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

Abstract

ABSTRACTLive recombinant attenuatedSalmonellavaccine (RASV) strains have great potential to induce protective immunity againstMycobacterium tuberculosisby deliveringM. tuberculosisantigens. Recently, we reported that, in orally immunized mice, RASV strains delivering theM. tuberculosisearly secreted antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP-10) antigens via theSalmonellatype III secretion system (SopE amino-terminal region residues 1 to 80 with two copies of ESAT-6 and one copy of CFP-10 [SopENt80-E2C]) afforded protection against aerosol challenge withM. tuberculosis. Here, we constructed and evaluated an improvedSalmonellavaccine againstM. tuberculosis. We constructed translational fusions for the synthesis of two copies of ESAT-6 plus CFP-10 fused to the OmpC signal sequence (OmpCSS-E2C) and amino acids 44 to 338 of antigen 85A (Ag85A294) flanked by the signal sequence (SS) and C-terminal peptide (CT) of β-lactamase (BlaSS-Ag85A294-BlaCT) to enable delivery via theSalmonellatype II secretion system. The genes expressing these proteins were cloned as an operon transcribed from Ptrcinto isogenic Asd+/MurA+pYA3681 lysis vector derivatives with different replication origins (pBR, p15A, pSC101), resulting in pYA4890, pYA4891, and pYA4892 for SopENt80-E2C/Ag85A294synthesis and pYA4893 and pYA4894 for OmpCSS-E2C/Ag85A294synthesis. Mice orally immunized with the RASV χ11021 strain engineered to display regulated delayed lysis and regulated delayed antigen synthesisin vivoand harboring pYA4891, pYA4893, or pYA4894 elicited significantly greater humoral and cellular immune responses, and the RASV χ11021 strain afforded a greater degree of protection againstM. tuberculosisaerosol challenge in mice than RASVs harboring any other Asd+/MurA+lysis plasmid and immunization withM. bovisBCG, demonstrating that RASV strains displaying regulated delayed lysis with delayed antigen synthesis resulted in highly immunogenic delivery vectors for oral vaccination againstM. tuberculosisinfection.