Influences Of Hepatitis B Virus Genotype On Serum Levels Of Hepatitis B Surface Antigen And Its Kinetics Under Antiviral Therapies

BackgroundHepatitis B surface antigen(HBsAg)is the marker of hepatitis B virus(HBV) infection.In life cycle of HBV infection,HBsAg is overexpressed compared to HBV virons.Loss or seroconversion of HBsAg is considered as the ideal and complete response to antiviral therapy,however the current approved regiments can barely achieve this ideal goal,even standard or pegylated interferon-alpha,lamivudine and adefovir can all decrease serum HBsAg levels.The loss of serum HBsAg was found in 3-8%patients treated with pegylated interferon-alpha and less than 2%patients with less than 2 years oral anti-HBV treatment.The undetermined issue for these patients is the kinetic changes of serum HBsAg levels under these therapies.Study profileThe thesis is consisted of six related and independent studies.The first study aimed to observe the collective kinetics of serum HBsAg levels in patients enrolled into clinical trials of adefovir or peg-interferon-alpha-2a.The second study was further carried out to profile changes of serum HBsAg in individual patient which achieved continuous decline of HBV DNA loads under the abovementioned treatments.To investigate the serum HBsAg changes with potent inhibition of HBV DNA replication,the third study was performed to understand the changes of serum HBsAg in HBeAg patients treated with 12 weeks lamivudine,and the candidate factors affecting serum HBsAg changes.To uncover serum HBsAg decline under long-term lamivudine therapy,patients achieving rapid and sustained HBV inhibition were investigated in the forth study.As the last four studies onging,it was found that baseline serum HBsAg might affect its changes and HBV genotype also played an important role,therefore the fifth study was conducted to find the possible factors affecting serum HBsAg levels in chronic hepatitis B patients.Finally,in vitro experiments,including transfection of replicative plasmids harboring Ba or C type HBV genome,or exchange constructs between HBV genotype BA and C.Materials and methodsPatientsPatients observed in the thesis were consisted of patients treated with adefovir, pegylated interferon-alpha-2a or lamivudine.Patients in the first and second study were treated with adefovir dipivoxil(10mg/day)in a multi-center phaseⅢtrial or pegylated-interferon-alpha-2a(180μg/week)in a global and multi-center phaseⅢtrial,respectively.Both trials treated patients with positive HBsAg over 6 months, HBeAg positive,serum HBV DNA load greater than half million copies/ml and no anti-HBV therapy prescribed within last three months.For the second study,patients were selected if they achieved a continuous serum HBV DNA load reductions during the 48 treatment weeks.Patients studied in the third study were continuously enrolled within below standards: HBsAg lasted over 6 months,naive to any anti-HBV therapy and HBeAg positive prior to treatment.Regiment was lamivudine l00mg/day and serum samples were collected before and after 12 weeks treatment.For the fourth study,patients in third study were selected as they had a rapid and sustained viral response to therapy(HBV DNA was lower than 1000 copies/ml at week24 and afterwards)and all were regularly followed-up with intervals from 3-6 months.For the fifth study,all patients suffered chronic hepatitis B with HBsAg in blood and elevated ALT and high HBV DNA loads(greater than 10,000 copies/ml).All were naive to antiviral therapy and without infection of HCV or HIV or addiction to alcohol.Quantification of serum HBsAgThe ARCHITECT HBsAg assay(Abbott Laboratories,Abbott Park,IL)is a two-step immunoassay,using chemiluminescent microparticle technology for the quantitative determination of HBsAg in human serum and plasma.The lower limit of detection of this assay is 0.05 IU/ml(around 0.2ng/ml),and a 1:500 dilution was necessary in samples with HBsAg titer greater than the upper detection limit of the assay(250 IU/ml).All procedures were performed as the manufacturer recommended.Assays for HBV genotypeHBV DNA was extracted from 100μl serum using QIAamp Blood DNA kit(Qiagen, Germany)according to manufacture's instructions and dissolved in 50μl water.A polymerase chain reaction based restricted fragment length polymorphism method was used to determine HBV genotype.For the confirmation of the genotyping results drawn from PCR-RFLP assay,nucleotide sequences analysis was then performed with direct sequencing of HBV surface gene.Plasmids and cell