Detoxified bacterial endotoxns. II. Preparation and biological properties of chemically modified crude endotoxins from Salmonella typhimurium

Martin, 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 heat-killed, phenol-preserved (HP) vaccine prepared from the same strain of S. typhimurium. Employing a 10 ld(50) challenge, the protective immunogenicity of the respective vaccines was determined by active immunized mouse protection tests. Although two 100 mug immunizing doses of the Boivin, RE, and the respective endotoxoid preparations varied in mouse protection (potassium methylate RE > Boivin > RE > boron trifluoride RE > potassium periodate RE), it was evident that, with the exception of the potassium methylate preparation, the HP vaccine yielded greatest protection against the 10 ld(50) challenge with S. typhimurium. Further mouse protection experiments suggested that the minimal immunogenic dose of the potassium methylate RE vaccine preparation was approximately 50 mug. These data indicated an approximate fivefold difference between the minimal pyrogenic dose (10 mug) 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.     

Pyrogenic Principle of the Ribonucleic Acid from the Yeast Candida utilis

Experiments were performed to characterize the pyrogenic principle of ribonucleic acid (RNA) from the yeast Candida utilis. It was shown that ribonuclease hydrolysis of the RNA does not lead to inactivation of the pyrogenicity. Pyrogenicity was, however, destroyed by treatment with sodium deoxycholate. On column chromatography with Biogel under sterile and pyrogen-free conditions, the pyrogenic principle of yeast RNA was eluted together with the RNA. After treatment of the RNA with ribonuclease, it was possible to separate the pyrogenic activity from the RNA (hydrolysis products) to a great extent. Column chromatography of Escherichia coli endotoxin showed that the endotoxin was eluted in the same fractions as the pyrogenic activity of yeast RNA. On the basis of the behavior of the pyrogen, it may very well be that the fever reaction is produced not by the nucleic acid but by pyrogenic contaminants of the RNA preparation.     

Relation of polysaccharide content to some biological properties of endotoxins from mutants of Salmonella typhimurium

Kessel, R. W. I. (Rutgers, The State University, New Brunswick, N.J.), Henry H. Freedman, and Werner Braun. Relation of polysaccharide content to some biological properties of endotoxins from mutants of Salmonella typhimurium. J. Bacteriol. 92:592-596. 1966.-Endotoxins were extracted by the phenol-water procedure from a variety of Salmonella typhimurium mutants with known differences in the composition of their cell wall polysaccharides. The lethality of these preparations for mice proved to be correlated with the complexity of the polysaccharide: endotoxin from the smooth parent strain and from rough strains with several sugars attached to the heptose-phosphate backbone were of high toxicity, whereas endotoxin from a mutant possessing only glucose attached to the heptose-phosphate backbone was less toxic, and endotoxin from a mutant possessing the backbone only was least toxic. All of these mutants yielded endotoxins that were equally capable of protecting mice against subsequent challenge with Pseudomonas aeruginosa. Material obtained from a heptoseless mutant by the phenol-water method proved to be neither toxic nor protective. The apparent dissociation of biological properties that can be achieved with the aid of endotoxin preparations from certain mutants is discussed in terms of possible mechanisms.     

Immunogenic endotoxin associated protein from a rough strain of Salmonella

Abstract A multimolecular complex of polypeptides found associated with the lipopolysaccharide endotoxin in Salmonella , reffered to as endotoxin-associated protein (EP), has been extracted from a rough strain of Salmonella typhimurium which does not synthesize 0 antigens. Since standard methods of extraction applicable to smooth strains of Salmonella were not successful for this rough strain, two modified procedures were developed. The resulting products were similar to smooth EP in terms of their biochemical, physical and mitogenic properties. When the immunogenicity of the rough EP was characterized by a protection assay in mice challenged with virulent Salmonella , it was found that the rough EP preparations were protective; however, they were not as active as the EP from a smooth strain of S. typhimurium .     

Immunogenic endotoxin associated protein from a rough strain of Salmonella

A multimolecular complex of polypeptides found associated with the lipopolysaccharide endotoxin in Salmonella, referred to as endotoxin-associated protein (EP), has been extracted from a rough strain of Salmonella typhimurium which does not synthesize 0 antigens. Since standard methods of extraction applicable to smooth strains of Salmonella were not successful for this rough strain, two modified procedures were developed. The resulting products were similar to smooth EP in terms of their biochemical, physical and mitogenic properties. When the immunogenicity of the rough EP was characterized by a protection assay in mice challenged with virulent Salmonella, it was found that the rough EP preparations were protective; however, they were not as active as the EP from a smooth strain of S. typhimurium.     

