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AIM:To investigate whether P28 derived from C3d canenhance the immune response to HBV-preS2/S induced bydirectly injection of naked plasmids containing variablerepeats of P28 and HBV-preS2/S in fusion form.METHODS:One to four copies of C3d-P28 coding gene,amplified by PCR and modified by restriction endonucleasesdigestion,were subcloned into a eukaryotic expressionvector pVAON33 to construct pVAON33-P28,pVAON33-P28.2,pVAON33-P28.3 and pVAON33-P28.4 (pVAON33-P28.[1-4]).HBV-preS2/S coding sequence was thenintroduced into the pVAON33-P28.[1-4] and identified byboth PCR and DNA sequencing.BALB/c mice were primedby intramuscular gene immunization with 100μg differentrecombinant plasmids on day 0 and were boosted bysubcutaneous inoculation with HBsAg protein (1 μg) 12wk post-priming.The levels and avidity of specific IgG insera collected at the indicated times from each group weredetermined by ELISA and NaSCN-displacement ELISA,respectively.RESULTS:HBsAg specific antibody response was elicitedin groups primed with plasmids pVAON33-S2/S-P28.[1-4]and pVAON33-S2/S.However,the response against HBsAgin the groups primed with pVAON33-S2/S-P28.[1-4] wassignificantly higher than that in pVAON33-S2/S group,thehighest level of the specific antibody response was observedin the groups primed with pVAON33-S2/S-P28.4 (P<0.01).After secondary immunization with specific antigen,theacceleration of antibody levels was significantly higher andfaster in the mice primed with DNA expressing preS2/S-P28fusions than that with DNA expressing preS2/S only (P<0.05).Interestingly,mice primed with DNA expressing preS2/S-P28.4 fusions maintained the highest levels of anti-HBsantibodies in all animals.The avidity assay showed that theavidity index (AI) collected at 18 wk from mice primed withpVAON33-S2/S-P28.3 and pVAON33-S2/S-P28.4 weresignificantly higher than that from preS2/S-DNA vaccinatedmice (P<0.01). CONCLUSION:Different repeats of C3d-P28 can enhanceboth humoral immune response and avidity maturation ofspecific antibodies induced by gene immunization,in whichfour copies of C3d-P28 may be necessary to achieve themost modest antibody response.
AIM: To investigate whether P28 derived from C3d canenhance the immune response to HBV-preS2 / S induced by direct injection of naked plasmids containing variable fractions of P28 and HBV-preS2 / S in fusion form. METHODS: One to four copies of C3d-P28 coding gene, amplified by PCR and modified by restriction endonucleases digestion, were subcloned into an eukaryotic expression vector pVAON33 to construct pVAON33-P28, pVAON33-P28.2, pVAON33-P28.3 and pVAON33-P28.4 (pVAON33- ] 1-4) and identified byboth PCR and DNA sequencing. BALB / c mice were primed by intramuscular gene immunization with 100 μg of different recombinant plasmids on day 0 and were boosted bysubcutaneous inoculation with HBsAg protein (1 μg) 12wk post-priming. These levels and avidity of specific IgG insera collected at the indicated times from each group were determined by ELISA and NaSCN-displacement ELISA, respectively .RESULTS: HBsAg specific antibody response was elic [1-4] and pVAON33-S2 / S. However, the response against HBsAgin the groups primed with pVAON33-S2 / S-P28. [1-4] wassignificantly higher than that in pVAON33-S2 / S group, thehighest level of the specific antibody response was observedin the groups primed with pVAON33-S2 / S-P28.4 (P <0.01) .After secondary immunization with specific antigen, the acceleration of antibody levels was significantly higher and faster in the mice primed with DNA expressing preS2 / S-P28 fusions than that with DNA expressing preS2 / S only (P <0.05). Interestingly, mice primed with DNA expressing preS2 / S-P28.4 fusions maintained the highest levels of anti-HBs antibodies in all animals.The avidity assay showed that the avidity index (AI) collected at 18 wk from mice primed withpVAON33-S2 / S-P28.3 and pVAON33- S2 / S-P28.4 weresignificantly higher than that from preS2 / S-DNA vaccinated mice (P <0.01). CONCLUSION: Different repeats of C3d-P28 can enhanceboth humoral immune response and avidity m aturation ofspecific antibodies induced by gene immunization, in which copies copies of C3d-P28 may be necessary to achieve themost modest antibody response.