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  • GASTROENTEROLOGY 2009;137:1593–1608 GASTROENTEROLOGY BASIC AND CLINICAL REVIEWS IN REVIEWS IN BASIC AND CLINICAL GASTROENTEROLOGY John P. Lynch and David C. Metz, Section Editors Hepatitis B Virus Resistance to Nucleos(t)ide Analogues FABIEN ZOULIM*,‡,§ and STEPHEN LOCARNINI *INSERM, U871, Lyon; ‡Université Lyon 1, Lyon; §Hospices Civils de Lyon, Service d’hépatologie et de gastroentérologie, Lyon, France; and Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia Patients with chronic hepatitis B (CHB) can be success- reducing morbidity and mortality. Therapeutic efficacy fully treated using nucleos(t)ide analogs (NA), but can be affected by factors such as the development of drug-resistant hepatitis B virus (HBV) mutants fre- adverse effects, poor patient compliance, previous treat- quently arise, leading to treatment failure and progres- ment with suboptimal regimens, infection with drug- sion to liver disease. There has been much research into resistant viral strains, and inadequate drug exposure be- the mechanisms of resistance to NA and selection of cause of pharmacologic properties of particular drug(s) these mutants. Five NA have been approved by the US and individual genetic variation. Interferon (conventional Food and Drug Administration for treatment of CHB; it or pegylated) and 5 other drugs that belong to the class is unlikely that any more NA will be developed in the of nucleos(t)ide analogues (NA) (lamivudine [LMV], ad- near future, so it is important to better understand efovir dipivoxil [ADV], entecavir [ETV], telbivudine [LdT], mechanisms of cross-resistance (when a mutation that and tenofovir [TDF]) have been approved for treatment mediates resistance to one NA also confers resistance to of CHB in many parts of the world.2 The NA inhibit another) and design more effective therapeutic strate- reverse transcription of the HBV polymerase, and this article gies for these 5 agents. The genes that encode the poly- reviews the emergence of resistance to this class of agents. merase and envelope proteins of HBV overlap, so resis- tance mutations in polymerase usually affect the Clinical Importance of Resistance hepatitis B surface antigen; these alterations affect in- Impact on Disease Severity fectivity, vaccine efficacy, pathogenesis of liver disease, The development of drug resistance begins with and transmission throughout the population. Associa- mutations in the polymerase gene, followed by an in- tions between HBV genotype and resistance phenotype crease in viral load, an increase in serum alanine amino- have allowed cross-resistance profiles to be determined transferase (ALT) levels several weeks to months later, for many commonly detected mutants, so genotyping and progression of liver disease3–5 (Figure 1). In patients assays can be used to adapt therapy. Patients that expe- with LMV resistance, the risk of increased serum ALT is rience virologic breakthrough or partial response to usually correlated with the duration of infection with the their primary therapy can often be successfully treated mutant virus.6 These patients are also at significant risk with a second NA, if this drug is given at early stages of of ALT flare, which may be accompanied by hepatic these events. However, best strategies for preventing NA decompensation.6 The emergence of LMV-resistant mu- resistance include first-line use of the most potent tations is reflected in histologic assessment of liver sam- antivirals with a high barrier to resistance. It is impor- ples.7 The detrimental effect of HBV drug resistance on tant to continue basic research into HBV replication clinical outcome was shown by a placebo-controlled trial and pathogenic mechanisms to identify new therapeutic of LMV in patients with advanced fibrosis.8 Patients suc- targets, develop novel antiviral agents, design combina- cessfully treated with LMV who maintained wild-type tion therapies that prevent drug resistance, and decrease HBV had a significantly lower risk of liver disease pro- the incidence of complications of CHB. gression compared with those who received placebo, but T he hepatitis B virus (HBV) is a DNA virus that replicates its genome via an RNA intermediate using reverse transcription1 (Supplementary Figure 1); chronic Abbreviations used in this paper: ADV, adefovir dipivoxil; CHB, chronic hepatitis B; ETV, entecavir; HBV, hepatitis B virus; LdT, telbi- vudine; LMV, lamivudine; NA, nucleos(t)ide analogs; TDF, tenofovir. infection with this virus can result in cirrhosis and hep- © 2009 by the AGA Institute atocellular carcinoma.2 Effective treatments have been 0016-5085/09/$36.00 developed for chronic hepatitis B (CHB), significantly doi:10.1053/j.gastro.2009.08.063
  • 1594 ZOULIM AND LOCARNINI GASTROENTEROLOGY Vol. 137, No. 5 GASTROENTEROLOGY BASIC AND CLINICAL REVIEWS IN serum levels of ALT.9 In some cases, the emergence of ADV resistance was also associated with acute exacerba- tion of disease and liver failure.10 Only limited data are available on the clinical outcome of patients who are infected with LdT-, ETV-, or TDF- resistant HBV, mainly because treatment adaptation, usually based on in vitro cross-resistance data, has been initiated during early stages of resistance, or treatment failure. Thus, the availability of antiviral drugs with com- plementary cross-resistance profiles (see below) has changed the management of patients with drug resis- tance, allowing physicians to prevent the worsening of clinical outcome resulting from the emergence of resis- tance. Incidence and Prevalence of Resistance LMV resistance increases progressively over the course of treatment; 14%–32% of patients become resis- tant to the drug each year after treatment was initiated, and more than 80% are resistant after 48 months of treatment5 (Table 1). The rate of emergence of LdT- resistant HBV is lower than that of LMV but is still substantial. In a phase III trial that compared LdT with LMV, genotypic resistance occurred in 4.4% and 21% of hepatitis B e antigen (HBeAg)-positive patients after 1 and 2 years of treatment, respectively, and 2.7% and 8.6% in HBeAg-negative patients, respectively11,12 (Table 1). The rate of selection for ADV-resistant virus is lower; Figure 1. Kinetics of drug resistance emergence. Panel A: Evolution of resistance occurs in approximately 2% of patients with viral load and ALT levels. After an initial drop in viral load following the HBeAg-negative CHB after 2 years of therapy (Table 1). initiation of antiviral therapy, virologic breakthrough may occur as a However, following 4 –5 years of ADV monotherapy, up consequence of antiviral drug resistance. It corresponds to the rise in serum HBV DNA levels of at least 1-log10 IU/mL compared with the to 30% of patients are found to be resistant.9,13 When lowest value during therapy (nadir value), in 2 consecutive samples 1 ADV has been used in patients who are resistant to LMV, month apart, in patients who have previously responded and have a primary ADV resistance as detected by genotype analysis good treatment compliance. It may be followed by an elevation in serum has been found in up to 20% of patients by 12 months ALT levels in patients who previously showed transaminases normal- after ADV therapy began.14 Recent trials of TDF reported ization under treatment. It may result in hepatitis flares and in worsening of liver histology. Panel B: Evolution of the viral quasispecies with re- that no resistance had developed by weeks 4815 and 96 of spect to primary and secondary resistance mutations. At the beginning treatment, although at week 72, the majority of viremic of therapy, wild-type virus is the major strain circulating in the patient’s blood, whereas viral genomes harboring polymorphic mutations may be detected. Because of the spontaneous error rate of the viral poly- Table 1. Cumulative Annual Incidence of Resistance for merase, primary resistance mutations are usually present at levels that Lamivudine, Telbivudine, Adefovir, Tenofovir, and are undetectable by conventional diagnostic techniques. At the time of Entecavir virologic breakthrough, viral genomes harboring primary resistance mu- tations start to emerge and become the dominant viral strains. The Resistance at year of therapy expressed as continuation of viral replication under the selective pressure of the drug percentage of patients may lead to the accumulation of additional mutations that increase the resistant mutant replication capacity (ie, secondary resistance muta- Drug and patient 1st 2nd 3rd 4th 5th 6th tions). population (y) (y) (y) (y) (y) (y) Lamivudine 23 46 55 71 80 — Telbivudine HBeAg-Pos 4.4 21 — — — — this effect was lost in patients who developed LMV- Telbivudine HBeAg-Neg 2.7 8.6 — — — — AdefovirHBeAg-Neg 0 3 6 18 29 — resistant mutant forms of the virus.8 Adefovir (LAM- Up to 20% — — — — — The kinetics of emergence of resistance to ADV are resistant) typically slower than those of LMV but follow the same Tenfovir 0 0 — — — — sequence of events: polymerase variants with the specific Entecavir (naïve) 0.2 0.5 1.2 1.2 1.2 1.2 resistance mutations can be detected initially, which is Entecavir (LAM 6 15 36 46 51 57 resistant) next followed by virologic breakthrough and then rising
