Multi-drug resistant hepatitis B virus (HBV) has been reported in hepatitis B patients who received sequential antiviral therapy. In vitro studies showed that HBV constructs with mutations resistant to lamivudine and adefovir have marked reduction in sensitivity to combination of lamivudine and adefovir, whereas constructs with mutations resistant to either drug remain sensitive to the other drug. We conducted this study to determine whether mutations conferring resistance to multiple antiviral agents co-locate on the same HBV genome in vivo and to describe the evolution of these mutations. Sera from six patients who had been found to have multi-drug resistant HBV mutations to lamivudine + adefovir, lamivudine + hepatitis B immunoglobulin (HBIG), or lamivudine + entecavir on direct sequencing were cloned after nested polymerase chain reaction (PCR). Analysis of 215 clones from 11 samples with multi-drug resistant mutations on direct sequencing showed that 183 (85%) clones had mutations to both therapies on the same genome; 31 clones had lamivudine-resistant mutants only. Clonal analysis of serial samples from three patients showed progressive evolution from all clones with lamivudine-resistant HBV mutations only to mixtures of clones that have multi-drug resistant mutations and clones that have lamivudine-resistant HBV mutations only, and ultimately all clones having multi-drug resistant HBV mutations. In conclusion, mutations conferring resistance to multiple antiviral agents co-locate on the same viral genome, suggesting that combination therapy directed against mutants resistant to each treatment may not be adequate in suppressing multi-drug resistant HBV. De novo combination therapy may prevent the emergence of multi-drug resistant mutants. (HEPATOLOGY 2006;44:703–712.)
Treatment of chronic hepatitis B is greatly improved with the availability of nucleoside/tide analogs (NA) such as lamivudine, adefovir, and entecavir.1–3 However, antiviral resistance has become an increasingly common problem during long-term treatment with NA.4, 5 Viral breakthrough associated with selection of antiviral-resistant hepatitis B virus (HBV) mutants is usually followed by biochemical breakthrough and in some instances hepatitis flares and liver failure.6–10 Therefore, treatment should be changed when viral breakthrough is detected. However, multi-drug resistant HBV has been reported in patients who received sequential treatment with NA monotherapies.7, 11–14
Treatment failure caused by drug-resistant HBV is not confined to NA. Mutation of the surface gene (S gene) has been attributed as the cause of recurrent hepatitis B in some patients who received hepatitis B immunoglobulin (HBIG) monotherapy to prevent HBV reinfection after liver transplantation.15–17 The most common mutation, glycine to arginine change at codon 145 of the small S protein (sG145R), has been shown to have decreased binding to hepatitis B surface antibody.18–20 More recently, case reports have been published of dual mutations in the polymerase and the S genes in patients who developed recurrent hepatitis B while receiving lamivudine and HBIG prophylaxis.21–24
Development of multi-drug resistance may have implications on the efficacy of rescue therapy, as in the case of multi-drug resistant human immunodeficiency virus.25, 26In vitro studies showed that HBV constructs with mutations resistant to lamivudine and adefovir have marked reduction in sensitivity to the combination of lamivudine and adefovir, whereas constructs with mutations resistant to either drug remain sensitive to the other drug.12 These findings suggest that if the antiviral-resistant mutations to lamivudine or adefovir are not present on the same viral genome, combination therapy with these two antiviral agents will still be effective in suppressing mutants that are resistant to one of these drugs. In contrast, if mutations resistant to the two drugs are present on the same viral genome, efficacy of combination therapy will be limited. Therefore, the distinction between these two scenarios is important in determining appropriate rescue therapy and in predicting its likelihood of success. However, no in vivo data have been published regarding whether these mutations exist on the same or different viral genomes.
Successive evolution of different patterns of resistant mutations has been reported during long-term lamivudine therapy.27, 28 The initial mutations are usually associated with decreased replication fitness compared with wild-type HBV; however, additional mutations that can restore replication fitness are frequently selected as treatment is continued.29–31 Successive evolution of mutations may result in a combination of mutations that have the greatest replication fitness as well as maximum drug resistance in patients with multi-drug resistant HBV.
This study was conducted to determine whether mutations conferring resistance to multiple antiviral agents are present on the same HBV genome in vivo and to describe the evolution of these mutations.
NA, nucleoside/tide analogs; HBV, hepatitis B virus; S gene, surface gene; HBIG, hepatitis B immunoglobulin; PCR, polymerase chain reaction.
