Journal of Hepatology
Volume 30, Issue 3 , Pages 536-550, March 1999

Antiviral chemotherapy for chronic hepatitis B infection: lessons learned from treating HIV-infected patients

  • Stephen Locarnini

      Affiliations

    • Corresponding Author InformationStephen Locarnini, Victorian Infectious Diseases Reference Laboratory (VIDRL), 10 Wreckyn Street, North Melbourne, Victoria, 3051, Australia. Tel: 61 3 9342 2614. Fax: 61 3 9342 2666.
    • ,
  • Chris Birch

Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia

Article Outline

 

After years of modest treatment success with the use of interferon alpha, antiviral chemotherapy for chronic hepatitis B virus (HBV) infection with nucleoside analogues now provides an alternative means for interrupting the progression of disease and even conceivably eradicating the virus from the infected host. Three factors have converged to provide important advances in anti-hepadnaviral chemotherapy:

1.(i) improved understanding of the immunopathogenesis of HBV infection;

2.(ii) the availability of standardised, reliable quantitative assays for serum HBV DNA; and

3.(iii) the range and type of potent anti-hepadnaviral drugs.

Almost identical statements were made about therapeutic options for the human immunodeficiency virus (HIV) over 2 years ago (1). Since then, confidence and optimism regarding the likelihood of suppressing and even eliminating HIV from infected individuals has waxed and waned. However, extremely important lessons have been learned from the HIV experience, and these are likely to have a significant impact on the treatment of HBV infection. Several reviews describing antiviral agents active against the HBV have recently been published 2., 3., 4. and this material will not be re-presented here. The focus and purpose of this review is to compare and contrast key virological, pathogenetic and chemotherapeutic issues in the treatment of these two viral infections so that new paradigms for managing chronic hepatitis B infection may be developed.

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The Experience to Date with HIV and HBV 

The pathogenesis of HIV 

Understanding of the pathogenesis of HIV-AIDS has advanced considerably in the last few years (5). The damage inflicted by HIV-1 is mainly brought about by active viral replication 1., 5.. An individual's risk for disease progression can now be assessed early in the course of the infection and the efficacy of antiviral therapies can now be determined rapidly and accurately. This has been due mainly to the advent of sensitive and reliable laboratory testing such as quantitative polymerase chain reaction (PCR) assays for monitoring HIV replication in vivo (5). Also, combinations of potent antiviral drugs have reduced the rate of emergence of resistance, the main reason for therapeutic failure, and have permitted the development of rational approaches to block and possibly eliminate HIV-1 from an infected person 1., 5..

The clinically latent period characteristic of HIV infection is in reality virologically dynamic. Nearly 1010 HIV virions are produced per day in some patients (6). A cycle of infection, destruction and reinfection of CD4+ T-lymphocytes sustains high-level virus replication 6., 7.. The majority of circulating virus (more than 99%) is derived from these cells (Fig. 1). The remainder is produced from latently infected, long-lived cells which become activated (7). Substantial numbers of HIV species containing pre-existing drug resistance mutations are present within this rapidly replicating pool of virus. These strains are thought to arise as a result of the error-proneness of the HIV reverse transcriptase (RT). Conservatively, one error occurs in every 10 000 nucleotides transcribed, and this is not corrected by exonucleolytic proof reading (8). As a result, a large number of HIV quasispecies exist in each individual. Some of these mutants have the potential to replicate in the presence of antiretroviral drug pressure, and it is recognised that HIV strains containing amino acid changes known to be associated with phenotypic resistance to protease inhibitors exist in patients who have never been treated with this class of drug (9). Because they represent only a fraction of the number of wild-type viruses present (10), such mutants are normally likely to be at a replicative disadvantage compared to wild-type virus. However, when a drug is used for the first time, they will be rapidly selected, despite their relatively reduced replicative fitness. This is a form of Darwinian evolution; mutations providing a survival advantage result in selection of populations acquiring and retaining that mutation.

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  • Fig. 1. 

    Diagramatic representation of the dynamics of HIV-1 infection in vivo (modified from Perelson et al. (44) with data added from Finzi et al. (17) as well as Wong et al. (18)). Shown in the centre is the cell-free HIV-1 population that is measured as plasma associated virus load (VL). Also shown at the top of the figure is the latently-infected CD4+ lymphocyte population, the memory CD4+ cells (RO45+), which is one of the important persistent reservoirs of HIV during HAART 17., 18..

