Detoxified Bacterial Endotoxins II. Preparation and Biological Properties of Chemically Modified Crude Endotoxins from Salmonella tphimurium'

MARTN, WILLIAM J. (University of Utah, Salt Lake City), AND STANLEY MARCUS. Detoxified bacterial endotoxins. II. Preparation and biological properties of chemically modified crude endotoxins from Salmonella typhimurium. J. Bacteriol. 91: 1750-1758. 1966.-Chemical modification of a crude endotoxin prepared by the Roschka-Edwards (RE) procedure from a strain of Salmonella typhimurium yielded products which were nontoxic for mice and had reduced fever effects in rabbits. A reduction in rabbit pyrogenicity of approximately 100 times was noted with a potassium periodate-treated RE preparation when compared with the parent RE preparation. Measured in a similar fashion, pyrogenicity of a potassium methylate-treated RE preparation was reduced by a factor of 10 while pyrogenicity of a boron trifluoride RE preparation was unchanged. All of these endotoxoids, including the parent RE preparation, showed little toxicity for mice. Immunogenicity was determined in mice by comparing Boivin, RE, and endotoxoid preparations with a heatkilled, phenol-preserved (HP) vaccine prepared from the same strain of S. typhimurium. Employing a 10 LD5w challenge, the protective immunogenicity of the respective vaccines was determined by active immunized mouse protection tests. Although two 100 lg immunizing doses of the Boivin, RE, and the respective endotoxoid preparations varied in mouse protection (potassium methylate RE > Boivin > RE > boron trifluoride RE > potassium peliodate RE), it was evident that, with the exception of the potassium methylate preparation, the HP vaccine yielded greatest protection against the 10 LDwo challenge with S. typhimurium. Further mouse protection experiments suggested that the minimal immunogenic dose of the potassium methylate RE vaccine preparation was approximately 50 MAg. These data indicated an approximate fivefold difference between the minimal pyrogenic dose (10 MAg) and the minimal immunogenic dose (50 Mug). These findings further suggest that potassium methylate RE vaccine preparations should be considered in the search for less toxic enteric fever vaccines. Several 0 antigen isolation methods have been described since that first reported by Boivin, Mesrobeanu, and Mesrobeanu (1). Their trichloroacetic acid extraction procedure enabled these investigators to isolate toxic and immunizing fractions from gram-negative bacteria. This method of isolating endotoxin from enteroI This paper is an essential portion of a thesis submitted to the Department of Microbiology, University of Utah, in partial fulfillment of the requirements for the Ph.D. degree. 2Present address: Enteric Bacteriology Unit, Communicable Disease Center, Public Health Service, U.S. Department of Health, Education, and Welfare, Atlanta, Ga. bacteriaceal organisms was followed by many other extraction procedures (11, 17, 19, 20, 22, 23). In addition, the selective reduction of one or more of the toxic properties of endotoxin with retention of antigenic, nontoxic factors of value in immunization of the host has also been the subject of much experimental effort (2, 3, 4, 14, 21). Recently, we described the acetylation of a crude endotoxin material obtained by the Roschka-Edwards (RE) method from a strain of Salmonella typhimurium (9). The results indicated that the preparation obtained had reduced pyrogenicity in rabbits and was not lethal in mice unless given in doses exceeding 1 g/kg of body weight. Moreover, experiments in mice with this 1750 on N ovem er 6, 2017 by gest http/jb.asm .rg/ D ow nladed fom VOL. 91, 1966 PROPERTIES OF CHEMICALLY MODIFIED CRUDE ENDOTOXINS same acetylated Roschka-Edwards (Acet-RE) preparation indicated a protective capacity comparable to that obtained with heat-killed, phenol-preserved vaccine employed from the same strain of S. typhimurium. In studying the effects of heat and chemicals on erythrocyte-modifying, antigenic, toxic, and pyrogenic properties of enterobacteriaceal lipopolysaccharides, Neter et al. (13) reported that, if lipopolysaccharides are exposed to a periodate solution, they lose theis antigenic specificity and toxicity but retain their pyrogenicity. More recently, the use of a variety of other chemical procedures for the purpose of detoxifying endotoxin preparations has been reported by Nowotny (15, 16) and by Johnson and Nowotny (5). Employing endotoxin isolated by the trichloroacetic acid method from Serratia marcescens, these investigators found that toxicity could be decreased or completely abolished by chemical methods, such as treatment with boron trifluoride, potassium methylate, or pyridinium formate. In addition, the ability to induce the second fever peak of the biphasic fever response characteristic of endotoxin was