Coexpression of double or triple copies of the rabies virus glycoprotein gene using a ‘self-cleaving’ 2A peptide-based replication-defective human adenovirus serotype 5 vector

Rabies virus glycoprotein (RVG) is a major structural protein and antigen of rabies virus that induces a highly immunogenic response. In the present study, we have used 2A self-cleaving sequence of the foot-and-mouth disease virus (FMDV) to express double or triple copies of the RVG from a single open reading frame derived from human adenovirus 5 (AdHu5). The recombinant adenoviruses produce similar virus titers, indicating that the insertion of double or triple copies of the RVG gene linked with the FMDV 2A sequence does not affect virus replication. The RVG was efficiently expressed by constructs containing the 2A sequence and retained its antigenic property. The 2A self-cleaving peptide mediated efficient generation of individual glycoprotein in transient expression assay and did not lead to an altered surface distribution of RVG. Flow cytometry demonstrated that the expression levels of RVG were improved in recombinant Ads carrying multiple RVG gene copies. We conclude that ribosome skipping induced by the FMDV 2A sequence is an effective strategy to express multiple glycoprotein genes of rabies virus in adenoviruses and 2A-containing recombinant Ads may represent an attractive alternative to other coexpression strategies for multiple gene expression.