  • November 2009 HBV AND RESISTANCE TO NUCLEOS(T)IDE ANALOGUES 1595 GASTROENTEROLOGY BASIC AND CLINICAL REVIEWS IN patients was given a combination of TDF and emtricit- therefore be selected rapidly out when using drugs that abine (Truvada; Gilead Sciences, Inc, Foster City, CA).16 exhibit cross-resistance.24 Thus, the stability and replen- Very low rates of genotypic resistance to ETV have been ishment of cccDNA is the stumbling block for eradicat- reported in treatment-naïve patients after 1 year (0.1%), 2 ing CHB infection with current antiviral agents.25–28 years (0.4%), 3 years (1.2%), 4 years (1.2%), 5 years (1.2%), A combination of host and viral factors determine viral and 6 years (1.2%) of therapy17,18 (Table 1). In contrast, in persistence and also NA resistance. Infected hepatocytes patients previously treated with LMV, the cumulative have a long half-life, contributing to HBV persistence in genotypic resistance rates are 6% (year 1), 14% (year 2), the liver.29 –32 Mathematical modeling showed that the and 32% (year 3) (Table 1), steadily increasing to almost half-life of hepatocytes varies from 30 to 100 days, de- 60% by year 6.17,18 pending on individuals’ immune response.33,34 HBV ge- nome variability during the chronic phase of the disease Principles of Resistance and determines the selection for viral resistant strains.20,35,36 Cross-resistance Clonal and pyrosequencing analysis of HBV genomes A major determinant in the slow kinetics of HBV have shown that single mutants exist in the overall viral clearance from infected cells is the presence of a replica- population of HBVs even before therapy begins. Viral tive form of the viral DNA termed covalently closed circular quasispecies within the same patient evolve during the DNA (cccDNA)19,20 (Supplementary Figure 1). During course of infection: different variants or mutants are chronic HBV infection, cccDNA is maintained in the selected at different stages of infection in response to the hepatocyte nuclei with a long half-life in infected cells.21 host immune response or antiviral therapy (Figure 2). Furthermore, it has been shown that antiviral therapy Different mechanisms are involved in the selection of with NA cannot prevent the initial formation of cccDNA, drug-resistant mutants during antiviral therapy.20,35 As indicating that persistent viremia during therapy leads to described above, a complex mixture of genetically distinct infection of new cells.22,23 The HBV cccDNA acts as a variants have a replicative advantage in the presence of reservoir for the reactivation of viral genome replication the selective pressure of NA therapy. A newly acquired or and is responsible for viral relapse after withdrawal of a preexisting mutation conferring a selective advantage antiviral therapy or those patients with CHB with im- to a variant will generate progeny virus, which is more mune suppression. It has also been shown in the wood- fit and can spread more rapidly in the liver, allowing chuck model of hepadnavirus infection that drug resis- the corresponding mutant to accumulate and become tance mutations are archived in cccDNA and may the dominant species in the liver in the presence of the Figure 2. Mechanisms of se- lection and emergence of HBV drug-resistant mutants. The main factors involved in the se- lection of escape mutants are: (i) the long half-life of hepatocytes and viral cccDNA; (ii) the HBV genome variability leading to a complex viral quasispecies and mutant archiving in cccDNA. The composition of the viral quasi- species evolves over time de- pending on the selective pres- sure including antiviral therapy and the host immune response. Escape mutants may then spread in the liver and become the dominant species depend- ing on their fitness (ie, their ca- pacity to replicate and dominate wild-type strain in the presence of antiviral pressure) and the rep- lication space available for their dissemination in the liver. Their selection results in treatment failure.
  • 1596 ZOULIM AND LOCARNINI GASTROENTEROLOGY Vol. 137, No. 5 GASTROENTEROLOGY BASIC AND CLINICAL REVIEWS IN antiviral drug.37– 40 The replacement of wild-type virus in mixture of viral quasispecies, each differing in 1 or more liver cells by a dominant mutant is a slow process; studies mutations.49 The probability that a mutation associated in animal models indicate that resistant mutants pre- with drug resistance is selected for during therapy also dominately infect uninfected cells (ie, replication space), depends on the potency of that drug.50 Replication fit- so the spread of the dominant mutant depends on the ness (defined as the ability to replicate under selection number of uninfected cells in which HBV can repli- pressure45) and the replication capacity of resistant iso- cate.24,41 It might take months for the immune system to lates can shape the pattern of primary vs secondary mu- remove hepatocytes that are infected with wild-type HBV tations that emerge.51 The availability of replication and for new hepatocytes to develop that are susceptible space for HBV also determines resistance; the liver can to infection by viral drug-resistant mutant HBV. The accommodate new transcriptional templates of cccDNA infectivity of the drug-resistant mutants can impact the only if uninfected cells are generated by normal liver speed of their selection; mutations in the genes that growth or hepatocyte proliferation or by direct loss of encode overlapping surface antigens can affect viral fit- cccDNA.52 The genetic barrier to resistance of the treat- ness and infectivity,42 virion release (because of intracel- ment regimen increases as the number of specific muta- lular retention of newly synthesized virus),43 virologic tions required for drug resistance increases.53 Finally, breakthrough (with slower kinetics of viral load in- antiviral drug resistance is also affected by host charac- crease),43 and vaccine prophylaxis.44 Finally, the level of teristics of virus-infected hepatocytes, immune response, resistance to a drug, usually conferred by specific muta- and genetic background. tion in the viral polymerase, not surprisingly affects the Cross-resistance is defined as resistance to drug(s) to fitness of the mutant. which a virus has never been exposed. From a cross- Antiviral drug resistance results from adaptive muta- resistance perspective, the 5 approved NA have been tions in the viral genome.45 HBV infection is character- placed, based on structural characteristics, into 3 groups: ized by very high levels of virus production and turnover, L-nucleosides (LMV and LdT), alkyl phosphonates (ADV producing more than 1011 virions per day.46 Further- and TDF), and D-cyclopentane group (ETV). Resistance more, the viral population in an infected person is highly and cross-resistance tend to be structure specific. heterogeneous.47 The high rate of HBV replication, com- Two types of mutations have been associated with bined with the high mutation rate (1 in every 105 nucle- treatment failure to NA: primary resistance mutations otide substitutions during each cycle of replication, (Figure 3 and Table 2), which are directly responsible for because of the error-prone nature of reverse transcrip- drug resistance, and secondary (compensatory) muta- tion48), results in patients with CHB having a diverse tions, which promote or enhance replication compe- Figure 3. Primary antiviral drug resistance mutations. Polymerase gene mutations conferring resistance to nucleos(t)ide analogs are depicted. Resistance to lamivudine (LMV) and telbivudine (LdT) is conferred by mutations in the YMDD motif within the C domain of the polymerase, ie, rtM204V or rtM204I, often associated with compensatory mutations in the B domain restoring a higher replication capacity, ie, rtL180M and/or rtV173L. Resistance to adefovir (ADV) is conferred by a rtA181V or rtA181T substitution or a rtN236T substitution. The rtA181V/T substitution can also confer decreased susceptibility to LMV and LdT. Resistance to entecavir (ETV) is conferred by a combination of mutations in the B, C, or D domain of the viral polymerase, in addition to a background of substitutions at position rt204. Resistance to tenofovir (TDF) may be conferred by amino acid substitution at position rt194, which needs to be confirmed.
