In the past decade, the number of drugs approved for the treatment of chronic hepatitis B in China has increased from one common interferon to six drugs including pegylated interferon, lamivudine, adefovir, entecavir, and telbivudine, and tenofovir as well as emtricitabine abroad. Although nucleoside (acid) analogs are easy to take and have fewer side effects than interferon, they often fail to obtain sustained viral suppression after discontinuation at the end of a 48-week course and prolong the course, which may even be indefinite. Long-term use of these drugs can be associated with an increased risk of viral resistance, and viral resistance and poor patient compliance are two of the most important reasons for failure of antiviral therapy for chronic hepatitis B.
Nucleoside (acid) drug treatment failure is classified as primary treatment failure (no response to initial therapy) and secondary treatment failure, which is defined as a decrease in serum hepatitis B virus (HBV) DNA concentration of less than 1× log10 IU/ml within three months after initiation of antiviral therapy, while secondary treatment failure is defined as effective initial antiviral therapy (serum HBV DNA concentration decreased by more than or equal to 1× log10 IU/ml within three months). Viral mutation and drug resistance are the most important causes of secondary treatment failure.
I. Characteristics of hepatitis B virus replication and the generation of its mutations
In the process of HBV DNA replication, it needs to go through a reverse transcription process, and because the viral reverse transcriptase lacks 3′-5′ nucleotide exonuclease activity, it cannot proofread the mismatched nucleotide (acid) acid, resulting in a natural replication error rate of HBV DNA about 10 times higher than other DNA viruses. HBV genes are constantly mutating during the replication process, resulting in quasispecies, a group of viral strains with very similar but not identical genetic sequences, often in untreated HBV-infected patients. Because of the overlapping reading frame characteristics of HBV DNA, most HBV DNA quasispecies result in a reduction in their replication capacity, and the dominant strain in a given setting is the quasispecies with the highest replication capacity under a given selection pressure.
The presence of a pool of HBV variant strains (quasispecies) under endogenous (host immune response) and exogenous (antiviral drugs or viral transmission process) selection pressure provides a survival advantage for HBV, allowing for the presence of variant escape strains prior to immune response (pre-C region or e antigen escape), prophylactic vaccines (vaccine escape), and antiviral drugs (viral resistance).
Resistance of HBV to antiviral drugs reflects a decrease in the susceptibility of the virus to drug inhibition, due to adaptive mutation of the virus under selective drug pressure. Two types of resistance variants have been identified: major resistance variants, which directly reduce viral susceptibility to drugs, and compensatory resistance, which may enhance viral replication, as major resistance variants tend to be accompanied by a reduction in viral fitness for replication.
The importance of compensatory drug-resistant variants lies in their ability to compensate for the deficiencies of drug-resistant variants in the gene pool of quasispecies memory. Signs of the emergence of drug-resistant variants include an increase in viral load, typically greater than 1 logIU/ml from the nadir, and/or the appearance of known genetic resistance markers in the viral polymorpha region, an increase in serum ghrelin, and ultimately a worsening of clinical symptoms.
II. Factors associated with the development of hepatitis B virus drug resistance
The development of HBV drug resistance depends on at least six factors.
(1) The number and rate of viral replication;
(2) the fidelity of the viral polymerase;
(3) Selection pressure of the drug;
(4) the total amount of hepatic replication space;
(5) Replication adaptability of drug-resistant viral strains;
(6) the genetic barrier of the drug.
1. The number and rate of viral replication The high rate of viral renewal due to high HBV replication results in circulating viral concentrations in the serum of chronically infected patients that are often greater than 108-1010 viral particles/ml. Assuming that the half-life of circulating virus is one day, new viral particles are also produced each day in excess of 1011 .The HBV genome has 3200 base pairs and a polymerase mismatch rate of 10-4 to 10-5/base/cycle, which results in a large number of genomes with mutations (quasispecies) in the circulating virus overall, so that each base may change daily.
The maintenance of stability of the dominant strain in the HBV quasispecies pool depends on specific selection pressures from the host’s innate and adaptive immune systems as well as on the survival and replication capacity of the virus itself.
2. Viral polymerase fidelity HBV mutation rates range from approximately 1.4 to 3.2 × 10-5 amino acid substitutions/sites/year, which is approximately 10 times that of other DNA viruses, consistent with RNA viruses such as retroviruses. Unlike cellular polymerases, HBV polymerase is a reverse transcriptase and lacks corrective activity. Due to the presence of HBV quasispecies pools, it is possible to have mutant strains with one or two mutations associated with drug resistance before antiviral therapy is administered.
