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 9760 (a strain with multiple attenuating mutations: ⌬asd ⌬alr ⌬dadB ⌬recF) was transformed with Asd ؉ and DadB ؉ plasmids specifying pneumococcal antigens PspA and PspC, respectively. Both plasmids were stable in 9760 for 50 generations when grown in nonselective medium. This was significantly (P < 0.05) greater than the stability seen in its recF

New technologies in developing recombinant attenuated Salmonella vaccine vectors

“…The DadB + vector can be used either alone (in a Δ alr Δ dadB mutant) or in conjunction with an Asd + plasmid (in a Δ asdA Δ alr Δ dadB triple-mutant strain) in S . Typhimurium to deliver multiple antigens (see below) [115]. …”

“…Our initial constructions showed that there was an inverse correlation between the amount of DadB produced for complementation of the Δ alr Δ dadB mutant when using DadB + vectors with different copy numbers and this attenuated the virulence of vaccine strains [115]. It was reported that low-copy-number plasmids are relatively more stable and induce greater levels of antigen-specific immune responses than their high-copy-number versions [121].…”

“…The presence of two plasmids in a single vaccine strain did not decrease synthesis of individual antigens. Although the Asd + plasmid was more stable than the DadB + plasmid, the stability of both plasmids was ~95% over 50 generations of growth and did not differ significantly, suggesting that antigen synthesis and delivery in vivo were not compromised in this system [115]. …”

“…Deletion of recF reduces recombination frequency between plasmids at least 10 times without compromising the virulence of the bacterium [132]. Furthermore, including a recF deletion mutation improved the stability of two balanced-lethal vectors, especially of the DadB + vector in a Salmonella vaccine strain with Asd + and DadB + vectors each specifying a different protective antigen [115]. The Salmonella vaccine strain carrying PspA and PspC by Asd + and DadB + vectors, respectively, induced higher serum and secretory antibody responses than the strain delivering a single antigen or than a mixture of two vaccine strains each specifying one protective antigen and offered superior protection against challenge with different serotypes of S. pneumoniae [115].…”

A Bivalent Typhoid Live Vector Vaccine Expressing both Chromosome- and Plasmid-Encoded Yersinia pestis Antigens Fully Protects against Murine Lethal Pulmonary Plague Infection

“…Encoding several foreign antigens on a single expression plasmid may lead to unacceptably large and unstable plasmids which spontaneously delete the desired coding regions, thereby compromising immune specificity (26,27). The use of several compatible plasmids for antigen expression in a single live vector vaccine may exacerbate the metabolic burden and again overattenuate the vaccine strain, leading to plasmid loss in the absence of selection (28). Finally, administration of several vaccine strains encoding individual antigens cannot guarantee equivalent antigen delivery from all strains, again potentially interfering with immune responses (29).…”