[C3d enhances the immune response against HBV-preS2/S following gene immunization in mice].
OBJECTIVE
To investigate whether C3d can enhance the immune response to HBV-preS2/S induced by direct injecting naked plasmid containing three copies of C3d and HBV-preS2/S in fusion form.


METHODS
HBV-preS2/S coding sequence was introduced into the eukaryotic expression vectors TR421 and TR421-C3d3 containing 3 copies of C3d respectively. PCR was used to identify the direction of insertion, DNA sequencing analysis was used on the positive clones. The recombinant plasmids TR421-preS2/S and TR421-preS2/S-C3d3 were transfected into the human breast cancer cells of the line 4T1 as a transient expression system. Semi-quantitative RT-PCR was used to detect the expression of HBV-preS2/S protein. TR421-preS2/S and TR421-preS2/S-C3d3 were injected into the anterior tibial muscles of female BALB/c mice, 6 in each group, 3 times at an interval of 3 weeks. Six mice were injected with blank plasmid TR421 as controls. The total blood was collected from the mice. ELISA was used to detect the level of specific anti-HBs antibody. The splenocytes of the immunized mice were collected and stimulated with HbsAg protein and then harvested to analyze the specific lympho-proliferative response by (3)H-thymidine incorporation assay. CR2 positive Raji cells were added with plasmid transfected and mouse anti-HBs serum. Flow cytometry was used to detect the integration of preS2/S-C3d3 fusion protein.


RESULTS
The 4T1 cells transfected with TR421-preS2/S and TR421-preS2/S-C3d3 effectively expressed HBV-preS2/S, with the expression level of TR421-preS2/S-C3d3 lower by 15.25% than that of TR421-preS2/S. Twelve weeks after immunization, the specific anti-HBs-IgG level (A(490nm)) was 0.81 +/- 0.09 in the TR421-preS2/S primed mice, significantly higher than that in the blank plasmid injected mice (0.49 +/- 0.02, P < 0.05); and the specific anti-HBs-IgG level (A(490nm)) was 1.24 +/- 0.29 in the TR421-preS2/S-C3d3 immunized mice, significantly higher than that of the TR421-preS2/S primed mice (P < 0.001). The lympho-proliferative response of the TR421-preS2/S primed mice was significantly stronger than that of the blank plasmid injected mice (P < 0.05) and the lympho-proliferative response of the TR421-preS2/S-C3d3 primed mice was significantly stronger than that of the TR421-preS2/S primed mice (P < 0.05). Surface binding of protein could be detected only in the TR421-preS2/S-C3d3 transfected supernatant. About 43.19% of the CR2 positive Raji cells integrated C3d fusion protein. TR421-preS2/S transfected supernatant and negative control did not show obvious integration.


CONCLUSIONS
C3d enhances the humoral and cell-mediated immune responses against HBV induced by gene immunization.
Guan
Qing-Dong
QD
Department of Immunology and Key Laboratory of Molecular Medicine of Ministry of Education, Fudan University, Center for Gene Immunization and Vaccine Research, Shanghai 200032, China.
Wang
Li-Xin
LX
Guo
Qiang
Q
Zhang
Jin-Ping
JP
Xu
Wei
W
Wang
Ying
Y
Xiong
Si-Dong
SD
D000818:Animals; D000917:Antibody Formation; D015933:Complement C3d / Q000235:genetics* / Q000276:immunology; D005260:Female; D006509:Hepatitis B / Q000517:prevention & control; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D011498:Protein Precursors / Q000235:genetics* / Q000276:immunology; D017464:Receptors, Complement 3d / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D014162:Transfection; D019444:Vaccines, DNA / Q000276:immunology
D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D017464:Receptors, Complement 3d; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; C050807:presurface protein 2, hepatitis B surface antigen; D015933:Complement C3d