Patients and Methods
Serum samples from six patients with chronic HBV infection were studied. These patients were participants in studies on HBV that included collection of blood samples for surveillance of drug-resistant HBV mutations. Institutional Review Board approval of the studies and written consent from patients were obtained. All patients had a history of viral breakthrough during treatment with lamivudine. Adefovir or entecavir was started after the diagnosis of lamivudine-resistant HBV and HBIG was added at the time of liver transplantation to prevent HBV reinfection post-transplantation. Serum samples at the time of viral breakthrough from these six patients were directly sequenced. Samples found to have mutations that are resistant to more than one HBV therapy were cloned. Serial samples from three patients (nos. 1, 2, and 3) were analyzed additionally to examine the evolution of HBV mutations.
HBV DNA was quantified using COBAS Amplicor HBV Monitor Assay (Roche, Branchburg, NJ), which has a lower detection limit of 200 copies/mL.
Nested Polymerase Chain Reaction and Direct Sequencing.
DNA extraction was carried out with QIAamp® DNA Mini Kit (Qiagen Inc., Valencia, CA) following the manufacturer's instructions. Nested polymerase chain reaction (PCR) was performed as described previously.7 The amplicons spanned domains A through F of the reverse transcriptase region of the HBV polymerase gene (rt1-rt280). PCR products were purified by QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's instructions. Purified HBV DNA was directly sequenced by the DNA sequencing core facility at the University of Michigan Medical Center, using the standard protocol for the ABI 3730xl DNA Analyzer (Applied Biosystems Co., Foster City, CA). The DNA sequences were aligned using Seqman™ II and EditSeq™ software (DNASTAR Inc., Madison, WI).
To determine whether the various drug-resistant mutations detected by direct sequencing co-locate on the same HBV genomes, PCR-amplified HBV DNA was cloned into pGEM T Easy Vector (Promega Co., Madison, WI) according to the manufacturer's instructions. Eighteen to 20 colonies with HBV insert were selected for each sample. The colonies were grown overnight at 37°C in 3 mL Luria-Bertoni broth mixed with ampicillin (100 μg/mL). Recombinant plasmid DNA was purified using the Wizard® Plus SV Minipreps DNA Purification System (Promega, Madison, WI) following the manufacturer's instructions. Purified plasmids were electrophoresed after digestion with restriction enzymes, Xba I and Pst I (Roche Diagnostics Co., Indianapolis, IN), and sequenced using primers, SP6 (5′-ATT TAG GTG ACA CTA TAG-3′) or T7 (5′-TAA TAC GAC TCA CTA TAG GG-3′). The sequences of all the clones from each sample were compared using MegAlign™ software (DNASTAR Inc., Madison, WI).
Clinical Characteristics of Patients.
Clinical characteristics of the six patients are described in Table 1; mean age was 45.3 (±10.8) years, and five were men. Two patients (nos. 1 and 2) received entecavir after viral breakthrough during lamivudine treatment. Patient 1 received combination of lamivudine and entecavir (initially 0.5 mg/d for 20 months and then 1 mg/d), whereas patient 2 stopped lamivudine when he was switched to entecavir (1 mg/d). Maximum viral suppression to 4.3 log10 copies/mL was achieved at month 23 of entecavir therapy in patient 1, and to 2.9 log10 copies/mL at month 32 in patient 2. Dual resistance to lamivudine and entecavir was detected by direct sequencing 26 and 36 months after initiation of entecavir in patients 1 and 2, respectively (Table 2). Patient 1 discontinued both therapies at month 34 because she wished to be pregnant. Patient 2 continued entecavir monotherapy and had increasing HBV DNA level to 5.0 log10 copies/mL at month 45.
Table 1. Characteristics of Patients
Abbreviation: NA, not applicable.
Detected 41 months after start of 3rd treatment.
Liver transplant recipient
Liver transplant recipient
Liver transplant recipient
Duration of prior lamivudine treatment (mo)
HBeAg/Ab at the start of 2nd course of antiviral treatment
Second antiviral treatment
Interval between onset of second course of treatment and first detection of multi–drug-resistant mutation by direct sequencing (months)
HBIG + LAM-resistant mutations and LAM-resistant mutations +ADV-resistant mutations
Patient 3 had received lamivudine for 7 months before transplantation. Despite combination prophylaxis of lamivudine and intravenous HBIG, this patient was diagnosed as having HBV reinfection 15 months after transplantation. Resistant mutations to both lamivudine and HBIG were detected at this time (Table 2). HBIG was stopped and adefovir was added, serum HBV DNA became undetectable, and lamivudine was discontinued after an overlapping period of 7 months. Viral breakthrough was detected 41 months after adefovir was initiated. Lamivudine was reintroduced after adefovir resistance was confirmed. However, mutations resistant to both adefovir and lamivudine were detected within 4 months of resumption of lamivudine.
