| Early from the end of 1960s, scientists have already begun working on anti-caries vaccinations. A lot of scholars have done much in immunity agents, immunity adjuvants and immunity approaches.Along with the development of immunity, molecular biology, gene engineering, and with much research in carious antigen of S. mutans (e.g. PAc, GTFs, GBP), several novel anti-caries vaccines have been acquired. This study intended to adopt GbpA/gbpA as immunity antigens, by using chitosan to replace the traditional adjuvants and to alter injectable vaccine model to mucosal vaccination. Compared with the injectable vaccines, the striking advantage of the mucosal vaccines is their potential to generate both a systemic and local immune responses. Further more, it is much more convenient to administer vaccine via mucosal surfaces. However, mucosal vaccination usually fails to induce ideal effect that we expected, especially with soluable antigens. Therefore, mucosal immunity adjuvants or carriers would play a key role in this process.Recently, a naturally occurring source of cationic polysaccharide chitosan as a delivery system has been used in peptides, proteins, DNA vaccines delivery and cell transfections and achieved ideal results. Chitosan has been considered for drug delivery applications in which attention has been focused on its absorption enhancing, controlled release and bioadhesive properties. Chitosan is bioadhesive and able to interact strongly with the nasal mucus layer and with the nasal epithelial cells. Hence, nasal chitosan drug formulations can prolong the residence time at the absorption sites and lead to the increased bioavailability of the vaccine in nasal membrane. Many studies have demonstrated that chitosanappears to increase cell permeability by affecting paracellular and intracellular pathways. Chitosan nano- and microparticles are also suitable for controlled drug release. Chitosan causes relatively mild and reversible effects on epithelial function and morphology. From the above review, it is concluded that chitosan and chitosan derivatives are promising polymeric excipients for mucosal drug and vaccine delivery. Chitosan carrier system has not yet used as anti-caries adjuvants. The aim of this study was to develop and characterize GbpA/gbpA-chitosan nanoparticles and to evaluate their immune efficacy in mice intranasally.This study consisted of three parts:Part One: Preparation and evaluation of GBD/pcDNA3.1-GBD -chitosan nanoparticles vaccinesFrom abundant literatures, we selected proper formulations and preparation of GBD/pcDNA3.1-GBD-chitosan nanoparticles vaccines. We prepared the blank chitosan nanoparticles system, GbpA-chitosan nanoparticles vaccine and two kinds of pcDNA3.1-GBD chitosan nanoparticles vaccines. The results indicated that GbpA/gbpA could be encapsulated to chitosan and form nanoparticles. They are about 50-100nm in size and spherical in shape, distributing in mass. The encapsulation efficiency and capacity of GbpA nanopaticles were respectively 99% and 43%. No plasmid was examined in the supernatant. No GbpA/gbpA was released at 4 during 1 month. CLSM visualization studies showed that GbpA was entrapped within the chitosan nanoparticles and not only associated with their outer surface as some people held. Supernatant GbpA and GbpA were analyzed by phase gel electrophoresis system and showed the same protein molecular level. The prepared anti-caries vaccines have high encapsulation efficiency , capacity and stability. GbpA did not decompose in the preparation processs.Part Two: Nasal vaccination study: assessment of local and systemic immune responsesFor the nasal vaccination study in mice, each group received vaccines in week 1, 3 and 4, and serum and salivary samples were collected in week 2, 4, 6 and 8. GBD-specific IgG antibodies and GBD-specific IgA were measured by an adapted method enzyme-linked immunosorbent assay (ELISA). The results indicated that chitosan-GbpA/gbpA nanoparticles as novel nasal deliveryvaccines could induce protective spe...
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