PREPARATION AND BIOLOGICAL PROPERTIES OF A CHEMICALLY MODIFIED ESCHERICHIA COLI ENDOTOXIN OF HIGH IMMUNOGENIC POTENCY AND LOW TOXICITY

Abstract

The preparation of a non-pyrogenic Boivin antigen by reductive treatment with LiAlH4 is described. Infrared spectroscopic studies showed that this procedure resulted in the elimination of esterbound fatty acid. The chemically modified product showed virtually no toxicity when tested for lethality in mice (LD50 > 1.0 mg) and no pyrogenicity in rabbits (0.1 mg); it also failed to cause leukopenia in rabbits (5 [mu]g) or intradermal inflammation in man (0.1 [mu]g). Comparative studies showed that reduction with LiAlH4 and controlled hydrolysis with LiOH gave products of different chemical and biological properties. Mild alkaline hydrolysis, while virtually abolishing immunogenicity in rats, caused only moderate reduction of the pyrogenicity for rabbits and lethal toxicity for mice. Immunization of rabbits with the detoxified material induced typical pyrogen tolerance, and immunization of mice caused the same increase of resistance to lethal challenge doses of regular endotoxin as was observed after immunization with the parent endotoxin. The formation of precipitating antibodies after immunization of rats with various endotoxin preparations was studied by Ouchterlony''s agar gel diffusion technique. The sera of rats immunized with the detoxified product produced the earliest, strongest and most completely cross reacting precipitin bands of all the antigens tested. Significant protection of rats against Escherichia coli induced pyelonephritis was observed after immunization with the non-toxic antigen.