Design Of A New Porcine Influenza Diarrhea Vaccine Adjuvant Based On N-2-HACC Nanoparticles And Its Immune Effect Stud

Porcine epidemic diarrhea(PED)is an acute,highly contagious intestinal disease caused by Porcine epidemic diarrhea virus(PEDV).Clinically,it is characterized by severe vomiting,watery diarrhea and dehydration.The mortality rate of infected pigs is extremely high,causing a very serious loss in the pig industry.At present,the main method for preventing swine epidemic diarrhea is vaccine immunization.Among them,the inactivated vaccine is one of the main methods of immunizing the vaccine.The vaccine has good stability,the immune prevention effect is remarkable,and the immune adjuvant can effectively improve the immune effect.However,there are still many problems with PEDV inactivated vaccines,such as lack of effective antigen presentation and processing,poor bioavailability of vaccines,and lack of effective immunological adjuvants.Research and development of safe and effective vaccine adjuvants are great significance for improving the immune effect of PEDV inactivated vaccines.Chitosan is biocompatible,low toxicity,and is the material of choice for vaccine delivery vehicles.Therefore,in this study,the natural degradable biopolymer material N-2-hydroxypropyltrimethylammonium chloride chitosan(N-2-HACC)and N-2-hydroxypropyltrimethylammonium chloride chitosan/N,O-Carboxymethyl chitosan(N-2-HACC/CMC)nanoparticles respectively were used as delivery vehicles,and the model antigen PEDV inactivated virus was entrapped.A new inactivated virus vaccine against PED was prepared,and relevant performance and immune effects of the prepared inactivated vaccine was studied.First of all,in this study,the natural degradable biopolymer materials N-2-HACC and N-2-HACC/CMC were used as raw materials,which were prepared by a small device developed by own research group to control the particle size of the nano-adjuvant.The synthesis conditions of the two kinds of nano-adjuvants were optimized.Then,PED inactivated virus was used as the model antigen,and two inactivated vaccines were prepared with the above nano-adjuvants: one is an inactivated vaccine(N-2-HACC/PEDV)prepared by using N-2-HACC nanoparticles as a nano adjuvant,and the other is an inactivated vaccine(N-2-HACC/CMC/PEDV)prepared by using N-2-HACC/CMC nanoparticles as a nano adjuvant.At the same time,this experiment evaluated the immune effects of the above two PED inactivated vaccines to screen out the best combination of vaccine adjuvants.Finally,we synthesized a nano-inactivated vaccine(N-2-HACC/PEDV/CMC NPs)encapsulating PED-inactivated virus with N-2-HACC as the aqueous phase,petroleum ether and liquid paraffin as the oil phase and CMC as the cross-linking agent,and evaluated the physicochemical properties of N-2-HACC/PEDV/CMC NPs.The results showed that:1)A small device for controlling the particle size of nano-adjuvant was prototyped,and the controllability of nanoparticles was realized;2)The optimal synthesis conditions for N-2-HACC nanoparticles were determined,namely 1 mg/m L N-2-HACC solution(3 m L),1.3 mg/m L β-GP solution(1 m L),2.3%(w/v)Span 80,oil-water ratio is 14(v/v),cross-linking 4min;3)The optimal synthesis conditions for N-2-HACC/CMC nanoparticles were determined,namely 1.7 mg/m L N-2-HACC(3 m L),1.3 mg/m L CMC(1 m L),3.0%(w/v)Span 80,oil-water ratio is 8(v/v),cross-linking 4min;4)PED inactivated vaccine was prepared by using PED inactivated virus as model antigen,and nano-adjuvant N-2-HACC nanoparticles and N-2-HACC/CMC nanoparticles were used as delivery vectors respectively.The results showed that The N-2-HACC/CMC/PEDV prepared by 1%(w/v)of the nano-adjuvant N-2-HACC/CMC is better than the N-2-HACC/PEDV prepared by the 5%(w/v)nano-adjuvant N-2-HACC,and the inactivated vaccines prepared by the two nano adjuvants have a longer duration of immunity than the PEDV commercial inactivated vaccine;5)The optimal process conditions for the preparation of N-2-HACC/PEDV/CMC NPs were determined,namely 1.8 mg/m L N-2-HACC(3 m L),1.6 mg/m L CMC(1 m L),3.0%(w/v)Span 80,1.6 m LPEDV,cross-linking for 4min;6)Encapsulated PEDV in N-2-HACC/PEDV/CMC NPs was intact,has good acid resistance,bile and enteric solubility,suitable for oral administration,and has well sustained release properties.