| Abstract|| |
Drug resistance can be considered as a natural response to the selective pressure of the drug. An increase in HBV DNA can be a good indicator of the presence of a resistant HBV mutant population. The nucleoside analogues Lamivudine, Adefovir, Entecavir etc. are oral drugs used for Hepatitis B viral infection. Resistance to HBV drugs arises due to mutations in the polymerase gene. The HBV polymerase can be divided into 4 domains: 1) the terminal protein, 2) the variable spacer domain, 3) the polymerase/reverse transcriptase and 4) the RNase. Drug resistance to Lamivudine is associated with mutations in the very conserved catalytic polymerase /reverse transcriptase domain, located specifically at a locus of four amino acids consisting of tyrosine-methionine-aspartate-aspartate, termed the YMDD motif at position 204:M204V/I. Adefovir resistance mutations are at amino acid residues 181,236/238:A181T/V and N236T/N238D. The Entecavir resistance mutations are at amino acid residues 184, 202 and 250 of the polymerase: T184X, S202I/G/L and M250L/V. There are several assays available that identify resistance mutation like polymerase chain reaction, real time PCR with specific probes, hybridization methods (line probe assay) and restriction fragment length polymorphism (RFLP).The best approach for patients with Lamivudine resistance is to continue Lamivudine and add Adefovir. Lamivudine is effective in suppressing serum HBV DNA levels in patients with Adefovir resistance. Entecavir resistance mutations are sensitive to Adefovir and Tenofovir. The careful selection of a first-line agent is essential to avoid the occurrence of resistance and the development of cross resistance to other agents. The most effective therapy of antiviral-resistant HBV is prevention through judicious use of nucleos(t)ide analogue therapy.
Keywords: Hepatitis B virus, drug resistance, nucleoside analog, polymerase inhibitor.
|How to cite this article:|
Hazam RK, Kar P. HBV drug resistance : Its relevance in clinical practice. Hep B Annual 2007;4:24-39
| Background|| |
Of the two billion persons worldwide who come into contact with HBV, approximately 6% of the global population (more than 350 million persons) fail to resolve acute viral infection and become chronic carriers, eventually placing between 15% to 25% of such individuals at risk for end-stage liver disease. The Hepatitis B virus (HBV), which belongs to the hepadnavirus family, is a small circular DNA virus containing a nucleocapsid and an envelope. HBV nucleocapsid contains a relatively small and incompletely double stranded 3.2 Kb DNA genome, viral polymerase and core protein. Its envelope is composed of viral surface proteins enclosed by a lipid membrane from host cells. An increase in HBV DNA can be a good indicator of the presence of a resistant HBV mutant population. Serum HBV DNA and HBeAg are two other key markers appearing precociously and are indicative of active viral replication.
The first therapeutic agent to be approved for hepatitis B was interferon-alpha (IFN-α), whose dual mode of action includes both antiviral and immunomodulatory effects. Unfortunately, extended IFN-αtreatment is expensive, injection-dependent, effective in no more than 15-25% of patients and associated with a wide spectrum of adverse reactions. However, it is the nucleoside analogue lamivudine that has become the gold standard worldwide for use in patients with chronic hepatitis B. The convenience of its relatively affordable cost, a one-pill-per-day regimen and the low incidence of side-effects has made it the preferred treatment for many patients. Lamivudine, the first nucleoside analogue introduced in 1998, heralded the era of oral therapy for HBV. Subsequently adefovir, a nucleotide analogue, became available in 2002, followed by the approval of entecavir in 2005 and finally Telbivudine in 2006. Another nucleoside analogue, Emtricitabine is currently in phase III clinical development. Drug resistance can be considered as a natural response to the selective pressure of the antiviral drug. Antiviral drug resistance depends on the viral mutation frequency, intrinsic mutability of the antiviral target site and the magnitude and rate of virus replication.
