I. Virological basis of drug resistance of nucleoside (acid) analogues
After HBV invades the human body, it binds to the receptor on the target cell membrane, sheds the envelope and enters the cytoplasm, sheds the nuclear capsid, and part of the double-stranded cyclic HBVDNA enters the target cell nucleus, and under the action of DNA polymerase, it uses the negative-stranded DNA as a template to extend the positive strand and repair the cleft region to form covalent closed-loop DNA (cccDNA). the replication of HBVDNA first uses cccDNA as The template is transcribed into 3, 5kb, 2, 4kb, 2, 1kb and 0, 7kb etc. by the action of host RNA polymerase.
HBV can replicate 1012 to 1013 copies every 24 h. Although HBV is a DNA virus, its replication process is not a direct DNA-DNA replication process, but an intermediate process of pre-genomic RNA, i.e. DNA-RNA-DNA replication process. In the process of reverse transcription of pregenomic RNA into negative-stranded DNA, HBV reverse transcriptase lacks a strict correction mechanism, resulting in a high nucleotide mismatch rate during HBV replication, which is about 1/105 between other DNA viruses and RNA viruses. this process and characteristics of HBV replication determine that there are differences between the genetic sequences of different HBV strains in the same patient. Thus, the virus in each patient is a dynamically changing viral population composed of viral strains with genetic sequence differences, i.e., HBV exists in quasispecies form.
The evolution of the HBV viral population is also in accordance with Darwinian evolutionary theory. Mutations at some loci can be lethal, and HBV that undergoes such mutations cannot survive. Mutations at some loci have no significant effect on their replicative capacity, but many loci mutations result in reduced or enhanced replicative capacity of the offspring virus. The relative proportion of viral strains with different genetic sequences in the viral population depends on the replication capacity of the viral strains themselves on the one hand, and is influenced by the selective pressure of the body’s immune system or drugs on the other.
Mechanism of action of nucleoside (acid) analogues: Upon entry into the body, they form triphosphate active components that bind competitively to the body’s natural deoxytriphosphate nucleoside to HBV polymerase. However, the DNA chain synthesis of HBV is terminated because the triphosphorylated nucleoside (acid) analogs do not possess the structure of natural dNTP, which is the mechanism by which nucleoside (acid) analogs inhibit HBV replication. However, if the sequence of HBV in the patient is mutated, resulting in the production of HBV polymerase with reduced binding to nucleoside (acid) analogs, then the mutated HBV is either not inhibited by nucleoside (acid) analogs or has a reduced inhibitory capacity. Therefore, under the continued application of nucleoside analogue therapy, the wild strain of virus continues to be suppressed because it is sensitive to nucleoside (acid) analogues; the mutant strain of virus gradually replaces the wild strain and becomes the dominant strain of HBV in the body because it has a certain replication capacity and is not sensitive to nucleoside (acid) analogues, thus leading to the resistance of patients to nucleoside (acid) analogues.
Second, nucleoside (acid) analog drug resistance variant-related concepts and nomenclature
(A) Concept of drug-resistant variants
Commonly used terms or concepts related to drug-resistant variants of HBV are.
1, primary non-response: refers to 12 weeks of nucleoside (acid) analogue treatment, the decline in HBVDNA load is less than 1log10IU/ml. primary treatment failure may be related to host, drug or virus and other factors: poor patient compliance, drug absorption disorders, poor ability to convert drugs into active ingredients in the body; weak antiviral efficacy of drugs or too small a therapeutic dose; HBV drug resistance occurs mutation, etc. can cause primary treatment failure.
2, virological breakthrough: refers to the process of treatment, two consecutive examinations 1 month apart, serum HBVDNA load than the lowest value after obtaining a response, the rise in value is greater than 1log10, virological breakthrough in patients with good compliance with treatment often suggests the emergence of drug resistance.
3, viral rebound: refers to patients who obtained virological response after treatment, although continued treatment, HBVDNA load increased ≥ 20,000IU/ml or higher than the pre-treatment level.