Patient 4 had received lamivudine for 11 months before liver transplantation. Post-transplantation, this patient continued to receive lamivudine; HBIG was administered intravenously during the first week and intramuscularly thereafter. HBV reinfection was diagnosed at month 20 after liver transplantation. Resistant mutations to both lamivudine (methionine to isoleucine at codon 204 in the reverse transcriptase region of HBV polymerase [M204I]) and HBIG were detected (Table 3). HBIG was stopped, and lamivudine was switched to adefovir. Serum HBV DNA levels have decreased and remained suppressed.
Table 3. Clonal Analysis of Samples From Three Patients With Multi-Drug Resistant Mutations by Direct Sequencing
Second Course of Treatment
Mutations on Direct Sequencing
Mutations on Clonal Analysis
Number of Clones (total)
Abbreviations: A, alanine; G, glycine; I, isoleucine; L, leucine; M, methionine; N, asparagine; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; ADV, adefovir; HBIG; hepatitis B immunoglobulin; LAM, lamivudine; Mo, month.
Patient 5 had viral breakthrough after 18 months of lamivudine treatment. Lamivudine was stopped, and the patient was switched to adefovir. Viral breakthrough occurred after 19 months of adefovir monotherapy; at this time, resistant mutations to both lamivudine (leucine to methionine at codon 180 of the reverse transcriptase region of HBV polymerase [L180M] +M204I) and adefovir (alanine to valine at codon 181 and glutamine to serine at codon 215 [A181V+Q215S]) were detected (Table 3).32 Adefovir was switched to tenofovir, and liver transplantation was performed subsequently. This patient was maintained on HBIG, lamivudine, and tenofovir and had undetectable hepatitis B surface antigen and serum HBV DNA at the last follow-up, 18 months post-transplantation.
Patient 6 had received lamivudine for 3 months before liver transplantation. Post-transplantation, he received a combination of lamivudine and intravenous HBIG during the first year and lamivudine monotherapy in the second year. At month 22 post-transplantation, HBV reinfection was diagnosed, and lamivudine-resistant mutation was detected. Lamivudine was continued and adefovir was added. Serum HBV DNA levels decreased from 9.7 to 4.7 log10 copies/mL after 12 months of lamivudine and adefovir combination therapy. Twenty-five months after the addition of adefovir, lamivudine-resistant mutations (leucine to isoleucine at codon 80 and valine to leucine at codon 173 [L80I+V173L] +M204I]) remained detectable and adefovir-resistant mutation (N238T, asparagine to threonine at codon 238) emerged (Table 3).32 Serum HBV DNA level increased from a nadir of 3.2 to 4.1 log10 copies/mL after the emergence of N238T despite continued treatment with lamivudine and adefovir.
Clonal analysis was performed on 16 samples from these six patients (Fig. 1). The timing of the samples studied are indicated in Tables 2 and 3.
Eleven samples showed dual-resistant mutations to lamivudine + adefovir, lamivudine + HBIG, or lamivudine + entecavir by direct sequencing. A total of 215 clones from these 11 samples were analyzed to determine whether the dual-resistant mutations co-locate, that is, were present in the same clone. Of the 215 clones analyzed, 183 (85%) clones had mutations resistant to both therapies in the same clone, 31 clones had mutations resistant to lamivudine only, and one clone from a follow-up sample 6 months after cessation of antiviral treatment (patient 1) showed wild-type sequence.
Five samples showed lamivudine-resistant mutations only on direct sequencing. Eighty-nine of 94 clones (95%) from these five samples confirmed the presence of lamivudine-resistant mutations only whereas the remaining five clones (from patients 1 and 3) had multi–drug-resistant mutations.
Evolution of Multi-Drug Resistant Mutations.
Evolution of multi-drug resistant mutations was examined in three patients (Table 2). Patient 1 received a combination of lamivudine and entecavir after lamivudine breakthrough (Fig. 2). At the start of entecavir therapy (month 0), all 18 clones had lamivudine-resistant mutations. Clonal analysis first detected entecavir-resistant mutations in 4 of 18 clones at month 20, 6 months earlier than direct sequencing. At this time, two clones had threonine to leucine substitutions T184L and two had serine to glycine substitutions S202G together with L180M and methionine to valine substitutions at codon 204 (M204V) (Table 2). As treatment continued, T184L became predominant whereas S202G was no longer detected. By month 32, all 20 clones had both entecavir- (T184L) and lamivudine-resistant (L180M+M204V) mutations. Six months after cessation of both treatments, T184L remained detectable in 12/20 clones in association with L180M+M204V whereas seven clones had lamivudine-resistant mutations only, and one clone had wild-type HBV sequence.
