, 24%) and a high HIV viral load (98 665 copies/ml). Adefovir was replaced by tenofovir disoproxil fumarate 300 mg a day (with didanosine and atazanavir/ritonavir) because of its dual activity against HIV and HBV, and the failure of adefovir dipivoxil to control the HBV load. A liver biopsy performed in April 2005 showed a stable level of fibrosis (F2) and a decreased level of viral activity (A1). The HBV load dropped steadily from 7.72 log in January 2005 to 4.2 log in April 2006. Transaminase levels decreased progressively to reach the normal range.
At the time of lamivudine breakthrough (September 2001), the direct sequencing of viral polymerase gene products showed the dominance of a classic L180M plus M204V mutant belonging to HBV genotype G. The whole HBV genome corresponding to this isolate was cloned in pTriex vector allowing the initiation of viral DNA synthesis in tissue culture after cell transfection following a methodology described elsewhere [2]. A polyclonal mixture of replicating HBV clones was transfected in Huh7 cells, and viral DNA synthesis was analysed 7 days post-transfection in the presence or absence of drugs. As shown in Figure 1b, the IC50 of lamivudine was increased more than 100 μmol (by comparison with a reference wild-type strain with an IC50 of 1.75 μmol).
At the time of adefovir breakthrough (June 2004), the direct sequencing of the viral polymerase gene showed the presence of an A181T substitution, which was the dominant viral population found in 10 of our 12 clones. All 12 clones also harboured a R217L substitution, a polymorphic site as revealed by the analysis of the HBV genome data bank. No amino acid substitution was found at position 236 on direct sequencing as well as in all 12 clones. The mixture of all 12 clones were transfected in Huh7 cells treated or not with nucleoside analogues, i.e. lamivudine, adefovir, or tenofovir. The analysis of intracellular viral DNA synthesis in these experimental conditions showed that the viral population was indeed resistant to adefovir with a 6.5-fold increase in the IC50 compared with the viral population isolated at the time of lamivudine resistance (Fig. 1b). By contrast, tenofovir was as active on the viral population isolated at the time of lamivudine resistance as on that obtained at the time of adefovir breakthrough, in concordance with the clinical evolution of the viral load after treatment adaptation.
To our knowledge this is the first case of adefovir resistance in HIV-HBV-co-infected patients [3]. The patient received adefovir dipivoxil after HBV became resistant to lamivudine, as expected with long-term monotherapy in co-infected patients [4], and a virological breakthrough was observed after 2 years of administration. Monitoring of the drug levels confirmed the observance to therapy, but more importantly viral genome sequence analysis revealed the presence of an unexpected mutation, A181T, in the viral polymerase gene, as the usual adefovir resistance mutations are A181V and N236T [5,6]. The in-vitro analysis of the phenotype of this mutant showed that it indeed conferred resistance to adefovir but not to tenofovir or lamivudine.
Whereas the in-vivo anti-HBV efficacy of tenofovir is known in co-infected patients with HBV lamivudine-resistant mutants [7,8], our observation shows that it may be also active at least against some adefovir-resistant strains, and may therefore represent a treatment option in patients with failure to adefovir therapy. This extends a recently published report on another HBV polymerase mutation, i.e. I233V, which was found to confer resistance to adefovir but not to tenofovir [9]. Moreover, we provide new evidence that after HBV treatment failure, add-on therapy with another drug lacking cross-resistance is a better option than the switching strategy [10]. In this clinical case, viral genome sequencing and phenotypic analysis of the HBV isolates provided insight into the understanding of HBV resistance, and may become part of the treatment monitoring algorithm to improve treatment efficacy in the near future.
1. Lacombe K, Massari V, Girard PM, Serfaty L, Gozlan J, Pialoux G, et al. Major role of hepatitis B genotypes in liver fibrosis during coinfection with HIV. AIDS 2006; 20:419-427. [Fulltext Link] [Medline Link] [CrossRef] [Context Link]
2. Durantel D, Carrouee-Durantel S, Werle-Lapostolle B, Brunelle MN, Pichoud C, Trepo C, Zoulim F. A new strategy for studying in vitro the drug susceptibility of clinical isolates of human hepatitis B virus. Hepatology 2004; 40:855-864. [Medline Link] [CrossRef] [Context Link]
3. Benhamou Y, Thibault V, Vig P, Calvez V, Marcelin AG, Fievet MH, et al. Safety and efficacy of adefovir dipivoxil in patients infected with lamivudine-resistant hepatitis B and HIV-1. J Hepatol 2006; 44:62-67. [Medline Link] [CrossRef] [Context Link]
4. Benhamou Y, Bochet M, Thibault V, Di Martino V, Caumes E, Bricaire F, et al. Long-term incidence of hepatitis B virus resistance to lamivudine in human immunodeficiency virus-infected patients. Hepatology 1999; 30:1302-1306. [Medline Link] [CrossRef] [Context Link]
5. Villeneuve JP, Durantel D, Durantel S, Westland C, Xiong S, Brosgart CL, et al. Selection of a hepatitis B virus strain resistant to adefovir in a liver transplantation patient. J Hepatol 2003; 39:1085-1089. [Medline Link] [CrossRef] [Context Link]
6. Angus P, Vaughan R, Xiong S, Yang H, Delaney W, Gibbs C, et al. Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase. Gastroenterology 2003; 125:292-297. [Medline Link] [CrossRef] [Context Link]
7. Lacombe K, Gozlan J, Boelle PY, Serfaty L, Zoulim F, Valleron AJ, Girard PM. Long-term hepatitis B virus dynamics in HIV-hepatitis B virus-co-infected patients treated with tenofovir disoproxil fumarate. AIDS 2005; 19:907-915. [Fulltext Link] [Medline Link] [CrossRef] [Context Link]
8. Benhamou Y, Tubiana R, Thibault V. Tenofovir disoproxil fumarate in patients with HIV and lamivudine-resistant hepatitis B virus. N Engl J Med 2003; 348:177-178. [Medline Link] [CrossRef] [Context Link]
9. Schildgen O, Sirma H, Funk A, Olotu C, Wend UC, Hartmann H, et al. Variant of hepatitis B virus with primary resistance to adefovir. N Engl J Med 2006; 354:1807-1812. [Medline Link] [CrossRef] [Context Link]
10. Zoulim F. Mechanism of viral persistence and resistance to nucleoside and nucleotide analogs in chronic hepatitis B virus infection. Antiviral Res 2004; 64:1-15. [Medline Link] [CrossRef] [Context Link]
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