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

Abstract

Attenuated Salmonella are useful oral vaccine vectors capable of carrying multiple heterologous antigen genes, but optimal expression of foreign antigens has not yet been achieved. We hypothesized that Salmonella phoP-activated genes, which are transcriptionally activated within antigen-processing macrophages, could prove useful for delivery of heterologous antigens to the immune system. We have created a suicide vector that allows the stable chromosomal insertion of heterologous antigen genes within the phoP-activated gene C (pagC) of Salmonella and permits the expression of heterologous antigens as fusion proteins between the first 84 amino acids of PagC and the chosen antigen. The Escherichia coli phoA gene encoding alkaline phosphatase was cloned into this vector; the resultant plasmid was used to construct Salmonella typhimurium strains that express PagC-alkaline phosphatase fusion proteins from a single chromosomal gene copy. Such strains were administered orally and i.p. as vaccines to BALB/c mice and compared with control strains expressing alkaline phosphatase constitutively. After 3 weeks, mouse sera were analyzed for IgG responses to S. typhimurium lipopolysaccharide and alkaline phosphatase. Remarkably, though all mice had comparable antibody responses to lipopolysaccharide, only mice immunized with strains bearing phoP-activated fusion genes had antibody responses to the heterologous antigen. We conclude that expression of a heterologous antigen from an S. typhimurium in vivo-induced promoter that is activated within macrophages markedly enhances the immunogenicity of a model antigen expressed from a single chromosomal gene copy.

Oral Vaccination withSalmonellaSimultaneously ExpressingYersinia pestisF1 and V Antigens Protects against Bubonic and Pneumonic Plague

“…To enhance the immunogenicity of passenger Ag expression by live Salmonella vaccines (35), various types of promoters have been tested to enhance passenger Ag expression. Such examples include the use of stationary phase inducible promoters, spv and dps, or macrophageinducible promoters to drive heterologous Ag expression in Salmonella vaccine strains to achieve greater immunity (26,27), or the use of a mucosal Ag-uptake adjuvant as an enhancer to strengthen immunogenicity (36). In this study, we focused on two factors for enhancing immunity against plague.…”

Enhanced Immunogenicity in the Murine Airway Mucosa with an AttenuatedSalmonellaLive Vaccine Expressing OprF-OprI fromPseudomonas aeruginosa

“…Despite protein expression at high quantities, these strains mounted only an occasional immune response. We therefore established an in vivo inducible protein expression system in order to better allow the carrier to cross the difficult gut epithelium barrier without the burden of foreign protein expression (6,16). By using the Salmonella P pagC promoter of the phoP/ phoQ regulon that is activated by the intracellular milieu in eukaryotic cells, our vaccine strains had an improved ability to colonize and persist in the GALT.…”

“…Escherichia coli Electro10 (Stratagene) was used for cloning. The oprFoprI fusion gene encoding Met-Ala-(His) 6 OprF 190-342 -OprI 21-83 from P. aeruginosa was cloned on pBR322-derived plasmids downstream of two constitutively active promoters (P yz [14] in pDB3 and P trc in pDB4) or the in vivo inducible Salmonella P pagC promoter (7,16) in plasmids pMW151, pMW209, and pMW210. These in vivo inducible constructs differed in the ribosomal binding site (33) upstream of oprF-oprI, resulting in differential in vivo OprF-OprI expression levels.…”

Salmonella-mediated oral DNA vaccination using stabilized eukaryotic expression plasmids

“…Expression from in vivo inducible promoters has been shown to enhance stable expression and immunogenicity of a number of heterologous proteins in Salmonella typhimurium. 4,5 Chromosomal integration of the expression cassette increased stability but lowered the level of antigen expression to a single copy. 6,7 Using the asd + / Dasd host lethal system, 8,9 only Salmonella carrying the expression plasmid can survive.…”