Recombinant avirulent Salmonella vaccine strains with stable maintenance and high level expression of cloned genes in vivo.

Salmonella typhimurium strains with deletion (delta) of the adenylate cyclase (cya) and cyclic AMP receptor protein (crp) genes are avirulent for mice and induce a high level of protective immunity to oral challenge with up to 10,000 times what would be a lethal dose of wild-type virulent S. typhimurium cells. This immunity begins as early as seven days after immunization and lasts for at least four months. S. typhimurium delta cya delta crp mutants stably maintain plasmids and give high-level expression of cloned gene products; in this they appear superior to other avirulent S. typhimurium strains. S. typhimurium delta cya delta crp strains with a delta asd mutation (abolishing production of aspartate beta-semialdehyde dehydrogenase), have an obligate requirement for diaminopimelic acid (DAP). This strain can be used in conjunction with plasmid vectors lacking antibiotic resistance markers but having the wild-type asd+ gene from Streptococcus mutans to complement the delta asd chromosomal mutation. The Asd+ plasmid vector can be used to express a diversity of colonization and virulence antigens from other pathogens. In the delta cya delta crp delta asd S. typhimurium vaccine strain, the plasmid is completely stable in the absence of any exogenous selective pressure either in vitro or in vivo.