Preparation Of Newcastle Disease DNA Vaccine Nanoparticles And Its Immune Effect

Newcastle disease (ND) is an acute, potent and highly contagious disease which is caused by Newcastle disease virus(NDV), it has always done great harms to the poultry industry. Although inactivated vaccine and attenuated vaccine have played an important role to prevent and to control ND in practice, conventional vaccine has some disadvantages including partial virus toxicity reservation, causing respiratory pathological changes, being difficult to distinguish between vaccine immunization and natural infection. Hence, developing ND engineering vaccine has important significance for ND prevention and cure. But present experimental results showed that DNA vaccine has to improve not only in immune effect and security but also in inoculation and delivery methods. Nanoparticle mucosal delivery systems use biodegradable materials and nanotechnology to improve the existing vaccine formulations and delivery methods in order to protect antigen, simplify vaccinate procedure, reduce vaccination times and enhance the immune efficiency.To formulate the vehicles used in this drug delivery strategy, we encapsulated plasmid DNA vaccine (pVAX1-F (o)) as a model drug in pDNA-PLGA-nanoparticles (pDNA-PLGA-NPs) through a water/oil/water (W/O/W) double emulsion-solvent evaporation—an established method of drug encapsulation. The formulation, preparation, influence factors, physicochemical characteristics, biological activity, cytotoxicity and immune effect of the pDNA-PLGA-NPs were evaluated. The results demonstrated that:1) We determined the optimization conditions of pDNA-PLGA-NPs:plasmid DNA (200μg) solution was emulsified into a PLGA solution (40mg/mL) by sonication for 30s using a probe sonicator at 50w energy output to form a W/O emulsion. The above emulsion was further emulsified into 2mL PVA (2%, w/v) using the sonicator as above for 60s to form a W/O/W emulsion. The emulsion was stirred 5h in 500r/min to evaporate chloroform, and the nanoparticles thus formed were recovered by centrifugation (3000g for 15min at 4℃), washed three times to remove PVA and unentrapped DNA, resuspended in sterile water and lyophilized;2) The results demonstrated that the pDNA-PLGA-NPs had been produced with morphous regulation, integrated profile, smooth surface, suitable size, good dispersion, high effective dosage, desirable encapsulate rate. The DNA loading in pDNA-PLGA-NPs was(91.8±0.3)%; the average particle size was(433.5±7.5)nm; the zeta potential was+2.7 mV; the polydispersity index (PDI) was 0.41;3) DNaseⅠdigestion test showed that pDNA-PLGA-NPs had the ability to protect DNA from DNaseⅠdegradation;4) The release behavior of pDNA-PLGA-NPs test in vitro demonstrated that pDNA-PLGA-NPs release was a continuous release process after initial burst release;5) The indirect immunofluorescence and Western-blotting results showed that the preparation method of pDNA-PLGA-NPs didn’t destroy the biological activity of plasmid DNA, which had expressed in vitro;6) The pDNA-PLGA-NPs mucosal delivery system was low toxicity and excellent safety of being satisfactory, which could be proved by a cytotoxicity test;7) The SPF chicken vaccination results showed that mucosal immunity by pDNA-PLGA-NPs used, the IgG and IgA contents of mucosal immune response positions were higher than intramuscular injection. In a word, the result proved that nanoparticles mucosal delivery system not only induced immune responses in mucosal location but also produced high systemic humoral and cellular immune responses.ND DNA vaccine encapsulated in PLGA nanoparticles had been assembled successfully with a further evaluation of its immunity effectiveness, which provided reference theoretically and experimentally for developing a safe, efficient and better performance gene delivery vector. Dose routing could be increased and vaccination times could be reduced but transport efficiency in cell could be increased and immunological effect could be enhanced by this pDNA-PLGA-NPs mucosal delivery system, which carried out the methanism of long-term effect of the genetic vaccine. The research was focus on improving the practical application of ND DNA vaccine in poultry industry and controlling ND explosion, which possessed significant theoretical practical sense and had a vast range of prospect in applications.