4, biochemical breakthrough: refers to the treatment to achieve serum ALT normalization, in the process of continuing treatment, ALT level increases and exceeds the upper limit of normal value. When the ALT level rises more than 5 times the upper limit of normal value is called hepatitis breakthrough.
5, primary drug resistance mutation: refers to the drug action target gene and its encoded amino acid mutation, resulting in the mutant virus strains to reduce the sensitivity of therapeutic drugs. For example, the mutant virus strain of rtM204V/I has a significantly reduced susceptibility to LAM. Although primary drug-resistant mutant strains have increased resistance to drugs, they also often lead to a decrease in the replication capacity of the mutant virus itself.
6. Secondary drug-resistant mutations: Due to the decreased replication ability of primary drug-resistant mutant strains, on the basis of primary drug-resistant mutations, the viral strains may also mutate at other loci, and these mutations may partially restore the replication ability of the mutant virus or may lead to a further decrease in the susceptibility of the mutant virus to drugs. For example, among the drug-resistant variants of LAM, rtM204V/I is the primary drug-resistant variant, and the often accompanying rtL180M variant is the compensatory drug-resistant variant.
7, genotypic resistance: refers to the detection of HBV variants that have been confirmed in phenotypic analysis studies in vitro to be associated with antiviral drug resistance.
8, Phenotypic resistance: HBV variants that have been confirmed to be detected by in vitro replication systems reduce their susceptibility to antiviral drugs. When the EC50 required to inhibit viral replication is increased by more than 100-fold compared to the wild strain is called highly resistant, 10-99-fold for moderate resistance, and 2-9-fold for mild resistance.
9, cross-resistance: HBV variants that are resistant to one nucleoside (acid) analogue are also resistant to one or more other nucleoside (acid) analogues. For example, LAM treatment occurs in rtM204I resistant variants, also have resistance to LdT.
10, multi-drug resistance: refers to when drugs with different targets for sequential or simultaneous treatment, HBV can occur in the target of different drugs resistant mutations, producing mutant strains of virus resistant to multiple drugs. If viral mutations occur at rtM204V/I and rtA181T/V after LAM treatment, the strain is resistant to both LAM and ADV.
Genetic mutations are the basis for viral resistance. Genetic variation occurs first in the clinic, followed by virological breakthrough and viral rebound, and then biochemical breakthrough. The interpretation of clinical viral resistance test results requires a comprehensive analysis in conjunction with changes in viral load, detection reagents and methods, and clinical manifestations.
(B) Nomenclature and writing format of drug resistance
HBV polymerase can be divided into four different functional regions: terminal protein, spacer region, and reverse transcriptase region. The known drug-resistant variants are all located in the reverse transcriptase region. 8 genotypes of HBV reverse transcriptase are composed of 334 amino acid residues, so the current internationally accepted HBV drug-resistant variants start from the rt 1st amino acid residue, and are written in the format of “rt – wild-type amino acid abbreviation – amino acid variation site relative to the starting point of the reverse transcriptase region – variation after the For example, rtM204V means that position 204 of the reverse transcriptase region is mutated from methionine (M) to valine (V). When there are more than two amino acid changes at the same site, that is, mixed HBV virus group, two amino acid changes should be listed at the same time.
Third, nucleoside (acid) analogs common resistance variant sites and the incidence of variation
1, lamivudine resistance-related variants: existing studies show that LAM common resistance-related variants are rtM204I/V±rtL180M variants, of which rtM204V mostly occurs in combination with rtL180M variants, and rtM204I variants can occur alone. The cumulative incidence of resistance at 1 to 5 years, calculated from published pivotaltrials of patients treated with LAM for nucleoside (acid) analogs, was 24%, 38%, 49%, 67% and 70%, respectively.
2. Variants associated with adefovir resistance: Available studies have shown that the common resistance-associated variants of ADV are the rtN236T and rtA181V/T variants, and the two loci variants can occur individually or in combination. The cumulative incidence of resistance in HBeAg-negative patients at 1 to 5 years was 0%, 3%, 11%, 18% and 29%, respectively, based on published data from pivotal clinical trials of patients treated with nucleoside (acid) analogs of ADV.
