Antitumor immunopreventive effect in mice induced by DNA vaccine encoding a fusion protein of alpha-fetoprotein and CTLA4.
OBJECTIVE
To develop a tumor DNA vaccine encoding a fusion protein of murine AFP and CTLA4, and to study its ability to induce specific CTL response and its protective effect against AFP-producing tumor.


METHODS
Murine alpha-fetoprotein (mAFP) gene was cloned from total RNA of Hepa1-6 cells by RT-PCR. A DNA vaccine was constructed by fusion murine alpha-fetoprotein gene and extramembrane domain of murine CTLA4 gene. The DNA vaccine was identified by restriction enzyme analysis, sequencing and expression. EL-4 (mAFP) was developed by stable transfection of EL-4 cells with pmAFP. The frequency of cells producing IFN-gamma in splenocytes harvested from the immunized mice was measured by ELISPOT. Mice immunized with DNA vaccine were inoculated with EL-4 (mAFP) cells in back to observe the protective effect of immunization on tumor. On the other hand, blood samples were collected from the immunized mice to check the functions of liver and kidney.


RESULTS
1.8 kb mAFP cDNA was cloned from total RNA of Hepa1-6 cells by RT-PCR. The DNA vaccine encoding a fusion protein of mAFP-CTLA4 was constructed and confirmed by restriction enzyme analysis, sequencing and expression. The expression of mAFP mRNA in EL-4 (mAFP) was confirmed by RT-PCR. The ELISPOT results showed that the number of IFN-gamma-producing cells in pmAFP-CTLA4 group was significantly higher than that in pmAFP, pcDNA3.1 and PBS group. The tumor volume in pmAFP-CTLA4 group was significantly smaller than that in pmAFP, pcDNA3.1 and PBS group, respectively. The hepatic and kidney functions in each group were not altered.


CONCLUSIONS
AFP-CTLA4 DNA vaccine can stimulate potent specific CTL responses and has distinctive antitumor effect on AFP-producing tumor. The vaccine has no impact on the function of mouse liver and kidney.
Tian
Geng
G
Departmentof General Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China. geng_tian707@hotmail.com
Yi
Ji-Lin
JL
Xiong
Ping
P
D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D060908:CTLA-4 Antigen; D019496:Cancer Vaccines / Q000494:pharmacology*; D045744:Cell Line, Tumor; D005260:Female; D007371:Interferon-gamma / Q000378:metabolism; D008113:Liver Neoplasms / Q000276:immunology / Q000517:prevention & control*; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids; D013154:Spleen / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology; D019444:Vaccines, DNA / Q000494:pharmacology*; D000509:alpha-Fetoproteins / Q000235:genetics / Q000276:immunology*
D015703:Antigens, CD; D000943:Antigens, Differentiation; D060908:CTLA-4 Antigen; D019496:Cancer Vaccines; C556707:Ctla4 protein, mouse; D019444:Vaccines, DNA; D000509:alpha-Fetoproteins; D007371:Interferon-gamma