title: Stable episomal expression system under control of a stress inducible promoter enhances the immunogenicity of Bacillus subtilis as a vector for antigen delivery.
abstract: Bacillus subtilis has been successfully engineered to express heterologous antigens genetically fused to surface-exposed spore coat proteins as a vaccine vehicle endowed with remarkable heat resistance and probiotic effects for both humans and animals. Nonetheless, the immunogenicity of passenger antigens expressed by B. subtilis spores is low particularly following oral delivery. In this work, we describe a new episomal expression system promoting enhanced immunogenicity of heterologous antigens carried by B. subtilis strains, either in the form of spores or vegetative cells, following oral or parenteral delivery to mice. Based on a bi-directional replicating multicopy plasmid, the gene encoding the B subunit of the heat-labile toxin (LTB), produced by enterotoxigenic Escherichia coli (ETEC) strains, was cloned under the control of the B. subtilis glucose starvation inducible (gsiB) gene promoter, active in vegetative cells submitted to heat and other stress conditions. The recombinant plasmid proved to be structurally and segregationally stable in both cells and spores under in vitro and in vivo conditions. Moreover, BALB/c mice orally immunized with B. subtilis cells or spores elicited enhanced anti-LTB systemic (serum IgG) and secreted (fecal IgA) antibody responses, thus, suggesting that antigen expression occurred during in vivo transit. These results indicate that the new episomal expression system may improve the performance of B. subtilis as a live orally-delivered vaccine carrier.
authors.affiliation: Department of Microbiology, Biomedical Sciences Institute, University of São Paulo, 05508-000 São Paulo, Brazil.
authors.forename: Juliano D
authors.initials: JD
authors.lastname: Paccez
authors.forename: Wilson B
authors.initials: WB
authors.lastname: Luiz
authors.forename: Maria E
authors.initials: ME
authors.lastname: Sbrogio-Almeida
authors.forename: Rita C C
authors.initials: RC
authors.lastname: Ferreira
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schumann
authors.forename: Luís C S
authors.initials: LC
authors.lastname: Ferreira
mesh_terms: D000284:Administration, Oral; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001412:Bacillus subtilis / Q000235:genetics* / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology*; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D004768:Enterotoxins / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D029968:Escherichia coli Proteins / Q000235:genetics / Q000276:immunology*; D005243:Feces / Q000737:chemistry; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D007070:Immunoglobulin A / Q000032:analysis; D007074:Immunoglobulin G / Q000097:blood; D007274:Injections, Intraperitoneal; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D014614:Vaccines, Synthetic; C075520:gsiB protein, Bacillus subtilis; C027538:heat-labile enterotoxin, E coli