Patient 2 was switched to entecavir monotherapy after lamivudine breakthrough (Fig. 3). At the start of entecavir therapy (month 0), all 20 clones had lamivudine-resistant mutations (L180M+M204V). Entecavir-resistant mutations were first detected after 36 months of entecavir treatment. At that time, 14 clones had isoleucine to threonine substitutions at codon 169 and threonine to alanine substitutions at codon 184 (I169T+T184A), three clones had S202G, and one clone had I169T+S202G, in association with lamivudine-resistant mutations. Two clones had lamivudine-resistant mutations only (Table 2). After 45 months of continued treatment with entecavir monotherapy, S202G became predominant, being present in 19 of 20 clones together with T184I in 18 clones; only one clone had I169T along with T184A. Lamivudine-resistant mutation L180M+ M204V persisted in all clones 45 months after lamivudine had been discontinued.
Patient 3 developed HBV recurrence after liver transplantation despite receiving lamivudine and HBIG (Fig. 4). At the time HBV reinfection was diagnosed, all 18 clones had lamivudine- (M204I) and HBIG-resistant mutations (sG145R) (Table 2). Adefovir was added and lamivudine was stopped 7 months later. After 41 months of adefovir treatment, viral breakthrough was diagnosed, and all 18 clones had adefovir-resistant mutation asparagine to threonine substitution at codon 236 (N236T). HBIG-resistant mutation (sG145R) remained detectable in one clone, but lamivudine-resistant mutation was not detected. Four months after reintroduction of lamivudine, lamivudine-resistant mutations (L180M+M204V) that were different from those found at the time of HBV reinfection were detected in all 20 clones. Twelve clones had additional compensatory mutation V173L, a valine-to-leucine substitution. However, adefovir-resistant mutation became less prominent, being present in only four clones. N236T was replaced by a different adefovir-resistant mutation P237H, a proline-to-histidine substitution, which has been described previously.32
Corresponding Changes in Overlapping Polymerase/S Genes.
Because the S gene of HBV overlaps the polymerase gene, mutations in one gene may result in changes in the other gene.33, 34 Table 4 shows the nonsynchronous changes affecting the overlapping gene. Several corresponding changes are genotype specific because of HBV DNA sequence polymorphisms.35
Table 4. Reciprocal Changes of P-gene and S-gene Observed During Antiviral Treatment
Location in P-gene
Location in S-gene
Observed Genotypes in This Study
Related Antiviral Therapy
Abbreviations: A, alanine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; L, leucine; M, methionine; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; HBIG, hepatitis B immunoglobulin; P-gene, polymerase gene; S-gene, surface gene.
These mutations involve polymorphic sites and vary according to hepatitis B virus genotypes.
This mutation is in the a determinant of the S gene.
Multi-drug resistant HBV mutants are becoming increasingly prevalent with sequential NA monotherapy.11, 13, 14, 36 Information on whether multi–drug-resistant mutations co-locate on the same viral genome is important in implementing appropriate rescue therapy. In this study, we demonstrated that among patients with multi-drug resistant HBV on direct sequencing, mutations conferring resistance to multiple antiviral agents co-locate on the same viral genome in more than 80% of clones; the remaining clones with resistance to only one drug had mutations to lamivudine only. A recent in vitro study showed that an HBV construct with resistant mutations to lamivudine and adefovir (L180M+M204V+ N236T) was associated with a 58.8-fold decrease in susceptibility to combination of the two drugs, whereas constructs with mutations to lamivudine (L180M+M204V) or adefovir (N236T) alone were associated with only 11- and 1.58-fold decreases in susceptibility.12 Our data suggest that the combination of two antiviral agents with activity against mutants resistant to each treatment may not be adequate in suppressing dual-resistant HBV.
The multi-drug resistant mutants were first detected by direct sequencing after a mean of 23.5 months (17.5 months for lamivudine- and HIBG- resistance and 26.5 months for resistance to two NA) of the second antiviral treatment. Our clonal analysis was expected to have a sensitivity of approximately 5% (1/20) based on an average of 20 clones analyzed from each sample. Using this method, multi-drug resistant mutations could be detected up to 6 months earlier than with direct sequencing, indicating that selection of multi-drug resistant HBV can occur very rapidly during sequential NA therapies. We acknowledge that the rapid selection of multi–drug-resistant HBV observed in this study may be related to the fact that three of the six patients had undergone liver transplantation. Enhanced HBV replication associated with immunosuppression as well as increase of replication space in the previously uninfected allograft liver may have facilitated the selection of drug-resistant mutations.37 We also included Q215S, P237H and N238T as adefovir-resistant mutations. These changes had been reported to be associated with adefovir resistance in one study.32 Although these mutations have not yet been validated by other investigators, both patients 3 and 6 had an increase of serum HBV DNA levels by approximately 1 log10 copies/mL when these mutations emerged.
