| Peste des petits ruminants (PPR) is a highly contagious viral disease caused byPeste des petits ruminants virus (PPRV), which affect goats, sheep and other wildruminants. In general, goat populations are particularly susceptible. This diseaseaccounts for significant economic losses of in local animal husbandry in outbreakingareas. At present, there is no effective treatment means to PPR. Although antibiotics tosome extent can prevent the second infection subsequently, it is not optimal choicewhen PPR outbreaks. Therefore, the prevention against PPR appears to be particularlyimportant. For most countries, the prevention against the PPR now still focuses ontraditional attenuated vaccine. Although this vaccine can induce the host to produceneutralizing antibodies against PPRV, it cannot distinguish natural infection fromvaccine animal serologically, which bring the impediment to serological investigationand affect study of epidemiology of this disease. In addition, PPRV like other memberof paramyxovirus is highly sensitive to temperature. Since the epidemic areas of thisdisease mainly distribute in the tropical and subtropical regions, strict facilities andprocedures are required for the transportation and preservation of the vaccine, whichextramly increase the expenses and the difficulties for the prevention against thisdisease.In this study, canine adenovirus type2(CAV-2) and adeno-associated virus (AAV)type1were used as vectors to express PPRV glycoprotein H or F, which generatedfour recombinant viruses of rCAV-2-PPRV-H, and rCAV-2-PPRV-F, rAAV-1-PPRV-Hand rAAV-1-PPRV-F, respectively. The immunogenicities and the bio-safety of theserecombinant viruses were evaluated by animal experiments.In the experiment described in part2chapter1of the thesis, compared with parental CAV-2strain, the two recombinant virus rCAV-2-PPRV-H andrCAV-2-PPRV-F had no obvious differences in growth properties in MDCK cells andthey grew to similar levels and reached peak titers of1×107.8TCID50/ml and1×107.3TCID50/ml, respectively. Firstly, the two strains of recombinant virusrCAV-2-PPRV-H and rCAV-2-PPRV-F were conducted in mouse models and theimmunogenicities of the recombinant viruses in mice were evaluated by detection ofhumoral and cellular immune response level. The results showed that therCAV-2-PPRV-H and rCAV-2-PPRV-F two recombinant viruses can effectivelystimulate mice to generate PPRV specific humoral and cellular immune responsesafter vaccined with the dosage of1×106TCID50. The PPRV specific ELISA antibodylevels were significantly higher in the animals treated with these two recombinantviruses than in controlled animal（sp<0.05）. The levels of PPRV neutralizing antibodyinduced by the recombinant viruses were in a simillar trend as that of ELISA specificantibody. The stimulating index of spleen lymphocyte proliferation in the micevaccined with rCAV-2-PPRV-H and rCAV-2-PPRV-F were2.39and2.45, respectively,which were significantly higher than that of controlled animals（p<0.05）.In order to boost the recombinant virus vaccines to be applied in field,immunization test in goats was carried out. Compared with mouse experiments, inpart2chapter2,141goats were involved in the immunization test, in which theimmunogenicities of rCAV-2-PPRV-H and rCAV-2-PPRV-F were further evaluated,the immune efficacies were compared between single strain and combinedimmunization. The results indicated that, compared with mouse, goats showed a muchstronger immunogenicity in3weeks after the first immunization with these tworecombinant vaccines. Both the OD value of ELISA specific antibody and twostrains of recombinant vaccines in goats have been showed stronger immunogenicitythree weeks after the first immunization andhe titer of neutralizing antibody againstPPRV in immunized goats reached the peak in3weeks after booster, which wassignificantly higher than controlled goats（p<0.05）. Lymphocyte proliferation assayshowed that, in goats, both rCAV-2-PPRV-H and rCAV-2-PPRV-F can stimulate PPRV specific cellular immune response in3weeks after booster. A difference inimmunogenicity was observed between these two recombinant vaccines.rCAV-2-PPRV-H showed a higher immunogenicity than rCAV-2-PPRV-F, in terms ofthe measurements of ELISA specific antibody and neutralizing antibody, enhancedthree weeks after booster, reaching the peak. Compared the two recombinant vaccines,PPRV neutralizing antibody induced by rCAV-2-PPRV-H is higher than that inducedby rCAV-2-PPRV-F in the same dose. With rCAV-2-PPRV-H, of the a singleimmunizationdosage of1×107.8TCID50was enough to seroconvert97.14%of thesubject goats, and PPRV neutralizing antibody titer in serum of immunized goats canremain the titer level of PPRV neutralizing antibody above1:10last for more than oneyear above the titer of1:10. There were no significant differences between theneutralizing antibody titers against PPRV induced by combined immunization of(tworecombinant vaccines) and single immunization with respect of the measurements inhumoral and cellular immune response of rCAV-2-PPRV-H. The results alsodemonstrated that oral inoculation of rCAV-2-PPRV-H or rCAV-2-PPRV-F failed toproduce PPRV specific neutralizing antibody, and no evidence was shown.Lymphocyte proliferation assay showed that rCAV-2-PPRV-H and rCAV-2-PPRV-Fboth can stimulate PPRV specific cellular immune response three weeks after boosterwith no significant difference. Toxic side effects caused by recombinant vaccineswere not observed during the animal immunization tests. The results demonstratedthat two recombinant vaccines had good bio-safety and immunogenicity, especiallythe rCAV-2-PPRV-H which can serve as a candidate for effective prevention of PPRand differentiating infected from vaccinated animals (DIVA).In part2chapter3, adeno-associated virus (AAV) type1was used as a vector togenerate two recombinant AAV, rAAV-1-PPRV-H and rAAV-1-PPRV-F, which expressPPRV glycoproteins H and F, respectively. H and F protein expression were detectedby Western blot. C57BL/6mice were immunized with rAAV-1-PPRV-H andrAAV-1-PPRV-F in prime/boost scheme at week0and4. The serum specificantibodies against PPRV were detected by ELISA and viral neutralization antibody test. IFN-γ and IL-2ELISpot assay were used to assess the T cellular response againstPPRV at12weeks after rAAV-1-PPRV-H and rAAV-1-PPRV-F immunized withrAAV-1-PPRV-H and rAAV-1-PPRV-F. The results showed that H and F proteinsexpression were detectable in cultured cells transduced with rAAV-1-PPRV-H andrAAV-1-PPRV-F, respectively. The two recombinant vaccines can signifficantlystimulate both humoral and cellular immune responses（p<0.05）, rAAV-1expressingPPRV-H could effectively elicit humoral immune response much higher than therAAV-1-PPRV-F group（p<0.05）, while in aspect of cellular immune responses thedifferences of cellular immune responses induced by these two recombinant vaccineswere not significant.In summary, this investigation successfully reveal the application of CAV-2andAAV-1viral vector in PPR recombinant vaccines producing system, which couldprovide a key technical procedure for practical production of PPR recombinantvaccine. |
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