Journal of Hepatology
Volume 44, Issue 2 , Pages 422-431, February 2006

Cellular and virological mechanisms of HBV drug resistance

  • Stephen Locarnini

      Affiliations

    • Research and Molecular Development, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Vic. 3051, Australia
    • Corresponding Author InformationCorresponding author. +32 9342 2637; fax: +3 9342 2666.
    • ,
  • William S. Mason

      Affiliations

    • Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA

published online 07 December 2005.

Article Outline

 

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1. Introduction 

Hepatitis B virus (HBV) exposure in childhood at a time when the immune system is not fully developed typically results in chronic infection. Chronic infection may also occur in adults, but the risk is much lower. These cases occur only in the setting of hepatitis B e antigen (HBeAg) positive infections. HBV is not cytopathic and can productively infect all hepatocytes. The immune response in chronically infected individual causes death of infected cells and the consequent liver disease, without eliminating the infection. Over the many decades of an infection that was acquired in childhood this chronic damage results in a 25% risk of death from cirrhosis or hepatocellular carcinoma. With more than 350 million people now chronically infected with HBV it has been estimated, without therapeutic intervention, that at least 60 million will die prematurely due to the infection. Not surprisingly then, there has been a great interest in developing effective antiviral strategies to prevent disease progression.

HBV replicates by reverse transcription. When the virus infects a hepatocyte, the 3kbp relaxed circular genome is converted in the nucleus to a covalently closed circular (ccc) DNA that is the template for viral RNA synthesis. The cccDNA, which exists as a viral minichromosome, does not replicate. Rather, viral DNA synthesis occurs within nucleocapsids formed in the cytoplasm, and new cccDNA is produced when this DNA is transported to the nucleus. Duck hepatitis B virus cccDNA synthesis is negatively regulated by the viral envelope proteins [1], which presumably sequester newly made DNA for release as virus, and it is assumed that this is also true for HBV. Thus, the amount of cccDNA is maintained at about 5–50 copies per hepatocyte. Since cccDNA appears to be stable in non-dividing cells new synthesis, once a final copy number is reached, is only expected to occur if hepatocytes divide or if new, uninfected hepatocytes appear in the liver. Cytokines, which have a major role in destabilizing HBV DNA synthesis [2] do not appear to have a significant effect on cccDNA stability [3], [4], [5], but a small effect has not been ruled out [6]. Thus, elimination of cccDNA appears to require inhibition of new synthesis combined with hepatocyte death and regeneration. Since hepatocytes are replaced primarily by the division of other hepatocytes, an obvious question is whether or not cccDNA survives mitosis. A single report suggests that it does [7], and results from a study of antiviral therapy in woodchucks chronically infected with woodchuck hepatitis virus, were consistent with this possibility [5]. However, this conclusion still remains controversial. In either case, a prolonged inhibition of viral DNA synthesis would be needed to clear the liver of cccDNA by any mechanism depending on cell division or cell death as the pathway for cccDNA removal.

Attempts in the early 1980s to block HBV replication using inhibitors of viral DNA synthesis were generally unsuccessful because of the poor efficacy and/or toxicity of available drugs, for example, as found with adenosine arabinoside [8], [9], [10], [11], [12], [13], [14], [15]. Moreover, with the lack at that time of tissue culture and in vitro systems for virus growth or viral polymerase expression, which were suitable for high throughput screening, antiviral compound libraries could not be tested against HBV. However, the development of polymerase inhibitors for the treatment of human immunodeficiency virus (HIV) infections spurred the testing of the same and related drugs for antiviral activities against HBV. While Zidovudine (AZT), one of the earliest anti-HIV nucleoside analogs, does not inhibit HBV replication in the liver [16], [17], only because it is not phosphorylated or salvaged there [18], another anti-HIV nucleoside analog, Lamivudine (3TC, a cytosine analog), is a strong inhibitor of HBV DNA synthesis [19], [20], [21] because it is phosphorylated in hepatocytes. Adefovir dipivoxil is a nucleotide analog, which is already monophosphorylated, and was found to moderately inhibit HIV replication. It is also active against HBV, but has been slower to gain widespread use because of early concerns about potential nephrotoxicity at higher doses [22], [23], [24], [25], [26], [27], [28]. Though these were the only two nucleos(t)ide analogs that received FDA approval for treatment of HBV carriers prior to 2005, Entecavir [29], [30], [31] recently received FDA approval and additional nucleosides are currently in phase III trials, including Clevudine [32] and Telbivudine (LdT) [33]. The major problem with nucleos(t)ide analog therapy is the expected one, emergence of drug resistance mutants (see Table 1). In retrospect, and by analogy to HIV, this was inevitable. Another problem is the possibility of cellular factors affecting antiviral efficacy. Both the cellular and viral factors associated with treatment failure will be considered in this review.

