Study On CpG Motif As Immunoadjuvant Of Tumor DNA Vaccine

Tumor gene vaccine was based on tumor-associated antigen (TAA) or tumor-specific antigen (TSA). The gene encoding TAA or TSA fragments inserted into eukaryotic expression vector, and then immunized animals with the recombinant vector. Antigen gene of purpose can be expressed in the host body, and induce tumor antigen-specific humoral and cellular immune responses to eliminate tumor cells. The effect of tumor gene vaccine was determined by the tumor antigen, carrier itself, vaccine adjuvants and the immunization program and their interactions.Adjuvant was used with antigen at the same time or in advance, which can enhance the body's immune response against the antigen, or change the type of immune response. Research on DNA vaccine adjuvant could improve DNA vaccine's ability to stimulate the body's immune response, as molecular biology and molecular immunology development, cytokines, CpG ODN, complement C3d and other bioactive molecules were discovered can be used as adjuvant.CpG motif was a class of repeat sequences, non-methylated cytosine guanine dinucleotide (CpG) as the core of the repeat sequences. CpG DNA could improve the activity of professional APC, enhance natural immunity and acquired immune responses. CpG DNA could activt dendritic cells, monocytes and macrophages directly, increased expression of costimulatory factors, thereby enhancing the processing and presenting of antigen on antigen presenting cells, levels of Thl type antigen-specific antibody and accelerate the antigen-specific CTL response. CpG motif was widely accepted as gene vaccine adjuvant. According to the difference of structure and stimulation in vitro on human peripheral blood mononuclear cells (PBMC), CpG could be divided into A, B, C types. A-type CpG could lead to antigen presenting cells maturation, stimulat dendritic cells (Plasmacytoid dendrtic cell, pDC) to secrete IFN-a, which strongly activate NK cells to produce IFN-y. B-type CpG was able to directly stimulate B lymphocyte proliferation which secrete large quantities of immunoglobulin, cytokines, and induce pDC maturation. C-type CpG was a newly discovered class of sequences, both A-and B-type features.CpG DNA could optimize DNA vaccine vector backbone, that was CpG immunostimulatory sequence inserting into the appropriate location of the vector. The position of CpG inserting into and the sequence number of CpG structure could influence the immune effect of the DNA vaccine. Studies have shown that, the backbone of DNA vaccine vector VR1012 insertde into CpG DNA, could significantly enhance immune response against plasmid DNA induced by the human immune cells. In a report,16 CpG motifs in the DNA vaccine can enhance the humoral immune response, while the import of 50 the same CpG motif has reduced the same immune response.Our laboratory have constructed DNA vaccine VR-MS which expressed tumor-associated antigen MUC I and Survivin fusion protein (MS), and adenovirus vaccine AD-MS. Studies have shown that VR-IL-2 which express IL-2 as adjuvant of immunity, and DNA prime-rAD boost immunization programs, can effectively produce specific cellular and humoral immune response in mice, and on immune mice treated with good results.To further enhance the immunogenicity of DNA vaccines, we insert 16 C-CpG immunostimulatory sequences into the backbone of VR-MS vaccine vector, construct tumor gene vaccine CpVR-MS, and examined whether it can increase immune effect and security of the vaccine. The results showed that compared with VR-MS/VR-IL-2, adding CpG DNA vaccine adjuvant can significantly enhance the level of humoral immunity. The level of cellular immunology could also be detected significantly increased by the Elispot method. When MUC I protein was the stimulus, the number of spots produced by Cp VR-MS vaccine is nearly twice of by VR-MS vaccine. In the therapeutic experiment, compared with VR-MS group, the inhibitory rate of Cp VR-MS vaccine did not significantly improve, but the life extension rate nearly doubled, which is a very valuable advance in cancer therapy.Bicistronic expression vector was a carrier with two ORF, one ORF can be inserted into a tumor antigen gene, another ORF inserted into adjuvant,. The vaccine used this kind of vector can improve the immune response, this method has been widely used in a variety of virus vaccine, and induced a higher immunogenicity. The most commonly used method of construction bicistronic expression vector is to be with two separate expression cassette promoter construct in a vector, and then transcript different mRNA and translate different proteins.In order to improve the safety of the tumor gene vaccines, and reduce production cost, we constructed bicistronic vector vaccine DV-IL-2-MS,that express IL-2 and MS. Compared with VR-MS/VR-IL-2, investigated bicistronic vector vaccine's efficacy and safety. The results show that bicistronic vector vaccine DV-IL-2-MS did not play on the optimal form of VR-MS/VR-IL-2, immune response and antitumor effect of vaccine DV-IL-2-MS did not achieve the VR-MS/VR-IL-2 levels.Reason may be to (1) The size of DV-IL-2-MS plasmid increased, and in the same injection dose, molar of DV-IL-2-MS plasmid was less than VR-MS, so the expression of MS antigen was reduced, immune effect was reduced too; (2) The size of vaccine DV-IL-2-MS was significantly increased, the difficulty of its uptake by muscle cells and entering nuclers was increased, so that the expression of MS antigen was decreased; (3) The expression of IL-2 was significantly reduced, IL-2 did not play the effect of immune enhancement.In summary, we insert 16 C-CpG immunostimulatory sequences into the backbone of VR-MS vaccine vector, construct tumor gene vaccine CpVR-MS, humoral and cellular immunity have been significantly improved, When MUC I protein was the stimulus, the number of spots produced by Cp VR-MS vaccine is nearly twice of by VR-MS vaccine. In the therapeutic experiment, compared with VR-MS group, the life extension rate of Cp VR-MS vaccine was nearly doubled, which is a very valuable advance in cancer therapy.