The pathogenesis of HBV 

In contrast to HIV, the pathogenesis of liver tissue injury in chronic hepatitis B infection, although closely related to ongoing active viral replication (11) is determined primarily by the host's immune responses 12., 13.. Under most conditions HBV is not directly cytopathic, and the lesion of chronic liver disease results from an inadequate or inappropriate host immune response directed at virus-infected hepatocytes (11). The major cytotoxic T-cell response to HBV appears to be directed to key epitopes within the hepatitis B viral core (HBcAg) and hepatitis B e antigen (HBeAg) 14., 15.. This activity has been identified as an important determinant of viral clearance and cell damage. The HBeAg is a toleragen (14) and the aim of removing the toleragenic effect of circulating serum HBeAg by chemosuppression in order to induce endogenous immuno-elimination may prove a useful strategy for future management of chronic hepatitis B. Furthermore, Milich (15) has proposed that the induction or maintenance of the chronic carrier state may be the result of an imbalance in the TH1 and TH2 cytokine responses specific to the HBcAg and HBeAg. A predominance of HBV-specific TH2-type cytokine responses during chronic infection would favour antibody production, whereas a predominance of TH1-type cytokine responses in acute infection would favour a cell-mediated immune response. Milich (15) has further suggested that a cytokine-focused therapy designed to shift a TH2-type cytokine response toward a TH1-type response may be beneficial in the treatment of chronic infection. The effects of cytokines such as interleukin 2 (a TH1-type cytokine) on the viral life-cycle as well as on the host's immune response clearly require further exploration (16).

The impact of antiviral therapy on HIV and HBV 

Clinical trials with highly active antiretroviral therapy (HAART) have shown that long-term suppression of HIV replication, indicated by plasma HIV RNA levels falling below the limits of detection, is now possible. Although HAART has not yet been shown to eliminate the virus from the body, since integrated HIV proviral DNA in activated, long-lived, memory CD4− T-lymphocytes is able to direct virus production, the viruses detected have a wild-type drug resistance profile 17., 18.. We can therefore be confident that the use of HAART can lead to the suppression of HIV replication and prevent resistance developing in some individuals. Nevertheless, as many as 50% of treated individuals do not achieve complete viral suppression (19). Many such patients have received prior antiretroviral drug therapy, and have received HAART outside of clinical trials, where compliance with a complex drug regimen is often less stringently monitored. These individuals are at risk for developing resistance.

Interferon alpha therapy, the only licensed treatment for chronic hepatitis B in most countries, has proven moderately effective but is often limited by dose-dependent side-effects 20., 21.. Most importantly, the majority of patients receiving interferon alpha will not clear the infection and will remain at risk for developing progressive liver disease including cirrhosis and hepatocellular carcinoma (HCC) (22). Similar clinical efficacy has been described with a second cytokine, thymosin α-1, but unlike interferon-alpha, treatment with this agent is not associated with serious adverse events 23., 24.. Clinical trials with the nucleoside analogues lamivudine (25), famciclovir (26), adefovir (27) and lobucavir (28) have been undertaken and show promising results in terms of maximal chemosuppression of viral load and association with minimal adverse side effects. However, monotherapy with this group of agents has failed to clear viral infection, and at least three challenges to the development of future effective therapy for chronic hepatitis B can now be defined:

1.(i) the persistence of viral covalently closed circular (CCC) DNA;

2.(ii) the role of extra-hepatocyte reservoirs; and

3.(iii) the emergence of drug-resistance.

During initial infection of the hepatocyte, the viral genomic DNA is converted to a minichromosome that functions as the transcriptional template of the virus. It is within this viral minichromosome that the viral DNA exists as a supercoiled or CCC DNA molecule (29). A major reason for the relapse seen after completion of antiviral therapy for hepatitis B infection was recognised more than 13 years ago by Omata and colleagues (30) who demonstrated that this CCC DNA form of the viral DNA was not eliminated by treatment with interferon-α. Similarly, hepadnaviral CCC DNA is also unaffected by nucleoside analogue therapy 31., 32. despite the reduction or elimination of all other viral DNA forms in the liver (reviewed in 33). A likely reason for this failure could be a long intrahepatic half-life of viral CCC DNA, which under normal circumstances may be as stable as host-cell DNA. Although, in vitro studies have determined a relatively short half-life of 3–5 days for duck HBV CCC DNA (34), the heterogeneity of the viral CCC DNA topoisomers (29) makes interpretation of these data difficult. Several studies have shown that long-term antiviral treatments have failed to eliminate viral CCC DNAs, which continue to persist and maintain their functionality in infected cells 35., 36.. Only famciclovir-penciclovir has demonstrated consistent and significant inhibition, but not elimination, of viral CCC DNA 37., 38.. Thus, the eradication of chronic HBV infection requires either the permanent inactivation of the viral CCC DNA species (33) or the elimination of each infected cell (39).

Viral dynamics, viral load, viral mutation and drug resistance 

HIV (40) and HBV (41) copy their genomes by reverse transcription, and it has been estimated that each undergoes approximately 3×10−5 mutations per nucleotide per replication cycle (42). The HBV has its genome organised into four open reading frames (ORF) with the polymerase gene overlapping the other three ORFs (41). Thus, a mutation in one ORF can affect the amino-acid sequence in another ORF (43), thereby restricting the number of viable mutants so generated. In HIV, the genome is linear and non-overlapping but splicing is required to produce mRNA for the envelope and regulatory proteins (40).

