Study On The Preparation Technology Of Peste Des Petits Ruminants Sustained-release Microcapsules Vaccine

Peste des Petits Ruminants(PPR) is a fetal disease caused by Peste des Petits Ruminants Virus(PPRV). The first outbreak of PPR in China was reported in Tibet in 2007, which threatens seriously livestock breeding industry in China. The main measure to prevent and control PPR is to inoculate the live attenuated vaccine, but there is a risk of reversing its virulence. Although the inactivated vaccine of PPR can overcome the shortage of live vaccine, the main drawback is weak immunogenicity and the adjuvant (mainly mineral adjuvant and oil adjuvant) can trigger the issue of animal-originated food safety. A potential solution is microcapsulation through which the virus antigen is encapsulated. Commercial microcapsules have been listed overseas, while the study on the veterinary sustained-release microcapsules vaccine just begains. Therefore, study on the preparation technology of PPR sustained-release microcapsules vaccine is urgently needed considering of the animal disease prevention and animal-originated food safety in China.In this dissertation, inactivated concentrated PPRV was chosen as an antigen model and natural biodegradable polysaccharides (alginate and chitosan) were used as the encapsulated materials. The feasibility of the preparation of veterinary sustained-release microcapsules vaccines by emulsification /internal gelation and spraying-complex coacervation method was further studied in detail.The optimal technological conditions of making the inactivated PPRV microcapsules vaccine by emulsification/internal gelation through orthogonal were studied. The results showed that the optional conditions were as follows:concentration of alginate was 2%, concentration of Span-85 was 2%, stirring speed was 400rpm, the matching of aqueous/oil phases ratio was 1:5. Three batches of inactivated PPRV microencapsulated vaccine were prepared under opimum conditions and were evaluated with evaluation system and method established. The average encapsulation efficiency of PPRV antigen in microcapsules was 75.1%. The microcapsules showed a normal distribution, whose average diameter was 88.5μm and 70.1μm respectively before and after lyophilization. After 30 days of each immunization, blood samples were collected from the mouse eyes. The antibodies to the three batches of inactivated PPRV microencapsulated vaccine and inactivated PPRV antigen were detected by indirect ELISA. The results showed that the inactivated PPRV encapsulated had good immunogenicity, even after the microcapsules vaccine were stored for 6 month at 37℃.The inactivated PPRV microcapsules vaccine was prepared by spraying-complex coacervation method through a single factor experiment. The optimal pH of microcapsules vaccine solution was 7.5. Three batches of inactivated PPRV microencapsulated vaccine were prepared under opimum conditions and evaluated with evaluation system and method established. The average encapsulation efficiency of PPRV antigen in microcapsules was 42%, and there was no significant difference in activity of inactivated PPRV before and after encapsulation.The animal experiment indicated that their neutralizing antibody titer reached to more than 1:20 at 30d after inoculating sheeps with two kinds of microcapsules vaccines respectively, even to 1:90-1:160, which was significantly higher than the live vaccine quality standards (1:10), although the neutralizing antibody GMT induced by two kinds of microcapsules vaccine was less than that of the traditional oil adjuvant vaccine. Moreover, the higher neutralizing antibody titer lasted more than 6-7 month, and showed good sustained- release effect.Thus those two kinds of new vaccines will have a good application prospect..