title: Construction of recombinant S-layer proteins (rSbsA) and their expression in bacterial ghosts--a delivery system for the nontypeable Haemophilus influenzae antigen Omp26.
abstract: This study has investigated the feasibility of a combination of recombinant surface layer (S-layer) proteins and empty bacterial cell envelopes (ghosts) to deliver candidate antigens for a vaccine against nontypeable Haemophilus influenzae (NTHi) infections. The S-layer gene sbsA from Bacillus stearothermophilus PV72 was used for the construction of fusion proteins. Fusion of maltose binding protein (MBP) to the N-terminus of SbsA allowed expression of the S-layer in the periplasm of Escherichia coli. The outer membrane protein (Omp) 26 of NTHi was inserted into the N-terminal and C-terminal regions of SbsA. The presence of the fused antigen Omp26 was demonstrated by Western blot experiments using anti-Omp26 antisera. Electron microscopy showed that the recombinant SbsA maintained the ability to self-assemble into sheet-like and cylindrical structures. Recombinant E. coli cell envelopes (ghosts) were produced by the expression of SbsA/Omp26 fusion proteins prior to gene E-mediated lysis. Intraperitoneal immunization with these recombinant bacterial ghosts induced an Omp26-specific antibody response in BALB/c mice. These results demonstrate that the NTHi antigen, Omp26, was expressed in the S-layer self-assembly product and this construct was immunogenic for Omp26 when administered to mice in bacterial cell envelopes.
authors.affiliation: Institute of Microbiology and Genetics, Vienna Biocentre, University of Vienna, 1090 Vienna, Austria.
authors.forename: Eva M
authors.initials: EM
authors.lastname: Riedmann
authors.forename: Jennelle M
authors.initials: JM
authors.lastname: Kyd
authors.forename: Adam M
authors.initials: AM
authors.lastname: Smith
authors.forename: Sara
authors.initials: S
authors.lastname: Gomez-Gallego
authors.forename: Katri
authors.initials: K
authors.lastname: Jalava
authors.forename: Allan W
authors.initials: AW
authors.lastname: Cripps
authors.forename: Werner
authors.initials: W
authors.lastname: Lubitz
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001433:Bacteriolysis; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D005822:Genetic Vectors; D006192:Haemophilus Infections / Q000276:immunology / Q000517:prevention & control; D018073:Haemophilus Vaccines / Q000008:administration & dosage* / Q000276:immunology; D006193:Haemophilus influenzae / Q000145:classification / Q000235:genetics / Q000276:immunology*; D007114:Immunization; D008297:Male; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D018073:Haemophilus Vaccines; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; C118414:OMP26 protein, Haemophilus influenzae; D011993:Recombinant Fusion Proteins; C088619:sbsA protein, Bacillus stearothermophilus