In the HIV-infected patient, the concentration of virus in blood is less than in lymphoid tissues and almost certainly reflects spillover from replication in that tissue. This large amount of virus turns over rapidly, with a virion half-life of hours in blood (see Fig. 1). Within a year after infection, each infected individual establishes a“set-point” or steady-state level of HIV RNA, which is usually between 102 and 106 copies/ml of plasma. This virus load largely determines the rate at which CD4 cells are subsequently lost 1., 44.. The set-point is a dynamic equilibrium of viral clearance (by the infected host's natural defences) and virus production. The half-life of most infected CD4 cells is approximately 1.5 days and 109 CD4HIV-infected cells are destroyed, and new cells generated, each day (Fig. 1) 5., 44.. The level of HIV replication, measurable as plasma HIV RNA, therefore drives the rate of immune destruction and can be used to predict the natural history of the disease (45). The major benefit of antiretroviral therapy in HIV-infected patients is the associated drop in plasma HIV-RNA, or alteration of the virological set-point downwards (45).

Similar kinetic studies have been performed in chronic hepatitis B infection and have shown that the half-life of HBV in plasma is 24 h, the total daily production of virus is 1011 virus particles and the overall total viral load is 2×1011 (46). The similarities to HIV infection are obvious. The half-life of infected hepatocytes has been calculated to be 10–100 days, depending on the degree of necro-inflammatory activity within the liver with a daily turnover of infected hepatocytes of 1–7% (46). Based on this model, it has been predicted that in the active phase of chronic hepatitis B infection, 12 months treatment with lamivudine should reduce the total body viral burden by a factor of about 1011, that is, virtual elimination of HBV (46). More recently, a short-term clinical trial using lamivudine to treat chronically infected individuals has suggested a half-life for serum HBV of 2–3 days (47) (see Fig. 2). This longer half-life significantly extends the duration of antiviral monotherapy required to eliminate HBV if the“active phase” disease model of Nowak and colleagues (46) is accepted. In both cases, high viral turnover combined with relatively high mutation rate and low to moderate cell turnover contributes to the generation of substantial viral genome diversity.

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  • Fig. 2. 

    Schematic summary of the dynamics of HBV infectionin vivo, showing the role that both hepatic and extra-hepatic sites play in the viral dynamics during chronic HBV infection. Modified from Lee et al. (103) and data from 46., 47..

Many factors are associated with the development of antiviral drug resistance. The virus itself, the drugs, and characteristics of the infected patient are all involved. Factors associated with HIV resistance include the kinetics of its production and clearance, and the inherent error-proneness of its RT. In addition, HIV possesses a structurally flexible protease and can alter the conformation of its substrates when under drug pressure. The potency of antiretroviral drug combinations, their pharmacokinetic properties (including their ability to reach reservoirs of HIV infection in sites such as the CNS), the likelihood of cross-resistance developing to other drugs and the prior antiretroviral drug experience of the treated individual are also important. Finally, whether the patient remains compliant in taking a large number of drugs, often at inconvenient times, also influences the development of resistance. Most, if not all, of these factors are likely to be relevant to the development of drug resistance by HBV.

In clinical trials of HIV drugs, most of the antiviral activity resulting from monotherapy is largely reversed within 4 weeks after the initiation of therapy. This reversal is associated with the emergence of drug-resistant mutants 48., 49.. These observations provide compelling evidence for a relationship between the loss of antiviral drug activity and the emergence of drug resistance, and they are consistent with the existence of drug-resistant subpopulations in patients before treatment (50). From the virus perspective, these mutations are rarely without cost. Compromises of replicative fitness appear to occur with most mutations in HIV (10) and HBV (51). However, in HIV a compromising mutation survives under the pressure of selection by, for example, protease inhibitors, by a second compensatory mutation that partly restores the original levels of viral replication (52). A similar scenario probably operates in HBV.

The swarm of genetic variants in any patient is a mixture of viruses with varying replication capacities and selective advantages under the changing conditions of host cell type, immune response or pressure from drug treatment. The fitness of the predominant population (master sequence) changes in response to changing selection pressures (53). Therapeutic strategies can only address these dynamics by suppressing virus replication to such a degree that the emergence of variants is prevented (54). (See Fig. 3).

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  • Fig. 3. 

    Graphical representation of the relationship between resistance selection versus antiviral activity which shows that increasing the antiviral effectiveness of therapy is accompanied by an increasing probability that drug-resistant mutants will be selected, but only up to a point (119). At high levels of antiviral activity, suppressing virus replication reduces the likelihood that resistance mutations will be selected, because replication is the process responsible for the emergence of the mutations (119). Reproduced with permission from Condra & Emini (119).

Multiple viral targets for antiviral inhibition: drugs available for treatment and the issue of cross-resistance 

HIV. Because of the importance of reverse transcription in the HIV replication cycle, the RT represents a prime target for chemotherapy. The RT inhibitors include the nucleoside analogues such as zidovudine (AZT), zalcitabine (ddC), didanosine (ddI), stavudine (d4T), and lamivudine (3TC) or the non-nucleoside reverse transcriptase inhibitors (NRTIs) such as nevirapine, delavirdine and efavirenz (Table 1). To show activity, the nucleoside analogue RT inhibitors (RTIs) must be first phosphorylated to the triphosphate form by cellular kinases. In this form they can inhibit RT activity by mechanisms including direct competition with the natural deoxynucleoside triphosphates (dNTPs) (55), and chain termination, whereby absence of the 3′-hydroxyl position on the ribose sugar prevents further addition of nucleotides.

