Dual-function vaccine for Pseudomonas aeruginosa: characterization of chimeric exotoxin A-pilin protein.
Pseudomonas aeruginosa is the major infectious agent of concern for cystic fibrosis patients. Strategies to prevent colonization by this bacterium and/or neutralize its virulence factors are clearly needed. Here we characterize a dual-function vaccine designed to generate antibodies to reduce bacterial adherence and to neutralize the cytotoxic activity of exotoxin A. To construct the vaccine, key sequences from type IV pilin were inserted into a vector encoding a nontoxic (active-site deletion) version of exotoxin A. The chimeric protein, termed PE64Delta553pil, was expressed in Escherichia coli, refolded to a near-native conformation, and then characterized by various biochemical and immunological assays. PE64Delta553pil bound specifically to asialo-GM1, and, when injected into rabbits, produced antibodies that reduced bacterial adherence and neutralized the cell-killing activity of exotoxin A. Results support further evaluation of this chimeric protein as a vaccine to prevent Pseudomonas colonization in susceptible individuals.
Hertle
R
R
Biotherapy Section, Laboratory of Molecular Biology, CCR, National Cancer Institute, Bethesda, Maryland 20892-4255, USA.
Mrsny
R
R
Fitzgerald
D J
DJ
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D001422:Bacterial Adhesion / Q000276:immunology; D001427:Bacterial Toxins; D001428:Bacterial Vaccines / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D005098:Exotoxins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D036781:Fimbriae Proteins; D015870:Gene Expression; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000276:immunology* / Q000502:physiology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014407:Tumor Cells, Cultured; D014611:Vaccination; D014614:Vaccines, Synthetic / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000907:Antibodies, Bacterial; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D005098:Exotoxins; D036781:Fimbriae Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A