title: Expression of a Porphyromonas gingivalis hemagglutinin on the surface of a Salmonella vaccine vector.
abstract: Live, attenuated Salmonella strains can serve as vectors for the delivery of recombinant vaccine antigens for development of oral mucosal vaccines. Various vaccine parameters can affect the immune responses elicited by Salmonella vectors, including the expression level, location and timing of expressed antigens. We have previously established immunogenic Salmonella enterica serovar Typhimurium strains which cytoplasmically express hemagglutinin B (HagB) of Porphyromonas gingivalis, a putative periodontal pathogen. In this study, we sought to determine whether the 39 kDa HagB protein could be stably expressed on the surface of an avirulent Salmonella vaccine strain. The hagB gene was cloned into an expression plasmid as a C-terminal fusion with Lpp-OmpA, a hybrid surface display system. High expression of Lpp-OmpA-HagB proved to be toxic to the vaccine strain, and it was necessary to introduce attenuating mutations in the trc promoter. Stable expression was obtained in transformants with promoter mutations that resulted in low levels of expression. The expression of Lpp-OmpA-HagB was confirmed by ELISA and Western blot. Localization to the outer membrane/periplasm was confirmed by transmission electron microscopy using immunogold labeling, surface labeling of whole mounts using electron microscopy, flow cytometry, and by quantitation of HagB in cytoplasmic, as well as inner and outer cell membrane fractions. When delivered orally in mice, the surface-expressing strain induced higher serum IgG and IgA responses to HagB than a cytoplasmic expressing strain, while responses in secretions were comparable. These results suggest that surface localization may differentially enhance the immunogenicity of antigens expressed by live, avirulent Salmonella vaccine vectors.
authors.affiliation: Department of Oral Biology, P.O. Box 100424, University of Florida, Gainesville, FL 32610, United States.
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Isoda
authors.forename: Scott P
authors.initials: SP
authors.lastname: Simanski
authors.forename: Latha
authors.initials: L
authors.lastname: Pathangey
authors.forename: Amy E S
authors.initials: AE
authors.lastname: Stone
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Brown
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D005434:Flow Cytometry; D005822:Genetic Vectors; D007114:Immunization; D037102:Lectins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D016253:Microscopy, Immunoelectron; D016966:Porphyromonas gingivalis / Q000378:metabolism; D011995:Recombination, Genetic; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D014613:Vaccines, Attenuated / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D037102:Lectins; D014613:Vaccines, Attenuated; C114923:hemagglutinin A, Porphyromonas gingivalis