TABLE 1. Antiretroviral agents currently in clinical use
Generic nameOther namesManufacturerPrincipal activities
Nucleoside reverse transcriptase inhibitor (RTI)
DidanosineddIBristol-Myers SquibbHIV-1 and HIV-2
Lamivudine3TCGlaxo WellcomeHIV-1 and HIV-2
Stavudined4TBristol-Myers SquibbHIV-1 and HIV-2
ZalcitabineddCHoffman-La RocheHIV-1 and HIV-2
ZidovudineZDVGlaxo WellcomeHIV-1 and HIV-2

Non-nucleoside reverse transcriptase inhibitor (NRTI)
DelavirdineDLVPharmacia & UpjohnHIV-1
NevirapineNVPRoxane Laboratories (Boehringer Ingelheim)HIV-1
EfavirenzDMP266Dupont MerckHIV-1

Protease inhibitor (PI)
IndinavirIDVMerckHIV-1 and HIV-2
NelfinavirNFVRocheHIV-1 and HIV-2
RitonavirRTVAbbott LaboratoriesHIV-1 and HIV-2
SaquinavirSQVHoffman-La RocheHIV-1 and HIV-2
Amprenavir141W94Glaxo-WellcomeHIV-1 and HIV-2

The NRTIs are a diverse group of compounds which bind to and inhibit the RT of HIV-1 strains by a noncompetitive mechanism with respect to dNTPs, template and primer (Table 1). The binding site occurs in a pocket in the palm region of the enzyme, adjacent to but not overlapping the catalytic site (56). Inhibition of RT is thought to involve a reduction in the flexibility of the enzyme that is necessary during viral DNA synthesis (56). The NRTIs do not inhibit HIV-2, presumably because of a lack of amino acid conservation at this site (Table 1).

The HIV protease recognises and cleaves at nine distinct sites on HIV precursor proteins. Under drug pressure, and when inhibition of replication is incomplete, HIV strains possessing protease mutations are selected for, and the processing capability of the enzyme is proportionately reduced as the number of mutations increases 57., 58.. Eventually this results in complete loss of proteolytic activity, even in the presence of compensatory mutations within the enzyme. HIV responds by mutating at secondary loci so as to regain some of its diminished replicative capacity. Mutations in the gag p7/p1 cleavage site of HIV strains derived from patients receiving indinavir therapy have now been reported (59). The flexibility of HIV at the level of the enzyme and its substrates highlights the difficulties in delivering effective therapy to infected patients.

When used as monotherapy, the RTIs, NRTIs and protease inhibitors are effective at producing reductions in HIV plasma load measurable as an increase in CD4+lymphocyte numbers 60., 61.. However, this is a transient response (days to months) because of the selection of resistant strains of HIV 62., 63.. Resistance occurs more rapidly in patients who are immunosuppressed (64). This is most likely related to the greater extent of HIV replication and, therefore, greater viral diversity, in patients with low CD4 counts.

The RTIs and NRTIs select for different mutant strains of HIV. Each of the NRTIs selects identical or near-identical mutations in the RT (65), effectively narrowing the number available for clinical use to one drug per patient. In the case of the RTIs, a range of mutations is selected by the well-established drugs (66). Although some mutations may be common (for example M184V selected by didanosine and lamivudine, and V75T selected by didanosine and stavudine), others are non-overlapping. The mutations selected by zidovudine are unique. It therefore remains possible to switch between different combinations of RTIs so as to avoid cross-resistance.

The protease inhibitors have a well-recognised ability to select for cross-resistance mutations, and both in vitro and clinical studies have revealed that extensive cross-resistance occurs between saquinavir, ritonavir and indinavir (67). The more-recently developed protease inhibitors (nelfinavir and amprenavir (formerly 141W94)) appear to select at least some of the same mutations (66). Approximately 60% of saquinavir-, ritonavir- or indinavir-resistant clinically derived strains are also phenotypically resistant to nelfinavir (67). Conversely, approximately 65% to 80% of nelfinavir-resistant strains are also resistant to saquinavir, ritonavir and indinavir (68). The end result is a situation similar to that which exists with the NRTIs; that is, the number of protease inhibitors available to individuals undergoing therapy is only one or, perhaps briefly, two, irrespective of which inhibitor is used first.

HBV. Two major targets for selective anti-hepadnaviral inhibition are presently recognised: the HBV DNA polymcrase and virus-specific post-transcriptional events. As with HIV, the HBV DNA polymerase enzyme also represents a prime target for chemotherapy. The HBV polymerase inhibitors used in clinical studies include the nucleoside-nucleotide analogues lamivudine (25), famciclovir-penciclovir (26), adefovir (27), lobucavir (28) and fluorothiacytidine (69). Many other nucleosides-nucleotides have been found to be active in pre-clinical studies (for review see 70). For most of these compounds, intracellular phosphorylation to the monophosphate form is mandatory for antiviral activity (71). The triphosphate form inhibits the viral enzyme competitively with respect to dNTP's and incorporation results in viral DNA chain termination as described above for HIV (55). The exception is the nucleotide analogue adefovir dipivoxil (also known as bis-POM PMEA), an acyclic phosphonate of adenine (72) which is active in the diphosphate form.

