Contribution of C3d-P28 repeats to enhancement of immune responses against HBV-preS2/S induced by gene immunization.
OBJECTIVE
To investigate whether P28 derived from C3d can enhance the immune response to HBV-preS2/S induced by directly injection of naked plasmids containing variable repeats 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 a eukaryotic expression vector 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 then introduced into the pVAON33-P28.[1-4] and identified by both PCR and DNA sequencing. BALB/c mice were primed by intramuscular gene immunization with 100 microg different recombinant plasmids on day 0 and were boosted by subcutaneous inoculation with HBsAg protein (1 microg) 12 wk post-priming. The levels and avidity of specific IgG in sera collected at the indicated times from each group were determined by ELISA and NaSCN-displacement ELISA, respectively.


RESULTS
HBsAg specific antibody response was elicited in groups primed with plasmids pVAON33-S2/S-P28.[1-4] and pVAON33-S2/S. However, the response against HBsAg in the groups primed with pVAON33-S2/S-P28.[1-4] was significantly higher than that in pVAON33-S2/S group, the highest level of the specific antibody response was observed in 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 with pVAON33-S2/S-P28.3 and pVAON33-S2/S-P28.4 were significantly higher than that from preS2/S-DNA vaccinated mice (P<0.01).


CONCLUSIONS
Different repeats of C3d-P28 can enhance both humoral immune response and avidity maturation of specific antibodies induced by gene immunization, in which four copies of C3d-P28 may be necessary to achieve the most modest antibody response.
Wang
Li-Xin
LX
Department of Immunology, Shanghai Medical College of Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, China.
Xu
Wei
W
Guan
Qing-Dong
QD
Chu
Yi-Wei
YW
Wang
Ying
Y
Xiong
Si-Dong
SD
D000818:Animals; D000917:Antibody Formation; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D015933:Complement C3d / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D018628:Gene Dosage; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics*; D017464:Receptors, Complement 3d / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000276:immunology*
D006514:Hepatitis B Surface Antigens; D010455:Peptides; D011498:Protein Precursors; D017464:Receptors, Complement 3d; D011993:Recombinant Fusion Proteins; C050807:presurface protein 2, hepatitis B surface antigen; D015933:Complement C3d