title: 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: 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 × 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.
authors.affiliation: Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, Arizona, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Xin
authors.forename: Soo-Young
authors.initials: SY
authors.lastname: Wanda
authors.forename: Xiangmin
authors.initials: X
authors.lastname: Zhang
authors.forename: Javier
authors.initials: J
authors.lastname: Santander
authors.forename: Giorgio
authors.initials: G
authors.lastname: Scarpellini
authors.forename: Karen
authors.initials: K
authors.lastname: Ellis
authors.forename: Praveen
authors.initials: P
authors.lastname: Alamuri
authors.forename: Roy
authors.initials: R
authors.lastname: Curtiss
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D000942:Antigens, Bacterial / Q000276:immunology*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D005260:Female; D007070:Immunoglobulin A / Q000378:metabolism; D007074:Immunoglobulin G / Q000097:blood; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D011018:Pneumonia, Pneumococcal / Q000517:prevention & control; D011994:Recombinant Proteins / Q000276:immunology; D012481:Salmonella Infections, Animal / Q000517:prevention & control*; D022562:Salmonella Vaccines / Q000276:immunology*; D012486:Salmonella typhimurium / Q000276:immunology; D014613:Vaccines, Attenuated; D014614:Vaccines, Synthetic
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D022562:Salmonella Vaccines; D014613:Vaccines, Attenuated; D014614:Vaccines, Synthetic