3. Selection pressure of drugs The chance of selecting resistance-associated mutations during treatment depends on the potency of the drug, and this chance can be expressed as a bell curve. Drugs with low antiviral potency do not exert significant selection pressure on the virus, and the risk of emergence of resistant strains is not high. Conversely, because mutation is dependent on viral replication, drugs that completely inhibit viral replication also give little chance for mutation to arise.
Because monotherapy only exerts antiviral effects to varying degrees at a single target site, it has a high chance of selecting for resistant variants. The ideal treatment regimen inhibits the virus at different stages of the viral life cycle, thereby significantly reducing the risk of resistance development. In the presence of drug selection pressure, drug resistance can only occur if viral replication is present.
4. Total hepatic replication space The replication space of HBV is the potential of the liver to accommodate new transcriptional templates or cccDNA molecules. This suggests that the eventual reception of viral variants is dependent on the attrition of the original wild virus strain and is influenced by other factors such as viral replication adaptations and hepatocyte proliferation and renewal. In the normal liver, hepatocyte renewal is slow, with a half-life of about 100 days. In inflammatory activity and toxicity, the half-life decreases to less than 10 days.
In fully infected livers, nascent HBV cccDNA molecules can be synthesized only during the generation of uninfected hepatocytes, which can be acquired by normal liver growth, proliferation and renewal of hepatocytes or depletion of wild-type viral cccDNA in infected hepatocytes.
5, replication fitness of drug-resistant virus strains Replication fitness can be defined as the ability to generate offspring under natural selection pressure, which is not measured by yield, but by in vitro co-infection competition assays, but this method is not applicable to HBV, because of the lack of suitable cell culture systems for HBV infection. Thibault et al. first reported the transmission of lamivudine-resistant HBV between patients; other groups found that lamivudine-resistant strains could coexist with wild-type HBV as co-dominant strains at least three months after drug discontinuation and with wild-type HBV as non-dominant strains approximately one year after drug discontinuation.
6, genetic barrier The genetic barrier of nucleoside (acid) drugs refers to the number of nucleotide mutations required for major drug resistance variants. For levonucleosides such as LMV and acyclic sulfate drugs such as ADV, only one mutation is required. For example, rtM204I results in LMV resistance while rtN236T causes ADV resistance. For ETV, a member of the cyclopentane class, at least three mutations are required: rtM180L and rtM204I plus one of rtI169, rtS184, rtS202, and rtM250.
7. Other factors Host factors affecting antiviral therapy include past drug history, compliance, host genetic factors (e.g., congenital metabolic defects) and the ability to efficiently convert nucleoside analogues to their active metabolites through a series of intracellular phosphorylations (remedial enzyme classes within hepatocytes). In addition, there are cryptic sites that may be beyond the reach of antiviral drug efficacy, and cccDNA, a key replication intermediate of HBV, is generally insensitive to conventional therapies.
Third, the specific resistance patterns of different nucleoside (acid) drugs
Currently, one class of drugs that have been marketed in China is levodeoxycytidine analogs including lamivudine (LMV) and telbivudine (LdT); the second class is acyclophosphates, adefovir (ADV); and the third class is cyclopentane-based drugs, which include deoxyguanosine analogs entecavir (ETV). Their chemical classification is emphasized because it may affect the patterns and pathways of resistance to nucleoside (acid) analogs.
LMV resistance variants LMV resistance variants are located in the catalytic region of HBV polymerase or the YMDD sequence known as the C region. the major resistance variants selected during LMV treatment are in the RT region, rtM204I/V/S (C region) with or without rtM180L (B region). Other resistance variants include rt181T/V. Compensatory variants occur in other regions of the HBV polymerase, such as rtL80V/I, rtV173L, and rtT184S. The incidence of resistance to lamivudine increases at a rate of 14 to 32% per year over the course of treatment. rtM204V/I, the predominant resistance variant of LMV, has cross-resistance with LDT, but not with ADV, but not with rtA181T. Of note is that rtM204V/I decreases susceptibility to ETV.
In in vitro experiments, LMV-associated drug-resistant mutations have reduced viral susceptibility to LMV by at least 100-fold and even more than 1000-fold. rtM204I mutations can be present alone, whereas rtM204V and rtM204S only accompany other mutations in the A or B regions. The molecular mechanism of lamivudine resistance is the replacement of the methionine in the YMDD motif of the polymerase by valine or isoleucine, whose ß-methyl causes a reduction in the lamivudine triphosphate binding space, creating a spatial barrier that prevents the binding of lamivudine triphosphate to HBV polymerase.
