| In China, more than 90% of the original liver cancers are hepatocellular carcinoma, and it counts for the second most cancer-caused death. Generally, patients seek therapy late, the therapeutic results are bad, the recurring-rate is high, and the therapeutic means are limited. Recently, genetic cancer vaccine has become a promising method in liver cancer treatment. Efficient therapeutic programs combining multiple genetic therapy methods have been developed, and the results were very encouraging. DNA vaccine, also called genetic vaccine, is made by cloning DNA fragment encoding carcinoma antigen into eukaryotic expression system. Later, immunization is performed by injection of the recombinant DNA. DNA vaccine encoding foreign protein can express certain antigen after direct injection of the DNA, and this antigen will induce antigen-specific humoral and cell-mediated immune responses. Genetic vaccine can express the antigen continuously, therefore continuously stimulate the immune system and induce comprehensive and long-lasting humoral and cell-mediated immune responses.During anti-tumor immunological responses, T-cells need two types of signals to be effectively functional, the tumor antigen signal and the cooperation stimulation signal. Since most tumors come from the tissues of the patients themselves, generally they cannot provide efficient antigen signal to induce CTL. Or in some tumors the related immune factors are mutated or down-regulated, so that they cannot convey normal functions. Since most tumors have very weak immunogenicities, immuno-stimulants, such as immuno-adjuvants, cytokines, costimulatory molecules, heat-shock protein need to be added to the vaccine to improve their immunogenicities.This study focuses on the hepatocellular carcinoma-associated antigens 661 (HCA661), also called TFDP3 or CT40, which belongs to the CT antigen family. In normal tissues, HCA661 is only constantly expressed in testis, and weakly expressed in pancreas. In tumor tissues, HCA661 is highly expressed in hepatocellular carcinoma, and weakly expressed in melanoma, so it is considered as a HCC-associated antigen. HCA661 protein has immunogenicity. It is able to induce antigen-antibody responses, stimulate the transformation from natural T cells to the cytotoxic T lymphocytes, and it has specific fetal effects. All these properties provide a new way for immunotherapy. Recent studies showed that HCA661 could block the transcription of endogenous E2F, therefore inhibit the E2F-mediated cell proliferation. We made a genetic vaccine using HCA661 to induce HCA661-specific immune responses after direct injection of the vaccine DNA into the muscle of the animal, thereby inhibit the growing of the tumor. For the adjuvant, we chose the C-terminal stimulatory domain of mtHSP70, which contains 7 essential amino acids, Q407, P408, V410, Q411, E420 and H424.This domain can largely improve the CD40L-induced DCs maturation and increase the phosphorylation of p38MAP in the CD40-HSP70 pathway. mtHSP70 is a CD40 ligand, which can rapidly and directly induce the secretion of multiple cytokins from macrophages and dendritic cells by interacting with CD40. It can also activate the humoral immune responses by activating CTL and CD4+Th cells through MHCI and MHCII pathways. The C-terminal stimulatory domain of mtHSP70, P407-426, has the complete immuno adjuvant function, but with a better performance by reducing the self-immune responses caused by the full length mtHSP70.This study applied multiple molecular techniques, such as PCR, restricted endonuclease digestion, ligation, etc., to construct a recombinant genetic vaccine encoding fusion protein of HCA661 and mtHSP70, P407-426. The construct was verified by PCR, endonuclease digestion and sequencing. In this construct, HCA661 is the target of the immunotherapy, and mtHSP70E help to improve the immunogenicity of the vaccine as an immunoadjuvant.To carry out the CTL fetal effects experiment, pCI-HCA661-EGFP was transfected into NIH3T3 cells by liposome transfection system, and expression of the protein was detected by visualizing the green fluorescence of the GFP. Stable expression cell-line was established by G418 screening. HCA661-EGFP mRNA and protein levels in transfected cells were examined respectively by RT-PCR and western blotting. The results showed that the recombinant pCI-HCA661-EGFP was stably expressed in the cells. This cell line was used to carry out the CTL fetal effects experiments, therefore provides a solid foundation for our following work.We also compared the immunological effects of pCI-HCA661-mtHSP70E and pCI-HCA661-mtHSP70C (the complete C-terminus of mtHSP70). The spleen and serum of mice injected with pCI-HCA661-mtHSP70E and pCI-HCA661- mtHSP70C were collected, and subjected to flow cytometry and MTT to examine the subgroups of T cells, the level of CD4+/CD8+, and the HCA661-specific lethal effects of the spleen cells. Western blotting, ELISA, and immunohisto- chemistry analysis were used to evaluate the levels of the polyclonal antibodies of HCA661 and mtHSP70E in mouse serum. Results: (1) flow cytometry results showed that, immunized mice (by pCI-HCA661-mtHSP70E and pCI-HCA661- mtHSP70C respectively) had significantly higher levels of CD3+, CD4+, CD8+ compare to the controls. The immunized mice also have significantly more T cells and higher CD4+/CD8+ values then the controls do. Furthermore, pCI-HCA661- mtHSP70E immunized mice have significantly higher CD4+/CD8+ values then the mice immunized with pCI-HCA661-mtHSP70C. (2) Specific lethal effect experiments showed that the immunized mice had higher CTL killing-effects than controls, while pCI-HCA661-mtHSP70E immunized mice had higher level than the pCI-HCA661-mtHSP70C immunized mice. (3) Western blot and immuno- histochemistry showed that immunized mouse serum contained anti-HCA661 antibody, while pCI-HCA661-mtHSP70E immunized mice had higher level of staining than the pCI-HCA661-mtHSP70C immunized mice under the same dilution condition. (4) ELISA showed that pCI-HCA661-mtHSP70E immunized mice had higher dilution, which means they have higher antibody concentration in their serum. (5) Immunized mice do not produce antibody against mtHSP70. These results indicate that: (1) Recombinant genetic vaccine pCI-HCA661-mtHSP70E and pCI-HCA661-mtHSP70C can effectively induce specific and unspecific immune response in mouse. (2) pCI-HCA661-mtHSP70E can induce higher level of immune responses than pCI-HCA661-mtHSP70C. (3) The C-terminal stimulatory domain of mtHSP70, P407-426 is a stronger immunoadjuvant, and can effectively reduce the self-immune responses caused by mtHSP70 whole protein.To summary, this study successfully constructed pCI-HCA661-mtHSP70E fusion protein. Furthermore, we verified that this recombinant gene is more efficient compare to the pCI-HCA661-mtHSP70C in inducing humoral and cell-mediated immune responses, which indicate that p407-425 can decrease the self-immune responses caused by mtHSP70, and is a stronger immunoadjuvant than mtHSP70C. We also established a mouse cell line, which can stably express HCA661, therefore provide a good foundation for our future work.
|
PDF Full Text Request