diminished; the preparations produced potent antisera except in the case of the boron trifluoride-treated material. These investigators also reported that these endotoxoids enhanced the nonspecific resistance of mice and rabbits against experimental infections. With these observations in mind, as well as for purposes of comparison, the chemical modification procedures of transesterification with boron trifluoride, deacylation with potassium methylate, and oxidation with potassium periodate were applied to the same crude endotoxin material obtained by the RE method from a strain of S. typhimurium. By use of the previously reported biological parameters of pyrogenicity, lethality, and immunogenicity, data were obtained which suggest the feasibility of using these chemical methods as potential procedures for producing a less toxic enteric fever vaccine. MATERIALS AND METHODS Experimental animals. Testing for pyrogenicity was done in rabbits that had been properly "screened" by methods previously reported (8). Rabbits which yielded positive fever results, and were used in more than one experiment involving pyrogenicity, were "rested" for 2 or more weeks as outlined by the United States Pharmacopeia XVl. Tests for immunogenicity and lethality were performed in white mice weighing between 20 and 25 g; deaths were recorded for a period of 1 week. Organism. A mouse-virulent strain of S. typhimurium originally obtained from the Utah State Department of Health was employed in all experiments. This organism has been maintained by transfer culture at monthly intervals on tryptose-phosphate-agar slants. It is incubated at 37 C for 24 hr and thereafter kept at room temperature. RE procedure. The method and handling of the parent RE preparation was described in a previous publication (9). To summarize, pooled washings of previously heat-killed S. typhimurium organisms were taken through three changes of absolute ethyl alcohol and acetone. The remaining sediment was dried at 37 C and then ground to a fine powder. This powder, consisting primarily of the "hulls" of these Salmonella organismns, was further disintegrated by the use of an ultrasonic disintegrating device. A 15-ml amount of water was added to 1,500 mg of this crude endotoxin and disintegrated (Sonifier, Branson Instruments, Stamford, Conn.) for 15 min under sterile conditions. The suspension was filtered (Millipore filter), and the resulting precipitate was dried overnight at 37 C. As previously reported, employing this procedure of ultrasonic exposure produced no visible changes in yield or in alteration of this crude endotoxin. Periodate procedure (13). The periodate-treated RE crude endotoxin was prepared as follows: 200 mg of the parent RE preparation was suspended in 160 ml of distilled water; 20 ml of 1.0 N sodium acetate buffer (pH 5.0) as well as 20 ml of 0.1 N potassium periodate solution were added. This material was kept in the dark for 24 hr at room temperature and then dialyzed in cellophane (Visking) casing against distilled water for 24 hr. Finally, the material was concentrated by pervaporation at room temperature and freeze-dried. The resulting product was a light, white, fluffy powder with a yield of 112 mg (56%). Although Neter and his associates employed 0.1 N sodium periodate solution in their procedure, it is of interest to note that the potassium periodate-treated RE material in identical concentrations reacted in a similar manner; i.e., our freeze-dried oxidation product was also insoluble in distilled water, but was soluble upon the addition of small amounts of 1.0 M NaOH and subsequent neutralization with dilute acetic acid. The soluble stock suspension of 50 mg/10 ml of saline was readily prepared and had a characteristic milky appearance. Potassium methylate procedure (15). The parent RE preparation was treated by extraction with anhydrous methanol, a procedure which removes small amounts of inert impurities but does not alter the biological properties of endotoxins (15). To 1 g of dry RE preparation, 250 ml of methanol was added, and the resulting suspension was refluxed on a hot-plate with continuous magnet-stirring for 60 min, cooled, and finally centrifuged in the cold at 2,500 rev/min for 10 min. The clear supematant fluid was drawn off, and the precipitate was dried in a vacuum desiccator for 48 hr. This "purified" RE preparation was subjected to the following chemical procedures. To 200 mg of the above described RE preparation, 40 ml of 0.02 M potassium methylate in anhydrous methanol was added, and the mixture was refluxed for 60 min. The insoluble residue was centrifuged in the cold at 2,500 rev/min for 10 min, washed with methanol, and dried in a vacuum desiccator. This preparation was a very fine, light powder which was ivory in color, and 175 1 on N ovem er 6, 2017 by gest http/jb.asm .rg/ D ow nladed fom