Recombinant Canine Distemper Attenuated Vaccine Virus Expressing Codon Optimized VP2 Protein Of Canine Parvovirus

Canine parvovirus disease, caused by canine parvovirus (CPV), is an acute, highly contagious and sometimes fatal enteritis in dogs of all ages and myocarditis in pups worldwide. The disease can cause a high mortality rate, up to 70% in pups. Now, canine parvovirus disease is already widely epizootic in our country and a serious threat to the kennel industry and fur animals industry. Currently, there is no effective treatment method against canine parvovirus disease, especially in pups, but vaccination is the most efficient strategy to prevent and control the disease. However, the dogs, vaccinated with the attenuated vaccine, sometimes may develop parvovirus-like diarrhea, which may due to the emergence of new antigen variation strains. And inactived vaccine can induce only a short-term specific immune response in dogs.With the development of reverse genetics, the nonsegmented negative-strand RNA viruses have become a new research focus to serve as live virus vaccine vector. Canine distemper (CD) is a worldwide infectious disease to many species of animals, caused by canine distemper virus (CDV). CD has caused great losses to dogs industry and fur animal industry in China. Inoculating CD attenuated vaccines is an efficient way to prevent CDV. Therefore, CDV-vectored canine parvovirus disease vaccine may serve as a bivalent vaccine to prevent canine parvovirus disease and CD, which will contribute to reduce the economic costs of dog vaccination and serve as vaccine to prevent the new antigenic type of CPV.The target protein gene with codons optimized for mammal usage will contribute to improve the protein expression in vitro, or in vivo. It is also a method to improve the immunogenicity of vaccines. In this study, we constructed a recombinant CDV, r20/8-CPVVP2opti containing a codon-optimized CPV VP2 protein gene, expressing the CPV VP2 protein by reverse genetics. The expression of CPV VP2 protein in BHK-21 cells, infected with r20/8-CPVVP2opti, was ascertained by western blot and indirect immunofluorescence assay. The r20/8-CPVVP2opti was proved to be of the same growth characteristics as r20/8 and approached peak titer of 10675 TCID50/mL. And, the production of CPV VP2 protein in BHK-21 cells, infected with r20/8-CPVVP2opti, is higher than that in BHK-21 cells infected with r20/8-CPVVP2, which is a recombinant CDV containing wild type CPV VP2 protein gene, expressing the CPV VP2 protein.To assess the immunogenicity of the recombinant virus r20/8-CPVVP2opti, four groups of eight 8-week-old BABL/c mice were i.m. inoculated with 0.1 mL 104.5 TCID50 of r20/8-CPVVP2opti, r20/8-CPVVP2, r20/8 or PBS, respectively.3 weeks post the initial injection, each group of mice received the same boost vaccination. Mice vaccined with r20/8-CPVVP2opti and r20/8-CPVVP2, respectively, developed a strong CPV HI antibody response. After receiving the second dose, the r20/8-CPVVP2opti or r20/8-CPVVP2 vaccinated mice showed signally boost responses to CPV HI antibody response. And, at 4 weeks after initial vaccination, the mean titers serum HI antibody from r20/8-CPVVP2opti and r20/8-CPVVP2 vaccinated mice were 6.33 and 5 log2, respectively, and there was significant difference in the HI antibody titers between the two groups. Meanwhile, r20/8-CPVVP2opti, r20/8-CPVVP2 and r20/8 induced a similar CDV neutralizing antibody response in mice. And, there was no significant difference among the three groups of mice on the CDV neutralizing antibody titers.These results demonstrate that the r20/8-CPVVP2opti is immunogenic for mice, which can serve as a bivalent vaccine candidate against canine parvovirus disease and canine distemper.