| Molecular Mechanism of Drug Resistance|| |
HBV polymerase protein is an enzyme that plays an essential role in viral replication. The HBV polymerase can be structurally and functionally divided into 4 domains: 1) the terminal protein, 2) the variable spacer domain, 3) the polymerase/reverse transcriptase and 4) the RNase. The polymerase domain which is the catalytic domain of the protein can be subdivided into several sub domains: A, B, C, D, E, F and G, as shown in the [Figure 1]:
Lamivudine, the first nucleoside analogue introduced in 1998, marked the new era of oral therapy for HBV. More recently, adefovir, a nucleotide analogue, became available in 2002, followed by the approval of entecavir in 2005. Lamivudine resistance has been the most extensively studied. Drug resistance to lamivudine is associated with mutations in the very conserved catalytic polymerase /reverse transcriptase domain of the gene, located specifically at a locus of four amino acids consisting of tyrosine-methionine-aspartate-aspartate, termed the YMDD motif. It is thought that lamivudine acts here by suppressing HBV replication. Given that HBV generates up to 10  virions/day and because of the selective pressure exerted by long-term administration of lamivudine on the virus, HBV mutants emerge. When mutations occur, the configuration of the wild-type YMDD motif becomes altered in such a way that the drug no longer successfully exerts its inhibitory action at that site. ,
Three key mutations in the polymerase gene have been shown to confer resistance to lamivudine, the first two include the substitution of methionine (M) by the amino acids isoleucine (I) or valine (V) in the YMDD motif (C domain) at position rtM204V/I. In the majority of cases, these mutations in the YMDD motif occur together with an additional compensatory mutation in the B sub domain,  namely the substitution of a leucine by methionine some 20 amino acids upstream from the YMDD domain at position rtL180M. Finally, the newly discovered mutant to adefovir (rtN236T) is located downstream from the YMDD motif in the D domain of the viral polymerase. LMV resistance by the HBV mutations at rtM204V/I is due to the changes in the van der Vaals forces that results in the repositioning of the antiviral agent as well as steric hindrance between the sulphur atom in LMV and the rtM204V/I side chain. Mutations affecting the YMDD locus typically alter the ability of the dNTP binding pocket to accommodate the nucleoside/nucleotide analogue. Thus, the primary mechanism of resistance appears to be by steric hindrance within the dNTP binding pocket. When the methionine(M) in the YMDD motif is replaced by valine(V) or isoleucine(I), the new amino acid side-chain projects into the dNTP binding site. Steric hindrance has been demonstrated for lamivudine when it was modeled into the dNTP binding pocket of the lamivudine-resistant mutations YIDD or YVDD. These alterations in the dNTP binding pocket can also hinder binding of the usual D-conformation nucleoside triphosphates. Other mutations associated with lamivudine-resistant HBV Pol occur outside the dNTP binding site. For example, the B domain changes rtV173L and rtL180M can occur in parallel with the rtM204V/I mutations as can the A domain change rtL80V/I.
The two major adefovir resistance mutations are A181T/V and N236T/N238D.  Additional adefovir mutations also reside in domain A, affecting residue 84 and 85 and other mutations have been identified between domains C and D, affecting residues 214 and 215. Similar to lamivudine resistance mutations, the A181T/V mutation is located near the triphosphate binding site. Conceptually, this mutation has a subtle effect on the triphosphate binding site conferring steric hindrance that makes the mutant strains resistant to adefovir. Another class of adefovir resistance mutations affecting residue 215 resides in the C and D interdomain region, which is distant from the nucleotide triphosphate binding site. It is not clear how the mutation at residue 215 results in adefovir resistance. It is speculated that this mutation may affect interaction with other domains, causing structural perturbation of the polymerase, making it less stable and representing a unique mechanism for resistance to adefovir.
The three identified entecavir resistance mutations are at amino acid residues 184, 202 and 250 of the polymerase: T184X, S202I/G/L and M250L/V , . A fourth mutation at residue I169 was also identified, but does not appear to contribute to resistance. To date, each of the identified entecavir resistance mutations have occurred only in the presence of preexisting lamivudine resistance mutations. Similar to the other nucleoside analogues, mutations in amino acid residues 184 and 202 occur fairly close to the triphosphate binding site, causing steric hindrance and conferring resistance. Of interest, the residue 250 is not close to the triphosphate binding site but is located in domain E, where the DNA/RNA template binds. It is possible that the M250V mutation affects the interaction of the growing nucleic acid strand with the nucleoside triphosphate and confers resistance to entecavir triphosphate.
Other resistance mutations
Telbivudine resistance mutations identified to date are similar to those associated with lamivudine resistance and occur at the classical YMDD domain with the M204I mutation.  Tenofovir treatment has been associated with the emergence of the A194T mutation. , It is interesting that despite the similarities in structure between adefovir and tenofovir, the associated resistance mutations differ somewhat for the two drugs.
| Monitoring HBV Drug Resistance|| |
There are several assays available that identify resistance mutation like polymerase chain reaction, real time PCR with specific probes, hybridization methods (line probe assay) and restriction fragment length polymorphism (RFLP). Several options are currently available to monitor HBV drug resistance and can generally be divided into genotypic assays and phenotypic methods.
(1) Genotyping Methods
Direct DNA Sequencing
Standard DNA sequencing technology provides highly accurate and complete DNA sequence information and is applicable to any part of the 3.2-kilobase HBV genome.  A serious handicap is the inability of sequencing to detect viral resistance even when the mutated virus still makes up a relatively large fraction (up to 30%) of the entire HBV population (i.e, mixtures of wild-type and mutant species). This limits its use for detecting upcoming resistance at an early stage. It also tends to be time-consuming and labor intensive, not readily adaptable to high-throughput screening and is amenable to analysis only by well-trained personnel.
