We have devised and constructed a biological containment system designed to cause programmed bacterial cell lysis with no survivors. We have validated this system, using Salmonella enterica serovar Typhimurium vaccines for antigen delivery after colonization of host lymphoid tissues. The system is composed of two parts. The first component is Salmonella typhimurium strain 8937, with deletions of asdA and arabinose-regulated expression of murA, two genes required for peptidoglycan synthesis and additional mutations to enhance complete lysis and antigen delivery. The second component is plasmid pYA3681, which encodes arabinoseregulated murA and asdA expression and C2-regulated synthesis of antisense asdA and murA mRNA transcribed from the P22 P R promoter. An arabinose-regulated c2 gene is present in the chromosome. 8937(pYA3681) exhibits arabinose-dependent growth. Upon invasion of host tissues, an arabinose-free environment, transcription of asdA, murA, and c2 ceases, and concentrations of their gene products decrease because of cell division. The drop in C2 concentration results in activation of P R, driving synthesis of antisense mRNA to block translation of any residual asdA and murA mRNA. A highly antigenic ␣-helical domain of Streptococcus pneumoniae Rx1 PspA was cloned into pYA3681, resulting in pYA3685 to test antigen delivery. Mice orally immunized with 8937(pYA3685) developed antibody responses to PspA and Salmonella outer membrane proteins. No viable vaccine strain cells were detected in host tissues after 21 days. This system has potential applications with other Gram-negative bacteria in which biological containment would be desirable.programmed cell lysis ͉ rPspA ͉ Rx1

S. typhimurium SL1344 and UK-1 mutants with deletions of the crp (cyclic AMP receptor protein) and cdt (colonization of deep tissues) genes have been constructed and characterized, and their levels of virulence and immunogenicity have been determined for BALB/c mice. All Crp ؊ Cdt ؊ and Crp ؉ Cdt ؊ mutants were avirulent, as mice survived oral doses of 10 9 cells without illness. All the mutants colonized the gut-associated lymphoid tissue efficiently, but capacities to colonize deeper tissues, such as those of the spleen and liver, and blood were significantly reduced for the Crp ؊ Cdt ؊ and Crp ؉ Cdt ؊ mutants compared with the Crp ؊ Cdt ؉ mutant and the wild-type parent strain. The Crp ؊ Cdt ؊ and Crp ؉ Cdt ؊ SL1344 strains induced complete protection, as all mice immunized with the mutants survived challenge with ϳ10 4 times the 50% lethal dose of the wild-type SL1344 strain. The Crp ؊ UK-1 strain was least attenuated yet induced the highest level of protective immunity against challenge with the wild-type UK-1 strain. The Crp ؉ Cdt ؊ and Crp ؊ Cdt ؊ strains, although totally attenuated, differed in immunogenicity to challenge with the highly virulent UK-1 parent, with the apparently hyperattenuated Crp ؊ Cdt ؊ strain inducing a lower level of protective immunity than the Crp ؉ Cdt ؊ strain. Nevertheless, these UK-1 Crp ؊ Cdt ؊ and Crp ؉ Cdt ؊ strains induced complete protective immunity to challenge with the less-virulent SL1344 wild-type strain. Taken collectively, the results indicate that the attenuation of a highly virulent S. typhimurium strain can yield a vaccine that induces excellent protective immunity to challenge with less-virulent S. typhimurium strains.

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