Table 1. Annual prevalent resistance rates for lamivudine, adefovir, entecavir, emtricitaline and telbivudine
DRUGResistance at year of therapy expressed as percentage of patients
1234
Lamivudinea23465571
Adefovirb03618
Entecavirc (naïve)00––
Entecavirc (LAM resistant)110––
Emtricitabinec9–1619–37––
Telbivudined4d–––

aFrom [45], [46].

bFrom [79].

cFrom [98].

dIn the LAM comparator arm, the percentage was only 8% based on a complex case definition of antiviral drug resistance/treatment failure. One would thus expect a comparable relative level of 10–12% based on genotypic resistance compared with Lamivudine (25% per annum).

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2. Cellular factors associated with resistance 

Drugs used for treatment of chronic hepatitis B virus infections are clearly able to overcome host factors at the recommended dose, at least in the vast majority of hepatocytes. What is less clear is how, or if, cell based drug resistance varies between hepatocytes, with virus persisting and replicating at high levels in small numbers of drug resistant cells. Some known mechanisms of resistance to nucleoside analogues are presented below, though it remains unclear if any of these mechanisms play a role in the response to anti-hepadnavirus drugs.

Nucleoside analogues are usually processed by the cellular anabolic pathways that salvage the corresponding nucleosides or nucleobases. Before nucleoside analogues can inhibit enzymatic functions of the HBV DNA polymerase, they must be transported across cell membranes and phosphorylated intracellularly to generate dNTP equivalents. Several membrane transporters mediate cellular nucleobase and (deoxy)nucleoside uptake and are distinguishable by different functional characteristics, including substrate specificities, ion dependency, and sensitivity to inhibitors (reviewed in Ref. [34]).

During nucleoside analogue activation, substrate recognition by cellular enzyme(s) that perform the initial phosphorylation is usually the slowest, most substrate-specific, and not surprisingly then, the most stringently regulated step. Mammalian genomes encode only four deoxynucleoside-specific kinases, which vary in substrate specificity, tissue distribution, cell-cycle dependency, and subcellular location (for review see [35]). All are encoded by nuclear genes. Deoxyguanosine kinase and thymidine kinase 2 are mitochondrial enzymes, which, together with deoxycytidine kinase, are expressed constitutively in most cells. These enzymes play an important role in the initial phosphorylation of nucleoside analogues. They also share sequence homology with the herpesvirus deoxypyrimidine (‘thymidine’) kinases and are characterized by much less stringent substrate specificities than the fourth deoxynucleoside kinase, thymidine kinase 1 (TK-1), expression of which is restricted to S-phase of the cell cycle. There is very little TK-1 expression in resting hepatocytes, probably accounting for the poor anti-hepadnaviral effect of the thymidine nucleoside analogue, AZT. Nucleoside monophosphate and diphosphate kinases, which exchange terminal phosphate groups between nucleotides, are relatively non-specific in their substrate preferences and perform the second and third phosphorylation, respectively (reviewed in reference [36]). Interestingly, the phosphorylation of the L-nucleosides from diphosphate to triphosphate metabolites is performed in the liver by 3-phosphoglycerate kinase, a glycolytic enzyme [37]. Finally, the rate-limiting initial phosphorylation reaction is bypassed by nucleoside analog phosphonates (e.g. PMEA or Adefovir), which are structurally analogous to (d)NMPs. Phosphonates are able to cross cell membranes because they are not substrates for the ubiquitous 5′-nucleotidases and are less polar than nucleotides. Diphosphates of phosphonates, rather than triphosphates, function as dNTP analogs.

