The Asd+-DadB+ dual-plasmid system offers a novel means to deliver multiple protective antigens by a recombinant attenuated Salmonella vaccine

ABSTRACT We developed means to deliver multiple heterologous antigens on dual plasmids with non-antibiotic-resistance markers in a single recombinant attenuated vaccine strain of Salmonella enterica serotype Typhimurium. The first component of this delivery system is a strain of S . Typhimurium carrying genomic deletions in alr , dadB , and asd , resulting in obligate requirements for diaminopimelic acid (DAP) and d-alanine for growth. The second component is the Asd + -DadB + plasmid pair carrying wild-type copies of asdA and dadB , respectively, to complement the mutations. To evaluate the protection efficacy of the dual-plasmid vaccine, S . Typhimurium strain χ9760 (a strain with multiple attenuating mutations: Δ asd Δ alr Δ dadB Δ recF ) was transformed with Asd + and DadB + plasmids specifying pneumococcal antigens PspA and PspC, respectively. Both plasmids were stable in χ9760 for 50 generations when grown in nonselective medium. This was significantly ( P recF + counterpart χ9590 and could be attributed to reduced interplasmid recombination in χ9760. Oral immunization of BALB/c mice with 1 × 10 9 CFU of χ9760 (carrying Asd + -PspA and DadB + -PspC plasmids) elicited a dominant Th1-type serum IgG response against both antigens and protected mice against intraperitoneal challenge with 200 50% lethal doses (LD 50 s) of virulent Streptococcus pneumoniae strain WU2 or intravenous challenge with 100 LD 50 s of virulent S. pneumoniae strain L81905 or intranasal challenge with a lethal dose of S. pneumoniae A66.1 in a pneumonia model. Protection offered by χ9760 was superior to that offered by the mixture of two strains, χ9828 (Asd + -PspA) and χ11026 (DadB + -PspC). This novel dual-plasmid system marks a remarkable improvement in the development of live bacterial vaccines.