Recombinant Canine Distemper Attenuated Vaccine Virusexpressing Glycoprotein Of Rabies Virus

Rabies is a widespread zoonosis caused by the rabies virus (RV) and causes acute, progressive, andfatal encephalitis. The disease is currently one of the deadliest infectious diseases and is almost100%fatal once the neurologic symptoms appear. Worldwide there are approximately55,000human deaths peryear due to rabies, mostly in developing countries of Africa and Asia. Since2003, the annual number ofhuman deaths has been growthing in our country and was above2,000. Now, rabies is a serious publichealth problem in our country. However, the exposure to rabid dogs is still the main cause of over99%of human deaths worldwide. For preventing human rabies, the most efficient strategy is to eliminate thedisease in dogs through vaccination. The manufacturing costs of routine rabies attenuated vaccines arelow, but their residual virulence or pathogenic mutations can sometimes cause the disease of vaccinatedanimals. Although, inactivated cell culture rabies vaccines are safe and effective, their manufacturingcosts are high. Therefore, effective, safe, and affordable rabies vaccines are still being sought.The development of reverse genetics has provided a powerful tool to create recombinantnonsegmented negative-strand RNA virus-based bivalent vaccine. Canine distemper (CD) is awidespread infectious disease of most carnivores, such as dogs, minks, foxes and so on, caused bycanine distemper virus (CDV), a morbillivirus of family Paramyxoviridae. Now, CD is a very seriousproblem for dogs industry and economic animals industry worldwide. However, vaccination of CDattenuated vaccines is an efficient method to prevent CDV infection. Therefore, CDV-based liveattenuated vaccine serve as a bivalent vaccine can prevent rabies and CD, which will contribute toimprove the rabies vaccine vaccination rate of dogs and the quality of immunization. It will save theeconomic costs of rabies vaccine immunization.In this study, we established a reverse genetics system of the attenuated CDV vaccine strainCDV/R-20/8. The rescued CDV, rCDV, was almost indistinguishable from the parental strainCDV/R-20/8. There are no difference in growth properties between rCDV and the parental wild virus inVero cells. And, the rCDV and the parental strain grew to similar levels and reached peak titers of106.5TCID50/mL at72h post-infection. To investigate the potential of CDV serve as a live vaccine vectorexpressing the foreign proteins, we generated a recombinant CDV, rCDV-EGFP, expressing theenhanced green fluorescent protein (EGFP) by using reverse genetics. The rCDV-EGFP has the similarbiological characteristics with parental rCDV, including high growth titer (10^6.5TCID50/mL) andexcellent adaptability and genetic stability in Vero cell. After serially passages10times in Vero cells, therecombinant CDV still kept a high level expression of EGFP. These results indicated that theCDV/R-20/8attenuated vaccine strain has the potential to serve as a live vaccine vector.CDV is a promising live viral vector to develop a bivalent vaccine against RV and CDV infection.In the present study, we generated a recombinant CDV, rCDV-RVG, expressing the RV vaccine strainERA G protein by using the reverse genetics. The rCDV-RVG grew to similar levels with rCDV and reached peak titer of10^6.25TCID₅0/mL at72h post-infection. This titer was approximately one-quarterof a log lower than that of rCDV. To assess the pathogenicity and immunogenicity of recombinant CDV,mice were injected intracerebrally (i.c.) with3×104TCID50in a volume of30μL or intramuscularly (i.m.)with105TCID50in a volume of100μL of the rescued viruses. All of the mice showed no apparent signsof disease after inoculation. There was no significant difference in the body weight changes among thethree i.m. inoculated groups or the three i.c. inoculated groups with rCDV-RVG, rCDV and PBS. Theseresults demonstrated that the insertion and expression of RV G gene did not increase the virulence of theCDV vector in mice. Mice inoculated i.m. with10⁵TCID₅0of the rCDV-RVG developed a strong RVneutralizing antibody response, which completely protected mice from challenge with a lethal dose ofstreet virus GX/09strain at3weeks post-vaccination. However, all control mice died of rabies afterchallenge. These results indicated that the rCDV-RVG is safe and immunogenic in mice.To further assess the immunogenicity of the recombinant virus rCDV-RVG, two groups of five3-month-old Beagle dogs were i.m. inoculated with1mL10⁶TCID₅0of rCDV or rCDV-RVG,respectively. At3weeks after initial vaccination, the dogs received a second vaccine dose, respectively.Dogs inoculated with the rCDV-RVG developed a strong and long-lasting RV neutralizing antibodyresponse. The mean titer of RV neutralizing antibodies was1.48IU at1week after the first dose androse to32.11IU at3weeks after the first dose. After receiving the second dose, the rCDV-RVGvaccinated dogs showed signally boost responses to RV neutralizing antibody. The mean titer of RVneutralizing antibodies for the group was96.71IU at2weeks after the second dose. RV neutralizingantibodies induced by rCDV-RVG can sustain for more than a year. At70weeks post-vaccination, themean titer of RV neutralizing antibodies was>0.5IU; the minimum NA titer required to protect animalsfrom challenge with RV street virus. These result suggested that two times vaccination of rCDV-RVGprovides effective protection for>1year in dogs. After receiving the third dose at70weekspost-vaccination, the rCDV-RVG vaccinated dogs showed substantial re-boost responses to RVneutralizing antibody. The mean titer of RV neutralizing antibodies for the group sharply increased to281.4IU at1week after the third dose. Meanwhile, rCDV and rCDV-RVG induced a remarkable CDVneutralizing antibody response in dogs at3weeks after the first dose. The titers of CDV neutralizingantibody for the two groups sharply increased after the second dose. After receiving the third dose at70weeks post-vaccination, all the rCDV-vaccinated and rCDV-RVG-vaccinated dogs showed substantialre-boost responses to CDV neutralizing antibodies. And, there was no significant difference in the peaksof CDV neutralizing antibody titers between the two groups.These results demonstrate that the rCDV-RVG is safe and immunogenic in dogs and a promising,novel and live bivalent vaccine candidate against CD and rabies.