The cytokines interferon alpha (IFN-α) and gamma (IFN-γ) as well as interleukin 2 (IL-2) and tumour necrosis factor alpha (TNF-α) have been found to inhibit HBV replication via a post-transcriptional mechanism (73) through at least two pathways. Interferon gamma and TNF-α treatment results in the climination of viral nucleocapsids and viral DNA replicative intermediates (pathway one), whilst the level of viral RNA is reduced via the second pathway. More recent studies have demonstrated that interleukin 12 (IL-12), an inducer of IFN-γ, can cause the disappearance of viral replicative intermediates from the liver and extra-hepatic sites of treated HBV-transgenic mice (74). Interferon alpha exerts its anti-hepadnaviral action through a combination of direct antiviral effects via the 2′–5′-oligoadenylate synthetase pathway (75) as well as by immune modulation 20., 21.. The mechanism of action of thymosin α-1 has not been defined, although it does appear to enhance TH1-type responses (76).

During interferon-alpha treatment, Gunther et al. (77) found frequent and rapid emergence of mutated pre-core sequences of HBV during interferon treatment from HBeAg-positive carriers who seroconverted to anti-HBe as a result of treatment. Most mutations detected newly emerging pre-core sequences that included silent point mutations, amino-acid changes, start and stop codons and frameshift mutations (77), some of which prevented HBeAg expression. However, in this pilot study of only 10 patients (77), the selection of these pre-core mutant viruses was not necessarily prognostic for virus elimination.

Monotherapy with lamivudine and famciclovir (see Table 2) has also resulted in the selection of HBV mutants which are resistant to these compounds (Table 3). The resistant mutants were first detected in the setting of orthotopic liver transplantation (OLT) where lamivudine or famciclovir was administered to patients either pre- or post-OLT in order to prevent or control recurrence of HBV infection 78., 79., 80.. The emergence of drug-resistant HBV mutants was not unexpected in light of the reports of lamivudine and famciclovir resistance in HIV and herpes simplex virus (HSV) infections, respectively 81., 82., 83..

TABLE 2. Anti-hepadnaviral agents currently in clinical studies
NameOther namesManufacturer
Cytokines
RoferonInterferon alpha-2aRoche Pharmaceuticals
Intron-aInterferon alpha-2bScherring-Plough,
WellferonLymphoblastoid InterferonGlaxo-Wellcome
ZadaxinThymosin alpha-1Sci-Clone

Nucleoside analogues
Lamivudine3TCGlaxo-Welcome
FamciclovirFCVSmithKline Beecham
Adefovirbis-POM-PMEAGilead Sciences
LobucavirBMS-180194Bristol Myers Squibb
FluorothiacytidineFTCTriangle Pharmaceuticals
TABLE 3. Mutations in the HBV polymerase protein associated with antiviral resistance
Polymerase Mutation1Effect on HBsAgReferences
Famciclovir-associated mutations
N422KA DomainI68N89
V519LB DomainE164D79, 89
L526MB Domainno change79, 89
L526VB Domainnot reported85
P523LB Domainno change79
A529TB DomainW172stop89

V553IC Domainnot reported90
S 565A S210R89
Lamivudine-associated mutations
Group 1
M550VC DomainI195M78
+ 84
L526MB Domainno change80, 93
Group 2
M550IC DomainW196S/W196L78
stop84, 80, 93

Other
S565P 210R86

Note: Examples of mutations reported in HBV polymerase protein associated with drug resistance to Lamivudine and Fanciclovir with the corresponding changes in HBsAg.

1 Amino-acid numbering based on consensus sequence where the methonine (M) in the YMDD motif is number 550 79., 90.. This table has been modified from (43).

In the case of both lamivudine and famciclovir, the appearance of resistant HBV mutants is associated with a rise in serum HBV DNA and ALT levels, indicating a breakthrough in suppressive therapy 78., 79., 80.. Molecular analysis of the viral DNA extracted from patient sera reveals that the mutations selected by lamivudine and famciclovir monotherapy are located within the viral DNA polymerase gene 79., 84.. In the case of lamivudine resistance, mutations are found within the tyrosine-methionine-aspartate-aspartate (YMDD) catalytic domain (domain C) 78., 79., 84., 85. (see Table 3). Mutation of the methionine (M) at amino acid position 550 to either isoleucine (I) 78., 80., 84., 86. or valine (V) 80., 84., 85. is sufficient to confer virus resistance to lamivudine. Two groups of mutations have been described. The more common mutation detected (group 1) is the M55OV change which is also associated with a domain B change, typically L526M. The M550I mutation appears to be selected in the absence of L526M (group 2) and is observed in approximately 20% of cases. Recently, a multi-centre study from Asia has reported that 14% of chronic hepatitis B patients treated with 100 mg of lamivudine for 12 months developed the M550I or M550V mutation (87). In Europe, Honkoop et al. (88) have estimated the prevalence of resistance to be 30% after 12 months of lamivudine therapy.