  • November 2009 HBV AND RESISTANCE TO NUCLEOS(T)IDE ANALOGUES 1597 GASTROENTEROLOGY BASIC AND CLINICAL REVIEWS IN Table 2. Cross-resistance Data for the Main Drug-Resistant Mutants and the Approved Antivirals Pathway Amino acid substitutions in the rt domain Lamivudine Telbivudine Entecavir Adefovir Tenofovir Wild-type S S S S S L-nucleoside M204I R R I S S L-nucleoside L180M M204V R R I S S Alkyl phosphonate N236T S S S R I Shared A181T/V I/R R S R I D-Cyclopentane (ETV) L180M M204V/I I169T V173L M250V R R R S S D-Cyclopentane (ETV) L180M M204V/I T184G S202I/G R R R S S I, intermediate; R, resistant; S, sensitive. tence.53 Compensatory mutations emerge because the Pathways of Resistance selection of resistance-associated changes in the viral The molecular mechanisms of resistance to drugs polymerase is usually associated with some cost in repli- for CHB have been recently reviewed in this Journal,55 cation fitness for the virus; these compensatory muta- and resistance tends to be NA structure (sugar residue) tions are important in the context of antiviral resistance specific, providing a structural framework for resistance because they “fix” the discriminatory primary drug-resis- selection. tant mutations into the genetic archive of viral cccDNA, the HBV minichromosome, thus providing “quasispecies L-Nucleoside-Associated Resistance memory.”49 The common mutations that confer resis- Resistance to LMV and LdT has been mapped to tance to LMV and LdT (eg, rtM204V/I rtL180M) con- the YMDD locus in the catalytic (C domain) of HBV Pol,56 fer cross-resistance to other L-nucleosides and reduce mediated primarily by the mutations rtM204I/V (domain sensitivity to ETV but not to ADV or TDF. Conversely, C) rtL180M (domain B) and rtA181T/V57 (Figure 3). mutants that are resistant to ADV (eg, rtN236T) and Compensatory mutations that increase viral replication TDF generally remain sensitive to L-nucleosides and levels can be found in other domains of the HBV Pol, ETV. Both the L-nucleosides (LMV and LdT) and alkyl such as rtL80V/I,58 rtI169T,59 rtV173L,60 rtT184S/G, phosphonates (ADV and TDF) also select for the muta- rtS202I, and rtQ215S.61 tion rtA181T/V, thereby making it a marker for multi- The mutations rtM204V/I do not confer cross-resis- drug resistance. Multiple mutations (eg, rtA184A/A/I/L, tance to ADV or TDF (Table 2), whereas rtA181T/V rtS202G/L, rtM250I/V) in addition to those that confer does. 61,62 The mutations rtI169T, rtT184S/G, and resistance to LMV and LdT (rtM204V/I rtL180M) are rtS202I/G contribute to ETV resistance but do not confer required for high-level resistance to ETV (see Table 2). significant resistance on their own59,63; rtM204V/I and Cross-resistance across NA groups (eg, rtA181T) (see Fig- rtA181T are cross resistant with all other L-nucleoside ure 3 and Table 2) might eventually be overcome by analogues tested including LdT (USA product insert; development of drugs that block stages of the viral life FDA, Washington, DC) (Table 2 and Figure 3). Muta- cycle distinct from those inhibited by NA (Supplemen- tions that confer LMV resistance decrease in vitro sensi- tary Figure 1). However, such drugs are unlikely to be- tivity of hepatocytes to the drug by 100- to more than come available for clinical use in the near future. Thus, it 1000-fold. rtM204I has been detected in isolation, but is important to understand more fully the molecular rtM204V and rtM204S are found only in association with mechanisms of NA resistance because, to optimize their other changes in the A or B domains.64 The pattern of pol use, we must develop methods for defining, detecting, sequence in which resistance mutations are usually and quantifying drug resistance and cross-resistance. detected include the following: (1) rtM204I, (2) rtL180M rtM204V, (3) rtL180M rtM204I, (4) rtV173L rtL180M rtM204V, and (5) rtL80V/I rtL180M Factors That Predispose to Resistance rtM204I; the dominance of a particular mutation se- There are several major risk factors for develop- quence is associated with HBV genotype.3,65 ment of resistance to NA, especially to LMV. These in- clude a high level of HBV DNA, high serum levels of ALT, Alkyl Phosphonate-Associated Resistance and high body mass index.3,5,54 Prior therapy with NA, as Resistance to ADV was initially associated with well as inadequate viral suppression during therapy, has mutations in the B (rtA181T/V) and D (rtN236T) do- also been shown to predict drug resistance.3,4,9,11 Trans- mains of the enzyme62,66,67 (Figure 3). These substitu- mission of drug-resistant mutants in newly infected pa- tions result in only a modest (3- to 8-fold) increase in the tients is also likely to predispose to more rapid resistance concentration of the drug required for 50% inhibition for once treatment is initiated, as it was shown for HIV viral replication in vitro (effective concentration [EC50]) infection. and are partially cross resistant with TDF (Table 2) al-
  • 1598 ZOULIM AND LOCARNINI GASTROENTEROLOGY Vol. 137, No. 5 GASTROENTEROLOGY BASIC AND CLINICAL REVIEWS IN most certainly because of their similar chemical struc- The first 3 pathways are associated with only 1 muta- tures.61 The mutation rtN236T does not significantly tion, whereas the fourth pathway requires at least 3 affect sensitivity to LMV, LdT, or ETV66,67 but decreases mutations for resistance. This “pathways of evolution the efficacy of TDF in vitro.68 rtA181T/V confer de- approach” facilitates understanding HBV evolution dur- creased susceptibility to ADV and TDF and are partially ing NA therapy and can be used to predict patient out- cross resistant to LMV62 and LdT (Table 2). Another comes and improve our understanding of cross-resis- mutation (rtI233V) has been recently identified that ap- tance patterns and profiles.80 peared to confer resistance to ADV.69 In clinical studies, the rtI233V mutation occurred in approximately 2% of all Multidrug Resistance patients with CHB,69 but the final significance of this Sequential monotherapy can promote selection mutation does require independent confirmation be- for multidrug-resistant (MDR) strains of HBV, especially cause other groups have not found an association be- when patients are sequentially treated with drugs with tween rtI233V and ADV resistance.70 –72 similar characteristics, such as with LMV followed by Tenofovir was originally approved for the treatment of ETV63,81 or LMV followed by ADV.37,68 Clonal analyses human immunodeficiency virus (HIV)-acquired immu- have shown that MDR usually occurs via the sequential nodeficiency syndrome (AIDS) and has also been used to addition of resistance mutations to the same viral ge- treat patients with HIV-HBV coinfection. Genotypic re- nome; mutants that arise from this selection process have sistance to TDF has been detected in several patients with full resistance to both drugs. Studies have shown that HIV-HBV coinfection; the substitution rtA194T (plus MDR strains arise if an “add-on” therapeutic strategy rtL180M rtM204V) has been associated with TDF resis- does not result in rapid and complete viral suppression, tance73; however, a recent report failed to confirm this74; especially if there is a large replication space available for therefore, further studies are needed. The detection of the mutants to spread (ie, necroinflammatory activity or rtA181T/V and rtN236T in patients failing ADV therapy high levels of serum ALT, resulting in hepatocyte prolif- resulted in reduced antiviral efficacy when patients were eration or need for a liver graft). A longitudinal clonal switched to TDF. Van Bommel et al75 demonstrated an and phenotypic analysis of variants in a patient with a intensification and consolidation of these ADV-resistant MDR strain of HBV after liver transplantation revealed clones following this switch and also reduced antiviral mutations in the overlapping polymerase and surface efficacy of TDF. genes that conferred resistance to both LMV and ADV as well as a decreased recognition of the virus by anti-HBs D-Cyclopentane-Associated Resistance antibodies.37 These findings emphasize the need to achieve complete viral suppression during antiviral therapy. Mutations in HBV polymerase associated with the Some specific single mutations confer MDR. This was emergence of ETV resistance have been mapped to the B shown with the rtA181V/T substitution, which