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A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like high-copy-number replication (pYA4534) was constructed for the regulated delayedin vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing theasdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal ΔasdA allele in live recombinant attenuatedSalmonella vaccines (RASVs) such asSalmonella enterica serovar Typhimurium strain χ9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers, respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/promoter of the P22cro gene (O/Pcro ) (PR ), which is negatively regulated by an arabinose-inducible P22c2 gene located on both the plasmid and the chromosome (araC PBAD c2 ). The absence of arabinose, which is unavailablein vivo , triggers replication to a high-copy-number plasmid state. To validate these vector attributes, theYersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. AnlcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression inSalmonella , flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of β-lactamase, and cloned into pYA4534 under the control of the Ptrc promoter to generate plasmid pYA4535. Our results indicate that the liveSalmonella vaccine strain χ9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge withY. pestis strain CO92 introduced through either the intranasal or subcutaneous route.

infection and immunity

Fine-Tuning Synthesis of Yersinia pestis LcrV from Runaway-Like Replication Balanced-Lethal Plasmid in a Salmonella enterica Serovar Typhimurium Vaccine Induces Protection against a Lethal Y. pestis Challenge in Mice