Construction and Immunogenicity Study of the Improved Non-replicating Vacc- inia Recombinant Co-expressing Rabies Virus Glycoprotein and Nucleoprotein

To improve the safety of recombinant vaccinia virus，a non-replicating vaccinia recombi- nant co-expressing rabies virus glycoprotein(RG) and nucleoprotein(RN) without a selectable marker was reconstructed. LacZ between fragment C and fragment K in non-replicating recombinant vaccinia virus VTKRG△CKlacZ was deleted and was substituted with rabies virus nucleoprotein by homologous recombination resulting in VTKRGΔCKRN. The recombinant vaccinia virus was screened by a two-step selection under, G418 and with White/Blue plaque color change as well as immunofluorescence. By Dot Blot analysis, RG gene and RN gene were shown to integrate stably into the non-replicating vaccinia recombinants even after 20 passages in CEF cells. The virulence of VTKRGΔCKRN was obviously less than that of VTKRG in naked mouse. The non-replicating characterization of VTKRGΔCKRN was also stable. It was proved that VTKRGΔCKRN could express RG and RN at the same time. VTKRGΔCKRN could elicit viral neutralizing antibody(VNA) similar to that of VTKRG by inoculated into the mice，and thus protect the animals from lethal challenge of rabies virus. It could elicit efficient viral neutralizing antibody(VNA) and protect the dogs from lethal challenge of rabies street strain SBD by intramuscular inoculation at dose of only 1.6×106PFU. This study confirmed the efficacy and safety of non-replicating recombinant vaccinia virus VTKRGΔCKRN.