Immunology and Immunopathology of the Intestines: Recombinant Avirulent Salmonella Vaccine Strains with Stable Maintenance and High Level Expression of Cloned Genes in Vivo

Abstract

Salmonella typhimurium strains with deletion (Δ) 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 Δcya Δcrp 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 Δcya Δcrp strains with a Δasd mutation (abolishing production of aspartate β-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 wildtype asd⁺ gene from Streptococcus mutans to complement the Δasd chromosomal mutation. The Asd⁺ plasmid vector can be used to express a diversity of colonization and virulence antigens from other pathogens. In the Δcya Δcrp Δasd S. typhimurium vaccine strain, the plasmid is completely stable in the absence of any exogenous selective pressure either in vitro or in vivo.