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 PE64⌬553pil, was expressed in Escherichia coli, refolded to a near-native conformation, and then characterized by various biochemical and immunological assays. PE64⌬553pil 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.Colonization of cystic fibrosis (CF) individuals with Pseudomonas aeruginosa represents a significant negative milestone in the progression of this disease. Once colonized, patients are subject to the damaging effects of various secreted virulence factors and to the inflammatory response of the host immune system. A key component of colonization is the adhesion of type IV pili to asialo-GM1 receptors on the surface of epithelial cells (26,45,48; for a review, see reference 21). Type IV pili are composed of pilin polymers arranged in a helical structure with five subunits per turn (19,41). The portion of the pilin protein responsible for cell binding is located near the C terminus (amino acids 129 to 142) in a -turn--turn loop subtended from a disulfide bond (5,6,23,36). A 12-or 17-aminoacid sequence (depending on the specific strain) in this loop interacts with receptors on epithelial cells. For CF individuals, the overproduction of the R domain of the mutant CF transmembrane conductance regulator can lead to an increased level of asialo-GM1 and, accordingly, an increased binding of P. aeruginosa (3,26,45). Functional studies of pilin have indicated that only the last pilin subunit (the tip) of a pilus interacts with epithelial cell receptors (31). To interfere with bacterial adhesion, anti-pilin antibodies will need to recognize residues that are normally located at the C-terminal loop of pilin (32). Structural studies have indicated that this loop is dominated by main chain residues; and this may explain why pilins from distinct strains bind the same receptor despite sequence variation and the presence of both 12-and 17-aminoacid loops. Generating antibodies to the C-terminal pilin loop may be useful in reducing or eliminating colonization (15,22,47). Table 1 lists the pilin loop sequences from several strains of P. aeruginosa.Pseudomonas exotoxin A (here called PE), a prominent virulence factor secreted by P. aeruginosa, is cytotoxic for mammalian cells by virtue of its ability to enter cel...