Biological activity of synthetic heptaacyl lipid A representing a component of Salmonella minnesota R595 lipid A

A synthetic lipid A (preparation 516), containing seven acyl groups and representing one component of natural free lipid A of Salmonella minnesota R595, has been investigated for biological activity in a number of endotoxin test systems. It was found that the synthetic preparation was, in typical in vivo endotoxin tests (lethality, pyrogenicity, Shwartzman reactivity) as well as in its antigenicity and macrophage activation capacity, significantly less active than natural Salmonella lipid A. However, in other in vitro assay systems (B-cell mitogenicity, complement activation, Limulus amoebocyte lysate gelation) it expressed similar activity as Salmonella lipid A.     

Relation of pyrogenic and emetic properties of enterobacteriaceal endotoxin and of staphylococcal enterotoxin

Martin, William J. (College of Medicine, University of Utah, Salt Lake City), and Stanley Marcus. Relation of pyrogenic and emetic properties of enterobacteriaceal endotoxin and of staphylococcal enterotoxin. J. Bacteriol. 87:1019-1026. 1964.-The pyrogenic, endotoxic fraction of enterobacteriaceal organisms, isolated in a relatively impure or purified state, is both pyrogenic in rabbits and cats as well as emeticogenic in cats in microgram amounts. Employing classical methods for preparation of enterobacteriaceal endotoxin from known enterotoxic strains of Staphylococcus aureus yielded preparations with less than one-hundredth the activity of the enterobacteriaceal material. However, a purified enterotoxin material from an S. aureus strain, with protein-like rather than polysaccharidelike properties, was found to be both pyrogenic and emeticogenic in microgram amounts. These results suggest the probability that the enterobacteriaceal pyrogenemetic substance(s) is a significant part of the whole cell, whereas the staphylococcal enterotoxin is elaborated as a metabolic product which apparently does not accumulate in the whole cell.     

The Escherichia coli efg gene and the Rhodobacter capsulatus adgA gene code for NH3-dependent NAD synthetase.

The essential gene efg, which complements ammonia-dependent growth (adgA) mutations in Rhodobacter capsulatus and is located at 38.1 min on the Escherichia coli chromosome, was found to code for NH3-dependent NAD synthetase. Crude extracts from a strain which overproduces the efg gene product contained up to 400 times more activity than crude extracts from the control strain, and the purified Efg protein possessed-NH3-dependent NAD synthetase activity. Glutamine-dependent NAD synthetase activity was found in crude extracts of E. coli but not in the purified enzyme, suggesting that it may be catalyzed by an additional subunit. An R. capsulatus strain carrying an adgA mutation was found to be deficient in NAD synthetase activity, and activity was restored by complementation with the E. coli gene. In accordance with the nomenclature proposed for Salmonella typhimurium (K. T. Hughes, B. M. Olivera, and J. R. Roth, J. Bacteriol. 170:2113-2120, 1988), the efg and adgA genes should now be designated nadE.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

The neutral fraction (neutral CPS-K) of Klebsiella pneumoniae capsular polysaccharide (CPS-K) from type 1, Kasuya strain, has already been reported as the active substance responsible for the strong adjuvant effect of CPS-K. The present results demonstrate that neutral CPS-K exhibits further common biological activities with lipopolysaccharide (LPS) isolated from Salmonella enteritidis. The intensity of the lethality in mice of neutral CPS-K by the intraperitoneal route is very similar to that of LPS. Its lethality for mice by the intravenous (i.v.) route is significantly stronger than that of LPS, because the degree of increase in the sensitivity to their lethality by i.v. challenge is smaller for LPS than for neutral CPS-K. The intensity of the pyrogenicity of neutral CPS-K in rabbits is approximately one-tenth of that of LPS as judged by the minimal pyrogenic doses and fever indices. The skin-preparatory potency of neutral CPS-K for the dermal Shwartzman phenomenon in rabbits is also approximately one-tenth of that of LPS compared on the basis of the minimal skin-preparatory doses. When injected i.v., neutral CPS-K exhibits a provocative effect on hemorrhagic reactions in skin sites prepared with neutral CPS-K or LPS.     

Heat-labile antigens of Salmonella enteritidis. II. Mouse-protection studies

Collins, F. M. (University of Adelaide, Adelaide, South Australia), and Margaret Milne. Heat-labile antigens of Salmonella enteritidis. II. Mouse-protection studies. J. Bacteriol. 92:549-557. 1966.-A number of extracts prepared from a virulent and an avirulent strain of Salmonella enteritidis were used to immunize mice. Living and alcohol-killed whole-cell vaccines were also used to compare the relative protective value of the various preparations. All mice were challenged intravenously with 100 to 1,000 ld(50) of S. enteritidis. Daily counts of the liver, spleen, and blood populations of vaccinated and control mice revealed that the challenge organism was rapidly eliminated only in those mice which had been immunized with a living vaccine. Immunization with extracts resulted in rapid clearance of S. enteritidis from the blood, but, after a delay of 24 to 48 hr, the bacterial populations increased until a maximal liver and spleen population of approximately 5 x 10(8) was reached. Between 55 and 100% of the immunized animals died, compared with 95 to 100% of the controls. With all four extracts, it was only the first antigenic fraction eluted from diethylaminoethyl cellulose which had any detectable effect on host resistance. The ineffectiveness of vaccines prepared with the various extracts or with whole killed bacteria relative to the protection observed after immunization with living organisms is discussed.     

