Genetic Polymorphism Of MxA Promoter Associated With HBeAg Seroconversion In Chronic Hepatitis B Patients Receiving Telbivudine Therapy

Background:Hepatitis B virus infection has become a serious public health problem. Chronic hepatitis B is a progressive, refractory infectious disease, the development of liver pathological changes led to cirrhosis and hepatocellular carcinoma is closely related to the sustained high viral load. Effective antiviral therapy can maximize long term suppression of hepatitis B virus, reduce inflammation and necrosis of liver cells and liver fibrosis, postpone and reduce the occurence of hepatic decompensation, liver cirrhosis, hepatocellular carcinoma and related complications, thereby improve the living quality of patients and prolong survival. Therefore, antiviral therapy is an important measure to improve the condition and prognosis of chronic hepatitis B.Major antiviral drugs for clinical application to the treatment of chronic hepatitis B are interferon and nucleoside analogues. The latter can inhibite HBV replication quickly and effectively, reduce the load of virus in the blood and liver; relieve liver tissue inflammation, necrosis and liver fibrosis. It has wide clinical application due to the advantage of convenient usage and good safety. In the EASL and AASLD guideline for management of chronic hepatitis B virus infection, Telbivudine was not listed as a first-line drug for the treatment of chronic hepatitis B, but with the high incidence of chronic hepatitis B in the Asia-Pacific region, it still has broad application market. Although effective inhibition of viral replication can improve clinical outcomes of CHB, the current antiviral drugs can not eliminate the hepatocyte nuclear HBV cccDNA thoroughly, as a result, viral replication rapidly returns to high levels after the antiviral treatment is discontinued and liver inflammation resumes. Durable suppression of viral replication depends on the recovery of the individual’s immune function. HBeAg and HBsAg are T cell-dependent antigens, the production of HBeAb or HBsAb from B cells requires the involvement of helper CD4+T cells. As a component of the particulate structure, HBcAg is a T cell-dependent and non T cell-dependent antigen, the occurence of anti-HBc in patients with chronic hepatitis B is common even with the the impaired cellular immunity. HBsAg or HBeAg seroconversion is a course of specific immune response, the emergence of the specific antibodies against the hepatitis B virus, namely HBsAb or HBeAb, may reflect the improved immune function.Ideal end point of antiviral therapy is HBsAg loss (with or without the emergence of HBsAb), the current available anti-HBV drugs, however, rarely achieve this goal. HBeAg seroconversion is a satisfactory treatment end point for HBeAg-positive chronic hepatitis B patients. Studies have shown that after the achievement of HBeAg seroconversion in HBeAg-positive chronic hepatitis B patients with antiviral therapy, liver injury can be improved and the occurrence of liver cirrhosis and hepatocellular carcinoma will be reduced, the further goal of HBsAg loss or seroconversion will be achieved. Therefore, HBeAg seroconversion can be used as a indicator of lasting efficacy and withdraw of antiviral therapy. Major treatment guidelines have adopted HBeAg seroconversion as a primary end point for antiviral therapy in patients with HBeAg-positive CHB. Although HBeAg seroconversion may develope to HBsAg seroconversion, some patients may come back to the status of HBeAg positive or anti-HBe negative because of impaired immune function. HBeAg loss without the occurence of HBeAb may lies in the suppression of HBV replication, which lead to the decrease or disappearance of corresponding secretory protein, reflects the suppression of virus rather than improved immune function. In some CHB patients, HBeAg expression will be terminated due to the mutation of the promoter region of pre C or C, and converted to HBeAg-negative hepatitis. Therefore, in addition to status of HBeAg seroconversion, serum HBV DNA and ALT levels should be evaluated for comprehensive assessment of treatment efficacy.Telbivudine plays antiviral activity by direct inhibition of HBV DNA polymerase. In order to achieve sustained suppression of viral replication, long term antiviral therapy is required for most patients; viral resistance becomes the most difficult clinical problem in the long-term course of treatment. Due to influence of many factors, such as infection route, duration of disease, gender, age, host genetic background, the degree of liver