Study On The Evolution Of Hepatitis B Viral Quasispecies And Its Clinical Significance During Nucleoside Analog Therapy

Chronic hepatitis B is one of the main threatens to global health. Over one third of the chronic hepatitis B virus (HBV) carriers are in China. Anti-HBV therapy is the core treatment of chronic hepatitis B. Among the available medications, Nucleoside Analogs (NA) are the main stream treatment drugs currently. As more and more novel NA being developed and saled in the market, more choices can be made in clinical practice. Entecavir (ETV) is one of those novel NAs, with strong effect on the inhibition of HBV replication, has been saled in the market recently. However, the long-term effect of ETV, as well as its comparison to current main stream NAs (e.g., lamivudine, LAM) on long-term anti-HBV effect and resistant rate, is still uncertain.Meanwhile, current clinical practice of anti-HBV treatment by NAs has resulted in a series of unanswerable questions, such as①Primary selection of NA for NA-na?ve patients,②Prevention and management of NA resistance,③Feasibility and strategy of combination or sequential NA therapy, and④Judgement of when to stop NA therapy. It has been noticed that NA resistance is the key of these questions. Thus, there are two main aspects in current clinical anti-HBV therapy with NAs. The one is to make full use of the antiviral effects of NAs, and the other is to avoid the emergence of drug resistance as far as possible. However, the lack of clinical laboratory evidence and the understanding of molecular virological course and the mechanism of drug resistance during NA therapy make the clinical anti-HBV treatment poorly guided. This is the key problem that limits the scientific and rational use of NAs in clinical antiviral therapy.The import of'quasispecies'into the field of virology has made it possible to understand the clinical course of NA resistance and immune escape from a completely new point of view. Studies on hypervarible viruses such as HCV and HIV have proved that these viruses exist in any single host as a population composed of various types of variants, and can adapt to the change of environment with their population selective advantage. Recent studies indicated that HBV also possesses a character of quasispecies, and the evolution of HBV quasispecies during NA therapy may be the course of drug resistance and the failure of anti-HBV therapy. However, these case studies is lack of systematic research deep into the mechanism of quasispecies evolution and its clinical significance. They were also lack of negative controls so that cannot reach to definitive conclusions. Meanwhile, current study method on HBV quasispecies relies on the accuracy of'PCR-cloning-sequencing'and has made this method the'Gold Standard'. There is still a lack of simple, accurate and cost-efficient method for quasispecies study.To answer these questions, 36 patients with chronic hepatitis B were included in this study, 19 with ETV and 17 with LAM na?ve treatment. Of the latter 17 patients, 14 received sequential ETV therapy. A 192-week follow-up was performed with dynamic detection of their serum HBV DNA and ALT levels. All serum samples were stored in the'Serum Bank'of our Institute, and were PCR-direct sequenced. 6 patients with clinical virologic breakthrough during NA therapy and 4 negative control patients without virologic breakthrough were selected for the study of quasispecies dynamics. All the 70 serum samples from these 10 patients were analyzed with the method of'PCR-cloning-sequencing'. Totally over 2,100 clones were sequenced. The target sequences obtained were aligned, compared and analyzed to study the mechanism of NA drug resistance and to understand the clinical course of NA therapy from a viral populational genetic point of view. In the 3rd part of this study, new method for the screening of HBV quasispecies containing the B, C, D and E domain of the polymerase gene rt area in HBV genome, which contained all the known NA resistant mutation loci, were set up. Discussion was made on the novel methodology for the detection of HBV quasispecies.Main Results1. Comparison of ETV and LAM on the effect of long-term HBV inhibition. ETV is superior to LAM on 1 year therapy (t=2.570, P=0.018), but not statistically superior to LAM on prolonged therapy (t60=1.570, P60=0.148; t72=1.432, P72=0.190; t96=1.663, P96=0.137). While no statistical difference between ETV and LAM on the effect of improvement of liver function. However, larger