Fine-Tuning Synthesis ofYersinia pestisLcrV from Runaway-Like Replication Balanced-Lethal Plasmid in aSalmonella entericaSerovar Typhimurium Vaccine Induces Protection against a LethalY. pestisChallenge in Mice

Abstract

A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like highcopy-number replication (pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal ⌬asdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain 9447. 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 P22 cro gene (O/P cro ) (P R ), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC P BAD c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of ␤-lactamase, and cloned into pYA4534 under the control of the P trc promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain 9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route.

Live Attenuated Salmonella Vaccines Displaying Regulated Delayed Lysis and Delayed Antigen Synthesis To Confer Protection against Mycobacterium tuberculosis

“…Briefly, suicide vectors were used to construct strain 11021 containing the nucleotide sequences designed to delete specific genes or introduce defined deletion-insertion mutations with modified promoter (P), SD, and start codon sequences (Table 1). RASV strain construction and characterization were performed as described previously (22,34,46,49,67,71). Maps of the deletions and deletion-insertion mutations have been described previously for ⌬(gmd-fcl)-26, ⌬pmi-2426, ⌬relA198::araC P BAD lacI TT, and ⌬asd27::TT araC P BAD c2 (49).…”

“…Stability of the recombinant plasmids was determined for approximately 50 generations of bacterial growth in LB medium under selective and nonselective (presence of DAP) conditions as described previously (22,32,57,67).…”

Live Attenuated Salmonella Vaccines against Mycobacterium tuberculosis with Antigen Delivery via the Type III Secretion System

“…Stability of the recombinant plasmids expressing the SopE Nt80 fusion proteins was determined for approximately 50 generations of growth under selective and nonselective conditions (presence of DAP), as described previously (74).…”

“…We have reported the advantages of using new-generation recombinant attenuated Salmonella vaccine (RASV) strains that are phenotypically similar to the wild-type strain at the time of oral vaccination as an alternative for vaccination (23,24,52,79). These RASV strains are able to colonize and persist in the lymphoid tissue without causing disease symptoms when carrying heterologous antigens, thereby inducing higher protective mucosal and systemic immune responses against a number of infectious diseases (27,45,47,48,68,74,83). Additionally, several approaches have been employed to improve the ability of Salmonella to survive in the gastrointestinal tract and to reach the lymphoid tissues.…”

“…The deletion of Salmonella genes that encode enzymes involved in the biosynthesis of the peptidoglycan layer of the bacterial cell wall (e.g., aspartate ␤-semialdehyde dehydrogenase [Asd]) allows the use of plasmid systems harboring the gene encoding this enzyme to be maintained without the use of antibiotic resistance markers (60). Additionally, use of plasmids with different copy numbers is used to attain a better balance between plasmid replication and the synthesis of heterologous protective antigens (45,60,74). A series of expression vectors harboring chimeric fusions between the antigen to be analyzed and different types of secretion signal sequences (e.g., a ␤-lactamase signal sequence to allow protein secretion into the periplasm or extracellular compartment) was constructed to enhance the immune responses to the antigens (45,81).…”