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AIM:Immune escape mutations of HBV often occur in thedominant epitope,the second-loop of the a determinantof hepatitis B surface antigen(HBsAg).To let the hostsrespond to the subdominant epitopes in HBsAg may be aneffective way to decrease the prevalence of immune escapemutants.For this reason,a man-made clone of HBV Sgene with the second-loop deletion was constructed.Itsantigenicity was evaluated by yeast expression analysisand DNA immunization in mice.METHODS:HBV S gene with deleted second-loop,aminoacids from 139 to 145,was generated using splicing byoverlap extension.HBV deleted S gene was then clonedinto the yeast expression vector pPIC9 and the mammalianexpression vector pcDNA3 to generate pHB-SDY and pHB-SD,respectively.The complete S gene was cloned into thesame vectors as controls.The deleted recombinant HBsAgexpressed in yeasts was detected using Abbott IMx HBsAgtest kits,enzyme-linked immunoadsorbent assay(ELISA)and immune dot blotting to evaluate its antigenicity in vitro.The anti-HBs responses to DNA immunization in BALB/cmice were detected using Abbott IMx AUSAB test kitsto evaluate the antigenicity of that recombinant proteinin vivo.RESULTS:Both deleted and complete HBsAg weresuccessfully expressed in yeasts.They were intracellularexpressions.The deleted HBsAg could not be detected byELISA,in which the monoclonal anti-HBs against thedeterminant was used,but could be detected by Abbott IMxand immune dot blotting,in which multiple monoclonal anti-HBs and polyclonal anti-HBs were used,respectively.Theactivity of the deleted HBsAg detected by Abbott IMx wasmuch lower than that of complete HBsAg(the ratio of samplevalue/cut off value,106±26.7 vs 1 814.4±776.3,P<0.01,t=5.02).The anti-HBs response of pHB-SD to DNAimmunization was lower than that of complete HBV S genevector pHB(the positive rate 2/10 vs6/10,4.56±3.52 mIU/mLvs27.60+17.3 mIU/mL,P=0.02,t=2.7).CONCLUSIONS:HBsAg with deleted second-loop of thedeterminant still has antigenicity,and can also raise weakanti-HBs response in mice to DNA immunization,suggesting that it is possible to develop a subdominant vaccine forpreventing infections of immune escape mutants of HBV.
AIM: Immune escape mutations of HBV often occur in the dominin epitope, the second-loop of the determinant of hepatitis B surface antigen (HBsAg). To let the hosts respond to the subdominant epitopes in HBsAg may be aneffective way to decrease the prevalence of immune escape mutants . For this reason, a man-made clone of HBV S gene with the second-loop deletion was constructed. Itsantigenicity was evaluated by yeast expression analysis and DNA immunization in mice. METHODS: HBV S gene with deleted second-loop, aminoacids from 139 to 145 , was generated using splicing byoverlap extension. HBV deleted S gene was then clonedinto the yeast expression vector pPIC9 and the mammalianexpression vector pcDNA3 to generate pHB-SDY and pHB-SD, respectively. The complete S gene was cloned into thesame vectors as controls. deleted recombinant HBsAgexpressed in yeasts was detected using Abbott IMx HBsAgtest kits, enzyme-linked immunoadsorbent assay (ELISA) and immune dot blotting to evaluate its antigenicity in vitro. he anti-HBs responses to DNA immunization in BALB / cmice were detected using Abbott IMx AUSAB test kit evaluation of the antigenicity of that recombinant protein in vivo .RESULTS: Both deleted and complete HBsAg weresuccessfully expressed in yeasts.They were intracellularexpressions. The deleted HBsAg could not be detected by ELISA, in which the monoclonal anti-HBs against the sterminant was used, but could be detected by Abbott IMxand immune dot blotting, in which multiple monoclonal anti-HBs and polyclonal anti-HBs were used, respectively. The activity of the deleted HBsAg detected by Abbott IMx wasmuch lower than that of complete HBsAg (the ratio of sample value / cut off value, 106 ± 26.7 vs 1 814.4 ± 776.3, P <0.01, t = 5.02). The anti-HBs response of pHB-SD to DNAimmunization was lower than that of complete HBV S genevector pHB (the positive rate 2/10 vs 6/10, 4.56 ± 3.52 mIU / mL vs 27.60 + 17.3 mIU / mL, P = 0.02, t = 2.7) .CONCLUSIONS: HBsAg with deleted second- loop of thedeterminant still has antigenicity, and can also also rai seweakanti-HBs response in mice to DNA immunization, suggesting that it is possible to develop a subdominant vaccine forpreventing infections of immune escape mutants of HBV.