Effect of acetylation (O-factor 5) on the polyclonal antibody response to Salmonella typhimurium O-antigen

Antibodies directed against lipopolysaccharide (LPS) O-antigen are often critical in the immune response to Gram-negative pathogens. Mice were orally immunized with isogenic strains of Salmonella typhimurium that differ only in a minor modification of O-antigen, namely acetylation, mediated by the oafA locus. To specifically examine the effect of acetylation on the antibody response to O-antigen, antibody titers were determined against both acetylated and unacetylated LPS by ELISA. In mice immunized with an oafA+ strain, the median titer against acetylated LPS was 32-fold higher than the titer against unacetylated LPS. Mice immunized with the oafA- strain had an 8-fold higher titer against unacetylated LPS. Thus, acetylation of O-antigen alters recognition by the vast majority of individual antibodies. This differential antibody recognition of O-antigen had a statistically significant correlation with protection against subsequent challenge with virulent S. typhimurium.     

Role of two outer membrane antigens in the induction of protective immunity against Francisella tularensis strains of different virulence

Abstract A crude outer membrane preparation from Francisella tularensis live vaccine strain was used to immunise mice. Immunised mice were completely protected from a F. tularensis challenge. We evaluated the role of two major outer membrane antigens in the induction of protective immunity, namely lipopolysaccharide and an outer membrane protein FopA . We presented FopA to the immune system using an aromatic amino acid dependent Salmonella typhimurium as a vector. Although mice mounted an immune response to cloned FopA no significant protection was induced. However, lipopolysaccharide-immunised mice were completely protected from a F. tularensis live vaccine strain challenge. No increase in LD₅₀ was observed using F. tularensis Schu4 as the challenge strain, although there was a significant increase in time to death. These data question the validity of the murine F. tularensis live vaccine strain model.     

Active and passive mouse-protecting capacity of Pseudomonas aeruginosa protein vaccines

Six different vaccines were prepared, each containing the soluble, practically lipopolysaccharide-free protein extract of 2 or 3 Pseudomonas aeruginosa strains. In active mouse protection tests the vaccines were shown to give protection against both homologous and heterologous serotype strains, and against strain PA-103 producing exotoxin A. In rabbits the vaccines were found to stimulate the production of protective antibodies demonstrable in a passive mouse protection test. The immune serum had a protective effect against the exotoxin A-producing strain PA-103, too. Toxicity of the vaccines was studied in mice (mouse weight gain test) and in rabbits (intracutaneous skin test and pyrogenicity). The vaccines were not or only slightly toxic.     

Detoxification of Salmonella typhimurium lipopolysaccharide by ionizing radiation

The efficiency of ionizing radiation in detoxifying the lethal determinant(s) of the lipopolysaccharide (LPS) of Salmonella typhimurium, S. enteritidis, and Escherichia coli in aqueous solution and associated with heat-killed S. typhimurium cells in suspension decreased with doses above 1 Mrad. The 50% end point of inactivation was more than 7.0 Mrad for heat-killed salmonellae and 4.8, 4.5, and 1.0 Mrad for the LPS of S. typhimurium, S. enteritidis, and E. coli, respectively. After exposure to 20 Mrad, S. typhimurium LPS retained a small portion of its lethal properties although the ld(50) was much greater than 9.5 mg per 20-g mouse. However, at -184 C, no inactivation of the lethal determinant(s) occurred after exposure to as much as 20 Mrad. This demonstrated the significance of the indirect effect and the mobility and formation of free radicals. At 22 C, the optical density at 400 mmu increased and the pH decreased with increasing radiation dose, but no qualitative changes were observed in the infrared spectrum. No change was observed in the pyrogenicity of S. typhimurium LPS; a slight decrease in antigenicity was revealed when 6 days, but not when 1 day, elapsed between vaccination and challenge in the mouse protection test. The results were interpreted as evidence of the existence of two or more lethal and antigenic determinants. The differential effect of radiation on these properties and on the pyrogenic component(s) probably are indicative of separate functional sites for lethal, antigenic, and pyrogenic activities.     