is respon- domain (rtI169T, rtL180M, and/or rtS184G), C domain sible not only for decreased susceptibility to the L-nucle- (rtS202I and rtM204V), and E domain (rtM250V) of HBV osides LMV and LdT but also to the alkyl phosphonates Pol (Figure 3). In the absence of the LMV-resistance ADV and TDF.43,62 This emphasizes the need for geno- mutations rtL180M and rtM204V/I, the mutation typic testing in patients with treatment failure to deter- rtM250V increases the median EC50 of ETV by 10-fold, mine the resistance mutation profile and tailor therapy to whereas the rtI169T, rtT184G, or rtS202I have only a the major viral strain circulating in the patient. Studies of modest effect on IC50 values.59,61,76 –79 Three other mu- the antiretroviral agents used to treat HIV have shown tations in HBV Pol (rtL180M rtM204V and either that drug resistance testing can be used to monitor re- rtT184G/S or rtS202I/G or rtM250V) are required for sponse to therapy and aide in the selection of new drug ETV resistance to develop (Figure 3). regimens for patients who have failed to respond to antiviral therapy.82 Mutational Pathways and Cross-resistance Eight codons in HBV polymerase are thus associ- ated with primary drug resistance to NA: 169, 180, 181, Detection and Monitoring of Resistance 184, 202, 204, 236, 250. These 8 codons have been shown Viral Load Assays to be involved in HBV antiviral drug resistance via 4 Measurement of viral load is indispensable for pathways of viral evolution80 (Table 2). (1) the rtM204V/I monitoring and confirming the presence of drug-resis- pathway for L-nucleosides; (2) the rtN236T pathway for tant virus because nearly all instances of resistance to NA alkyl phosphonates; (3) the rtA181T/V pathway, which is are initially identified by a sustained rise in viral load that shared between the L-nucleosides and alkyl phospho- occurs despite continuing antiviral therapy. The sensitive nates; and (4) the D-cyclopentante/entecavir pathway HBV DNA assays that are currently in use will detect (rtL180M rtM204V I169T T184S/G/C S202C/G/ rising viral loads because of drug-resistant virus even I M250I/V). when the emergence of the drug-resistant HBV popula-
  • November 2009 HBV AND RESISTANCE TO NUCLEOS(T)IDE ANALOGUES 1599 GASTROENTEROLOGY BASIC AND CLINICAL REVIEWS IN tion is slow. Because factors other than drug resistance Hybridization (for example, poor patient compliance and/or pharma- Examples of hybridization-based genotyping cogenomic factors) can affect viral load, it cannot be methods, which can detect single nucleotide mismatches automatically assumed that rising loads are indicative of include the following: drug resistance because drug-resistant HBV can only be confirmed by genotyping and/or phenotyping. (1) Mass spectrometric (matrix-assisted laser-desorption ionization time of flight mass spectrometry [MALDI- Genotyping TOF MS]) analysis of small DNA fragments that can identify mutants that constitute as little as 1% of the To identify potential genotypic resistance, the nu- total viral population.86 cleotide and deduced amino acid sequence of the HBV (2) The commercially available line probe assay, (INNO- polymerase isolated from the patient during virologic LiPA, Innogenetics, Ghent, Belgium) which relies on breakthrough should be compared with the sequence of the differential hybridization of particular targets to HBV isolated from a pretherapy sample from the same a series of short membrane-bound oligonucleotide patient.83 When pretherapy samples are not available for probes to discriminate between wild-type sequences analysis, sequence data at the time of virologic break- and those of known drug-resistant mutants.87 LiPA through should be compared with consensus published assays can detect developing viral resistance when the sequences(s) of the same HBV genotype.72 mutants responsible constitute only a minor fraction Genotyping relies on either DNA sequencing or hy- of the total viral population (5%–10%), an advantage bridization. Sequencing-based methods include standard in cases in which there is a high risk of disease population-based polymerase chain reaction (PCR), clon- progression.88 ing of PCR products, and restriction fragment-length (3) DNA chip technologies. Sequencing with microchip- polymorphism analyses. Direct PCR-based DNA sequenc- based technology using oligonucleotide microarrays ing can detect a particular mutant only if it is present has the clear advantage of improved sensitivity as well 20% of the total quasi species pool.61 Cloning can as ability to detect “new” mutants.89 These assays are overcome this problem, but analysis of large numbers of relatively easy to perform for the simultaneous detec- clones is required. Viral mutants that constitute as little tion of a multitude of unique mutations as well as as 5% of the total population can be detected by restric- recognized polymorphisms.90 tion fragment-length polymorphism analyses, but sepa- rate sets of endonuclease reactions must be designed One of the main limitations of all hybridization-based specifically for each (and known) mutant of interest. methods is their specificity: new sets of specific probes are These methods are labor intensive, require highly skilled required for every mutant, and natural sequence variabil- personnel, and are not suitable for high-throughput ity in regions of interest reduces their discriminatory screening. They are used only for “in house” or “home- power and specificity. Furthermore, sequence context and brew” assays; with the exception of the TRUGENE geno- secondary structures in the target can affect sensitivity, typing test developed by Visible Genetics (Siemens and minor subpopulations (those constituting less than Healthcare Diagnostics, Tarrytown, NY), few have been 10% of the total population) may escape detection. For detection of known and “new” mutants, genotyping us- commercialized or approved by regulatory bodies. ing oligonucleotide microarrays appears to be the only Pyrosequencing viable alternative to direct sequencing, but, because the number of clinically relevant HBV mutants is still rela- Pyrosequencing is a new sequencing method that tively small and the technology is specialized and expen- relies on the detection of DNA polymerase activity by sive, it will be some time before they become cost-effec- measuring the pyrophosphate (PPi) released by the addi- tive. tion of a dNMP to the 3= end of a primer. It allows determination of the sequence of a single DNA strand by In Vitro Phenotypic Assays synthesizing a complementary strand, 1 base pair at a Several approaches have been developed to per- time, and detecting which base was added at each step. form in vitro phenotypic analysis of the resistant mu- Currently, the main limitation of pyrosequencing is that tants identified in vivo in patients. These assays are crit- the maximum length of individual sequencing runs are ical to determine the role of a given mutation profile in shorter than those obtainable with conventional chain drug resistance as well as to determine the cross-resis- termination sequencing methods. Pyrosequencing is cur- tance profile of those mutants. These approaches include rently the fastest and probably most sensitive (0.1%) viral polymerase enzymatic assays, cell lines permanently method available for detecting small subpopulations of expressing HBV resistant mutants, and cell culture mod- resistant virus84,85 and is likely to become the method of els in which the viral genome of resistant mutants is choice in the near future, particularly if the associated transferred for the analysis of viral replication and drug instrumentation becomes more affordable. susceptibility; these assays have been reviewed recently.91
