In vitro and in vivo genetic stability of the rabies ERA glycoprotein expression cassette of AdRG13, a recombinant adenovirus vaccine against rabies

Abstract

The genetic stability of the rabies glycoprotein (G) expression cassette of AdRG1.3 was examined using both in vitro and in vivo models. For the in vitro study, the AdRG1.3 vaccine was serially passaged 20 times in 293 cell culture and from the twentieth passage a total of 67 AdRG1.3 virus clones were obtained. The G gene expression cassette (including an SV40 polyadenylation signal sequence) along with flanking human adenovirus type 5 sequences were amplified from these clones using PCR to generate an amplified product approximately 2.25 kb long. These products were then purified and subjected to DNA sequence analysis. No changes were observed in the 1870 nucleotide sequence window (containing both the G gene (1572 nt) and the SV40 polyA sequence (132 nt)) of any of the 67 clones. Findings show that the G gene insert is stably expressed in a conformationally appropriate form by the recombinant HAd5 vector. The genetic stability of the G gene cassette of AdRG1.3 was also evaluated upon in vivo passage using five independent series of cotton rats (Sigmodon hispidus). From the fifth in vivo passage of AdRG1.3, a total of 105 virus clones were obtained from these five independent series of animals. The complete G gene expression cassette was amplified from these clones by PCR and sequenced as for the in vitro study; no base changes were observed in the targeted 1870 nucleotide sequence window of any of these clones. Therefore, these results show that the G gene expression cassette of AdRG1.3 remains stable within the adenovirus vector upon passage of the vaccine both in cell culture and in animals. (Abstract shortened by UMI.)