3. Variants associated with entecavir resistance: The current findings show that ETV resistance-associated variants are amino acid substitution variants at at least one of the three loci rtT184, rtS202 or rtM250 in combination with the rtM204V+rtL180M variant. Cumulative resistance from 1 to 5 years based on published data from pivotal clinical trials of ETV in patients treated with nucleoside (acid) analogs for primary treatment.
4. Variants associated with resistance to telbivudine: The current findings show that the common resistance-associated variant of LdT is rtM204I. Other sites such as rtA181V/T are still controversial. The cumulative incidence of resistance at year 1 and year 2 based on published data from pivotal clinical trials of LdT in patients treated with primary nucleoside (acid) analogs was 4% and 22%, respectively.
It should be noted that the populations and designs of chronic hepatitis B involved in each of these drugs are different.
Detection and analysis of drug-resistant variants of hepatitis B virus
(a) Commonly used genotypic drug resistance detection techniques
1, PCR product direct sequencing: is the HBV genome of reverse transcriptase region amplification after direct sequencing analysis method. PCR product direct sequencing method can detect known and possible unknown drug resistance variant sites, is one of the most commonly used genotypic drug resistance detection methods. keefffe et al. believe that the PCR product direct sequencing method should be used as the gold standard for genotypic drug resistance detection. The disadvantage of this method is that it is less sensitive and can only be detected when the mutant strain exceeds 20% of the HBV quasispecies pool.
2, polymerase chain reaction-restriction fragment length polymorphism: this method has strong sensitivity and can detect drug-resistant variants of 5% of the HBV quasi-species pool, and has been used by many laboratories at home and abroad in the detection of LAM drug-resistant variants, and recently the ADV drug-resistant variant detection method based on this technology has also been established in China. However, PCR-RFLP can only detect known, single-site variants, and it is a simple, rapid and inexpensive method for monitoring a few drug-resistant variants. However, with the successive introduction of various nucleoside (acid) analogues and the continuous emergence of HBV resistance variants, this method will not be able to perform the detection of multi-locus variants.
3, reverse hybridization method: INNO-LiPA method based on this technology has been approved for clinical testing in foreign countries, and currently can detect drug resistance sites including LAM, ADV, ETV and LdT common loci. The method can detect variant strains accounting for 5% to 10% of the quasi-species pool of HBV samples, so the sensitivity is good, but it can also only detect known site variants, in addition, the test is expensive, it is difficult to be widely used in clinical testing in China.
4, real-time PCR: the method is easy to operate, and can detect drug-resistant variants with a variation rate of less than 10%. The disadvantage is that only known sites can be detected; at the same time, for each site of different variants need to synthesize the corresponding probe, with the increase in the number of nucleoside (acid) analog drug resistance sites, the corresponding synthesis of the probe cost increases. Domestic SFDA-approved real-time PCR kits have been used for clinical testing of rtM204V/I variants.
5, gene chip: also known as DNA chip, DNA microarray, with the advantages of rapid, efficient, sensitive, parallelization and automation, can detect known variant loci. In addition, with the progress of gene chip high-throughput sequencing technology, can also be used to detect unknown variant loci. At present, domestic microarrays for clinical testing of nucleoside (acid) analogues are under development.
6, restriction fragment mass spectrometry polymorphism technology: this technology is a combination of PCR-RFLP technology and matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology, which is highly sensitive and can detect a number of variant strains of less than 1% of the quasi-species pool of HBV, but it is also only able to detect known locus variants and is expensive, making it difficult to promote its application in the clinic.
(B) In vitro phenotyping
In vitro phenotypic analysis is the “gold standard” for confirming genotypic resistance and is often used to evaluate the level of resistance at half effective concentration. The principle is to introduce the whole genome of HBV containing the drug-resistant variant to be detected into a cell line of hepatocyte origin, then add nucleoside (acid) analogues of different concentration gradients into the cell culture medium, and after a certain incubation time, detect the replication of HBVDNA under the effect of drugs, calculate the EC50, and determine the sensitivity of the variant to the nucleoside (acid) analogues by comparing it with the EC50 of the wild strain of virus. sensitivity.
