Use of the nirB Promoter to Direct the Stable Expression of Heterologous Antigens in Salmonella Oral Vaccine Strains: Development of a Single–Dose Oral Tetanus Vaccine

Abstract

Plasmid pTETnir15, which directs the expression of the non-toxic immunogenic fragment C of tetanus toxin from the anaerobically inducible nirB promoter, was introduced into the Salmonella typhimurium aroA aroD live oral vaccine strain BRD509. The resulting strain, designated BRD847, was used to vaccinate orally BALB/c mice and was tested for plasmid stability and its ability to protect against a lethal tetanus toxin challenge. pTETnir15 was stably inherited by bacteria growing or persisting in the tissues of immunized mice whereas another BRD509 derivative, designated BRD753, harboring plasmid pTET85 which directs fragment C expression from the tac promoter, was highly unstable. Mice immunized with a single oral dose of BRD847 developed high levels of circulating anti-fragment C antibodies and were solidly protected against tetanus toxin challenge. Mice immunized with a single oral dose of BRD753 developed no detectable anti-fragment C antibodies. After boosting, antibodies were detected, but the mice were only partially protected against tetanus toxin challenge. Thus the use of an in vivo inducible promoter such as nirB may be a generally applicable approach to obtaining the stable in vivo expression of heterologous antigens in Salmonella vaccine strains.

Macrophage-inducible expression of a model antigen in Salmonella typhimurium enhances immunogenicity.

“…Here we have shown that foreign antigen expression driven by macrophage-inducible promotors leads to a dramatic humoral response to an antigen expressed from a single chromosomal copy. Our results utilizing Pag-alkaline phosphatase fusion antigens are comparable to previously published murine data for multicopy plasmid-encoded heterologous antigens (3)(4)(5) and superior to those for single chromosomal copies of malarial and bacterial antigens (7)(8)(9). In our experiments, even 3-to 10-fold greater constitutive expression was unable to induce humoral responses approaching those induced by phoP-regulated expression.…”

“…Other environmentally regulated promotors postulated to be expressed in vivo, including those induced by low iron concentrations or anaerobiasis, have been utilized for expression of heterologous antigens in plasmid expression systems in Salmonella (3,35). Therefore, as another control, Shiga-like toxin I-B (SLT-IB) expressed under control of an ironregulated promotor was integrated at the pagC chromosomal locus in strain SL3261.…”

“…Attenuated Salmonella have been widely investigated as live oral vaccine vectors capable of stimulating humoral, cell-mediated, and mucosal immunity in mammalian hosts (1), but optimal expression of heterologous antigens within Salmonella has not yet been achieved (2). In most vaccine strains, foreign immunogens have been constitutively expressed from plasmid-encoded antigen genes (3)(4)(5)(6). Difficulties associated with these vaccines have included the instability of plasmids within mammalian hosts and toxic effects of overexpressed heterologous proteins on bacterial vectors, leading to poor immunogenicity despite high levels of antigen expression.…”

Intestinal villous M cells: An antigen entry site in the mucosal epithelium

“…The recombinant S. typhimurium BRD 847 strain used in the immunization study is a double aroA aroD mutant that expresses the nontoxic, immunogenic 50-kDa ToxC fragment of tetanus toxin from plasmid pTETnir15 under the control of the anaerobically inducible nirB promoter (rSalmonella-ToxC) (30). For the control, rSalmonella that are not expressing ToxC were adopted.…”

Anti-flagellin antibody responses elicited in mice orally immunized with attenuated Salmonella enterica serovar Typhimurium vaccine strains

“…Additionally, peptides, particularly those encoding B-cell epitopes, may be expressed by attenuated Salmonella strains as hybrid proteins following genetic fusion to bacterial proteins. This approach may improve both the stability and the immunogenicity of target antigens delivered orally in live bivalent vaccines (Dougan et al 1987, Chatfield et al 1992.…”