title: Expression of Neisseria meningitidis class 1 porin as a fusion protein in Escherichia coli: the influence of liposomes and adjuvants on the production of a bactericidal immune response.
abstract: High level expression of meningococcal class 1 protein was achieved in Escherichia coli using the p-GEMEX-1 vector, in which the protein was expressed in inclusion bodies (IB), as a fusion with the bacteriophage T7 gene 10 capsid protein. The fusion protein (FP) was engineered with a factor Xa protease site between the gene 10 and class 1 protein, but treatment with the enzyme resulted in cleavage at additional sites within the class 1 protein. Since it was not possible to remove the leader protein, the intact FP provided an alternative antigen for immunization. Antisera raised to FP, solubilized from IB and incorporated into liposomes, generated a subtype-specific response which was weakly bactericidal for meningococci. In order to remove any possible effect of E. coli LPS present in IB, the FP was further purified by SDS-PAGE and incorporated into liposomes, either alone or in combination with the adjuvants monophosphoryl lipid A or muramyl dipeptide. The incorporation of adjuvants in liposomes resulted in stimulation of the overall immune response to FP, but the resulting antisera were not bactericidal. However an effective bactericidal response was obtained with the purest preparation of FP in liposomes, without any additional adjuvants, revealing that attempts to increase further the immunogenicity of such antigens must not be at the expense of interfering with optimal protein folding.
authors.affiliation: Molecular Microbiology Group, University of Southampton Medical School, Southampton General Hospital, U.K.
authors.forename: S J
authors.initials: SJ
authors.lastname: Ward
authors.forename: D
authors.initials: D
authors.lastname: Scopes
authors.forename: M
authors.initials: M
authors.lastname: Christodoulides
authors.forename: I N
authors.initials: IN
authors.lastname: Clarke
authors.forename: J E
authors.initials: JE
authors.lastname: Heckels
mesh_terms: D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000276:immunology; D000276:Adjuvants, Immunologic; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D017123:Bacteriophage T7 / Q000235:genetics; D001681:Biological Assay; D015153:Blotting, Western; D002213:Capsid / Q000235:genetics; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008050:Lipid A / Q000031:analogs & derivatives / Q000276:immunology; D008070:Lipopolysaccharides / Q000302:isolation & purification; D008081:Liposomes / Q000276:immunology*; D008854:Microscopy, Electron; D009345:Neisseria meningitidis / Q000235:genetics* / Q000276:immunology*; D018272:Porins / Q000235:genetics* / Q000276:immunology*; D021382:Protein Sorting Signals / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology*
chemical_list: D000276:Adjuvants, Immunologic; D000907:Antibodies, Bacterial; D008050:Lipid A; D008070:Lipopolysaccharides; D008081:Liposomes; D018272:Porins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000119:Acetylmuramyl-Alanyl-Isoglutamine; D015951:Factor Xa; C048436:monophosphoryl lipid A