disease and viral load, treatment response was different in patients with telbivudine. To obtain the optimal response to antiviral treatment, it is necessary for the implementation of individualized treatment based on the specific circumstances of the patient in the process of standardization of treatment, which can improve the long term antiviral treatment efficacy and reduce resistance. According to multivariate logistic regression analysis, GLOBE clinical research data showed that the2year HBeAg seroconversion rate was52%in HBeAg-positive subjects with baseline HBV DNA level<9log10copies/mL, ALT≥2×ULN and achievement of negative serum HBV DNA at24week, which showed that half of the patients who have the best treatment response to telbivudine can not achieve HBeAg seroconversion after2years of treatment. Some patients still can not achieve HBeAg seroconversion even with negative serum HBV DNA for a long time, the difference may be related to the immune function of host. Studies have shown that high levels of serum IL-10and IL-12in patients with HBeAg-positive CHB were associated with early spontaneous HBeAg seroconversion, and the level of cytokines were affected by gene promoter polymorphisms, which suggest that host immune function, to some extent, was affected by host genetic background.MxA is an interferon induced natural immune protein. In vitro studies have confirmed that MxA protein has antiviral activity against hepatitis B virus. By the use of a model of HBV/MxA transgenic mice lacking a functional IFN-α/β receptor, researchers found that an MxA-dependent moderate inhibitory effect on HBV expression was observed in female HBV/MxA mice, in which MxA downregulated viral HBeAg and capsid protein expression and HBV replication by decreasing the synthesis of HBV DNA replicative intermediates. Furthermore, this effect was not associated with change of steady-state levels of HBV RNAs. The results showd that, in vivo, MxA per se was able to reduce HBV expression in a post-transcriptional mechanism.Latest study demonstrated that in HepG2.2.15cells, MxA GTPase independently suppressed the production of HBsAg and HBV DNA without changing the level of HBcAg and the distribution of HBV mRNA. MxA significantly reduced the level of the encapsidated pregenomic RNA. Through its central interactive domain, MxA can interact with HBcAg, which lead to the accumulation of the proteins in perinuclear compartments. MxA-HBcAg interaction significantly affected the dynamics of HBcAg by immobilizing HBcAg in the perinuclear structures. The results suggestd that antiviral activity against HBV of MxA was involved in a mechanism of MxA-HBcAg interaction that may interfere with core particle formation. On the other hand, the chronicity of HBV infection result from continuous HBV replication indicates that HBV may antagonize the antiviral activity of MxA. HBV replication in HepG2cells can inhibit the interferon-inducible MxA gene transcription and protein expression In vitro. In co-transfection experiments, MxA promoter activity was inhibited by HBcAg and HBeAg expression vector. In addition, HBV preC/C proteins interacted directly with the MxA promoter, as shown by electrophoretic mobility shift assays. These results demonstrate a mechanism that HBV probably uses to downregulate an element of the IFN-induced host antiviral responses, which accounts for the impairment observed in HB V-infected patients.Current research about polymorphisms (SNPs) of MxA gene promoter has focused on-88G/T (rs2071430) and-123C/A (rs17000900). SNP-88G/T (rs2071430) located in an analogical interferon-stimulatedresponse element (ISRE) sequence of MxA gene promoter. T allele of this locus represents higher homology of ISRE motif and probably displays stronger effect. Studies results show that the single nucleotide polymorphism of the MxA gene promoter region, especially the-88G/T polymorphism, is not only associated with the disease progress of hepatitis C virus infection and the therapeutic effect of IFN, but also with HBeAg seroconversion in patients with chronic hepatitis B receiving interferon therapy, which indicate that SNP of MxA gene promoter plays an important role in mechanism of IFN treatment response and can be used as a reliable predictor for treatment response.With the development of individualized antiviral therapy, predictors associated with treatment response, especially host genetic factors have attracted research’s attention. Previous studies paid more attention to the response to interferon therapy, and failured to eradicate the influence of virological factors