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Viral features of lamivudine resistant hepatitis B genotypes A and D
  Hepatology Volume 39, Jan 2004, Issue 1, Pages 42-50
Bernhard Zöllner 1 *, Jörg Petersen 2, Elisabeth Puchhammer-Stöckl 3, Josef Kletzmayr 4, Martina Sterneck 2, Lutz Fischer 5, Matthias Schröter 1, Rainer Laufs 1, Heinz-Hubert Feucht 1. 1Institute of Medical Microbiology and Immunology, University of Hamburg, Germany 2Department of Internal Medicine and Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg, Germany 3Institute of Virology, University of Vienna, Austria 4Department of Internal Medicine, Devision of Nephrology and Dialysis, University of Vienna, Austria 5Department of Hepatobiliary Surgery, University of Hamburg, Germany
Viral differences among lamivudine resistant hepatitis B (HBV) genotypes have not been yet investigated. Therefore, we analyzed the characteristics of these viral strains in vivo.
Forty-one patients carrying lamivudine resistant HBV were enrolled. Twenty-six patients (63%) carried resistant HBV genotype A (group A) and 15 patients (37%) carried resistant HBV genotype D (group D).
The rate of reverse transcriptase 204I mutants was significantly higher in group D (67%) compared with group A (19%), whereas rt204V mutants (81% in group A vs 33% in group D; P = .006) and rt180M mutants (81% in group A vs 40% in group D, P = .015) prevailed in group A.
The median time of shift from rt204I to rt204V mutants was significantly shorter in group A (4 months in group A, >12 months in group D, P < .001). Additional resistance associated mutations were detected exclusively in group D (P = .004).
In a multivariate analysis, HBV genotype (P = .039) and pretreatment serum HBV DNA (P = .001) were independently associated with emerging rt204I or rt204V mutants, respectively.
Serum HBV copy numbers after emergence of resistance were higher in group A (mean log10 6.99 copies/ml; range 3-9) compared with group D (mean log10 6.1 copies/ml; range 3.3-8; P = .04).
There was no difference between both groups regarding core promoter/precore mutations, viral turnover, and number of flares or disease progression during follow-up.
In conclusion, the mutational pattern during selection of lamivudine resistant HBV strains differs between genotypes A and D. This may have consequences for a salvage regimen initiated for treatment of lamivudine resistant HBV.
The emergence of drug resistant hepatitis B virus (HBV) during lamivudine treatment for chronic hepatitis B is a major problem with an incidence of 14-36% after 1 year of treatment. This frequency increases to 38%, 49%, and 66% after 2, 3, and 4 years of treatment, respectively. Lamivudine resistant HBV is characterized by amino acid variations in the reverse transcriptase domain of the HBV polymerase. In particular, an exchange of the methionine within the YMDD motif by an isoleucine or a valin (rtM204I/V mutants) is associated with lamivudine resistance. Breakthrough of these drug-resistant HBV mutants leads to a viral rebound to baseline levels, to a decrease in the rate of loss of hepatitis B e antigen (HBeAg), a high rate of relapses of serum alanine transaminase (ALT) levels, and worsening liver histology. Therefore, the emergence of viral resistance is one of the critical issues in the long-term outcome of patients treated for chronic hepatitis B. On the other hand, lamivudine resistant HBV is considered to have reduced viral fitness due to less replication efficiency in vitro and lower ALT levels in vivo as compared with baseline levels. This led to the recommendation to continue lamivudine treatment despite the emergence of resistant variants as long as benefit to the patient is maintained. Taken together, it would be useful to identify factors which are associated with a better outcome of lamivudine treatment after the emergence of resistance.
In previous studies we found that, compared with HBV genotype A, genotype D showed a significantly better response with respect to HBV DNA decrease during the first 12 months of lamivudine treatment as measured by quantitative real-time polymerase chain reaction (PCR). Also, the incidence of lamivudine resistance after 18 months was lower in patients carrying HBV genotype D. Since differences between HBV genotypes B and C have been demonstrated to influence the response during treatment with interferon alfa, there is increasing evidence that HBV genotypes are potential viral factors that influence the outcome of antiviral therapy for chronic hepatitis B.This prompted us to investigate whether lamivudine resistant HBV variants with genotypes A and D, which are the most prevalent genotypes in our cohort, exhibit different viral features in vivo. We retrospectively analyzed the pattern of resistance-associated mutations, core promoter (CP)/precore mutations, virus levels, and viral turnover of lamivudine resistant hepatitis B genotypes A and D.
