Preparation Of Newcastle Disease DNA Vaccine O-2'-HACC Nanoparticles And Evaluation Of Mucosal Immune Effect

Newcastle disease(Newcastle Disease,ND)is caused by Newcastle disease virus(Newcastle Disease Virus,NDV).ND is an acute and highly contiguous acianinfectious disease of poultry.The main clinical symptoms are respiratory tract and digestive tract,characterized by fever,difficulty breathing,diarrhea,mucosal bleeding and necrosis,longer course of disease is also associated with neural symptoms.ND caused severe economic losses to the industry poultry.Now vaccination is an effective way to control ND.The most commonly used are theconventional vaccines,but the live vaccine and inactivated vaccine have many drawbacks.Live vaccines have some limitations,including the need for biocontainment during production,safety concerns due to the possibility of reversion.A major problem with the use of inactivated vaccines is that they generally have poor immuno genic ity and can cause disease if they are not completely inactivated.DNA vaccines represent a promising technology because of their safety,genetic stability,ease of production,non-requirement for cold chain,and activation of innate immunity pathways.But intramuscular injection was the primry route for DNA vaccine administration.Many studies indicated that after intramuscular injection,plasmid DNA is easy to be degraded by nuclease and it is difficult for the vaccines to move through cell membranes,so only a small amount reaches antigen-presenting cells(APCs)to induce immune responses.Biodegradable material has no toxicity,biocompatibility,biodegradability,release the advantages are widely used in the delivery system.These disadvantages could be avoided when the nanopartciles mucosa immunity delivery system was built by biodegradable materials.The pFDNA-O-2’-HACC-NPs were prepared according to a polyelectrolyte complex method.1)The pFDNA-O-2’-HACC-NPs were produced with good morphology,high stability,an average diameter of(202.3±0.52)nm,encapsulation efficiency of(90.74±1.10)%,loading capacity of(49.84±1.20)%,and a Zeta potential of(50.8 ± 8.21)mV;2)DNase I digestion test showed that pFDNA-O-2’-HACC-NPs had the ability to protect DNA from DNase I degradation;3)The in vitro release assay showed that the plasmid DNA was sustainably released from the pFDNA-O-2’-HACC-NPs,up to(82.97± 2.30)%of the total amount;4)Cell transfection test indicated that the vaccine expressed the F gene in cells and maintained good bioactivity;5)Storage stability tests showed that no significant changes in the appearance of nanoparticles storage at 37℃,20℃,4℃,-20℃ for 3 weeks,and have good stability;Storage at-20 ℃ for 2-3 months,still can induce strong immune response;6)The safety of mucosal immunity delivery system of the pFDNA-O-2’-HACC-NPs was also tested in vitro by cell cytotoxicity and in vivo by safety test in chickens;7)In vivo immunization showed that SPF chickens immunized intranasally experimental results showed that the pFDNA-O-2-HACC-NPs vaccines induced more tong-lasting IgG antibodies,IgA antibody levels,IL-2,IL-4 and IFN-y secretion than intramuscular injection,immune chicken spleen lymphocyte transformation rate have increased significantly,and protective efficacy was superior to intramuscular injection.8)pFDNA-O-2 ’-HACC-NPs can stimulate CD4+,CD8+ production,and in group were significantly higher than others in the corresponding injection group,showed that the cytokine induced expression of mucosal immune was significantly higher than those in the mucosal immune.ND DNA vaccine encapsulated in O-2’-HACC nanoparticles had been prepared,he mucosal delivery system had implemented the sustained release of plasmid A in vivo and induced mucosal immune,which could protect antigen form adation,increase the enhance the immune effect This study lays a foundation for further development of mucosal vaccines and drugs encapsulated in O-2’-HACC oparticles.