In vitro growth and stability of recombinant rabies viruses designed for vaccination of wildlife.

Three live rabies virus (RV) recombinant vaccine candidates, SPBNGA, SPBNGA-Cyto c (+), and SPBNGA-GA, were examined for their production levels and stability. Maximum production levels up to 10(10) infectious particles/mL were achieved using bioreactor technology. All virus lots exhibited thermostability profiles typical for RV vaccines and were non-pathogenic for intracranially inoculated immunocompetent mice. Moreover, sequence analysis indicated high genetic stability in all three RVs during 10 consecutive passages in newborn mice. This analysis revealed no change in the extra RV G gene in the SPBNGA-GA vaccine or in the cytochrome c gene in the SPBNGA-Cyto c (+) vaccine. Moreover, no changes were detected in the G gene codon for Glu333, which renders the virus non-pathogenic. However, after the fifth passage, a mutation resulting in an Asn194 --> Lys194 exchange emerged in the G genes of all three RVs. This mutation was associated with a modest increase in pathogenicity in SPBNGA and SPBNGA-Cyto c (+), but not in SPBNGA-GA, which contained the mutation in only one of its two G genes and which remained non-pathogenic. These results demonstrate the feasibility of producing RV vaccines that remain highly stable even after multiple passages.