population should be included for the verification of these results.2. Drug resistance of ETV was significantly fewer than that of LAM during the 96-week mono-NA therapy (Pearsonχ2=6.490, P=0.016<0.05).3. Drug resistance rate of ETV-na?ve-treated patients was significantly lower than that of LAM-pretreated patients (Pearsonχ2=8.291, P=0.036<0.05).4. In patients with virologic breakthrough, a complicated yet regular evolution of HBV quasispecies was observed, while HBV quasispecies population remains stable in patients without virologic breakthrough. Drug resistant variants were always selected out as minor strains at first, and accumulated in HBV quasispecies population, and replaced primary dominant quasispecies before or at the time point of virologic breakthrough.5. Patients with drug resistant variants all emerged virologic breakthrough, while patients who have emerged virologic breakthrough were not necessarily infected with rug resistant HBV variants.6. Once dominated variants can archive in HBV quasispecies population as monor variants, and can evoled to multi-NA resistant variant under sequential NA pressures.7. A novel ETV resistant mutation, rtV190A, was observed. This mutation can combine with several other reported ETV mutations to form a rtM204V/L180M/T184L/V190A combined multi-NA resistant mutation.8. Combined rtM204V/L180M variant can be selected during LAM therapy, and possesses a selective advantage over rtM204I or rtM204I/L180M variants. Therefore, the dominant quasispecies was always rtM204V/ L180M at the time point of virologic breakthrough in patients mix-infected with these variants. We named this phenomenom'VM breakthrough'.9. Single rtM204V variants can be selected by LAM mono-therapy. Combined rtM204V/L180M variant can emerge on the basis of these variants. With the existence of this variant, ETV resistant variants, e.g. rtM204V/L180M/S202G, rtM204V/L180M/T184L, and rtM204V/L180M/T184L/V190A etc, can be selected during sequential ETV therapy. Therefore, LAM-ETV sequential therapy can beat the'genetic barrier'of ETV step by step. We named this the'Ladder Effect'of LAM-ETV sequential therapy.10. We found that drug resistant variants can be detected as minor quasispecies 12~36 weeks before virologic breakthrough.11. 60 sequencing results was randomly devided into 10 groups. Each group has 10, 15, 20…55 clone sequences. Chi-square (χ2) test indicated that to represent the proportion of dominant qusispecies in HBV population as exactly as to study 60 clones in one serum sample, 15 clones should be studied (χ2=2.700,P=0.100>0.05), while to discover minor qusispecies as exactly as to study 60 clones in one serum sample, 30 clones should be included (χ2=2.411,P=0.120>0.05).From these results, we draw conclusions as below:1. With a significantly lower drug resistance rate, ETV is safer than LAM for long-term treatment of chronic hepatitis B.2. Mechanism of HBV resistance to NA: NA therapy can select drug resistant variants from HBV quasispecies population. These variants can accumulate in HBV population and gradually replace the previous dominant wild type HBV strain, and change the susceptibility of HBV population to certain NAs.3. There are still unknown NA resistant mutations. These mutations may locate outside the B, C, D, E domain of the P gene rt area of the HBV genome, and can combine with known NA resistant mutations.4. LAM-ETV sequential therapy can lead to the failure of the'genetic barrier'of ETV. Therefore, direct initiation of antiviral drugs with stronger efficacy of HBV inhibition and lower resistance rate is recommended instead of a'gradually better'strategy.5. The rtM204V/L180M combined variant has a strong potency of evolving into ETV resistant variants. Therefore, once rtM204V/L180M variant detected in HBV quasispecies population, ETV is not recommended6. NAr quasispecies can be detected 12– 36 weeks earlier than virologic breakthrough. Therefore, routine analysis of HBV quasispecies is urgently needed in clinical labs to guide clinical practice of anti-HBV treatment. Analysis of HBV quasispecies should be conducted at least every 3 months.7. Once increasing drug resistant variants are detected, other sensitive drug should be introduced as soon as possible.8. To determine the composition and distribution of HBV quasispecies in a certain serum sample accurately and cost-efficiently, at least 15 or 30 clones, according to study requirement, should be included with a method of'PCR-cloning-CSGE or SSCP/HDA- sequencing'.