The use of nanoparticles as a delivery system for a specific antigen could solve many limitations of mucosal vaccine applications, such as low immunogenicity, or antigen protection and stabilization. In this study, we tested the ability of nasally administered chitosan nanoparticles loaded with glycoprotein B of murine cytomegalovirus to induce an immune response in an animal model. The choice of chitosan nanoparticle type was made by in vitro evaluation of sorption efficiency and antigen release. Three types of chitosan nanoparticles were prepared: crosslinked with tripolyphosphate, coated with hyaluronic acid, and in complex with polycaprolactone. The hydrodynamic size of the nanoparticles by dynamic light scattering, zeta potential, Fourier transform infrared spectroscopy, scanning electron microscopy, stability, loading efficiency, and release kinetics with ovalbumin were evaluated. Balb/c mice were immunized intranasally using the three-dose protocol with nanoparticles, gB, and adjuvants Poly(I:C) and CpG ODN. Subsequently, the humoral and cell-mediated antigen-specific immune response was determined. On the basis of the properties of the tested nanoparticles, the cross-linked nanoparticles were considered optimal for further investigation. The results show that nanoparticles with Poly(I:C) and with gB alone raised IgG antibody levels above the negative control. In the case of mucosal IgA, only gB alone weakly induced the production of IgA antibodies compared to saline-immunized mice. The number of activated cells increased slightly in mice immunized with nanoparticles and gB compared to those immunized with gB alone or to negative control. The results demonstrated that chitosan nanoparticles could have potential in the development of mucosal vaccines.

• MeSH
• adjuvancia imunologická MeSH
• aplikace intranazální MeSH
• chitosan * chemie   MeSH
• glykoproteiny MeSH
• imunizace MeSH
• imunoglobulin A MeSH
• Muromegalovirus * MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nanočástice * chemie   MeSH
• slizniční imunita MeSH
• vakcíny * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-D-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite, N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other His.

• MeSH
• acetylglukosamin chemie   metabolismus   MeSH
• chromatografie afinitní MeSH
• endotoxiny biosyntéza   izolace a purifikace   MeSH
• Escherichia coli metabolismus   MeSH
• glutaminfruktosa-6-fosfáttransaminasa (izomerizující) biosyntéza   izolace a purifikace   MeSH
• kultivační média chemie   MeSH
• myši MeSH
• proteiny tepelného šoku HSP70 biosyntéza   izolace a purifikace   MeSH
• průmyslová mikrobiologie metody   MeSH
• rekombinantní proteiny biosyntéza   izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH