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 wildtype 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: Δasd Δalr ΔdadB ΔrecF) 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<0.05) greater than the stability seen in its recF+ counterpart Χ9590 and could be attributed to reduced interplasmid recombination in Χ9760. Oral immunization of BALB/c mice with 1 × 109 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 (LD50s) of virulent Streptococcus pneumoniae strain WU2 or intravenous challenge with 100 LD50s 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. © 2012, American Society for Microbiology.