An additional difficulty when using direct DNA sequencing of a PCR product is to know whether a given set of mutations occurs on the same molecule or in a different clonal subpopulation. This obstacle can theoretically be circumvented by sequencing multiple clones from a given sample. But unless a sufficient amount of clones are analyzed, minor subpopulations may remain undetected. , The technique is quite laborious and is not adaptable for large-scale use.
This methodology provides a means of overcoming some of the aforementioned limitations. RFLP methodology is as accurate as direct DNA sequencing,  but unlike sequencing, can detect samples with mixed virus populations containing mutant virus making up 5% to 10% of the virus population. , However, the procedure is generally long and tedious (multiple PCRs, multiple enzyme digestions) and requires skilled personnel since a specific endonuclease reaction has to be developed for each separate mutation to be analyzed.
Various types of assays exist that make use of probe-product hybrids. For instance, fluorometric real-time PCR with the Light-Cycler Assay can be used to detect resistant variants. Samples differing by only one nucleotide can be readily distinguished. However, natural sequence variability in places where the fluorescence probes bind can lead to a lower melting temperature of the probe without necessarily being associated with a resistance mutation. Moreover, for unequivocal detection of lamivudine resistance, the variant subpopulation may have to comprise at least 5% to 10% of the total population. Another test that employs mixed hybridization-sequencing-PCR ("minisequencing") technology involves the extension of multiple oligonucleotide primers with fluorescent ddNTPs by means of a DNA polymerase. 
An alternative approach involves differential hybridization to a preexisting panel of membrane-bound probes as illustrated by the commercially available INNO-LiPA HBV DR line probe assay. The test makes use of a series of short immobilized oligonucleotide probes to discriminate between different nucleic acid fragments (down to single nucleotide mismatches), thereby enabling identification of wild-type or mutant variants. Importantly, the test can detect variations early during the emergence of viral resistance even when the variant represents only a minor fraction of the total viral population. , This is especially relevant for patients at high risk for disease progression. In the consecutive patient samples presented, the YIDD mutation (rtM204I) was detected about 40 weeks after the start of lamivudine therapy, some 10 and 12 weeks before the increases in viral load and ALT, respectively. Subsequently, the YIDD mutation was replaced by a YVDD mutant. This latter mutation was seen to persist (over 40 weeks) after lamivudine treatment was halted.
The limitation of such hybridization-based methods (melting curve analysis, line probe assays) lies in their single-base discrimination. Specificity can be influenced by the sequences neighboring a polymorphic site or by possible interference from secondary structures. With the rise of new mutations, such hybridization-based assays must be updated accordingly.
Possible future methods for detection of resistance could make use of technologies such as fluorescence (relying on secondary reporter systems) , or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). , They offer a highly sensitive means to detect unidentified HBV variants-even when comprising only a small percentage of wild-type/mutant mixtures. While fast and accurate, these methods require the use of well-trained technicians and expensive equipment and have not been optimized for high-throughput use.
Microchip-based tests can also be envisaged for HBV resistance testing, as they may be well suited for the study of multiple mutations on the same genome.  However, as the number of clinically relevant mutants is presently relatively limited, the development of microarray-based assays for HBV resistance will probably not be cost effective unless combined with other tests.
(2) Indirect Methods
Quantification of HBV DNA (viral load assays)
Aside from its role as a 'baseline' indicator of HBV infectivity, the measurement of viral load is an indispensable means for monitoring and confirming resistance to lamivudine therapy. Indeed, resistance to antivirals is still generally defined clinically by a rise in serum HBV DNA levels during antiviral therapy.
(3) Phenotyping Methods
These methods detect drug resistance based on the use of molecular or cellular techniques or using animal models. Such systems are useful for studying HBV replication, cellular accumulation of covalently closed circular DNA and for characterizing HBV mutants. However, their use is labor-intensive and not amenable to high-throughput testing or commercialization.
| Management of Patients with Drug Resistant HBV|| |
Antiviral Drug-Resistant HBV
For optimal management of patients with antiviral drug-resistant HBV, knowledge of past history of HBV treatments and virologic response to those treatments, patterns of mutations detected at the time of virologic breakthrough and in vitro data on antiviral activity of various HBV nucleos(t)ide analogues against HBV isolates that harbor the mutations detected is required.
