Preparation Of Nanoemulsion-encapsulated Tumor Antigen Protein Vaccine And Study On Its Character And Antitumor Immunity

Tumor cells express antigens that can be recognized by the host'simmune system. These tumor-specific antigen (TSA) can generatetumor-specific cytotoxic T lymphocyte (CTL) and damage tumor cell.Today, tumor vaccine has played an important role in prevention andtherapy of tumor and has been regarded as the most attractive method.The melanoma antigen 1 (MAGE-1) was the first reported tumor specificantigen, which can induce specific CTL and show potency in tumorimmunotherapy. As molecular chaperone, HSP participates in processingand presentation of tumor antigen and plays an important role in elicitingantitumor immunity. Our former work has proved that MAGE-1 andHSP70 fusion protein can efficiently elicit MAGE-1-specific antitumorimmunity and enhance the potency of MAGE-1 protein vaccine.However, protein vaccine still has some serious limitations. First, theactivation of antigen-specific CTL responses requires the delivery ofsignals from CD4+T cells, monocytes and granulocytes recruited at the site of vaccination. Such signals may not be provided by antigen protein alone. Second, Proteins are unstable, high molecular weight, hydrophilic, complicated in structure, larger in size, they are harder to deliver by conventional ways as compared to classical molecules. The main barriers to successful delivery of proteins are enzymatic barriers and absorption barriers imposed by gastrointestinal tract. So it is important to change the formulator of proteins to maximize physical and chemical stability, prolong biological half-life, increase absorption and minimize metabolism.In this research, the MAGE-1-HSP70 fusion protein and superantigen SEA were mixed in a suitable ratio as complex protein vaccine; then this vaccine was captured in nanoemulsion which is a kind of protein drug carrier. The character of this nano-vaccine was investigated. Their humoral and cellular antitumor immunity were evaluated and the therapeutic effects against established tumor expressing MAGE-1 were observed.1. BSA-FITC-Nanoemulsion was prepared by magnetic stirring methods. The shape and size of NE-BSA-FITC were observed under electron microscope and the mean size of NE-BSA-FITC was 25 ±10 nm. The encapsulation rate and stability were determined by Sephedex-G100 chromatography. The result showed that the encapsulation rate was 91% and NE-BSA-FITC has a good stability. Then nanoemulsion was cocultured with Murine BMDCs in vitro and macrophages (M~) in vivo, after 30min the uptake of NE-BSA-FITC byDCs and M? was detected by FCM. The results of FCM showed that both cells can take in more NE-BSA-FITC than free BSA-FITC. The NE BSA-FITC was located in plasma of DCs when observed by laser confocal microscopy. These results showed Nanoemulsion can be efficiently uptaked by DCs and macrophage, so it can be promised as an efficient carrier of APCs-targeted antitumor vaccine. 2. The expression plasmids of pet-MAGE-l-HSP70 and pet-SEA were constructed and the BL21(DE3) containing the plasmid was induced. The fusion proteins were purified using Ni-NTA Resin column. Those two proteins were mixed in different ratio from 1000:1 to 1:1. Then the C57BL/6 mice were immunized using different groups of complex protein vaccine and spleen lymphocytes were isolated. The quantity of MAGE-1 specific T lymphocytes was identified by ELISPOT assay. The result demonstrated that the spleen lymphocytes, isolated from the complex protein (which ratio was 100:1) immunized mice, had significant more MAGE-1 specific T cells (p<0.05) than in those groups immunized with MAGE-1-HSP70 or SEA only. The results of LDH release assay showed that spleen lymphocytes from the complex protein (which ratio was 100:1) immunized mice had a higher cytotoxicity to B16-MAGE-l(p<0.05). The therapeutic effects of the complex protein vaccine to the tumor bearing mice demonstrated that MAGE-1-HSP70 and SEA complex protein vaccine could strongly delay the growth of B16-MAGE-l(p<0.05) when compared with MAGE-1-HSP70 or SEA only. The results above showed that complex protein vaccine of MAGE-1-HSP70 and SEA can enhance the potency of MAGE-1 proteinvaccine.3. Nanoemulsion-Encapsulated complex protein vaccine(MAGEl-hsp70 and SEA) was prepared and its shape and size were detected by electron microscope. The mean size of NE complex was 25±10 nm, the encapsulation rate was 89%. C57BL/6 mice were immunized intraperitoneal with NE complex vaccine, free complex vaccine, blank NE and PBS as negative control. The anti-tumor immunity was detected by Enzyme-linked immunospot assay( ELISPOT) and Cytotoxicity assays. Then the therapy effect of NE complex vaccine was detected among the mice which had bearing B16-MAGE1 melanoma. ELISPOT and Cytotoxicity assays showed the immunization using NE complex vaccine enhanced the frequency of splenocytes secreting Y -IFN significantly (p<0.05) when compared with immunization using free complex vaccine alone. Cytotoxicity assays showed the MAGE1 B16 lysis of CTLs from mice immunized with NE complex vaccine was greater than that from mice immunized with free protein vaccine. The tumor masses formed in the mice immunized with NE complex vaccines markedly smaller(p<0.05) than those immunized with free complex vaccines. The mice planted tumor of B16-MAGE-1 had significant longer survival times, after therapy with NE complex vaccine (P<0.05). These mice had average survival times of 72+4 days, and survival extended ratios of 76.5% respectively, which are significant higher than the groups treated with free complex vaccine and PBS. These results showed that Nanoemulsion had novel characters, and Nanoemulsion-Encapsulated complex vaccine could...