(iii) Virtual phenotype analysis
The prerequisite for virtual phenotype analysis is the establishment of a database of HBV drug-resistant variants containing interrelated clinical information, genotypes and phenotypic drug resistance information. When a sequence to be analyzed is submitted to the database, the database will look for the HBV sequence that best matches the sequence and infer the drug resistance variants of the sequence to be analyzed based on the clinical and drug resistance testing of the matching sequence. As an adjunct to the study of phenotypic resistance, virtual phenotyping cannot replace in vitro phenotypic testing, but will facilitate the monitoring of known resistance variants and the discovery of new variants. Currently, available databases include the Australian.
V. Clinical management of hepatitis B virus drug-resistant variants
(i) Predictors of hepatitis B virus drug-resistant variants
A variety of factors may be associated with the odds of occurrence of HBV resistance to nucleoside (acid) analogs, including the type of nucleoside (acid) analog applied, HBVDNA load at the time of initial treatment, a basis of liver fibrosis/cirrhosis, and previous antiviral treatment with nucleoside (acid) analogs. In addition, male patients, high body mass index and alcohol abuse are also high risk factors for resistance mutations in antiviral therapy. However, an increasing number of studies suggest that early virological response status is an important predictor of the incidence of drug resistance.
(B) Prevention strategies for drug-resistant mutations of hepatitis B virus
1. Rational selection of indications for nucleoside (acid) analogue antiviral therapy: nucleoside (acid) analogs are not recommended for people with immune tolerance or inactive HBV infection, especially those who are younger, if they do not need to receive immunosuppressive or chemotherapeutic drugs. For the first appearance of active chronic HBV infection, especially in younger people, the decision to apply nucleoside (acid) analogues should be made carefully by fully analyzing their predisposing factors.
2. Rational selection of antiviral treatment regimen: The treatment regimen is recommended to refer to the Chinese Guidelines for the Prevention and Treatment of Chronic Hepatitis B. For patients with indications for antiviral therapy, if nucleoside (acid) analogs are used, try to use drugs with strong antiviral effects and low incidence of drug resistance variation; at the same time, be sure to understand the previous antiviral treatment, including the application of nucleoside (acid) analogs, treatment response and drug resistance variation, in order to select drug therapy without cross-resistance. In addition, single drug sequential therapy should be avoided as much as possible to avoid the occurrence of multi-drug resistance.
3, improve patient compliance: during antiviral therapy with nucleoside (acid) analogs, it should be repeatedly emphasized to follow the doctor’s instructions to take the medication on time and in sufficient quantity. Analysis of clinical trial data shows that more than 30% of virological breakthroughs are caused by poor patient compliance. In any case, a gradual dose reduction dosing regimen is wrong and will significantly increase the risk of drug resistance.
4, standardized monitoring of HBVDNA and genotypic drug resistance, timely adjustment of treatment regimen: HBVDNA load is the most important indicator of drug resistance monitoring during the application of nucleoside (acid) analogue antiviral therapy. HBVDNA levels should be tested regularly during treatment. A large number of clinical trials have shown that early virological response is an important predictor of the incidence of drug resistance, so both the APASL and EASL guidelines recommend adjusting treatment regimens based on early virological response to improve efficacy and reduce the incidence of drug resistance.
The existing guidelines do not recommend genotypic resistance testing as a routine test for nucleoside (acid) analogue antiviral therapy Genotypic resistance testing should be performed in patients who experience virologic breakthrough during nucleoside (acid) analogue therapy; unless there is clear evidence that the primary patient is infected with HBV from a patient receiving nucleoside (acid) analogue antiviral therapy. Genotypic resistance testing of patients on primary treatment is generally not advocated.
(C) Recommendations for clinical management of patients who have developed drug-resistant variants
For a small number of patients with normal pre-treatment ALT and mild inflammatory or fibrotic lesions on liver histology.