Preparation Of A Novel M Cell-targeting Mucosal Vaccine Against CVB3-induced Myocarditis

Mucosal immune system acting as an entry ports for antigens are important to viral and pathogens attacks. The specific mucosal immune responses induce in mucosal surface is very important in anti-virus infection in the body. Thus researchers are interested in mucosal vaccine development. Due to their inadequate capacity to induce strong immune response and tendency to induce immune tolerance, only a handful of mucosal vaccines are available for people. Therefore, how to improve the efficacy of mucosal vaccine has been an important question.The M cells, specifically located on the follicle-associated epithelium (FAE) of the Peyer’s patch (PP) follicle, have fewer microvillus, more micorfold and the basolateral of M cell is uncontinuous in which lymphocytes are allowed passing freely. The special structure of M cell is benefit for antigens uptake and delivered. It allows the antigens rapidly transport by M cell from luminal to the underneath APC (antigens processing cells) such as DCs. M cell plays a key role in mucosal immune system. And it provides new strategies for oral mucosal vaccine.1. Compared with vital function of M cells, there are fewer M cells in mucosal surface. Thus we development a M cell-targeting deliver system. Glycoprotein-2 (GP2) is specially expressed in both human and mouse M cells. It has been found that FimH, a component of type I pili expressed on E. coli, is the ligand of GP2, which can interact with GP2 protein on the apical area of M cells. Thus, we introduced FimH protein into the CS-pVPl vaccine system to prepare a novel M-cell targeting vaccine CS-FimH-pVPl for better prevention against CVB3-induce viral myocarditis. Results so far indicate:Contraction of M-cell targeting vaccine CS-FimH-pVPl and characterization of physicochemical properties.The FimH protein carboxyl are activated by EDC, and then interact with chitosan for 24h. In this way, about 60% of the FimH protein has been coupling with chitosan. Then we prepare the M-cell targeting particles CS-FimH-pVP1 by co-acervation method. The diameter of CS-FimH-pVPl particles were about 200-400nm. And we find the M-cell delivery system can protect the DNA from the DNase1 degradation.2. CVB3-spectific immune response and immunoprotection of mice to oral immunization of M-cell targeting vaccine CS-FimH-pVP1.BALB/c mice, ages 6 to 8 weeks, were divided into four groups with oral immunization by different vaccines (CS-FimH-pVP1/CS-pVP1/CS-FimH-vector /CS-vector). In CS-FimH-pVP1 group, the CVB3-specfic fecal sIgA titers and avidity index were significantly higher than control group. Then we detected the T cell proliferation, specific CTL activity and IFN-y-producing T cell of mice immunized with CS-FimH-pVP1. The results showed that CS-FimH-pVP1 has increased levels of T cell proliferation, specific CTL activity and IFN-y-producing T cell in MLN. On the contrary, there is no significant increases of T cell proliferation, specific CTL activity and IFN-y-producing T cell in spleen. We can come to a conclusion that M-cell targeting vaccine CS-FimH-pVPl could induce CVB3-specific mucosal immune response but not systemic immune response.We had detected the immunoprotection of mice to oral immunization of M-cell targeting vaccine CS-FimH-pVP1. The weight change, serum CK and CK-MB of mice in CS-FimH-pVPl group is not obvious when challenged with 3LD50 CVB3. Histopathogical observation of heart tissue showed that there is reduced infiltration of lymphocytes and myocardial injury in CS-FimH-pVP1 compared with CS-pVP1 group. Viral titer of mice immunized with CS-FimH-pVP1 has a dramatic decline, indicating that the immune response induced by CS-FimH-pVPl can clean the viral effective. Echocardiographic Assays showed that no damage in heart functions of mice immunized with CS-FimH-pVPl. And the CS-FimH-pVPl vaccine can greatly increase mice survival rates.3. The mechanism research of CVB3-specific immune responses induced by the M cell targeting mucosal vaccine CS-FimH-pVP1.In this part, first we successfully construct the in vitro M-cell model. And we evaluate the translocation of CS-FimH-pVP1 and CS-pVPl in M-cell model. The results showed that the translocation amount of CS-FimH-pVPl is higher than CS-pVPl and it is time dependent. According to the immunofluorescent assay we found that there is more co-colocation of CS-FimH-pVP1 and GP2 in vitro and in vivo compared with CS-pVP1. These results indicated that the higher translocation of CS-FimH-pVP1 is mediated by FimH protein.Finally, combining the above results, we have successfully constructed the M-cell targeting vaccine CS-FimH-pVP1 and effective CVB3-specific immune response and immune protection can be induced by CS-FimH-pVP1 vaccine. This may be new strategies which provide a new immunology treatment of viral myocarditis induced by CVB3.