  • 1600 ZOULIM AND LOCARNINI GASTROENTEROLOGY Vol. 137, No. 5 GASTROENTEROLOGY BASIC AND CLINICAL REVIEWS IN All these assays have been useful to demonstrate the role were observed when week 24 viral load was 1000 cop- of a given polymerase gene mutation, observed in pa- ies/mL ( 200 IU/mL) compared with patients with a tients with treatment failure, in the development of an- lower viral load at the same time point, whatever their tiviral drug resistance. They are also important to deter- initial HBeAg status. mine the cross-resistance profile of the main resistance ADV was shown to suppress viremia levels with a mutations (see Table 2)51,92 and help develop clinical slower effect by comparison with other nucleoside ana- management algorithms.53,93,94 Further useful informa- logs, ie, LMV, ETV, LdT, or TDF. Therefore, the week 48 tion is also generated on viral fitness, which is an impor- time point may be used for predicting resistance to ADV tant determinant in the process of understanding the therapy.9 It has been demonstrated, in HBeAg-negative patterns and profiles of resistant mutant selection in the patients treated with ADV for 192 weeks, that patients patient. with HBV DNA levels greater than 1000 copies/mL ( 200 IU/mL) after 48 weeks of therapy had a higher risk Clinical Aspects of Resistance of developing ADV resistance at week 192. With ETV and TDF, the rate of undetectable HBV Definitions DNA after 1 year of therapy is much higher, reaching 67% All patients receiving NA therapy for CHB should and 74% in HBeAg-positive patients and 90% and 91% in be closely monitored for virologic response and break- HBeAg-negative patients, respectively.15,97,98 Because the through during treatment and for durability of response rate of viral suppression increases over time with these 2 and viral relapse after treatment has stopped.53 Serum antiviral drugs, the timing of treatment adaptation de- HBV DNA should be tested every 3 months during treat- pends on the kinetics of viral load decay, especially in ment.93 Failure of antiviral therapy of CHB may follow patients starting from very high viral load who may need different directions, which rely on specific mechanisms additional weeks of therapy to reach undetectable HBV and therefore have clinical implications in terms of treat- DNA by PCR testing. Thus, persisting low viremia/pla- ment adaptation. Thus, it is important to distinguish teau levels should lead to treatment adaptation to max- between primary nonresponse, partial virologic response, imize viral suppression and minimize the subsequent risk and virologic breakthrough because of antiviral drug of resistance.99 resistance (Figure 1). Virologic breakthrough: viral rebound. Virologic Primary nonresponse. The failure to achieve a breakthrough typically results from the emergence of 1-log10 copies/mL (or 1.0 log10 IU/mL) decline in viral drug-resistant viral strains. It is defined by an increase of load after 12 weeks of therapy is considered as a primary at least 1-log10 copies/mL (or 1.0-log10 IU/mL) com- nonresponse.53,93,94 It may be due to a problem of com- pared with the lowest value (or nadir) during treatment, pliance or the medication may not exhibit its antiviral confirmed by a second test, in a treatment compliant activity in a given patient. Suboptimal response has been patient.53,93,94 Depending on the mutation profile se- shown to be due to host pharmacologic effect and/or to lected by the drug, viral load increase may be slow, mak- patient compliance but not to a reduced susceptibility of ing the diagnosis of rebound difficult. It usually follows viral strains to ADV as measured in vitro by phenotypic the detection of genotypic resistance (Figure 1), ie, detec- assay.70 With more potent antiviral drugs, this phenom- tion of resistance mutations.3,53,93 In the absence of treat- enon seems to be less frequent. When a primary nonre- ment adaptation, the rise in viremia levels may be fol- sponse is identified, antiviral treatment should be mod- lowed in subsequent weeks or months by an increase in ified to prevent disease progression and subsequent risk ALT levels (biochemical breakthrough) and subsequently of emergence of populations of drug-resistant mutants. progression of liver disease (clinical breakthrough). The week 12 time point of therapy is therefore important to determine the antiviral activity of the treatment regi- Treatment of HBV Drug Resistance men and assess treatment adherence. Partial response. A partial response corresponds Primary nonresponse. A primary nonresponse is to the failure to achieve a viral load decline to a threshold observed more frequently in patients treated with ADV that translates to an improvement in liver histology and (approximately 10%–20% of patients) than in those to a minimum risk of resistance.95 The most recent Eu- treated with other NA, probably because patients are ropean Association for the Study of the Liver clinical inadequately dosed.70 Patients who do not respond to practice guidelines recommended to achieve undetectable ADV should be rapidly switched to TDF or ETV therapy. HBV DNA during therapy; therefore, partial response was A primary nonresponse to LMV, LdT, ETV, or TDF is defined by detectable HBV DNA on real-time PCR assay observed only rarely2; in these patients, it is important to during continuous therapy.96 determine the level of compliance. If a patient with a The antiviral response at week 24 of therapy has been primary nonresponse to these drugs is compliant, analy- found to be a predictor of resistance in patients treated sis of HBV NA-resistance mutations can identify alter- with LdT or LMV.11,12 Higher rates of resistance at 2 years nate treatment strategies96 (see Table 2).
  • November 2009 HBV AND RESISTANCE TO NUCLEOS(T)IDE ANALOGUES 1601 GASTROENTEROLOGY BASIC AND CLINICAL REVIEWS IN Partial virologic response. Partial virologic re- added but would depend on the profile (refer to sponses have been observed with all NA used in CHB. Table 2). Again, it is important to check for compliance. There are 2 strategies for treating patients who have a partial viro- Note that the safety of some combinations in the logic response to LMV, ADV, or LdT at week 24: change longer term is presently unknown and that add-on ther- apy is not always successful in achieving adequate viral to a more potent drug (ETV or TDF) or add a more inhibition (PCR undetectability). potent drug that does not share cross-resistance. Teno- fovir should not be added to ADV therapy if the patient is infected with an HBV mutant that is resistant to ADV Preventing Resistance (ie, rtA181T/V rtN236T) because these drugs belong to The spread of drug-resistant HBV mutants can be the same chemical group of NA, the alkyl phospho- reduced by avoiding unnecessary drug use, choosing nates.15,96,99,100 drugs and combinations more carefully, and continually Virologic breakthrough. Virologic breakthrough monitoring or carrying out targeted surveillance for drug in compliant patients is related to viral resistance. Resis- resistance.45 tance is associated with prior treatment with NA or, in Because of the unusual replication strategy used by treatment-naïve patients, with high baseline levels of HBV, viral populations are genetically heterogeneous, so HBV DNA, a slow decline in HBV DNA levels, and partial even treatment-naïve patients have drug-resistant mu- virologic response to treatment. Resistance should be tants that constitute only a minor component of the identified as early as possible, before ALT levels increase, population in the absence of selection pressure from by monitoring HBV DNA levels and if possible identify- antiviral drugs. A majority of patients may not require ing the NA resistance profile; the therapeutic strategy can antiviral therapy. Several professional bodies (including be determined based on this information. Clinical and the American Association for the Study of the Liver, the virology studies have demonstrated the benefit of an early European Association for the Study of the Liver, the (as soon as viral load increases) adaptation of treat- Asian Pacific Association for the Study of the Liver, and ment.95,96,101 In cases of resistance, an appropriate rescue the National Institutes of Health) publish regularly up- dated guidelines to assist clinicians with recognition, therapy should be initiated that has the most effective diagnosis, prevention, and management of CHB: these antiviral effect and minimal risk for selection of MDR are unanimous in recommending that therapy should be strains. Therefore, adding a second drug that is not in the considered for patients with only more active or ad- same cross-resistance group as the first is the recom- vanced liver disease and others most likely to respond in mended strategy. the context of defined treatment end points. Treatment Table 2 shows the cross-resistance data for the most algorithms have been developed