Patients, Study Design, and Definitions
In a retrospective study, all consecutive patients were analyzed who underwent lamivudine monotherapy for chronic hepatitis B for at least 12 months between December 1995 and February 2003 at the University Hospitals of Hamburg and Vienna. Seventy-nine patients were included. The median time of treatment in these patients was 18 months (range 12-60). Thirteen patients (16%) had undergone renal transplantation, 15 patients (19%) had received a liver transplant, and 51 (65%) were immunocompetent patients. Forty-one patients (53%) developed lamivudine resistant HBV during a median treatment duration of 12 months (range 6-29 months). Lamivudine resistance was defined as detection of a rt204I or a rt204V HBV variant with or without additional resistance associated mutations within the HBV polymerase. These 41 patients (35 men and 6 women) were further analyzed. All patients had been positive for hepatitis B surface antigen (HBsAg) for at least 6 months before treatment. They had received 100 mg lamivudine daily and continued lamivudine therapy after emergence of resistance. Serum samples were obtained every 3 to 4 months during treatment before resistance and after resistance had emerged. In case of a positive result for serum HBV DNA in the polymerase chain reaction (PCR), the HBV polymerase was sequenced for resistance associated mutations by population-based sequencing. In our analysis, the first sample of each patient after breakthrough of lamivudine resistant HBV was investigated cross-sectionally, respectively. After the first detection of resistant HBV, patients were followed for 12 months during continuous lamivudine treatment. The endpoint of this follow-up was set as disease progression (defined as ALT > 2x the upper limit of normal (ULN) and serum HBV DNA levels >log 7/ml in at least 2 samples making it necessary to change antiviral treatment). Flares were defined as ALT level >10x ULN. For calculation of the turnover of resistant HBV, HBV DNA was quantified in the final serum sample with lamivudine sensitive HBV and compared with the HBV DNA concentration detected in the first sample with resistant HBV.
Patterns of Resistance Associated Mutations
There were significant differences between group A and D with respect to the mutational pattern within the YMDD motif. Codon rt204I mutants (n = 15) were more frequent in group D (n = 10), while rt204V mutants (n = 26) were more prevalent in group A (n = 21; P = .006). Also, a higher incidence of the rt180M substitution was found in group A (81% vs. 40% in group D; P = .015). Additional mutations within the polymerase gene were found in 5 patients of group D (3 patients with rtV173L, 2 patients with rtA200V), but in none of group A (P = .004). A multivariate analysis revealed that HBV genotype (P = .039) and pretreatment serum HBV DNA (P = .01) were independently associated with the emerging mutational pattern at codon rt204. HBV genotype A had a 6-fold higher probability of selecting a valin substitution at this codon compared to genotype D (95% CI 1.1-33.6, P = .039).
At baseline, the HBV DNA levels did not differ significantly between genotypes A and D. However, after resistance had emerged the HBV DNA level was significantly higher in group A. The mean serum HBV copy number was log10 6.99 copies/ml (range 3-9) in group A compared with log10 6.1 copies/ml (range 3.3-8) in group D (P = .04). After stratification for the mutational patterns at codons rt180 and rt204, a significant difference could not be observed for HBV copies between groups A and D. The viral turnover of resistant variants was not different in group A and D (5.5 x 105 virions/ml/day vs. 2 x 105 virions/ml/day; P = .2). Also, the adjusted turnover values did not differ significantly (3.3 x 105 virions/ml/day in group A and 1 x 105 virions/ml/day in group D).
Patients were followed for 12 months while under lamivudine. Samples (n = 126) were available in median intervals of 4 months (range 1-9). Seven patients were lost for follow-up. Hence, a total of 34 patients were evaluated (genotype A: n = 22; genotype D: n = 12). Twenty-one patients carried a rt204V mutant (genotype A: n = 17, genotype D: n = 4) and 13 patients were infected with a rt204I mutant (genotype A: n = 5, genotype D: n = 8). The mean level of serum HBV DNA was significantly higher in group A during the 12 month period (P= .04). There was no difference in the number of flares between both groups (group A: n = 1, group D: n = 0). Also, the rates of disease progression were similar in both genotype groups (genotype A: n = 8, genotype D: n = 5). Disease progression was independent of the mutational pattern at codon rt204: 5 of 13 patients (38%) carrying a rt204I mutant and 8 of 21 patients (38%) infected with a rt204V mutant had disease progression during the follow-up period. Loss of HBeAg was not detected in any patient after 12 months. Relapse of HbeAg occurred in 1 patient of group D who was infected with a rt204I mutant.