By contrast, HBV polymerase mutations selected during famciclovir monotherapy are not located within the YMDD motif but in an upstream region identified as the template binding domain (domain B) (79). A valine (V) to leucine (L) change at amino acid position 519 (V519L) and a leucine (L) to methionine (M) change at position 526 (L526M) represent the two most common mutations characterised to date 79., 89., 90. (Table 3). The location of these mutations corresponds broadly to those found in penciclovir-resistant HSV DNA polymerase (83). Interestingly, the two dominant famciclovir-selected mutations have also been detected in some lamivudine-resistant mutants, whose reduced sensitivity to famciclovir is therefore not surprising (91). Methods for the rapid detection of genotypic and phenotypic changes in the HBV polymerase are urgently required for future monitoring of the efficacy of antiviral therapy 3., 43., 51., 91., 92., 93..

Reservoirs of infection and the pharmacokinetics of antiviral drugs 

Both the oral and tissue bioavailability of individual antiretroviral drugs are increasingly being seen as important factors in determining whether development of resistance will develop. The ability of HIV to create reservoirs of infection in sites such as the brain has major implications for successful therapy. In general, the RT and protease inhibitors currently available do not achieve levels in the central nervous system (CNS) that are equivalent to those in plasma. For example, the plasma to cerebrospinal fluid (CSF) ratios of stavudine, didanosine, zidovudine and lamivudine are 40%, 23%, 17% and 6%, respectively 94., 95., 96., 97.. It is likely that CSF drug levels overestimate the amount of drug present in brain extracellular fluid, since the two compartments may not intermix freely (98). A blood-brain barrier model utilising bovine brain microvessel endothelial cells has shown the permeability of key antiretroviral drugs to be in the order nevirapine ⪢ amprenavir ⪢ didanosine ⪢ stavudine ⪢ zalcitabine ⪢ zidovudine ⪢ indinavir ⪢ saquinavir (99), in line with clinical studies. The essential element in achieving penetration of antiretroviral drugs into any sequestered site of replication must be that the concentration attained extracellularly significantly exceeds the 100% inhibitory concentration. If this does not occur, resistant mutants will be selected in these sites.

HBV can replicate in cells other than hepatocytes (100) and this extra-hepatocyte reservoir of virus may be the source of reactivation or exacerbation of disease following successful drug-driven eradication of virus from the liver 70., 101. or natural immune clearance (102). It has been shown that ganciclovir (101) and penciclovir 37., 38. do not appreciably affect the duck HBV load in extra-hepatocyte sites such as bile-duct cells and pancreatic islets. Furthermore, treatment of ducks infected with duck HBV using either penciclovir or lamivudine has produced minimal effects on virus replication at these sites after treatment, whereas virus in hepatocytes was essentially cleared 37., 103.. To date, only adefovir dipivoxil has been shown to suppress viral replication in extra-hepatocyte and extra-hepatic reservoirs (104). Thus, these sites plus the lymphoid compartment (100) represent substantial reservoirs for seeding of virus back to hepatocytes, even during treatment (see Fig. 2). Knowledge of the metabolic basis for the uptake, activation and processing of antiviral agents such as nucleoside-nucleotide analogues 70., 71., 72. is essential for improved understanding of the efficacy of these compounds as antihepadnaviral agents. The ability of an antiviral agent to inhibit HBV replication in all cells harbouring virus is, therefore, an important consideration when developing therapeutic approaches to chronic hepatitis B infection (105).

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Future Directions in Treating HBV 

There are valuable lessons to be learned for HBV from the therapeutic approaches used to manage HIV infection. The key features of the strategy are:

1.(i) to decrease viral load,

2.(ii) to decrease rate of disease progression,

3.(iii) to decrease rate of emergence of drug-resistant virus; and

4.(iv) to use combination chemotherapy to effect (i) to (iii) above.

(i) To decrease viral load 

Monotherapy with currently approved antiretroviral drugs typically produces a 1–2-log reduction in plasma HIV-1 RNA levels, and reductions of 2–3 logs can be achieved with multiple combinations of these agents (106) (see Fig. 4). Recent studies have shown that both the degree and the duration of inhibition of HIV replication are related to subsequent clinical outcome (54), with a strong positive correlation between inhibition of viral replication and the slowing of clinical progression (107). To date, the best responses have been seen in drug-naive individuals.

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  • Fig. 4. 

    A theoretical dose-response effect analysis of HAART on HIV-1 RNA viral load in plasma. Four phases of elimination of virus are represented. An initial rapid decrease (phase 1), which reflects the direct effect of HAART on the main reservoir of productivity infected CD4+ lymphocytes (see Fig. 1). A second phase represents elimination of virus from the macrophage-monocyte infected population (phase 2). The third phase represents eventual elimination of thosecells which are chronically infected, long half-life memory CD4+ (RO45+) lymphocytes 17., 18.. The final phase of elimination represents that residue of virus currently sequested in sanctuaries such as the nervous system.

