Construction of exogenous multiple epitopes of helper T lymphocytes and DNA immunization of its chimeric plasmid with HBV pre-S2/S gene

AIM: To design and construct an exogenous multiple epitope of helper T lymphocytes (HTL), and to evaluate its effect on anti-HBs response through DNA immunization. METHODS: Artificial HTL epitope, PADRE and four other HTL epitopes from different proteins were linked together using splicing by overlap extension to generate exogenous multiple epitopes of HTL, MTE5. pcMTE5 and pcHB were generated by cloning MTE5 and fragments of HBV pre-S2/S gene into mammalian expression plasmid pcDNA3. Four chimeric plasmids were constructed by cloning MTE5 into the region of pre-S2 gene (Barn HI), 5' terminal of S gene (HincII, Xba I) and 3' terminal of S gene (Acc I) of pcHB respectively. BALB/c mice were used in DNA immunization of the recombinant plasmids. Anti-HBs was detected using Abbott IMx AUSAB test kits.RESULTS: The sequences of MTE5 and the 6 constructs ofrecombinant plasmids were confirmed to be correct by DNA sequencing. The anti-HBs response of the coinoculation of pcHB and pcMTE5 was much higher than that of the inoculation of pcHB only (136.7+69.1 mIU/mL vs 27.6+17.3 mIU/mL, P<0.01, t =-6.56). Among the 4 chimeric plasmids, only the plasmid in which MTE5 was inserted into the pre-S2 region had good anti-HBs response (57.54±7.68 mIU/mL), and had no significant difference compared with those of pcHB and the co-inoculation of pcHB and pcMTE5. CONCLUSION: Exogenous multiple epitopes of HTL had immune enhancement when they were co-inoculated with pre-S2/S gene or inoculated in the chimeric form at a proper site of pre-S2/S gene of HBV. It might suggest that it was possible to improve hepatitis B vaccine using exogenous multiple epitopes of HTL. The antibody responses were very low using DNA immunization in the study. Thus, the immune enhancement effect of exogenous multiple epitopes of HTL has to be confirmed and the effect on overcoming the drawback of the polymorphism of HLA II antigens should also be evaluated after these chimeric plasmids are expressed in mammalian cell lines.

AIM:To design and construct an exogenous multiple epitope of helper T lymphocytes(HTL),and to evaluate its effect on anti-HBs response through DNA immunization. METHODS:Artificial HTL epitope,PADRE and four other HTL epitopes from different proteins were linked together using splicing by overlap extension to generate exogenous multiple epitopes of HTL,MTE5.pcNTE5 and pcHB were generated by cloning NTE5 and fragments of HBV pre-S2/S gene into mammalian expression plasmid pcDNA3.Four chimeric plasmids were constructed by cloning NTE5 into the region of pre-S2 gene(bam HI),5' terminal of S gene (HincⅡ,Xba Ⅰ)and 3' terminal of S gene(Acc Ⅰ)of pcHB respectively.BALB/c mice were used in DNA immunization of the recombinant plasmids.Anti-HBs was detected using Abbott INx AUSAB test kits. RESULTS:The sequences of MTE5 and the 6 constructs of recombinant plasmids were confirmed to be correct by DNA sequencing.The anti-HBs response of the co- inoculation of pcHB and pcMTE5 was much higher than that of the inoculation of pcHB only(136.7±69.1 mlU/mL vs 27.6±17.3 mIU/mL,P<0.01,t=-6.56).Among the 4 chimeric plasmids,only the plasmid in which MTE5 was inserted into the pre-S2 region had good anti-HBs response (57.54±7.68 mIU/mL),and had no significant difference compared with those of pcHB and the co-inoculation of pcHB and pcMTE5. CONCLUSION:Exogenous multiple epitopes of HTL had immune enhancement when they were co-inoculated with pre-S2/S gene or inoculated in the chimeric form at a proper site of pre-S2/S gene of HBV.It might suggest that it was possible to improve hepatitis B vaccine using exogenous multiple epitopes of HTL.The antibody responses were very low using DNA immunization in the study.Thus,the immune enhancement effect of exogenous multiple epitopes of HTL has to be confirmed and the effect on overcoming the drawback of the polymorphism of HLA Ⅱ antigens should also be evaluated after these chimeric plasmids are expressed in mammalian cell lines.