The Mechanism Of Low HBsAg Level In Chronic Hepatitis Patients

Objective:The sera from chronic hepatitis B(CHB) patients contain a large amount of hepatistis B surface protein(HBsAg) produced by hepatitis virus(HBV).Usually the level of HBsAg reaches up to thousands of international units per millimeter(IU/ml) of the serum.However,the clinical studies also observed that the titers of HBsAg in some of HBV carriers with active HBV replication(HBV DNA≥10~4 copies/ml) are less than 250 IU/ml.In this thesis,we mainly focus on the mechanism for the decreased titers of Hepatitis B surface antigen(HBsAg) in chronic hepatitis B patients.Methods:The titers of HBsAg were determined by the ABBOTT ARCHITECT HBsAg QT assay kit loaded on ARCHITECT i 2000SR system,and the levels of HBV DNA were measured using real-time PCR kit(Daan co.).After analyzing the serum samples from 1783 CHB patients,we picked up those with less than 250 IU/ml of HBsAg and collected the clinical and biochemical data from the patients.HBV DNA was extracted from sera using phenol/chloroform,then amplified and sequenced to identify the dominant mutation patterns within the surface gene(S gene).From 4 representative serum samples that possess mutations in the S gene,full-length HBV genomes were amplified and sequenced to obtain the dominant patterns.Moreover,the full-length HBV genomes from above samples were cloned into pUC19 vectors so that individual clones were sequenced and compared with the dominant pattern of each sample.The S genes harboring dominant mutations were further cloned into pXF3H vectors with HA tags at their N-termini.Furthermore,these plasmids containing HA tagged wild type or mutant S genes were transfected into Huh7 and Hela cell lines.The reactivity of wild-type HBsAg(wtHBsAg) and mutant HBsAg(mtHBsAg) in the super medium was measured using the ARCHITECT HBsAg QT assay kit.Meanwhile,the levels of intracellular and extracellular HBsAg were detected by Westernblot using the antibody against HA tag.Results:There were 35 out of 1783(1.87%) CHB patients experiencing lower than 250 IU/ml of HBsAg as well as greater than 1×10~4 copies/ml of HBV DNA.Based on analysis of the S gene sequences,samples from 15 out of 16 patients enrolled in this study have point mutations in the S gene,resulting in amino acid changes.These mutations include some prevalence sites such as sT120T,sT126N,sQ129N,sG130R, sT131N and sM133T.Furthermore,some mutations were rarely observed in previous studies,including sL95W,sV96G,sL98V,sS117T,sA157D,sW196L and sF220L.One deletion mutation in the S promoter region(nt2976-3174) was found in 13 out of 15 individual clones isolated from the S13 patient.The HBV S gene bearing T220A mutation that converts Leu-22(UUA) to stop codon(UAG) was identified in the serum sample from S15 patient.The full-length HBV genomes from 4 patients were successfully amplified and cloned.The dominant mutation of S gene was sQ129N in S11 sample and sT/I126N combined with sT131N in S14 patient.After transfection of pXF3H plasmids containing these S gene mutants into Huh7 cell lines,Enzyme-linked immunosorbent assay using the antibody against wtHBsAg demonstrated that the expression level of mtHBsAg in the supernatants was 2-4 times lower than that of wtHBsAg,whereas the total amounts of mtHBsAg detected by Westernblot using HA antibody were much more than that of wtHBsAg.Meanwhile,the glycosylation of mtHBsAg was also higher than that of wtHBsAg.Furthermore,the examination of mtHBsAg in the cell lysates revealed that these HBsAg mutants were not recognized by the polyclonal antibody against wtHBsAg.Conclusion:Although the level of HBsAg in the sera of most(＞90%) CHB patients are above 250 IU/ml,we observed 1.87%of 1783 CHB patients had low titer of HBsAg(＜250 IU/ml) and high copies of HBV DNA(＞10~4 copies/ml) in the same time.The mutations in the S gene definitely contribute to this phenomenon.The point mutation to asparagine in the S gene causes increased glycosylation of HBsAg which may alters the protein conformation and demolishes the antigenicity of HBsAg.This interferes with the normal diagnosis based on immunological assays and is responsible for the escape of HBVs observed in the S11 and S16 patient.Other contributors such as the preS2/S promoter deletion,stop codons before major hydrophilic region in S gene etc.should be further investigated.Taken together,various factors result in the low titers of HBsAg in some CHB patients.