Pivotal studies characterising viral dynamics in HIV-1 infection (44) demonstrate that more than 99% of the virus present in blood originates from recently infected cells rather than long-lived chronically infected cells orfrom latently infected cells (memory cells) recently activated to produce virus (see Fig. 1). Thus, 30–50% of the virions present in the plasma of an infected person at any one time are produced by CD4 cells infected the previous day 1., 5..

Virological measurement of plasma HIV RNA levels at any point in time provides an insight into the degree of virus replication, which in turn determines the rate of CD4 T-lymphocyte destruction 108., 109.. Changes in plasma HIV RNA levels predict response to antiretroviral drug therapy and the failure of that therapy (110). It makes sense, therefore, that virus load is both a predictor of clinical outcome and a disease marker, and this is supported by clinical trials using drug regimens which achieve only modest reduction in plasma virus levels but achieve significant (i.e. approximately 50%) decreases in the relative risk of death (108). Treatment strategies involving HAART result in substantially greater reduction in virus load than the above studies, and are therefore almost certain to lead to decreased rate of disease progression and increased survival.

Viral load testing in chronic hepatitis B utilises three approaches: quantitative PCR (Roche Monitor), hybridisation-antibody-capture assay (Digene) and the branched chain DNA (bDNA) assay (Chiron). A detailed comparison of the performance characteristics of these assays was carried out by Krajden and colleagues (111), who showed that the bDNA and hybrid capture assays had the largest dynamic range, but the quantitative PCR from Roche Monitor was more sensitive (by at least 2 orders of magnitude) for measuring plasma HBV DNA. However, the clinical utility of all these ultra-sensitive HBV DNA assays requires further development in order to define more accurately the dose-response effects of antiviral therapy in chronic hepatitis B, including predictive or prognostic responses (112).

More recently, Dean and co-workers (113) have successfully developed a quantitative PCR for detecting HBV CCC DNA in liver biopsy samples. Since studies using duck HBV have shown that the half-life of DHBV CCC DNA in vitro is about 4–5 days (34), if viral replication can be fully inhibited over the long term, viral CCC DNA and thus infection should be eradicated. In patients receiving nucleoside analogue therapy for HBV, monitoring of intra-hepatic levels of the replicative intermediate of HBV, including the normally recalcitrant CCC DNA form, should provide a useful guide to the efficacy of treatment and the likelihood of a long-term, sustained response.

(ii) To decrease rate of disease progression 

Coincident with the rapid fall in plasma HIV-1 RNA in treated patients, there is an increase in the number of circulating CD4 cells 1., 5., 44., although the cell count often does not reach a value similar to that present before infection. This suggests that full immune restoration does not occur, and that only a subset of cells are replaced. What is not clear at this stage is whether T-cell responses lost as a result of HIV infection may be regained, even if new rounds of virus infection can be stopped with antiviral therapy.

Nucleoside analogue therapy has been associated with mitochondrial toxicity (114), which in chronic hepatitis B has been manifested as myopathy, neuropathy, lactic acidosis, pancreatitis and hepatic failure (115). Pre-clinical studies with long-term treatment using penciclovir in ducks (38) and similar clinical studies with lamivudine (116) have to date not identified any signs of mitochondrial toxicity. In fact, in the clinical study by Honkoop and colleagues (116), liver histology improved during lamivudine therapy. Also, a significant decrease in the activity of nuclear-DNA-encoded enzymes was found whilst there was no effect on mitrochondrial DNA synthesis. Nucleoside analogues such as lamivudine probably restore mitochondrial enzyme activity in chronic hepatitis B, due to the drug-related decreases in both viral replication and associated general hepatocellular injury as a consequence of therapy. Similar improvement in liver histology and decrease in necro-inflammatory activity has been reported by Lai and colleagues (117).

(iii) To decrease rate of emergence of drug-resistant virus 

Several strategies can be used to reduce the possibility that resistance will arise during therapy. Complete suppression of HIV replication as early as possible is essential. The HIV population is at its most homogeneous with respect to pre-existing resistance mutations in individuals recently infected with the virus (118), so this phase of the infection is crucial in terms of initiation of HAART. Irrespective of the duration of infection, maximisation of the therapeutic index through use of drugs of the highest possible potency at their maximum tolerated dose is necessary (Fig. 3). Combination chemotherapy with drugs known to produce synergistic interactions should also be used. This contributes to the overall potency of the therapy. If the inhibitors used are directed at multiple targets and select for multiple, non-overlapping resistance mutations, the genetic barriers to resistance will also be strengthened. Underpinning each of these strategies is the absolute need for compliance. Any significant alterations to the dosing schedule associated with the currently used antiretroviral drugs will inevitably result in the development of resistance.

As noted above, the two guiding principles in preventing the development of resistance with HIV are:

1.(i) maximise antiviral activity; and

2.(ii) maximise the genetic barriers to resistance (119).

Both are linked to the level and rate of viral replication (see Fig. 3). For the first principle, the aim is to use the highest possible doses of the most potent drugs (119). For the second, the aim is to avoid sequential monotherapy as this selects for the emergence of multiply resistant virus (110). Thus, combinations of drugs for which the development of resistance requires multiple non-overlapping viral mutations should be employed. In addition, drugs active at multiple targets should be used. The only way to prevent the emergence of resistance is to achieve complete suppression of viral replication (1).

