Effects Of Chitosan On The FMDV-DNA Vaccine For The Mucosal Immunization

Effects of Chitosan on the FMDV-DNA vaccine for the mucosal immunization Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, including cattles, pigs and sheep. OIE reported the highest murrain is FMD at 2002. It has become a dangerous of the development of stockbreeding. Scientists have made effort to get many vaccines such as conventional inactivated vaccine, protein vaccine and DNA vaccine. DNA vaccine was concerned because it has many characters such as safety, stability, low costing. It also has the ability to active simultaneous system immunoreaction and cells immunoreaction. Secretory immunoglobulin A (S-IgA) antibodies at mucosal system play an importantrole as a first line of defense against microorganisms that infect via mucosal surfaces. Thus, an important goal in halting the spread of mucosal surface transmitted diseases is the development of vaccines that induce local production and secretion of pathogen-specific, neutralizing S-IgA antibodies in mucosal surface. Systemic immunization routes do not generally induce secretion of specific S-IgA or protective immunity in mucosal tissues. Mucosal immune responses are initiated by uptake of antigens from mucosal surfaces into organized lymphoid tissues located in the mucosa or in nearby lymph nodes, where antigen-specific B cells are generated. Mucosal immunization with a variety of vaccines has been shown to induce disseminated secretory immune responses via the common mucosal immune system. However, the responses often are variable, transient, and low in magnitude. So the efficiency is still the primary problem of mucosal immunization. Therefore, it has been necessary to utilize delivery vehicles and adjuvants to potentiate immune responses to these vaccine antigens. One of several approaches which being investigated for effectiveness in augmenting immune responses to purified antigens is the use of chitosan as a vehicle for antigen delivery. Chitosan (CHI) is a polysaccharide and has been demonstrated as a potential gene encapsulation and delivery system. It could forms polyelectrolyte complexes with DNA and have the characters of biocompatibility, low immunogenicity, none cytotoxicity. So CHI was be researched as a nonviral gene delivery system. CHI as a DNA vaccine delivery system because it could forms nanoparticles complexes with DNA and this particles could entry cells. To get more uniformity particles, we depolymerized CHI got chitosan oligomers (DCH) with NaNO2. Due to various concentrations of NaNO2, the depolymerized chitosan will be arranging from high to low in oligomeric forms with low to high concentration of NaNO2 and designated as L-dechitosan(LDC), M-dechitosan(MDC) and H-dechitosan(HDC). DCH encapsulated plasmid DNA and formed particles. Electrophoratic mobility shows that the higher degree of depolymerization, the worse encapsulation capacity for plasmid is. Transmission electron microscopy (TEM) and Scanning electron microscopy (SEM) pictures showed DCH-DNA particles more uniformity than CHI-DNA. The diameters of the nanoparticles are about 100 nm. Zeta potentional result elementarily showed that DCH could form smaller particles with plasmid DNA than commercial chitosan. To test the stability, DCH-DNA complexes were treated with DNase and gastric juice. The result indicates that DCH have the ability of protect plasmid from DNase and gastric juice digestion. The expression of GFP has observed by fluorescence microscope after HEK293 cells were transfected with DCH-pEGFP-N3 nanoparticles for 48 hours. The phenomena indicated that DCH is an efficacious transfection reagent. DCH-pCMV-βwere administrated to KunMing White mice intranasal and intranoral. 7 days later the expression of plasmid pCMV-βcould seen after tissues stained byX-gal. Mice were immunized intranasal, intraoral, intrarectum and intravaginal with CHI-pcD-VP1 and DCH-pcD-VP1 (pcD-VP1). Plasmid pcD-VP1 is a DNA vaccine of FMDV (foot and mouth disease virus) made by our lab. SIgA antibodis of many mucosal surfaces and serum were tested. Results shows antibody of CHI-pcD-VP1 and DCH-pcD-VP1 groups increased. T cell proliferation of spleen T cells and Peyer's patches(PP)T cells demonstrated that CHI and DCH activated T cells at PP but spleen.