The liver comprises a mixed cell population composed of many cell types including endothelial, bile duct, and Kupffer cells as well as hepatocytes. The liver plays an important role in nucleoside homeostasis and probably contains all types of nucleoside transporters, but how they are distributed amongst hepatic cell populations is unknown. Under normal physiological conditions, most liver cells are quiescent, with DNA synthesis being restricted mainly to replacement and repair of mitochondrial and nuclear genomes respectively. Consistent with the slow rate of hepatocyte turnover, the activities of enzymes for deoxynucleoside salvage and dNTP synthesis are minimal, whereas the activities of enzymes of nucleoside and nucleotide catabolism are high (reviewed in Ref. [18]).

Recently several identified mechanisms for host-mediated resistance to nucleobase, nucleoside, and nucleotide analogs have been described. Increased efflux of nucleobase, nucleoside, and nucleotide analogs via two major families of efflux pumps: Pglycoprotein—(Pgp) and members of the multi-drug resistance associated proteins (MRP 1–9). Two transmembrane transporters have been shown to confer resistance to the cytotoxic effects of nucleoside analogs in cells, which have been engineered to overexpress either of these transporters (MRP4 and MRP5) in vitro [38], [39]. MRP4 and MRP5 have been shown to export antiviral drugs including Adefovir and Tenofovir, but it remains to be seen whether the same mechanism(s) operate in vivo and whether they can result in host-mediated resistance to Adefovir/tenofovir and/or other anti-HBV drugs. At least three separate cellular enzymes have been shown to be capable of excising DNA chain-terminated β-D- or β-L- nucleoside analogs from the 3′ end of DNA in vitro. These include: (1) the tumor suppressor protein p53, which possesses associated 3′→5′ exonuclease activity [40]; (2) the apurinic/apyrimidinic exonuclease APE1, a well-characterized DNA base excision repair protein [41], [42]; (3) a cytosolic 3′→5′ exonuclease which has been purified from human lymphocytes [43]. The p53-associated and cytosolic 3′→5′ exonuclease activities demonstrate a substrate preference for β-D- over β-L- nucleoside analogs, and APE1 does show a preference for the β-L- conformation. HBV DNA synthesis takes place in viral nucleocapsids and it not known whether nascent strands are accessible by these enzymes. Lastly, the in vitro expression in human cell lines of a recently identified ubiquitous cytoplasmic nucleotidase (named cN-I) has been shown to confer an almost 300-fold increase in resistance to toxicity resulting from exposure of cells to dideoxycytidine and some of its derivatives [44].

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3. HBV resistance to nucleoside/nucleotide analogs 

It was originally hoped that drug resistance might not be as serious a problem with HBV as with HIV because the overlapping open reading frames (ORFs) of HBV appear to constrain the number of viable mutations that can arise, with the mutation frequency following infection with cloned virus estimated at about 10−4 per nucleotide. Nonetheless, though drug resistant virus emerges more slowly during monotherapy of HBV infections than of HIV, drug resistance with Lamivudine, the most widely used of the two antivirals now available, is a major problem. Lamivudine resistant mutants of HBV emerge as a predominant species in 24% of chronic carriers after a year of therapy and in over 70% of carriers after four years of monotherapy [45], [46]. Moreover, the relatively slow emergence of drug resistant mutants as compared to HIV probably reflects differences not just in mutation rates, but to an even larger extent, in the biology of the two viruses. HIV infects a population of cells with a rapid rate of turnover. In contrast, HBV infects a self-renewing cell population, hepatocytes, with a low rate of turnover even in chronic carriers, probably of the order of a few percent per day. Therefore, the spread of the expected low number of naturally occurring drug resistant HBV mutants is prevented initially by the absence of uninfected target cells, and resistance apparently emerges only as inhibition of wild-type virus replication leads over one or many cycles of hepatocyte division to the generation of virus free cells; that is, replication space [47].

Sequencing of Lamivudine resistant HBV strains has revealed a similarity to Lamivudine resistant HIV. The DNA polymerase domain of HBV is shown in Fig. 1. By convention, amino acids are numbered not from the beginning of the polymerase ORF at the terminal protein, but from ∼amino acid 350, since differences in the length of the ORF between virus isolates map mostly upstream. This system, in which numbering starts from the beginning of the DNA polymerization domain (rt1 to rt344 typically), provides a universal basis for numbering and comparing drug resistant mutants in clinical isolates from different HBV genotypes [48].

  • View full-size image.
  • Fig. 1. 