cultureReplicative plasmids were 1.24 genome HBV which harbor common genotype Ba and C constructed in puc-19.The exchange strategy were respectively based on genotype Ba and C,the exchange fragment were region coded large surface antigen (Type 1)or major surface antigen(Type 2)and surface promoter 2(Type 3)region which was responsible for HBV small surface gene transcription. Huh7 cell lines were cryopreserved in our lab and cultured with DMEM supplemented with 10%FBS and antibiotics and 5%CO2.Huh7 cells were seeded into 6-well plates with half million cells in each well.Supernatants and cells were collected at 72 hours after transfection for HBsAg quantitation.Statistical analysisLevene's test is adapted to determine homogeneity of variance between groups,data at serial time points were compared with repeated measure in general linear model. Fisher's exact test was used to compare catogorized variables,and univariate or multivariate analyses under general linear model were used to compare means of continuous variables.Linear regression was adapted to analyze factors affected serum HBsAg levels and binary logistic regression was used to determine the independent factors contributing to patterns of serum HBsAg changes.Profile analysis was also used to observe changes of HBsAg levels between genotypes.All data calculations and analysis were performed with SPSS 13.0.Statistical significance was considered as p value less than 0.05.Results1.In the first study,54 patients were enrolled into two independent clinical trials: 31 patients in adefovir trial and 23 patients in pegasys trial.Compared to baseline,serum HBsAg levels at treatment week 12,24 or 28(adefovir)and 48 were all significantly decreased.However,decline of serum HBsAg levels after treatment week 12 were not significant.Meanwhile,serum HBV DNA loads were also decreased predominantly during first 12 treatment weeks.In both treatment groups,ALT evolution seemed to be paralleled with HBV DNA during therapy.Collectively,serum HBsAg changes with two phases:rapid decline phase during first 12 weeks and a slowdown phase afterwards.2.Thirty-nine patients achieved continuous declines of HBV DNA loads under 48 weeks adefovir(n=22) or pegasys(n=17) therapy.Three patterns of serum HBsAg changes under either treatment were most frequently found in these patients while serum HBV DNA continuously decaying.Biphasic pattern were found in 43.6%patients who got an obvious decline of serum HBsAg during first 12 weeks and no significant decline were observed afterwards.The second were assurgent pattern which was describted as an elevated serum HBsAg levels at week 12 and maintained aftermath.The third was wavy pattern which consisted of serum HBsAg decline during first 12 weeks and rebound during followed interval.Regiment(p=0.886) or HBeAg loss(p=0.868) was not associated with these patterns.3.Eighty-seven patients were enrolled into the lamivudine study.HBV genotype analysis showed 43 patients were infected with HBV genotype B and C, respectively,and one was remained undetermined.All patients got decline of serum HBV DNA loads from average 8.56 lg copies/ml at baseline to 4.10 lg at week 12;the mean reduction of HBV DNA was 4.46 with a range from 0.75 to 7.80 lg copies/ml.Meanwhile,serum HBsAg were also decreased significantly from 3.74(1g IU/ml) at baseline to 3.40(average reduction,p<0.001).For HBV genotype B patients,83.7%achieved serum HBsAg decline,however only 51.2%for HBV genotype C(36/43 vs.22/43,p=0.001).Over 30%(n=28) patients even got elevated serum HBsAg levels as HBV DNA decreased significantly with average reduction of 3.93 lg copies/ml ranged from 0.75 to 7.78.Binary logistic regression analysis showed that HBV genotype B infection (odds ratio=4.083,95%confidence interval=1.362-12.236,p=0.012) and high serum HBsAg level at baseline(odds ratio=2.587,95%confidence interval=1.259-5.315,p=0.010) were independent factors contributed to decline of serum HBsAg during 12 weeks treatment. 