title: DNA immunization with recombinant HCV E2 expression plasmids.
abstract: DNA-based immunization, the delivery of plasmid DNA by direct inoculation, is a newly developed method of vaccination. Besides of many advantages, DNA immunization was demonstrated to generate weaker antibody and CTL responses than did protein and live attenuated vaccines, respectively. To circumvent this shortage, several methods were tested such as using different vector, changing injection mode, and co-expressing cytokines, etc. The studies showed that many factors could greatly affect the immune responses to DNA vaccine. The aim of this study is to compare different vectors for the presentation of the HCV E2 to generate immune responses by using DNA-based immunization. Four expression plasmids, with different promoter types and with or without signal sequences were constructed, which encode C-terminally truncated E2 (384-660) with HBV preS1  21-47  tag fused to its N-termini. Transient expression in HeLa cells showed that only recombinant plasmids with signal sequence could be expressed and properly processed, and the product could be secreted into medium. Protein expression level is slightly higher in plasmids with CMV promoter than with EF1alpha promoter. After immunization of C57BL/6 mice, all the recombinant constructs could elicit both anti-preS1 and anti-E2 antibodies. But only pCMV Sec-S1E2t660 with both CMV promoter and signal sequence could induce high-level and long-lasting antibody, showing that it is a good vaccine candidate. Possible reasons for the different immune responses to these constructs were discussed.
authors.affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China. wangyuan@server.shcnc.ac.cn
authors.forename: Jun
authors.initials: J
authors.lastname: Zhu
authors.forename: Chun-Lin
authors.initials: CL
authors.lastname: Wang
authors.forename: Li-Xin
authors.initials: LX
authors.lastname: Zhu
authors.forename: Yu-Ying
authors.initials: YY
authors.lastname: Kong
authors.forename: Yuan
authors.initials: Y
authors.lastname: Wang
authors.forename: Guang-Di
authors.initials: GD
authors.lastname: Li
mesh_terms: D000818:Animals; D000906:Antibodies / Q000097:blood; D005260:Female; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000276:immunology / Q000378:metabolism; D019444:Vaccines, DNA / Q000276:immunology*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D000906:Antibodies; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus