Live attenuated bacteria provide the potential to replace traditional needle-based vaccination with an orally administered vaccine. The heterologous antigen gene is usually transformed as a multi-copy plasmid into the bacterial cell, but plasmids in live bacterial vaccine strains are often unstable, so an alternative approach is to integrate the single-copy antigen gene into the bacterial chromosome. We report a comparison between the chromosomally integrated and the plasmid-borne Bacillus anthracis protective antigen gene in live Salmonella enterica serovar Typhimurium, using the Operator-Repressor Titration (ORT) system to ensure stable plasmid maintenance. These studies demonstrate that the stabilised plasmid approach of gene expression produced greater amounts of antigenic protein, which in turn resulted in higher antibody responses and levels of protection in mice.