to assist in identification frequent resistant HBV variants.92,96 Treatment adapta- of suitable candidates for treatment and to determine tion should be performed accordingly and is summarized when to initiate treatment. as follows: Because drug-resistant mutant HBV populations are established and expand through replication, antiviral ● LMV resistance: add TDF (add ADV if TDF not therapy, once initiated, should aim to suppress viral rep- available); lication as completely and rapidly as possible. The lower ● ADV resistance: it is recommended to switch to TDF risk of resistance to TDF and ETV (compared with LMV, if available AND add a second drug without cross- LdT, and ADV) supports their use as first-line therapy, resistance. If an rtN236T substitution is present, add especially in patients who have received liver transplants LMV, ETV, or LdT or switch to TDF plus emtricit- and those with cirrhosis or decompensated liver disease abine. If an rtA181V/T substitution is present, it is because development of drug resistance is more likely to recommended to add-on ETV or to switch to TDF precipitate clinical deterioration in these groups. plus ETV or TDF plus emtricitabine (as a single Combination chemotherapy is being used more fre- tablet: Truvada); quently to treat CHB. It is effective when the appropriate combinations are employed and can reduce the risk of ● LdT resistance: it is recommended to add TDF (or drug resistance. Although HBV mutants that are resis- ADV if TDF is not available); tant to single drugs exist before therapy starts and can ● ETV resistance: it is recommended to add TDF; evolve rapidly in patients, HBV mutants with MDR are much less likely to exist before treatment. Ideally, drugs ● TDF resistance: primary resistance to TDF has not used in combination should have different mechanisms been confirmed so far. It is recommended that geno- so that they have additive/synergistic effects. Combina- typing and phenotyping be done by a reference-type tion therapy using NA with a complementary cross-resis- laboratory to determine the cross-resistance profile. tance profile prevents the development of resistance but Entecavir, LdT, LMV or emtricitabine could be does not have increased antiviral effects, compared with
  • 1602 ZOULIM AND LOCARNINI GASTROENTEROLOGY Vol. 137, No. 5 GASTROENTEROLOGY BASIC AND CLINICAL REVIEWS IN single-drug therapy.102 Use of interferon in combination with NA is probably the next logical step. Although initial clinical trials of such combinations were disap- pointing, results from later trials are more encouraging. However, the added benefit of the combination tends to be lost after treatment cessation.103,104 Combinations of L-nucleosides are unlikely to be more effective than therapy with single L-nucleosides and can have antagonistic effects (because they compete for cel- lular activation mechanisms and viral targets). The lack of cross-resistance of HBV mutants to LMV and ADV observed in vitro (except for rtA181T/V) and in some clinical studies indicates that these drugs could be effec- tive in combination. Preliminary data also support the use of ETV in combination with ADV or TDF, but de- finitive recommendations will require further clinical tri- als and cost-benefit studies. Each patient’s response to treatment should be moni- tored carefully so that drug resistance can be detected early, before viral breakthrough and disease progression. Assays for serum levels of HBV DNA and ALT should be performed 3– 6 months after therapy begins, to check for efficacy and compliance; lack of compliance is the most common cause of primary treatment failure. Additional assays, performed at 6-month intervals during the first 2 years of treatment, are recommended for patients with mild liver disease. Patients should then be assessed for viral load and ALT level every 3 months after 2 years of therapy: this is the time during which the probability of developing resistance increases. The consequences of re- sistance appear more rapidly and can become life-threat- ening in patients with advanced disease; these patients should be tested for viral load and ALT level every 3 months. Once the viral load increases to 1.0-log10 IU/ mL, HBV Pol should be sequenced to identify resistance Figure 4. Management flow chart for first virologic breakthrough/par- mutations and determine the next therapeutic approach, tial virologic response. based on cross-resistance information (Figure 4). Infectivity and Public Health Aspects does not affect HBsAg. The mutation selected by ADV The gene that encodes the HBV polymerase over- and/or LMV/LdT at rtA181T typically results in a stop laps with the gene that encodes the viral envelope, and so mutation in the envelope gene (sW172stop) (Figure 5B), mutations in the overlapping reading frame can change and the ADV resistance mutation at rtA181V results in a both proteins (Figure 5A). The nucleotide change that concomitant change sL173F. Mutations that result in a alters codon rt204 (rtM204I/V) in the polymerase gene stop codon mutation in the envelope gene, such as those confers resistance to LMV, LdT, and ETV and also results for LMV, LdT (rtM204I and rtA181T), and ADV in a nonsynonymous change in the gene encoding the (rtA181T), are usually found in the presence of a low hepatitis B surface antigen (HBsAg), directly in the over- percentage of wild-type HBV to ensure rescue of the lapping region. The rtM204V mutation typically results mutant by the wild-type to allow viral packaging and in the substitution sI195M in HBsAg, whereas the release of the defective variant.43 The ETV resistance rtM204I change can cause sW196S, sW196L, or a termi- mutation rtI169T results in a change at sF161L. This nation codon (Figure 5B), depending on codon useage. mutation, along with sE164D, is located within the re- The mutation rtL180M is synonymous in HBsAg, but gion that is defined as the “a” determinant, which in- rtV173L results in sE164D, and this combination of cludes amino acids 95 to 165—the major antibody neu- mutations is found in up to 20% of cases of LMV resis- tralization domain of HBV. tance.3,65 The ADV resistance mutation rtN236T overlaps Thibault et al published the first case report of primary with the stop codon at the end of the envelope gene and infection with LMV-resistant HBV 105 (rtL180M
  • November 2009 HBV AND RESISTANCE TO NUCLEOS(T)IDE ANALOGUES 1603 GASTROENTEROLOGY BASIC AND CLINICAL REVIEWS IN Figure 5. Impact of drug resis- tance mutations in the viral poly- merase gene on the overlapping surface gene. Panel A: Physical map showing the impact of drug resistance mutations in the viral polymerase gene on the enve- lope gene. Resistance mutations may therefore result in viral enve- lope changes leading to altered virion secretion, altered infectiv- ity, and escape to anti-HBs anti- bodies. Panel B: Antiviral drug- associated HBsAg changes. The main amino acid substitu- tions in the viral polymerase and their corresponding changes in the envelope proteins are shown. rtM204V), which was associated with a typical bout of Several studies have reported mutations in HBsAg that acute hepatitis and an incubation period of 2–3 months. alter its antigenicity. Torresi et al observed that the LMV The level of viremia was lower than that usually observed resistance mutations rtV173L rtL180M rtM204V (pro- during the acute phase of hepatitis B, and the virus was ducing sE164D and sI195M in HBsAg) resulted in re- cleared. The acutely infected individual had not been duced binding of antibody to this antigen,106 although previously vaccinated against HBV. the reduction was not as great as that caused by the