LDT resistance variants Tebivudine is the L-enantiomer of the natural thymidine deoxynucleoside, and the resistance site is similar to that of lamivudine, both occurring in the YMDD region, with the rtM204I substitution being the most frequently occurring variant.
ADV resistance variants Adefovir resistance was initially found to be associated with rtA181T and N236T mutations in the polymerase B region and D region. Adefovir resistance variants are less common than LMV resistance, with the incidence of resistance after two years of dosing being approximately 2, three years 4, four years 18 and five years up to 29. rtN236T does not significantly affect viral susceptibility to lamivudine, but rtA181T/V mutant strains can be partially cross-resistant to lamivudine. Another variant in the reverse transcriptase region (rtI233V) has also been shown to be associated with ADV resistance. Clinical studies have shown that the rtI233V variant occurs in nearly 2 of all CHB patients, but the exact role of this variant in ADV treatment failure or non-response is currently unclear.
ETV resistance variants Entecavir resistance initially occurs only in patients resistant to lamivudine, and its emergence is associated with mutations in viral polymerase genes, mainly rtI169T or rtS184G in region B, rtS202I in region C, and rtM250V in region E. In the absence of lamivudine resistance, rtM250V increases the IC50 9-fold, whereas rtT184G + rtS202I has no such effect. In the absence of lamivudine resistance, rtM250V increased the IC50 9-fold, whereas rtT184G + rtS202I did not; in the presence of rtL180M and rtM204V variants, the IC50 could be increased more than 100-fold. Primary resistant variants of ETV have recently been reported in primary care patients. The incidence of resistance in the first year of entecavir in primary care patients is very low, and at five years it is only 1.3. However, in patients who have previously received lamivudine, the resistance rate four years after switching to entecavir is as high as 40.
The mechanism of resistance to the rtT184G combined with the rtS202I variant is a conformational change that includes a change in the geometry of the nucleotide-binding region and a change in the binding of the polymerase to the template DNA located near the YMDD motif. The molecular mechanism of resistance to rtM250V is a change in the binding between the DNA template strand, the primer strand, and the newly enrolled dNTP.
IV. Problems caused by genetic overlap between the polymerase and S regions
The HBV viral outer membrane (S antigen) gene overlaps completely in the polymerase gene, so that nucleoside (acid) drug resistance variants lead to alterations in the S antigen. The genetic overlap between polymerase and S-antigen is important because common lamivudine-resistant variants such as (rtV173L+rtL180M+rtM204V) have important and significant alterations in S-antigen (sE164D+sI195M), resulting in a significant decrease in binding to S-antibodies (vaccine-related) in in vitro assays. Similarly, rtA181T alone or accompanied by rtN236T can be found in more than 40 cases in patients failing adefovir treatment. rtA181T variants in the Rt region can lead to Sw172 (stop codon) alterations in S antigens that overlap with them.
Warner and Locarnini et al. found that this HBV variant has a defective secretion function, where viral particles are retained intracellularly, and that this variant inhibits the secretion of viral particles from wild strains of HBV. The clinical significance of these studies is that the virological definition of resistance (increase in HBV DNA from a nadir of more than 1 logIU/ML in two consecutive samples separated by more than one month) no longer applies when this variant is (co-)selected. rtA181T emerged and viral load only gradually increased from a nadir over a 12-month period. Therefore, genotyping and polymerase region sequencing should be performed in addition to viral load observation when patients are on antiviral therapy.
Conclusion
HBV resistance to nucleoside (acid) analogs is essentially a screening of mutant strains in HBV quasispecies under pressure of drug selection. The long half-life of cccDNA and the long lifespan of infected hepatocytes lead to the need for long-term treatment and monitoring of patients with chronic hepatitis B. During this period, appropriate drugs and treatment regimens should be selected to prevent or reduce the emergence of virus-resistant mutant strains and improve antiviral efficacy. The complex pattern of drug-resistant variants in the polymerase region of HBV that have emerged, and the appearance of numerous compensatory variants, have forced the choice of compromise strategies for subsequent remedial therapy.
Further research is needed on how to properly characterize viral load, HBV genotype, and sequence assays of the polymerase region, and there is a great need for available and more extensive interactive database programs to provide a basis for remedial therapy. If viral replication can be effectively suppressed over time, viral load will be reduced to a level where the emergence of new drug-resistant virus quasispecies is unlikely.