The possible achievement of complete suppression of viral replication for a sufficient time to overcome the problem of long-lived infected cells suggests that the infection could be eradicated from some individuals. If eradication is possible for either HIV or HBV, then aggressive early treatment would appear to be essential. In HBV, this would be during the immunotolerant phase of disease 20., 22. and in HIV immediately after the seroconversion illness or following initial diagnosis 120., 121.. This is based on a classic clinical tenet from the treatment of infectious diseases, that the earlier one treats an infection with antimicrobial drugs, the better the results (93). This underpins the battle cry “Hit it early and hard” (118).

If eradication cannot be achieved, then proper implementation of a strategy of long-term chemosuppression must consider several competing factors (1):

1.(i) The premature use of potent antiviral regimens involves a financial commitment, risk of adverse side effects, and considerable patient inconvenience.

2.(ii) Treatment can be considered premature only if the pathological process is sufficiently reversible to justify delay.

3.(iii) The clinical practice of using the next available drug as monotherapy whilst the patient progressively deteriorates with multidrug-resistant virus is ideally suited to select for more resistant virus and preclude the benefits of potent combination therapy.

It should not be forgotten then, that once treatment is initiated, the physician needs to be aware that:

1.(i) drug-resistant isolates pre-exist 50., 122.: they are selected, not induced by the therapy; and as a result,

2.(ii) the viral quasispecies present at initiation of treatment are committed to an evolutionary pathway that cannot be re-traced (1).

(iv) Use of combination chemotherapy 

From the preceding discussion, it is clear that no single compound possesses all the properties required of an ideal antihepadnaviral agent, so there is a strong case for combination chemotherapy 123., 124.. Also, no drug currently exists which is capable of effectively blocking viral replication in all infected cell-types as well as inhibiting viral CCC DNA generation and processing.

Possible combination approaches to consider could include a cytokine and a nucleoside analogue. The theoretical rationale for this is that the nucleoside analogue would inhibit HBV replication, reduce viral burden in most compartments, and thus possibly restore the hepatitis B carrier's response to the effects of the administered cytokine. However, suppression of HBV by the nucleoside analogue could be too profound, resulting in no immunological target for the CTL's. This is a real problem as most nucleoside analogues studied to date have reduced the level of intra-hepatic necro-inflammatory activity during therapy.

Kruger et al. (125) have used the combination interferon alpha and famciclovir to successfully treat HBV-associated polyarteritis nodosa unresponsive to prednisolone. In contrast, Heathcote et al. (126) demonstrated no signficant difference between interferon-alpha monotherapy, lamivudine monotherapy or the combination of lamivudine and interferon-alpha, with all three treatment regions resulting in approximately 20–25% HBeAg seroconversion. At the histological level, the combination was potentially antagonistic in terms of improvement in histological activity index (HAI) scores.

Lau and colleages (127) have also used the combination approach with thymosin α-1 and famciclovir in immunotolerant Asian hepatitis B carriers, and found significant HBeAg loss after treatment. Thymosin α-1 is better tolerated than interferon alpha and is typically associated with a more gradual and sustained post-treatment alanine aminotransferase normalisation and HBV DNA clearance, properties which make it a useful alternative to interferon alpha in combination with a nucleoside analogue.

The combination of at least two different nucleoside analogues has a solid theoretical basis: as each would have different pathways for activation to the triphosphate form, there would be successful competition with different dNTP's for the HBV polymerase and each would ideally have separate sites of action such as reverse-transcription priming and chain elongation. There is the problem of possible selection for cross-resistant virus (e.g. lamivudine group 1 mutations and famciclovir sensitivity) as well as possible additive toxicity. Studies performed to date include the in vitro combination of penciclovir and lamivudine which acted at least additively and possibly synergistically to inhibit duck HBV DNA replication and blocked viral CCC DNA generation and processing (123). Unfortunately, group 1 lamuvidine resistant HBV isolates do not appear to be sensitive to famciclovir (see above and Table 3). However, adefovir appears to inhibit replication of the major types of HBV resistant to both famciclovir and lamivudine (92). These data suggest that combination therapy utilising two or more nucleoside analogues would, at a minimum, be effective in reducing viral burden for prolonged periods, at a sustainable level without substantial risk for selection of resistance.

Finally, any therapeutic protocol should include agents which can modify the ineffective and/or inappropriate immune response to HBV-infected hepatocytes. By doing this, we should be in a position to develop a therapeutic protocol that can successfully control HBV and effectively reduce the global viral burden of this important pathogen with its associated morbidity and mortality.

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Acknowledgements 

The authors wish to thank Julian Druce, Jo Dean and Tim Shaw for editorial assistance. Also, we are grateful to Drs. Condra and Emini for permission to use Figure 3, and Dr. Jai-Yee Lee for use of a modification of Figure 2.

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PII: S0168-8278(99)80118-4

Journal of Hepatology
Volume 30, Issue 3 , Pages 536-550, March 1999

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