    The location of Major drug-resistant mutations on the HBV polymerase. According to convention and for consistent identification of mutations conferring resistance to antiviral nucleos(t)ide analogs, amino acids are numbered from the beginning of the Pol/RT (rt1 to rt344) domain [48]. Mutations associated with resistance to Lamivudine, Adefovir, and Entecavir are indicated. [This figure appears in colour on the web.]

Attempts to understand the molecular basis of Lamivudine resistance have involved molecular modelling in order to build a plausible 3D structure for the HBV DNA polymerase using the crystal structure of the HIV polymerase as a template [49], [50]. Thus, by analogy, it appears that HBV polymerase domains A, C and D form the dNTP binding domain and catalytic center, that the B domain is involved in template binding, and that the E domain binds to the primer strand [49], [50]. HBV Lamivudine resistance results from a rtM204I or rtM204V change in the YMDD motif at the catalytic center in domain C, and appears to be due to steric hindrance of proper binding of the Lamivudine triphosphate. The rtM204V change is always, and the rtM204I change sometimes, accompanied by an upstream rtL180M transition in the B domain. Either mutation in the YMDD motif can by itself confer Lamivudine resistance [51], [52], [53], [54], [55], [56]. The rtL180M transition in the B domain enhances replication efficiency, particularly of the rtM204V (YVDD) mutant, possibly by restoring binding efficiency to natural dNTPs and, with the YVDD mutant, increasing the Ki for inhibition by Lamivudine [57], [58], [59], [60], [61], [62]. Structural modelling suggests that the rtL180M amino acid change is able to alter the catalytic site by interaction with rtP92, which is adjacent to the dNTP binding domain [50]. Additional amino acid changes upstream of the B domain have been found in Lamivudine treated patients (rtT128N and rtW153S) (Fig. 1) and have been reported to enhance the Lamivudine resistance of the rtL180M/rtM204V mutant [63]. However, it is unclear whether this result can be generalized to other isolates of HBV because, in this study, in contrast to others, the rtL180M mutation decreased, rather than enhanced, the drug resistance of rtM204V. A potential problem in generalizing the significance of mutations other than rtM204V/I and rtL180M is that different isolates of HBV are used in different laboratories, which may change the sequence requirements to achieve Lamivudine resistance. In addition, amino acid changes in pol in patients receiving antiviral therapy, in addition to those in Fig. 1, are often observed in clinical studies, but not always subjected to further characterization.

An additional problem in understanding the range of mutants found during the setting of drug failure is that the DNA polymerisation domain overlaps the viral envelope gene, particularly S, and changes in pol can alter the envelope sequence, and vice versa. Therefore, virus fitness may require a compromise between fitness of the envelope and fitness of the DNA polymerase. An interesting example of this has recently been reported [64]. HBV with an rtA181V/T mutation in the B domain of the polymerase emerged as the predominant Lamivudine resistant species in several patients, despite the fact that the mutation can introduce a stop codon into the overlapping S gene (rtA181T/sW172*). This mutant has also been found in the HBV pol from patients in whom Adefovir has been failing as therapy [65]. An equivalent mutant is usually the first to emerge in WHV infected woodchucks treated with Lamivudine [5], [66]. In both cases, emergence of the mutant can be inferred to require co-infection of hepatocytes with a second virus, perhaps wild type, that produces a wild type S protein. A more serious concern is raised by the fact that the polymerase B domain and other upstream regions that could influence drug resistance overlap the region of the S gene encoding the external hydrophilic domain of the S protein, which includes virus neutralizing epitopes. Mutations in this region of S, particularly in the ‘a’ determinant, encoded by a region of the genome just upstream of the B domain in pol, occur in vaccine escape variants that sometimes emerge in vaccinated individuals [67], [68]. Thus, the concern has been raised that vaccine escape variants may enhance resistance to nucleoside analogs such as Lamivudine and Adefovir, and that nucleoside analog resistant viruses may also, coincidently, be vaccine escape variants. To date, this has not been proven, though a possible example has been reported: A sP120T mutant in the ‘a’ determinant of S, one of the main targets of virus neutralizing antibodies, was found in liver transplant patients who had received hepatitis B immune globulin (HBIG) during transplantation [63]. The same nucleoside change produced an rtT128N mutation in the polymerase. During post-transplantation treatment with Lamivudine and HBIG, the triple mutant rtT128N/rtL180M/rtM204V emerged. Unlike the double mutant, the triple mutant appeared to be Lamivudine dependent, with more DNA synthesis occurring in the presence than absence of the drug. The key clinical finding was that when the Lamivudine was stopped, the patient viral load dropped significantly [63]. The mechanism by which Lamivudine dependence could occur requires elucidation.