  • 1604 ZOULIM AND LOCARNINI GASTROENTEROLOGY Vol. 137, No. 5 GASTROENTEROLOGY BASIC AND CLINICAL REVIEWS IN mutant sG145R, compared with wild-type HBV. These Future Directions and Conclusion results were confirmed and extended by Sloan et al using Very few new drugs are being developed to treat cell-derived HBVs.107 HBV; therefore, it is important to continue research into Very few in vitro studies have been performed to study mechanisms of pathogenesis and resistance and to iden- the infectivity of the NA-resistant mutants. The combi- tify new therapeutic targets. Small molecule inhibitors nation of polymerase and surface gene mutations might that are directed against multiple HBV targets should result in viruses that exhibit a reduced fitness that trans- improve viral clearance and prevent resistance (see Sup- lates to differences of selection kinetics. However, these plementary Figure 1). For example, virus entry into the studies are hampered by the challenges of working with cell can be inhibited111,112 using pre-S1 peptides, which primary human hepatocytes, the only cellular system mimic the envelope protein domain involved in virus-cell available for these investigations. Bartholomew et al in- membrane interaction. These peptides prevented HBV fected primary human hepatocytes with HBV using se- entry into cultured hepatocytes and inhibited subsequent rum samples collected from patients before the start of viral infection and spread in the HBV animal model of treatment with LMV and after viral breakthrough.108 severe combined immunodeficient mice.112 Combination Their results demonstrated that the viral strains isolated of these peptides with NA could prevent the infection of at the time of viral breakthrough were resistant to LMV new cells when viral load is suppressed by NA and in- but could still infect hepatocytes. The hepatocyte progen- crease the rate of clearance of infected cells as well as preventing further de novo infections. Ongoing preclin- itor cell line HepaRG has specific hepatocyte functions ical and clinical studies will determine their efficacy and and can be infected by HBV; primary cultures of normal safety. human hepatocytes were also used in infectivity studies Reagents have been tested that target cccDNA or steps of clinical isolates, especially of drug-resistant strains.109 in its formation and processing, but these have been These studies showed that mutations in the HBV poly- found to have cytotoxic effects. Agents that modify epi- merase and overlapping surface genes can impair repli- genetic regulation of cccDNA transcriptional activity are cation capacity, virion secretion efficiency, and infectiv- being investigated in experimental models.113 Viral pre- ity;42 some of these mutant HBVs escaped antibody genome encapsidation and capsid formation also repre- recognition and could therefore mediate breakthrough sent potential targets. Phenylpropenamide derivatives infection in vaccinated individuals and escape detection and heteroaryl-pyrimidines are known to inhibit the rep- by commercial diagnostic kits. The effect of these substi- lication of wild-type and LMV-resistant mutant genomes tutions on HBV infectivity was shown to be a critical in hepatoma cell lines,114 –117 although clinical trials were determinant of which resistant mutants would spread not conducted because of formulation problems. AiCuris more rapidly in the liver and dominate other HBV vari- Pharmaceuticals (Wuppertal, Germany) has developed ants.42 the heteroaryl-pyrimidines molecules further as non-nu- In chimpanzees, Kamili et al44 challenged the immu- cleoside inhibitors of HBV core protein dimerization that nity induced by a commercial hepatitis B vaccine against prevent nucleocapsid formation.116,117 The heteroaryl- a tissue culture-derived HBV clone that contained 3 poly- pyrimidines prevented HBV infection in an animal mo- merase mutations (rtV173L, rtL180M, rtM204V) and del116 and represent a potentially important new advance substitution mutations in the overlapping region that in chemotherapy. encodes the envelope/HBsAg (sE164D, sI195M). Immu- Viral morphogenesis and egress are also useful targets. nologic evidence of HBV infection and replication was Iminosugars, which modulate the glycosylation status observed in the vaccinated chimpanzees after challenge and conformation of envelope proteins, can decrease the with the mutant as well as after rechallenge with serum- production of infectious particles in vitro.118 They were shown to have an antiviral effect in the woodchuck derived wild-type HBV, despite robust humoral and cel- model of hepadnavirus infection.119 Therapeutics might lular anti-HBV immune responses to the vaccine. The also be developed to modulate the innate response of observed infection by the mutant form of HBV, despite infected hepatocytes,120,121 dendritic cell activation,122 or the presence of high levels of HBV antibody (which were the adaptive immune response.123–125 Induction of sus- considered to be protective), are consistent with clinical tained immunologic control of HBV infection could al- reports of breakthrough infections in anti-HBs-positive low for timed cessation of NA administration. patients who are infected with escape HBV mutants.44 Ideally, treatment for CHB should begin at diagnosis; Therefore, “antiviral drug-associated potential vaccine es- this is not feasible because of limitations of drugs. Clin- cape mutants” have the potential to jeopardize the hep- ical trials and concurrent improvements in diagnostic atitis B immunization program110 (Supplementary Fig- technology ensure that treatment options and expert ure 2). As more HBV mutants arise that have resistance to opinion on patient management will continue to evolve. different antiviral agents, the effects on the antigenicity Many laboratories are genotyping and phenotyping HBV of the HBsAg protein will need to be established. mutants to delineate patterns of resistance and cross-
  • November 2009 HBV AND RESISTANCE TO NUCLEOS(T)IDE ANALOGUES 1605 GASTROENTEROLOGY BASIC AND CLINICAL REVIEWS IN resistance. These data will improve the design of new 5. Lai CL, Dienstag J, Schiff E, et al. Prevalence and clinical corre- therapeutic strategies and maximize the benefits of anti- lates of YMDD variants during lamivudine therapy for patients with chronic hepatitis B. Clin Infect Dis 2003;36:687– 696. viral agents. Resistance-testing methodologies vary, and, 6. Lok AS, Lai CL, Leung N, et al. Long-term safety of lamivudine although few direct comparisons have been made, in treatment in patients with chronic hepatitis B. Gastroenterology vitro phenotype testing (if possible, in combination with 2003;125:1714 –1722. genotype testing) seems superior to conventional geno- 7. Dienstag JL, Goldin RD, Heathcote EJ, et al. Histological out- type or virtual phenotyping testing. This is especially true come during long-term lamivudine therapy. Gastroenterology 2003;124:105–117. for analysis of HBV isolated from patients who have 8. Liaw YF, Sung JJ, Chow WC, et al. Lamivudine for patients with already been treated with several drugs, in whom multi- chronic hepatitis B and advanced liver disease. N Engl J Med ple mutations are more likely to have become fixed in the 2004;351:1521–1531. genetic archive. Drug resistance testing of HBV isolates is 9. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, et al. Long-term currently performed only in a few specialized “reference- therapy with adefovir dipivoxil for HBeAg-negative chronic hepa- titis B for up to 5 years. Gastroenterology 2006;131: type” laboratories, but it should become routine as more 1743–1751. sensitive, reliable, high-throughput, and accurate meth- 10. Fung SK, Andreone P, Han SH, et al. Adefovir-resistant hepatitis ods are developed, along with clinically useful algorithms B can be associated with viral rebound and hepatic decompen- for interpretation. In particular, standardization of tests sation. J Hepatol 2005;43:937–943. and definition of resistance/susceptibility thresholds or 11. Lai CL, Gane E, Liaw YF, et al. Telbivudine versus lamivudine in “cut-offs” that can be used to correlate laboratory results patients with chronic hepatitis B. N Engl J Med 2007;357: 2576 –2588. with clinical observations and outcomes are urgently 12. Liaw YF, Gane E, Leung N, et al. 2-Year GLOBE trial results: needed. telbivudine is superior to lamivudine in patients with chronic Methods for assessing the relative replication capacity hepatitis B. Gastroenterology 2009;136:486 – 495. of HBV mutants are being developed and will prove 13. Marcellin P, Chang TT, Lim SG, et al. Long-term efficacy and useful in selecting therapy and tailoring individual pa- safety of adefovir dipivoxil for the treatment of hepatitis B e antigen-positive chronic hepatitis B. Hepatology 2008;48:750 – tient management. In cases in which treatment failure 758. cannot be attributed to patient noncompliance or emer- 14. Lee YS, Suh DJ, Lim YS, et al. Increased risk of adefovir resis- gence of resistant virus, host factors might be involved. It tance in patients with lamivudine-resistant chronic hepatitis B is important to monitor viral load throughout therapy so after 48 weeks of adefovir dipivoxil monotherapy. Hepatology that the treatment strategy can be modified in cases of 2006;43:1385–1391. 