Biosafety Evaluation And Immunization Of Recombinant Canine Adenovirus Type-2 Expressing The Glycoprotein Of Rabies Virus In Dogs

To evaluate the biosafety and the field immunogenic effectiveness ofthe recombinant canine adenovirus type-2 expressing the rabies virusglycoprotein, the following trials have been fulfilled.The biosafety of the recombinant canine adenovirus type-2 expressingthe rabies virus glycoprotein was evaluated according to the Rules ofManagement on Agricultural Transgenic organisms. Forty 3-month-oldunvaccinated dogs were split into two groups, 20 in each group. Group 1was intramuscularly injected with 1 ml (106.25 TCID50 /0.1ml) and Group 2was orally inoculated with 2 ml (106.25 TCID50 /0.1ml), respectively. At thevaccination and after, the dog sera were collected weekly or biweekly andthe following items were evaluated: the genetic stability of the recombinantvaccine in cell passage and in dogs;the existence and sustaining of therecombinant virus in the body and in the feces of the immunized dogs;thecontamination of the exogenous (i.e., the glycoprotein) gene in theenvironment or in the area of experiment;the toxic effect of therecombinant virus on dogs;the neutralizing antibody and cellular immunitylevel in dogs;and the kinetics of the neutralizing antibody.Results showed that the growth characteristic of the recombinant virusand the vector virus was identical, no the pathogenicity was observed indogs after injection of the recombinant virus. After 43 passages in MDCKcells and 6 passages in dogs, no deletion or rearrangement of the foreigngene in the recombinant virus was detected. The recombinant virus couldbe shed from the feces of the immunized dogs but no antigen was detectedin any tissue or organs from the immunized dogs. No contamination of therecombinant virus and the glycoprotein gene was found out in theenvironment. Increasing the amount of the recombinant virus did no harmto the dogs and no toxic effect was observed. Neutralizing antibody wasdetectable monthly after the intramuscular and oral vaccination,respectively. The antibody could persist for 54 weeks with an effectivelevel of protection. All the above demonstrated that the recombinant viruswas safe to both the dogs and the environment.Five hundreds 3-month-old unvaccinated dogs were split into twogroups, 250 in each group. Group 1 was intramuscularly injected with 1 ml(106.25 TCID50 /0.1ml) and Group 2 was orally inoculated with 2 ml (106.25TCID50 /0.1ml) of the recombinant vaccine, respectively. The dog serawere collected weekly before and after vaccination. Western blot analysiswas performed to test the specific antibody. The hemagglutinationinhibition test was carried out to monitor the antibodies against the vectorvirus. The neutralizing antibody and cellular immunity level in dogs wasassayed by FAVN.Results showed that the glycoprotein of rabies virus that expressed inthe recombinant virus induced an effective immunological response in dogsas indicated by the western blot analysis. High level of antibody againstCAV-2 was also produced in dogs. The neutralizing antibody level, assayedby FAVN, ranged from 0.33IU-4.5IU. The sero-conversion ratio reached to87.5% after one time of vaccination.The results demonstrated that the recombinant canine adenovirustype-2 expressing the rabies virus glycoprotein is not only safe, but alsoeffective. This has provided important data for the application of therecombinant vaccine.