The results of our study show for the first time that the emergence of resistance-associated mutations during lamivudine treatment follows different patterns in HBV genotype A and D. In previous studies it was shown that the first mutation which appears during selection of lamivudine resistant HBV is an isoleucine exchange at codon rt204 (rtM204I) within the YMDD motif of the HBV polymerase. If lamivudine treatment is continued, the isoleucine is replaced in most patients by a valine (rtM204V), often in parallel with a methionine substitution at codon rt180 (rtL180M). Since the rtM204I variant is replaced completely by the rtL180M/rtM204V variant, this succession has been interpreted as a gain of viral fitness for resistant HBV in vivo. After emergence of resistance, the rtL180M/rtM204V mutant has been described to represent a stable viral population during continuous lamivudine treatment. In our investigation, the frequency of the rtM204I mutation was significantly higher in group D, while the rtM204V mutation was more prevalent in group A. It can be concluded from this cross-sectional analysis that HBV genotype A has a preference for a valin at codon rt204 (YVDD mutant) during the emergence of lamivudine resistance. This is further confirmed by the longitudinal observation that the mean time for a shift of the rtM204I mutant to a rtM204V mutant was significantly shorter in genotype A carriers. Our data strongly indicate that the rtL180M/rtM204V variants represent the most stable virus population in HBV genotype A infection after emergence of resistance. However, hepatitis B genotype D populations persist for long periods with a stable rtM204I mutation as the predominant viral strain. In this context, it is interesting to note that in a recent study, involving exclusively HBV genotype D carriers, 22/30 (73%) patients were infected with a rtM204I variant, a rate similar high to that found in our study (67%). Taken together, the succession of resistance-associated mutations from rtM204I to rtM204V appears to occur mainly in genotype A carriers, but to a significantly lesser extent in genotype D carriers.
The underlying mechanism for these different prevalences of the mutational patterns in HBV genotypes A and D is unclear. HBV genotypes show the greatest genetic diversity in the surface gene. The surface gene, however, overlaps with the reading frame of the HBV polymerase. Hence, mutations within the polymerase gene may change the antigenicity of the HBsAg in different ways in genotypes A and D. This could have impact on the succession of resistance-associated mutations. This was postulated by our group using Chou-Fasman predictions for the hydrophobicity of the HBsAg in lamivudine sensitive and resistant HBV with genotypes A and D, respectively. In this analysis, the HBsAg of genotype D became more hydrophilic after the emergence of lamivudine resistance at codon 198 which affects a conformational region of the HBsAg protein. This was not found for the HBsAg of resistant hepatitis B genotype A. In a recent study, it has been shown that lamivudine selects for mutations that change the antigenicity of the HBsAg, leading to reduced binding of HBsAg to anti-HBs antibodies. We conclude that, besides other reasons, immunological effects may have great impact on the succession of resistance-associated mutations.
The incidence of lamivudine resistance has been demonstrated to be higher in patients who are positive for HBeAg compared with HbeAg negative patients.[4] A loss of HBeAg is often due to the emergence of HBV variants with CP/precore mutations. It has been shown in vitro, that precore mutants can compensate for the replication deficiency in lamivudine resistant HBV. These mutations have also been shown to be associated with genotype D. However, our in vivo data suggest that CP/precore mutations in lamivudine resistant HBV are neither associated with certain mutations within the HBV polymerase nor with higher HBV-DNA yield. Hence, CP/precore mutations appear to have no influence on the mutational pattern arising in the HBV polymerase during lamivudine treatment in vivo.
The most prevalent HBV genotype in North America and Europe is genotype A. An important aspect of our study is that this genotype is associated with complex mutational patterns during lamivudine treatment inducing cross-resistance to other antiviral drugs, such as the rtL180M mutation. This mutation has been shown to be a key mutation associated with famciclovir resistance.[38] Hence, it can be expected from the epidemiological data that the prevalence of patients carrying HBV with mutations inducing cross-resistance will increase dramatically in regions in which lamivudine is used widely as a monotherapeutic agent. In particular, the ability to transmit rtL180M/rtM204V mutants even to persons receiving high-dose lamivudine[39] raises concerns about the use of lamivudine as a first-line drug. This may apply particularly to patients infected with HBV genotype A. However, studies involving a greater number of patients are required to assess the optimal strategy for the initial treatment of chronic hepatitis B.
In conclusion, the results of this study show for the first time that viral differences exist in vivo between lamivudine resistant hepatitis B genotypes A and D. In particular, the HBV genotype seems to determine the emerging pattern of resistance-associated mutations, the stability of the mutations at codon rt204, and the level of viremia after selection of lamivudine resistant HBV. Our data suggest that the selection of resistant HBV during lamivudine treatment is a multifactorial process in which the replicative capacity of the virus (as measured by pretreatment HBV DNA), the host immune response (reflected by pretreatment ALT values), and the HBV genotype play important roles. The different mutational patterns of resistant HBV genotypes A and D may have impact on treatment strategies for lamivudine resistant HBV.
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