Toxoids of Pseudomonas aeruginosa exotoxin-A: photoaffinity inactivation of purified toxin and purified toxin derivatives

Abstract

For the preparation of greatly detoxified but highly immunogenic toxoids, two enzymatically active, low-toxicity derivatives of Pseudomonas aeruginosa exotoxin-A were further inactivated by photoaffinity labeling. These derivatives were formed during toxin purification, when a relatively crude toxin preparation was concentrated by ammonium sulfate precipitation and subsequently dialyzed. These derivatives, designated peak-1 protein (PK-1) and peak-2 protein (PK-2) were antigenically indistinguishable from native toxin, but had isoelectric points (5.00 and 4.90, respectively) that were different from that of the native toxin (4.95). Although the enzymatic activities and molecular weights of PK-1 and PK-2 were similar to those of native toxin, their toxicities were greatly reduced (ca. 500-fold). Photoaffinity labeling of fully active toxin-A, purified by a process which limits the formation of these derivatives, decreased its enzymatic activity (ca. 30-fold) and toxicity (ca. 100-fold). Likewise, photoaffinity labeling of purified PK-1 and PK-2 decreased their enzymatic activities and toxicities (ca. 30-fold and 100-fold, respectively) and, thus, yielded toxoids that were ca. 50,000-fold less toxic than unpurified native toxin. These toxoids were irreversibly detoxified and highly immunogenic during 9 months of storage at 4 degrees C.