Study On The Efficacy And Evolutionary Patterns Of HBV In Patients With Chronic Hepatitis B During Nucleot(s)ide Analog Therapy

Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. In recent years, treatment of chronic HBV has been improved, with the availability of nucleoside/nucleotide analogues (NUCs) such as lamivudine (LAM). Unfortunately, the clinical benefit is rarely sustained under long-term treatment due to the selection of lamivudine resistant mutants, which occur at an annual rate of 14-32%. Adefovir (ADV) has an antiviral profile with potent activity against lamivudine-resistant strains, making it useful as a rescue treatment for lamivudine resistant patients. However, suboptimal responses have also been reported after adefovir rescue therapy, and its efficacy for patients with different genotypes and different LAM-resistant mutations remains unclear.Most studies of HBV drug resistance have focused on analysis of the A to D domain of the HBV DNA polymerase gene after detection of antiviral-resistant HBV mutants, and the drug-resistant mutational pattern that occurs in this region of the gene has been well characterized. The full HBV genome contains four partially overlapping open reading frames (ORFs) and mutations in the reverse transcriptase (RT) domain can affect the amino acid sequence of the surface protein. Previous studies have found that the immune-escape variants probably appear after antiviral-resistant variants emerge; this might be responsible for the exacerbation of chronic hepatitis B in some patients. Based on these findings, dynamic changes within other regions (especially in the immune-targeted surface and core antigen) might also be informative, and comparison of the full genome is necessary.Like other RNA viruses, HBV shows quasispecies distribution in infected patients. Thus, to permit chronic infection management, the evolution and distribution of HBV should be monitored. These studies about the dynamic evolution of HBV quasispecies during treatment of patients with commonly used NUCs focused on the HBV DNA polymerase gene and did not investigate the mutational pattern of the full HBV genome. Several more recent studies have reported the mutational pattern of the full HBV genome. However, the HBV full genome sequence was examined by direct sequencing, not by cloning. Thus, the evolutionary pattern of complete HBV quasispecies remains unclear.The present study investigated patterns of lamivudine-resistant mutations in 212 patients with lamivudine-resistance and evaluated the effect of these mutations on the antiviral response to LAM+ADV combination therapy. 1 033 sequences of HBV RT region derived from the patients with NUCs therapy were used to elucidate the evolution pattern and positive selection in HBV population. Meanwhile, we used full genome sequences generated from patient sera to assess the quasispecies evolutionary dynamics of five chronic hepatitis B patients who underwent sequential NUC therapies, as well as of two untreated patients with acute flares.Main Results1. The duration for LAM resistance in patients with genotype C was significantly longer than those with genotype B (P=0.036). Mean HBV DNA level after 3 months of LAM+ADV combination therapy was lower than that in LAM resistance (P<0.001). 180 patients (84.9%) achieved ALT normalization and 15 patients (11.6%) achieved HBeAg seroconversion. There was no difference in HBV DNA level, ALT normalization and HBeAg seroconversion rate between patients with genotype B and C.2. rt204 mutation was detected in 193 patients. L180M was more often found in patients with M204V than those with M204I (76/80 vs. 28/113,P<0.001). M204I+L180M combined mutation presented more in patients with genotype C (P=0.002), and M204V/I+V207L was detected more in genotype B (P=0.005). There was no difference in the incidence of M204V+L180M, M204V/I+229 and M204V/I+S213T mutation between patients with genotype B and C. Multi-mutations were detected in 42 patients (19.8%).3. The presence of the rt180?rt229?rt213 did not significantly affect viral load reduction and ALT normalization during combination therapy. The proportion of ALT normalization for patients carrying M204V/I combined with V207L was lower than those without V207L (P=0.001).4. Mean nonsynonymous (dN)/synonymous (dS) ratio?for genotype B sequences was 0.26; rt204 and rt222 were identified as positively selected sites. Mean?for genotype C sequences was 0.19; rt204 and rt180 were identified as positively selected sites.5. Significantly different selection was identified in six codons between genotype B and C sequences, including rt182 (P=0.08), rt209 (P=0.09), rt211 (P<0.001), rt226 (P=0.04), rt235 (P=0.08) and rt238 (P=0.02).6. In the three treated patients who received lamivudine as initial antiviral therapy, nucleotide polymorphism and nonsynonymous divergence decreased at lamivudine breakthrough but increased after rescue therapies. Conversely, two other treated patients showed distinct change in divergence during adefovir-telbivudine sequential therapies.7. Untreated subjects exhibited increased polymorphism and divergence in the preC/C region at hepatitis flare.8. Divergence of different HBV coding regions showed different changing trend in a single host and within different patients.9. All treated patients showed NUCs resistant mutations, three of which showed multi-drug mutations. Four of the treated patients presented amino acid changes in the“a”determinant during NUC therapy. In untreated patients, most of the variations were located in the preC/C gene.10. All of the treated subjects showed amino acid changes within the known T-cell or B-cell epitopes in the surface or core antigen, most of which were accompanied by mutations in the RT region. Co-variations in the core promoter, the preC region and the known epitopes of the preS gene, accompanied by RT mutations, were not rare.From these results, we draw conclusions as follows:1. LAM and ADV combination therapy is effective for patients with genotype B and C and for patients with different LAM-resistant patterns.2. There are differences for the LAM-resistant patterns between patients with different genotypes. Patients who receive LAM as initial therapy have risk to develop multi-mutations.3. Purified evolution prevail HBV RT region for patients who receive NUCs therapy, with a small portion of codons under positive selection. There is difference in selection pressure between patients with genotype B and C.4. The distribution of genetic variability of HBV shows remarkably different patterns between NUC-treated and untreated subjects and within patients receiving different NUCs therapies.5. The underlying mechanisms of hepatitis flare differ between NUC-treated and untreated patients.6. NUC-resistant mutations can cause amino acid changes within epitopes of other HBV regions and that the characteristics of HBV immunogenicity may change when NUC resistance emerges.