Rearrangement of the genes of vesicular stomatitis virus eliminates clinical disease in the natural host: new strategy for vaccine development.

Gene expression among the nonsegmented negative-strand RNA viruses is controlled by distance from the single transcriptional promoter, so the phenotypes of these viruses can be systematically manipulated by gene rearrangement. We examined the potential of gene rearrangement as a means to develop live attenuated vaccine candidates against Vesicular stomatitis virus (VSV) in domestic swine, a natural host for this virus. The results showed that moving the nucleocapsid protein gene away from the single transcriptional promoter attenuated and ultimately eliminated the potential of the virus to cause disease. Combining this change with relocation of the surface glycoprotein gene yielded a vaccine that protected against challenge with wild-type VSV. By incremental manipulation of viral properties, gene rearrangement provides a new approach to generating live attenuated vaccines against this class of virus.

Moving the glycoprotein gene of vesicular stomatitis virus to promoter-proximal positions accelerates and enhances the protective immune response.

Vesicular stomatitis virus (VSV) is the prototype of the Rhabdoviridae and contains nonsegmented negative-sense RNA as its genome. The 11-kb genome encodes five genes in the order 3'-N-P-M-G-L-5', and transcription is obligatorily sequential from the single 3' promoter. As a result, genes at promoter-proximal positions are transcribed at higher levels than those at promoter-distal positions. Previous work demonstrated that moving the gene encoding the nucleocapsid protein N to successively more promoter-distal positions resulted in stepwise attenuation of replication and lethality for mice. In the present study we investigated whether moving the gene for the attachment glycoprotein G, which encodes the major neutralizing epitopes, from its fourth position up to first in the gene order would increase G protein expression in cells and alter the immune response in inoculated animals. In addition to moving the G gene alone, we also constructed viruses having both the G and N genes rearranged. This produced three variant viruses having the orders 3'-G-N-P-M-L-5' (G1N2), 3'-P-M-G-N-L-5' (G3N4), and 3'-G-P-M-N-L-5' (G1N4), respectively. These viruses differed from one another and from wild-type virus in their levels of gene expression and replication in cell culture. The viruses also differed in their pathogenesis, immunogenicity, and level of protection of mice against challenge with wild-type VSV. Translocation of the G gene altered the kinetics and level of the antibody response in mice, and simultaneous reduction of N protein expression reduced replication and lethality for animals. These studies demonstrate that gene rearrangement can be exploited to design nonsegmented negative-sense RNA viruses that have characteristics desirable in candidates for live attenuated vaccines.