To investigate the antitumor immune responses induced by MAGE-3 DNA vaccine, the recombinant mammalian expression plasmid pcDNA3.1/MAGE-3 was constructed by ligating MAGE-3 gene, which was amplified by RT-PCR, and the pcD-NA3.1 + vector. The recombinant plasmids were transfected into B16 cells by liposome, the expression of MAGE-3 was checked by RT-PCR, immunocytochemistry and Western blot. Then, 100 ug recombinant plasmids were injected intramuscularly per C57BL/6 mouse on 0, 10 and 20 days, with pcDNA3.1 + plasmid and PBS as controls. Splenocytes CTLs, the level of antibodies against MAGE-3 the changes of the T lymphocyte subsets and the levels of cytokines were checked after 3 times immunization. As a result, the mice immunized with pcDNA3.1/MAGE-3 plasmid can produce MAGE-3 specific immune response. The CTLs kill activities against B16/MAGE-3 cells was 51.08 +/- 7.41%, and had significant difference (P < 0.01) compared with that of pcDNA3.1 + group (8.44 +/- 1.89%) and PBS group (5.76 +/- 1.75%). The titre of antibody against MAGE-3 was 1:15, while controls were negtive. The number of CD4 + CD8 + and the levels of IFN-gamma IL-2 increased significantly after immunization with pcDNA3.1/MAGE-3 plasmid as compared with those of control groups (P < 0.01). It is concluded that the pcDNA3.1-MAGE-3 DNA vaccine are able to induce both cellular and humoral immune responses in vivo.
Animals ; Antibodies, Neoplasm ; blood ; Antigens, Neoplasm ; biosynthesis ; genetics ; immunology ; Cancer Vaccines ; biosynthesis ; immunology ; Female ; Melanoma, Experimental ; prevention & control ; Mice ; Mice, Inbred C57BL ; Neoplasm Proteins ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; T-Lymphocyte Subsets ; immunology ; Vaccination ; Vaccines, DNA ; biosynthesis ; genetics ; immunology

Transduction of cytokine gene into tumor cells is a promising method of tumor therapy, but the value is limited by accompanying side effects. To focus antitumor immune response to tumor antigen-specific CTL, we developed an antitumor vaccine by transfecting modified IL-2 gene in a membrane-bound form (mbIL-2) into B16F10 melanoma cells. The mbIL-2 clone showed reduced tumorigenicity and metastatic ability, and inhibited metastasis and prolonged the survival of mice against B16F10 cells. The inhibition of B16F10 metastasis by mbIL-2 was accompanied by the increment of CD8+ T cells. The metastasis of mbIL-2 clone was significantly increased in the CD8+ T cell-depleted mice, but not in CD4+ T cell depleted mice. Spleen cells immunized with the mbIL-2 clone showed higher CTL activity towards B16F10 cells than those immunized with control cells. The size of CD8+ T cell population in the lung of mice injected with the mbIL-2 clone was markedly greater than that of mice injected with B16F10 cells, but there was no detectible change in CD4+ and CD8+ T cell populations of lymph nodes and spleen. These results suggest that when the mbIL-2 clone is introduced into the blood stream, it migrates mainly to lung and activates CD8+ T cells in situ, possibly by direct priming. Such a tumor vaccine may ameliorate the toxic side effects encountered with conventional cytokine gene therapy.
Animals ; CD4-Positive T-Lymphocytes ; CD8-Positive T-Lymphocytes/immunology ; Cancer Vaccines/*immunology ; Female ; *Genetic Engineering ; Interleukin-2/*genetics/metabolism ; Lung Neoplasms/*immunology/secondary/therapy ; Lymphocyte Activation ; Melanoma, Experimental/genetics/*immunology/*prevention & control ; Mice ; Mice, Inbred C57BL ; Research Support, Non-U.S. Gov't ; Spleen/immunology ; Survival Rate ; T-Lymphocytes, Cytotoxic/immunology ; Vaccination