Adefovir has a much different resistance profile than Lamivudine. In general Lamivudine resistant virus remains sensitive to Adefovir [60], [62], a result attributed to the fact that Adefovir is much less bulky as compared to Lamivudine. Adefovir resistance is uncommon in the first year of therapy (<1%) [23], [27], [69], [70] but does increase to over 25% by 5 years. The most common genotypic change occurs in the downstream D domain (Fig. 1), also involved in dNTP binding [65]. This rtN236T change does not significantly affect sensitivity to Lamivudine, but the rtA181T/V and rtV214A/rtQ215S mutations are partially cross-resistant to Lamivudine (see Table 2).

Table 2. Antiviral sensitivity of drug-resistant HBV in vitro
Lamivudinea, bAdefovircClevudined, eTelbivudinefEntecavirg, h
HBV POL CHANGE-Fold resistance-Fold resistance-Fold resistance-Fold resistance-Fold resistance
Wild-type1.01.01.01.01.0
L180M1.70.5>120121
M204I>1000.7>12023630
L180M+M204V>1000.2>12013330
A181T/V2–61–5NANANA
N236T3–87–104.72.40.67
I169T/M250V>10001.0NA>100>1000
T184G/S202I>10002.0NA>1,000>1000
V214A/Q214S10-207-10NANANA

a[93];

b[99];

c[65];

d[93];

e[100];

f[101];

g[99];

h[102].

Recent reports indicate that Entecavir resistance in patients that had previously developed resistance to Lamivudine is the consequence of additional mutations in the B and E domains along with those mutations already in the B and C domain that were required for Lamivudine resistance (Fig. 1) [31]. All of these mutations appear to be necessary for resistance to Entecavir (see Table 2).

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4. Emergence of drug resistant virus as prevalent species 

As discussed previously, Hepadnavirus DNA synthesis results in about one error per 104–105 nucleotides [71], [72], [73]. Thus, in a fully infected liver of 1012 hepatocytes each containing at least one copy of cccDNA, there will be a minimum of 107 variants with, for instance, the M204I mutation in the polymerase active site. Immune pressure over time may cause a selection for hepatocytes that no longer support virus replication, so that the total virus load drops, but the fraction of drug resistant virus created by de novo mutation of the wild type should remain constant as long as wild type virus continues to replicate. Assuming the mutation in pol does not inactivate the envelope proteins, emergence of this mutant virus, when replication of the wild type is inhibited by Lamivudine, should be unavoidable, depending only on the rate of appearance of uninfected hepatocytes, with a possible contribution from super-infection of hepatocytes already infected with the wild type. The exception may occur when there is a spontaneous episode of acute hepatitis which, in association with the antiviral agent, causes HBeAg to anti-HBe seroconversion with loss of replicating virus and normalisation of serum ALT. However, the more typical case is a progressive emergence of drug escape mutants in about 20% of patients per year (Table 1), the rate probably depending in individual patients primarily on the rate of hepatocyte turnover during therapy, with a higher rate of hepatocyte turnover during therapy increasing the rate of emergence of the Lamivudine resistant virus.

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5. Liver disease after emergence of drug resistant mutants 

In contrast to interferon-alpha (see below), nucleoside analogues exert a selective pressure on HBV replication that leads to the emergence and expansion of drug-resistant or treatment escape mutants. This has considerable clinical significance. The cumulatative prevalence of resistance to Lamivudine and Adefovir over 4 years and the other antiviral agents Entecavir, Emtricitabine and Telbivudine over 1–2 years is summarised in Table 1.

Clinical studies have shown that emergence of Lamivudine-resistant HBV can be associated with marked viral rebound, increases in serum ALT levels [74] hepatitis flares, and uncommonly, hepatic decompensation and death from liver failure [75], [76]. Persistent infection with Lamivudine resistant HBV is associated with blunting of the histological response during years 2 and 3 of treatment and with an increased risk of hepatic flares with associated increased risk for decompensation in patients with cirrhosis during years 4 and 5 [75], [76]. In Lamivudine resistant patients who have advanced fibrosis, treatment confers only an intermediate level of protective benefit in forestalling disease progression [77]. Acute exacerbations of hepatitis are common once Lamivudine resistant HBV emerges as the dominant quasispecies [74] and this is probably because the YMDD/YVDD/YIDD locus can act as a CD-8+ CTL epitope in HLA-A2 positive individuals [78]. In the setting of advanced disease, such flares can be fatal [77].