on treatment response. Studies on host genetic influence on nucleoside drugs induced HBeAg seroconversion are sparse. For the hypothesis that MxA gene polymorphisms of different individuals may exert an important impact on the incidence and antiviral efficacy of chronic hepatitis B, in the premise of equalization virological factors, study foucus on the relationship between MxA gene polymorphism and nucleoside based HBeAg seroconversion may reflect the impact of the host genetic background on immune function, which will contribute to a better understanding of the influence of host genetic background on treatment response.Objective:To explore the association between MxA gene promoter polymorphisms and telbivudine induced HBeAg seroconversion in HBeAg-positive CHB patients, reveal the impact of host genetic background on antiviral therapy response and provide theoretical basis for the application of individualized treatment.Methods:1. Ninety one patients with HBeAg-positive chronic hepatitis B who participate in telbivudine optimized treatment clinical studies (baseline HBV DNA level<9log10copies/mL, ALT≥2×ULN and maintain undetectable HBV DNA level from week24to week104of treatment) were enrolled in this study. According to treatment response at week104, subjects were divided into complete response group (CR, HBeAg seroconversion and normal ALT level) and non-complete response group (NCR, failed to obtain HBeAg seroconversion). Demographic and baseline clinical indicators between the two groups were compared;2. MxA gene promoter SNPs (rs2071430and rs17000900) were selected as candidate loci. Forward and backward primers were designed to amplify target segment according to MxA gene promoter sequence, and genotypes of candidate SNPs were detected by direct sequencing. Differences of loci genotype distribution in complete response group and non complete response group were compared to find the positive locus that was associated with telbivudine induced HBeAg seroconversion at week104;3. Haploview was used to analysis of the linkage disequilibrium between two SNPs of MxA gene promoter. PHASE was used to construct potential haplotype according to the genotype of two SNPs. Frequencies of different haplotype were compared between CR and NCR groups;4. Study subjects were divided into four groups according to the stratified criteria: age (<30years or≥30years old) and rs2071430genotype (TT/GT or GG). HBeAg seroconversion rate was calculated for each group and distribution of different hierarchical groups were compared;5. To find the independent factors that associated HBeAg seroconversion, impact factors that associated with HBeAg seroconversion from univariate test were analysed by unconditioned logistic regression analysis and adjusted for age, gender, hepatitis B virus genotypes;6. Cox proportional hazards models were used to estimate the association between rs2071430genotypes and HBeAg seroconversion during antiviral therapy. The likelihood ratio test was used, and adjusted for age, gender, and HBV genotype;7. Study subjects were divided into two groups according to the type of rs2071430genotype (TT/GT and GG), demographic and baseline clinical indicators between the two groups were compared;8. MxA promoter fragment containing different rs2071430allele was inserted into pGL3-Basic vector to construct allele-specific recombination expression vector, and the inner control plasmid pRL-TK were co-transfected into HepG2or HuH7cells. Under the stimulation of different concentration of interferon a-2a (100IU/ml or1000IU/ml), relative luciferase expression activity of different allele recombinant vector was compared;9. Biotin-labeled probes containing different rs2071430allele were synthesized. Nucleoprotein was extracted from HepG2cells with interferon α-2a (1000IU/ml) stimulate for1hour. In vitro EMSA experiment to explore the interaction between probe and nucleoprotein, and binding specificity between probes and nucleoprotein was verified by cold probe competition experiments. The binding capacity between allele-specific probe and nuclear protein was analysed by Quantity One software quantitative analysis.Results:1. According to definition of treatment response,50cases were complete response group and the other41cases were non-complete response group. In univariate analysis, rs2071430genotype and age were found associated with HBeAg seroconversion. Compared with the rs2071430GG genotype, individuals carrying the T allele were more accessible to HBeAg seroconversion (co-dominant model: OR=2.13,95%CI=1.20-6.65, P=0.016). The average age of CR group was