Biosafety Evaluation And Field Trial Of Rabies Virus Glycoprotein And Nucleoprotein "Bivalent" DNA Vaccine In Dogs

To evaluate the biosafety and the actual immune effect of the Rabies Virus Glycoprotein and Nucleoprotein "bivalent"DNA vaccines-pVGN, the following trials have been fulfilled. 1 The biosafety of the DNA vaccines-pVGN was evaluated that based on Rules of Management on Agricultural Transgenic organision. 40 unvaccined dogs were immunized with a dose of 200μg plasmid each time at 0, 7 and 21 days respectively. The biosafety was evaluated by means of observation on the development of immune responses in immunized dogs. Two pairs of primers were designed and synthesized according to the sequences of Kanamycin-resistant gene, Rabies virus glycoprotein and nucleoprotein cDNAs to monitor the transfer of the Kanamycin-resistant gene,detect the distributions and the persistence of plasmid in main organs, the injected tissues as well as the gonads and the fetus tissues to analyze the influences of vaccine on the pregnant dogs and their puppies. A 9mer random primers and the plasmid template were used to produce the probes labeled with the 32P, Southern was used to detect whether the plasmid had integrated with chromosome DNA. The pathological effect in the main organs was observed. The results showed that this DNA vaccine could induce excellent humoral immune response in the immunized dogs without any abnormal clinical responses observed. Transfer of the Kanamycin-resistant gene to the normal intestinal flora did not occur, and this plasmid was not excreted into the surroundings through intestinal flora, urine and saliva either. The plasmid could detected in heart,spleen,kidney and the injected tissues, and persisted for 18, 14-18,14 and 26 weeks respectively, while it could not be detected in the liver. The vaccine had no influence on the pregnant dogs and their puppies because it was not detected in the gonads and fetus tissues. No integration of the plasmid DNA with the genomic DNA was found. Also, there was no pathological effect observed in the main organs. 2 The humoral response was also assayed by the method of indirect ELISA besides the evaluation on the biosafety, and the dogs were randomly divided into five groups by different immunization methods to evaluate the influence on the humoral response. Five groups were intramuscular immunized, intramuscular immunized, intramuscular and auricle intracutaneous immunized, auricle intracutaneous immunized, the neck intracutaneous immunized. The results showed the seroconvertion ratio reached to 100% after the third immunization. Compared with preimmunization, the differences of antibody of the second and third immunization were all very evident(P<0.01), the difference between second and third immunization was not eviden(tP>0.05)as well as among the five groups, but the antibody level of the neck intracutaneous injection group were a little higher than the others. 3 The field trial was to evaluate the actual effect of this bivalent DNA vaccine pVGN. 302 dogs were randomly divided into 2 groups of which 201 dogs were immunized by intramuscular injection, while 101 dogs were immunized by intramuscular and auricle intracutaneous injection with a dose of 300μg plasmid each time at 0, 14 and 35 days respectively. Indirect ELISA and Western blot were used to assay the specific antibody and to detect the composition of the antibody respectively, the neutralization antibody titers were determined by indirect immunofluorescent test and...