15. Marcellin P, Heathcote EJ, Buti M, et al. Tenofovir disoproxil partial response or virologic breakthrough. fumarate versus adefovir dipivoxil for chronic hepatitis B. N Engl The choice of the first-line agent(s) is very important; J Med 2008;359:2442–2455. the goal is to delay the development of resistance and to 16. Snow-Lampart A, Chappell BJ, Curtis M, et al. Week 96 resis- preserve treatment options over the long-term of treat- tance surveillance for HBeAg-positive and negative subjects with ment for CHB. The development of therapeutics that chronic HBV infection randomized to receive Tenofovir DF 300 mg QD. In 59th Annual Meeting of the American Association for complement the effects of NA is warranted; new treat- the Study of Liver Diseases, San Francisco, CA, October 31– ment strategies must be aimed at increasing viral sup- November 04, 2008. Hepatology, 2008. pression and promoting virologic clearance and ensuring 17. Tenney DJ, Rose RE, Baldick CJ, et al. Long-term monitoring the prevention of drug resistance and its complications. shows hepatitis B virus resistance to entecavir in nucleoside- naive patients is rare through 5 years of therapy. Hepatology 2009;49:1503–1514. Supplementary Data 18. Tenney DJ, Pokornowski KA, Rose RE, et al. 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  • 1608 ZOULIM AND LOCARNINI GASTROENTEROLOGY Vol. 137, No. 5 GASTROENTEROLOGY BASIC AND CLINICAL REVIEWS IN 103. Marcellin P, Lau GK, Bonino F, et al. Peginterferon alfa-2a alone, 118. Lazar C, Durantel D, Macovei A, et al. Treatment of hepatitis B lamivudine alone, and the two in combination in patients with virus-infected cells with -glucosidase inhibitors results in pro- HBeAg-negative chronic hepatitis B. N Engl J Med 2004;351: duction of virions with altered molecular composition and infec- 1206 –1217. tivity. Antiviral Res 2007;76:30 –37. 104. Lau GK, Piratvisuth T, Luo KX, et al. Peginterferon alfa-2a, 119. Block TM, Lu X, Mehta AS, et al. Treatment of chronic hepad- lamivudine, and the combination for HBeAg-positive chronic navirus infection in a woodchuck animal model with an inhibitor hepatitis B. N Engl J Med 2005;352:2682–2695. of protein folding and trafficking. Nat Med 1998;4:610 – 614. 105. Thibault V, Aubron-Olivier C, Agut H, et al. Primary infection with 120. 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Antiviral resistance mutations combination of CpG-induced cytokines with lamivudine against potentiate hepatitis B virus immune evasion through disruption hepatitis B virus replication in vitro. Antivir Ther 2009;14:131– of its surface antigen a determinant. Antivir Ther 2008;13: 135. 439 – 447. 123. Das A, Hoare M, Davies N, et al. Functional skewing of the 108. Bartholomew MM, Jansen RW, Jeffers LJ, et al. Hepatitis B virus global CD8 T-cell population in chronic hepatitis B virus infec- resistance to lamivudine given for recurrent infection after or- tion. J Exp Med 2008;205:2111–2124. thotopic liver transplantation. Lancet 1997;349:20 –22. 124. Lopes AR, Kellam P, Das A, et al. Bim-mediated deletion of 109. Gripon P, Rumin S, Urban S, et al. Infection of a human hepa- antigen-specific CD8 T cells in patients unable to control HBV infection. J Clin Invest 2008;118:1835–1845. toma cell line by hepatitis B virus. Proc Natl Acad Sci U S A 125. Dunn C, Brunetto M, Reynolds G, et al. Cytokines induced 2002;99:15655–15660. during chronic hepatitis B virus infection promote a pathway for 110. Clements J, Coghlan B, Creati M, et al. Global control of hepa- NK cell-mediated liver damage. J Exp Med 2007;204:667– 680. titis B infection: does the potential for antigenic change of the virus in the face of antiviral treatment affect the global hepatitis B immunization programme? Bull WHO 2009. In press. Received June 19, 2009. Accepted August 28, 2009. 111. Gripon P, Cannie I, Urban S. Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral Reprint requests surface protein. J Virol 2005;79:1613–1622. Address requests for reprints to: Fabien Zoulim, MD, INSERM UNIT 112. Petersen J, Dandri M, Mier W, et al. Prevention of hepatitis B 871, 151 Cours Albert Thomas, 69003 Lyon, France. email: virus infection in vivo by entry inhibitors derived from the large fabien.zoulim@inserm.fr; fax: 33 472 681971. envelope protein. Nat Biotechnol 2008;26:335–341. 113. Pollicino T, Belloni L, Raffa G, et al. Hepatitis B virus replication Acknowledgments is regulated by the acetylation status of hepatitis B virus The authors thank Tania Candy for editorial help in the cccDNA-bound H3 and H4 histones. Gastroenterology 2006; manuscript preparation. 130:823– 837. 114. King RW, Ladner SK, Miller TJ, et al. Inhibition of human hepa- Conflicts of interest titis B virus replication by AT-61, a phenylpropenamide deriva- The authors disclose the following: F.Z. has received consulting tive, alone and in combination with ( )b-L-2=,3=-dideoxy-3=-thia- and speaker honoraria from Gilead Sc, Bristol Myers Squibb, cytidine. Antimicrob Agents Chemother 1998;42:3179 –3186. Novartis, and Roche and has received research support from Gilead 115. Delaney WEt, Edwards R, Colledge D, et al. Phenylpropenamide Sc and Roche. S.L. has received consulting and speaking honoraria derivatives AT-61 and AT-130 inhibit replication of wild-type and from Gilead Sciences, Bristol-Myers Squibb, Roche Pharmaceuticals, lamivudine-resistant strains of hepatitis B virus in vitro. Antimi- Abbott Diagnostic, and Evivar Medical Pty Ltd. crob Agents Chemother 2002;46:3057–3060. 116. Weber O, Schlemmer KH, Hartmann E, et al. Inhibition of human Funding hepatitis B virus (HBV) by a novel non-nucleosidic compound in Supported by the European Community (ViRgil network of a transgenic mouse model. Antiviral Res 2002;54:69 –78. excellence; ViRgil LSHM-CT-2004-503359; to F.Z.) and grants from 117. Deres K, Schroder CH, Paessens A, et al. Inhibition of hepatitis the National Agency for Research against AIDS and Hepatitis B virus replication by drug-induced depletion of nucleocapsids. (ANRS; to F.Z.) and funding from the US National Institutes of Science 2003;299:893– 896. Health (RO1 AIO60449 and RO1 AI071820; to S.L.).
  • November 2009 HBV AND RESISTANCE TO NUCLEOS(T)IDE ANALOGUES 1608.e1 Supplementary Figure 1. The heptatitis B virus replication cycle and the potential antiviral targets. The Figure shows all the major steps required in the viral replication cycle. HBV is able to infect hepatocytes, successful infection defined by the formation of covalently closed circular (cccDNA). Linear genomes have a high propensity to integrate into host DNA; viral genome integration is not required for viral genome replication. Following formation of cccDNA), viral messenger RNA are transcribed, including pregenomic RNA (pgRNA), the template for reverse transcription of viral DNA. In the cytoplasm, pgRNA can be bound by the viral reverse transcriptase and packaged into viral nucleocapsids, where reverse transcription takes place. Following completion of the minus strand DNA and partial completion of the plus strand DNA, virions are formed via budding into the endoplasmic reticulum. Nucleoside(tide) analogs mainly inhibit the viral minus strand DNA synthesis (reverse transcription) and plus strand DNA synthesis (DNA dependent DNA polymerase activity) within viral nucleocapsids. These drugs do not affect directly viral cccDNA, which is maintained in the nucleus of infected cells, therefore requiring long-term treatment for a sustained control of viral replication. All other steps of the viral life cycle are potential targets for antiviral therapy.
  • 1608.e2 ZOULIM AND LOCARNINI GASTROENTEROLOGY Vol. 137, No. 5 Supplementary Figure 2. Public health significance of antiviral drug associated potential vaccine escape mutants (modified from Clements et al, 2009110). Antiviral drug-associated potential vaccine escape mu- tants (ADAPVEMs) have the potential to infect naïve but also immunized people and may therefore jeopardize both the antiviral treatment effi- cacy and the hepatitis B immunization program.