Nucleos(t)ide Analogue Resistance and Rescue Therapy
Lamivudine or Telbivudine Resistance
Lamivudine and telbivudine are L-nucleosides and mutations that cause resistance to one drug are associated with cross-resistance to the other. A recent pilot study in patients infected with lamivudine-resistant HBV reported that adefovir monotherapy resulted in similar rates of viral suppression when compared with combination therapy of lamivudine and adefovir.  However, the combination of lamivudine and adefovir has been shown to be more effective in preventing adefovir resistance vs. adefovir monotherapy. , Tenofovir has also been shown in clinical studies to be effective at suppressing lamivudine-resistant HBV , but tenofovir is not yet approved for treatment of hepatitis B. It has been demonstrated from in vitro studies that the activity of entecavir against lamivudine-resistant HBV is significantly lower when compared with its activity against wild-type HBV. , Clinical trials have also shown that a higher dose of entecavir was necessary for effective suppression of lamivudine-resistant vs. wild-type HBV.  However, despite the use of a higher daily dose (1.0 mg/day vs. 0.5 mg/day in patients without lamivudine resistance), a 48-week course of entecavir resulted in undetectable serum HBV DNA in a lower percentage of HBeAg-positive lamivudine-refractory patients compared with nucleos(t)ide-naive patients (19% vs 67%, respectively). , The weaker antiviral activity of entecavir against lamivudine resistant HBV accounts for the higher rate of entecavir resistance in lamivudine-refractory patients vs. nucleos(t)ide-naive patients (42% vs ~1% after 4 years of treatment, respectively).  Available data indicate that the best approach for patients with lamivudine resistance is to continue lamivudine and add adefovir. In patients with suboptimal viral suppression and those with the rtA181T mutation, tenofovir may be substituted for adefovir. Entecavir is not an optimal treatment for lamivudine-resistant HBV patients; if entecavir is chosen as rescue therapy, lamivudine treatment should be stopped. Based on in vitro data and the detection of rtM204I in patients with telbivudine resistance, the same approach used for managing lamivudine resistance can also be applied to patients with telbivudine resistance.
Adefovir-resistant mutations are partially cross-resistant with tenofovir. Clinical data regarding rescue therapy for patients with adefovir-resistant HBV are limited. Case reports have suggested that lamivudine is effective in suppressing serum HBV DNA levels in patients with adefovir resistance. ,, However, the durability of response, particularly in patients with previous lamivudine resistance, is unknown. Furthermore, reemergence of lamivudine-resistant mutations in this population has been observed within 3-6 months of reintroducing lamivudine.  Tenofovir (300 mg/day) has been reported to result in decreases in serum HBV DNA levels in patients with adefovir-resistant HBV, possibly because of the higher dosage than that used for adefovir in clinical practice (ie, 10 mg/day). However, the efficacy of tenofovir in this setting is likely limited due to in vitro evidence of cross resistance with adefovir. In vivo efficacy of entecavir in patients with adefovir resistance has been confirmed,  but experience to date is limited. Based on limited clinical data, the best approach to treat patients with adefovir resistance is to add lamivudine, telbivudine or entecavir. Entecavir may be a better option for patients with previous lamivudine resistance and in patients with the rtA181V/T mutation.
From in vitro studies it is seen that Entecavir resistance mutations are sensitive to adefovir and tenofovir, , whereas in vivo data demonstrating the efficacy of adefovir has been reported in only 1 patient. There are insufficient data to support recommendations on the treatment of patients with entecavir resistance. Addition of adefovir or tenofovir to continued entecavir appears to be the only option, but the efficacy of this approach remains to be confirmed.
Sequential treatment with nucleos(t)ide analogue monotherapy has resulted in the sequential selection of mutations conferring resistance to the initial therapy and the subsequent rescue therapy.  In most instances, effective rescue treatment is not feasible because cross-resistance exists among most of the approved therapies and combination therapy with nucleos(t)ide analogues to which the virus has developed resistance appears to have limited efficacy. 
| Conclusions|| |
The emergence of resistance to antiviral agents for the treatment of chronic HBV can be associated with exacerbation of hepatitis, hepatic decompensation, reduced HBeAg seroconversion and worsening of liver disease. Levels of HBV DNA suppression can be a predictor of drug resistance. The knowledge of in vitro cross-resistance profiles between different nucleos(t)ides has provided the rationale for selecting second-line agents for patients who developed drug resistance. However, the careful selection of a first-line agent is essential to avoid the occurrence of resistance and the development of cross resistance to other agents. The most effective therapy of antiviral-resistant HBV is prevention through judicious use of nucleos(t)ide analogue therapy. The most potent drug with the lowest rate of genotypic resistance should be administered and compliance reinforced. The treatment should be modified in patients with primary nonresponse.
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Department of Medicine, Maulana Azad Medical College, New Delhi-110 002
Source of Support: None, Conflict of Interest: None