Resistance to Adefovir is uncommon in the first two years of therapy but increases steadily thereafter [79] (see Table 1). Not as much is known about longer-term effects on disease progression with Adefovir resistant HBV, as patients are usually switched or rescued with another drug very quickly. In a recent study by Lok and colleagues [80] involving eight patients with Adefovir resistance, HBV DNA levels increased to greater than 5 log10 copies/ml in serum and hepatic decomposition occurred in 2 patients, one of whom died. All of these patients had pre-existing Lamivudine resistance. Salvage therapy with Lamivudine, Entecavir or Tenofovir was given to 7 patients resulting in a reduction of HBV DNA by greater than 3 log10 copies/ml in 3 patients.

The main conclusions that have been clearly established over the last 5–6 years is that drug-resistant HBV is not a ‘benign or attenuated virus’. Furthermore, this selection and dominance in drug-treated patients is associated with disease progression and the benefits initially obtained with drug treatment in the first phase are quickly lost when resistance emerges [46], [76].

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6. Immunotherapy of chronic HBV infection 

Under normal circumstances, HBV is generally regarded as non-cytopathic. The liver disease associated with persistent infection is considered to come from inadequate and/or inappropriate host immunological responses. Initial therapies for chronic hepatitis B utilized regimens of interferon-alpha in the hope of promoting or enhancing these immunological responses.

Conventional interferon-alpha has suboptimal pharmacokinetics, resulting in fluctuating drug levels and low response rates in chronic hepatitis B [81]. Pegylated interferon, prepared by attaching polyethylene glycol molecules to either interferon alpha 2a or interferon alpha 2b, is more effective than standard interferons, producing reductions in the serum HBV DNA level of 3–4 log10/ml. [82], [83], [84]. This increased antiviral efficacy is probably due to a more pronounced effect on viral replication through the JAK-STAT pathway activating RNaseL and PKR as well as a greater immunomodulatory action since HBeAg-positive patients treated with pegylated interferon alpha 2a demonstrate treatment induced ALT flares and higher rates of HBeAg seroconversion (24%) [85] than conventional interferons (12%) [81].

Forty-eight weeks of pegylated interferon alpha 2a in HBeAg-positive patients, with a twenty-four week follow up typically results in HBV DNA suppression with HBeAg seroconversion rate of 30% versus 19% with Lamivudine monotherapy [82] and up to 3% actually achieve HBsAg serovoncersion compared to 0% with Lamivudine.

In HBeAg negative patients, normalisation of serum ALT and HBV DNA suppression to below 400 copies/ml occurs in around 20% of patients treated with pegylated interferon alpha 2a and 7% of patients treated with Lamivudine alone. Loss of HBsAg can occur in around 2–3% of interferon-alpha treated patients but 0% of Lamivudine treated individuals [83]. Unfortunately, in both HBeAg-positive and HBeAg-negative CH-B, there was no advantage in terms of a sustained virological benefit in treatment outcome in the follow-up period (i.e. sustained virological response) when Lamivudine was combined with pegylated interferon-alpha 2a [82], [83].

One of the limitations in using interferon therapy is that response rates vary according to HBV genotype. This does not appear to be an issue with nucleoside analogue therapy [86]. Typically, patients with genotype A demonstrate the highest rates of HBeAg loss or seroconversion following interferon therapy as compared with the rates associated with genotypes B, C and D (52, 30, 31 and 22% respectively for seroconversion) [82].

To date, no specific mutation in the HBV genome has been conclusively associated with interferon-alpha treatment failure, and host rather than viral differences in response have not been ruled out. However, in vivo, a correlation between basal core promoter (BCP) mutations and poor IFN-α response has been reported from Asia for patients infected with genotypes B and C [87]. In European patients, HBV genotype A appears more sensitive to IFN-α than genotype D [88]. Gunther and collegues [89] have shown that IFN-α treatment is associated with the emergence of novel pre-C sequences, some of which prevent HBeAg expression. In vitro, HBV genotype C HBV with the G1896A precore stop codon mutation was reported to be ‘resistant’ to the antiviral effects of IFN-α [90].