4.There were 45 patients which achieved rapid and sustained suppression of HBV DNA and completed at least 3 years lamivudine treatment.Among them 21 were infected with HBV genotype B and 24 with genotype C.Even rapid and sustained inhibition of HBV DNA replication was gained,the possibility of HBsAg loss was rare(2.2%,n=1),however meanwhile HBeAg loss were found in 26/45 patients in this pilot group.Profile analysis showed that the trends of serum HBsAg changes were comparable between patients with or without HBeAg loss(p=0.991).Two frequent patterns,biphasic pattern(n=25)and maintaining pattern(serum HBsAg levels changed little during 3 years therapy, n=14)of serum HBsAg changes were found.Similarly,binary logistic analysis uncovered that HBV genotype C infection(odds ratio=8.206,95%confidence interval=l.070-62.948,p=0.048)and low level of serum HBsAg at baseline (odds ratio=0.020,95%confidence interval=0.002-0.743,p=0.040)were independent factors predicting the occurrence of maintaining pattern.5.In this cross-sectional investigation,there were 218 chronic hepatitis B patients. Among them there were 181 male patients,160 patients were HBeAg positive, and 121 and 97 patients were infected with HBV genotype B and C,respectively. Between genotypes,gender distribution,patients' age,ALT levels,HBV DNA loads and HBeAg status were all comparable,except serum HBsAg levels which were much higher in genotype B than in C patients.Univariate analysis and linear regression both showed that HBV DNA loads,gender and HBV genotype were independent factors affected serum HBsAg levels in patients who were naive to anti-HBV treatment.Taking regard of standard coefficient,HBV DNA loads was the most important one having impacts on serum HBsAg levels.6.For each paired transfection,HBsAg levels in supernatant and in cells were higher in genotype B transfectant than in C,both p values were less than 0.001. Six exchanged plasmids were successfully constructed,and the transient transfection showed that surface promoter 2 which is responsible for the transcription of small surface gene was the dominant region determining HBsAg expression levels.Conclusions1.For HBeAg positive patients,anti-HBV therapy including adefovir,pegasys and lamivudine can reduce serum HBsAg levels.In patients achieved continuous HBV inhibition by adefovir or pegasys,serum HBsAg changes,clollectively with two phases:rapid decline phase during first 12 treatment weeks and a flat-like phase with little reduction of serum HBsAg.2.For individual patient,profile analysis showed that serum HBsAg changes were not always paralleled with HBV DNA declines.Elevated serum HBsAg levels at week 12 compared to baseline were found in about 25%patients(assurgent pattern),and serum HBsAg levels rebounds after mild reduction in first 12 weeks(wavy pattern)were found in another 25%patients even meanwhile serum HBV DNA loads continuously declined.Somehow,the biphasic pattern which means serum HBsAg and HBV DNA have parallel changes were more frequent.Therefore these multiple patterns of serum HBsAg changes indicated that some certain host or viral factors have significant influences on changes of HBsAg levels under anti-HBV therapy.3.Short term lamivudine treatment can decrease HBV DNA loads dramatically, however meantime serum HBsAg decline were observed in about 30%HBeAg positive patients.For patients with HBV genotype B,changes of serum HBsAg were paralleled with those of HBV DNA,which was not found in patients with HBV genotype C.Regression analysis showed that serum HBsAg declines were more in patients with high baseline HBsAg levels and HBV genotype B infection.4.In patients achieved rapid and sustained inhibition under long term lamivudine therapy,serum HBsAg changes presented as an L-like profile,however even with complete inhibition,HBsAg loss occurrence was rare.For individual patient,two patterns of serum HBsAg change were found:biphasic pattern and maintaining pattern(serum HBsAg levels maintained and little changed during 3 year treatment).Patients with low baseline serum HBsAg levels and HBV genotype C infection much more tended to have a maintaining pattern.Herein HBV genotype and HBV DNA loads as well,were considered as the viral factors affecting changes of serum HBsAg concentrations.5.Cross-sectional study disclosed that serum HBsAg levels were positively correlated with serum HBV DNA loads,and serum HBsAg levels were higher in male patients than female,higher in genotype B patients than in C.Multivariate analysis and linear regression confirmed that HBV DNA loads,HBV genotype and patient's gender were independent to influence serum HBsAg levels in patients na(?)ve to anti-HBV treatment.However,the contribution of HBeAg status to serum HBsAg levels needs further investigation.6.After transient transfection of replicative plasmids harboring HBV genotype B or C,extracellular and intracellualr HBsAg levels were both found higher for HBV genotype b than for C.7.Exchanged plasmids were transfected into Huh7 cells,and it was surface promoter 2 which was responsible for small surface gene transcription determines HBsAg expression levels.