Evolution of resistant mutations with progressive selection for mutants that confer greater degree of resistance or mutants that restore replication fitness has been reported in patients receiving long-term lamivudine therapy.30, 31 In this study where patients received sequential therapies, we demonstrated progressive evolution from all clones with lamivudine-resistant HBV mutations only to mixtures of clones that have multi-drug resistant mutations and clones that have lamivudine-resistant HBV mutations only, and ultimately all clones having multi-drug resistant mutations. In addition, we found that the predominant mutation to a specific antiviral therapy also evolves during the course of treatment. For example, patient 1, who was receiving lamivudine and entecavir initially, had two different entecavir-resistant mutations: T184L and S202G on different clones, but T184L became dominant during continued treatment. Conversely, patient 2, who was receiving entecavir monotherapy, had predominantly I169T and T184A mutations initially, but T184I and S202G became dominant during continued treatment. Whether the subsequent mutation pattern conferred greater resistance to entecavir or improved replication fitness of the mutant remain to be determined. Whether the difference in dominant entecavir-resistant mutations between patients 1 and 2 is related to the continued presence or absence of lamivudine treatment is also unclear. Studies are ongoing to compare the replication capacity and drug susceptibility of these mutants.
Cessation of antiviral therapy has been suggested to lead to disappearance of drug-resistant mutations.38 However, other investigators have cautioned that drug-resistant mutations are archived and are rapidly selected upon re-introduction of antiviral therapy.39 In this study, lamivudine-resistant HBV mutation was not detected in patient 3 by clonal analysis 34 months after lamivudine was stopped. Nevertheless, lamivudine-resistant mutations re-emerged within 4 months after reintroduction of lamivudine, indicating that treatment to which the virus had been previously resistant has limited efficacy even after it had been withheld for several years. Persistence of lamivudine-resistant mutations in patients who are switched to entecavir is worrisome because resistance to entecavir is greatly enhanced in the presence of lamivudine-resistant mutations.13 In this study, lamivudine-resistant mutations remained detectable in all 20 clones from patient 2 up to 4 years after withdrawal of lamivudine. Our finding raises concerns about the long-term efficacy of entecavir in patients with lamivudine-resistant HBV.
Because of overlap of the HBV surface and polymerase genes, antiviral-resistant polymerase gene mutations can result in amino acid changes in the surface protein, leading to alteration of S epitope and decreased binding to HBIG.20, 40 We found that mutations conferring resistance to lamivudine, adefovir, and entecavir all can result in amino acid changes in the HBV surface protein (Table 4). Whether all these changes decrease binding to HBIG has not been determined. However, some of the S gene changes such as glutamic acid to aspartic acid sE164D (attributable to lamivudine-resistant V173L in the P gene) are located just downstream of the immunodominant “a” determinant and may affect binding to HBIG.20, 40 Decreased efficacy of HBIG in a patient with lamivudine- (or other antiviral) resistant mutations is of grave concern in liver transplant patients. Other studies have found that HBIG-resistant S gene mutations result in changes in the P gene that restore replication fitness of lamivudine-resistant HBV mutants, leading to severe hepatitis and rapid progression to liver failure.22, 23 Although adefovir and entecavir are associated with lower rates of drug resistance compared with lamivudine, close monitoring of serum HBV DNA levels during antiviral treatment is crucial, particularly in liver transplant patients. With the availability of more antiviral therapies for hepatitis B, confirmation of genotypic resistance and characterization of resistant mutations are of increasing importance in the selection of rescue therapy.
In conclusion, we showed that mutations conferring resistance to multiple antiviral agents are present on the same viral genome, suggesting that the combination of two antiviral agents with activity against mutants resistant to each treatment may not be adequate in suppressing dual-resistant HBV. Sequential antiviral therapy leads to selection of multi-drug resistant HBV and evolution of mutations during continued treatment may select for mutants with increased replication fitness or maximal viral resistance. Thus, antiviral therapy should only be administered after careful balance of benefits versus risks, de novo combination of potent antiviral agents with low risk of resistance should be encouraged, and viral response should be carefully monitored.