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7. Strategies to overcome resistance 

Currently, interferon, Lamivudine, Adefovir, and Entecavir can all be considered as first-line therapy for individuals with non-cirrhotic liver disease. In the context of rescue or salvage therapy, mutations that confer resistance to Lamivudine confer cross-resistance to other L-nucleosides and reduce sensitivity to Entecavir but generally not to Adefovir (Table 2). Conversely, mutants that are resistant to Adefovir generally remain sensitive to L-nucleosides and Entecavir (Table 2). Multiple mutations are required for high-level resistance to Entecavir. However, these generalizations will probably not be the case as more and more Lamivudine resistance appears with mutations in other parts of the polymerase such as the A domain (rtL80) [91].

In the future, combination chemotherapy will almost certainly be used to treat chronic hepatitis B, especially in patients with advanced disease. Provided that appropriate drug combinations of antiviral agents that act at least additively and preferably synergistically are used, this approach yields well-recognized benefits, including reduction of the risk of drug resistance. This is because even though the pre-existence or rapid evolution of viral mutants with the potential to resist individual drugs is almost guaranteed by high HBV loads and turn-over in vivo, the pre-existence or evolution of multidrug-resistant mutants is much less likely. Drugs used in combination should be chosen which have different mechanisms or sites of action and act additively or synergistically. Unfortunately, in the management of chronic hepatitis B, the nucleoside/nucleotide analogues have essentially similar mechanisms and sites of action. Among nucleoside analogs now in phase III trials, slightly different patterns of resistance are emerging. Clevudine (L-FMAU) shares cross resistance with Lamivudine. Resistance is conferred in cell culture assays by rtL180M/rtM204V, rtL180M/rtM204I, and rtM204I (Fig. 1) [92], [93]. However, unlike with Lamivudine, rtM204V does not confer resistance but this mutation, by itself, has not been found in patients. Cross-resistance is also seen in the woodchuck model [94]. Resistance to Telbivudine (LdT) appears to be much less frequent than to Lamivudine [95], but detailed studies have not yet been published.

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8. Conclusion 

Several new antiviral nucleosides, some with so far low incidence of resistance, are now being evaluated. Experience with HIV and HBV suggests that none of these will be suitable as a monotherapy. Combination therapies for patients with chronic hepatitis B employing multiple nucleoside analogs have not yet been developed. Attempts to target other steps in the replication pathway such as core assembly [96], [97] have not yet reached phase I clinical trials.

Antiviral drug resistance now poses a major problem in the management of patients with chronic hepatitis B. The probability that viral resistance will develop is directly proportional to the grade of selection pressure and the diversity of quasispecies. Sufficiently potent inhibition of HBV replication should be able to prevent the development and emergence of new drug resistant variants at least in the medium term (3–5 years), mainly because mutagenesis is replication dependent. Provided multi-site mutation is required for resistance, reducing the chance that drug resistant virus is present prior to therapy, and if viral replication can be sufficiently suppressed by treatment, viral production will theoretically decline to a point where the creation of new quasispecies with the potential for resisting drug treatment is no longer likely. Substantial data is now appearing in the literature that this level is below 104copies/ml, irrespective of HBeAg status.

Whether this short-term end point also translates to other benefits such as HBeAg seroconversion, sustained virologic suppression with histologic improvement, or even HBsAg seroconversion, is presently unknown. However, a reasonable clinical goal at present is the application of this concept via the optimization of combination therapies, analogous to the highly active antiretroviral therapy regimens used for HIV infection with the minimization of the toxicity associated with such multiple therapies.

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Acknowledgements 

W.S.M. acknowledges support from the National Institutes of Health and the Commonwealth of Pennsylvania. We are grateful to Drs. Allison Jilbert (Institute of Medical and Veterinary Science, Adelaide, Australia), Raymond Schinazi (Emory University, Atlanta, GA), Christoph Seeger (Fox Chase Cancer Center, Philadelphia, PA) and Tim Shaw (VIDRL) for helpful advice during the preparation of this review.

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PII: S0168-8278(05)00775-0

doi:10.1016/j.jhep.2005.11.036

Journal of Hepatology
Volume 44, Issue 2 , Pages 422-431, February 2006

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