younger than NCR group, the difference between two groups was statistically significant (27.3±6.6vs31.3±9.3, t=-2.293, P=0.025);2. The pairwise disequilibrum measure of rs2071430and rs17000900showed that the two SNPs of MxA gene promoter were in linkage disequilibrium (r2=0.34). Four kinds of haplotype were constructed by PHASE software, and the frequencies of haplotype between the CR and NCR group was not statistically significant (χ2=4.250, P=0.119). In dominant model, unconditioned logistic regression analysis adjusted for age and gender factors showed that individual AT haplotype distribution between the CR group and NCR group was not statistically significant (P=0.071); 3. Study subjects were divided into four groups according to stratified criteria, namely population Ⅰ (rs2071430TT/GT genotype and age<30years old), population Ⅱ (rs2071430TT/GT genotype and age≥30years), population Ⅲ (rs2071430GG genotype and age<30years old), population Ⅳ (rs2071430GG genotype and age≥30years). HBeAg seroconversion rate of each population was75%(24/32),50%(7/14),43.4%(10/23) and40.9%(9/22), respectively. The difference between population Ⅰ and population Ⅲ, population Ⅳ was statistically significant respevtively.(χ2=5.633, P=0.018;χ2=6.375, P=0.012);4. After binary unconditioned logistic regression adjusted for age, gender, HBV genotypes, the association between rs2071430genotype and telbivudine induced HBeAg seroconversion still keep statistically significant. Compared with the rs2071430GG genotype, individuals carrying the T allele have significantly higher probability of HBeAg seroconversion (dominant model:OR=2.48,95%CI=1.03-5.97, P=0.042), while the association between age and telbivudine induced HBeAg seroconversion closed to marginal statistical significance after regression analysis (P=0.058);5. Cox proportional hazards regression model analysis showed that after adjustment for age, gender, and HBV genotypes, compared with patients patients rs2071430GG genotype, patients carrying the T allele had higher accumulated HBeAg seroconversion rate after discontinuation of telbivudine treatment at week104and the difference was statistically significant (P=0.024);6. Study subjects were devided into two groups according to rs2071430genotype (TT/GT and GG), the results showed that patients carrying T allele have higher proportion of hepatitis B virus genotype B, the distribution in two groups was statistically significant (χ2=5.082, P=0.024). Compaired with non-T allele and non-HBV genotype B patients, patients with T allele and HBV genotype B had higher HBeAg seroconversion rate at week104, but the difference between the two group was not statistically significant (χ2=0.345, P=0.557);7. Relative luciferase activity was detected when rs2071430allele-specific recombinant expression vector and inner control plasmid pRL-TK were co-transfected into HepG2cells. The results showed that relative luciferase activity of different allele recombinant vector had no significant at medium condition (t=1.268, P=0.223). When the co-transfection system was treated with different concentration of interferon α-2a (100IU/ml or1000IU/ml), the expression activity of MxA-T-pGL3-Basic recombinant vector was significantly higher than MxA-G-pGL3-Basic vector (t=3.227, P=0.005;1=2.825, P=0.012, respectively). Similar results can be observed in HuH7cells (t=2.565, P=0.021; t=3.035,P=0.008);8. In vitro EMSA experiment showed that rs2071430can combine with the nucleoprotein (extracted from HepG2cells after interferon α-2a stimulating for1hour) to form a DNA-nucleoprotein complex. Competition experiments showed that the binding of biotin-labeled probes with nucleoprotein can be inhibited by corresponding cold probes (400times), which suggested that biotin-labeled probe with different allele binded to the same type of nuclear protein. Quantity One quantitative analysis showed that the binding band corresponding to the rs2071430T allele was much more intense than that corresponding to the G allele (90%increase), suggesting that under the stimulation of interferon, rs2071430T allele probe have stronger binding ability to nucleoprotein than G allele probe.Conclusion:1. MxA gene promoter SNP rs2071430is associated with HBeAg seroconversion at week104after discontinuation of telbivudine treatment, patients carrying T allele have higher HBeAg seroconversion rate;2. Rs2071430T allele motif of MxA gene promoter has stronger binding activity to nucleoprotein and show a stronger promoter activity;3. Genetic variants of MxA gene may affect telbivudine induced HBeAg seroconversion in CHB patients.