The concentration, physical state, and purity of bacterial endotoxin affect its detoxification by ionizing radiation

Increasing concentrations of a highly purified bacterial lipopolysaccharide preparation, the U.S. Reference Standard Endotoxin, were exposed to increasing doses of ionizing radiation from a 60Co source. At identical radiation doses both the structural change and Limulus amebocyte lysate (LAL) reactivity were progressively smaller with increasing concentrations of the lipopolysaccharide in an aqueous medium. Under the experimental conditions used, there was a linear relationship between the endotoxin concentration and radiation dose for the structural changes. In contrast to endotoxin in aqueous medium, endotoxin irradiated in its dry state showed no decrease in LAL reactivity and rabbit pyrogenicity. Endotoxin exposed to radiation in water in the presence of albumin showed a much smaller decrease in LAL and pyrogenic activities than expected. The results show that the concentration, physical state, and purity of endotoxin influence its structural and functional alteration by ionizing radiation.     

Tolerance and Other Biological Properties of Vibrio parahaemolyticus Endotoxins

Biological properties of endotoxins prepared from three strains of Vibrio parahaemolyticus were compared with reference to commercially prepared Salmonella typhi endotoxin. Endotoxin assays performed in rabbits included dermal Shwartzman reactivity, pyrogenicity, heat stability, and ability to induce tolerance as well as cross-tolerance. Mice were used for endotoxin LD₅₀ determinations. Results showed V. parahaemolyticus endotoxins were similar to that of S. typhi strain O901. Induction of tolerance to V. parahaemolyticus strain 11590 endotoxin resulted in complete cross-tolerance to S. typhi endotoxin, and vice versa. Partial cross-tolerance to S. typhi endotoxin was demonstrated with rabbits rendered tolerant to endotoxin from V. parahaemolyticus strains Sak-3 and FC1011. Absorption spectra, nitrogen, phosphorus and carbohydrate analyses revealed additional similarities between endotoxins from V. parahaemolyticus and endotoxin from a member of the Enterobacteriaceae.     

Hepatic drug-metabolizing enzyme system and endotoxin tolerance: structural requirement of LPS in induction of an early tolerance

The alteration of hepatic drug-metabolizing enzyme activities in mice given Salmonella endotoxin by single or multiple intraperitoneal injections was investigated. An essentially the same biphasic, early and late phase, endotoxin tolerance was observed in the animals receiving a single injection of endotoxin or repetitive daily injections. The results of reciprocal cross tolerance tests using lipopolysaccharide and free lipid A preparations derived from Salmonella minnesota, Salmonella typhimurium, E. coli, Pseudomonas aeruginosa, and Chromobacterium violaceum suggested that lipid A moiety plays an important role in the induction of early endotoxin tolerance to endotoxin response.     

Measurements of lipopolysaccharide (endotoxin) in meningococcal protein and polysaccharide preparations for vaccine usage

Lipopolysaccharide (LPS, i.e. endotoxin) present in meningococcal outer-membrane protein and polysaccharide preparations made for vaccine use was quantitated by a silver-stain method following SDS-PAGE. The reactivities of LPS in the preparations were also measured by rabbit pyrogenicity and Limulus amoebocyte lysate (LAL) assay. Although rabbit pyrogenicity and LAL assay are more sensitive than the silver stain method, the latter provided an actual amount of LPS present in the protein or in the polysaccharide. For a meningococcal protein preparation, rabbit pyrogenicity showed about one-tenth, and even less by LAL assay, of the actual amount of LPS. This is because protein-bound LPS in meningococcal protein preparations is about 10-fold less active in causing fever in rabbits, and 20- to 40-fold less active in the gelation of LAL than the same amount of a purified free LPS which is generally used as a reference in quantitating LPS in these two assays. As for the small amount of LPS present in a meningococcal polysaccharide preparation, similar LPS content was obtained when measured by the three methods suggesting that the LPS is not bound to the polysaccharide in contrast to that in the proteins mentioned above. The purified meningococcal LPS was pyrogenic in rabbits at 1 ng/kg.     

Monoclonal antibodies to Salmonella lipopolysaccharide: anti-O-polysaccharide antibodies protect C3H mice against challenge with virulent Salmonella typhimurium

The present investigation reports the production of monoclonal antibodies to antigenic determinants of the O-polysaccharide of Salmonella typhimurium lipopolysaccharide (LPS), and assesses the effectiveness of these antibodies in protecting C3H mice against the lethal effects of Salmonella infection. Hybridomas were generated by fusing spleen cells from (BALB/c X A/J)F1 (CAF1) mice hyperimmunized by i.v. injection with acetone-killed S. typhimurium SR-11 with X63-Ag8.653 murine myeloma cells. Hybridomas producing antibodies reactive with S. typhimurium SR-11 whole cells were subcloned, and seven of the resulting clones as well as one previously described clone were selected for use in the studies reported here. Monoclonal antibodies from these eight clones were of the IgG1 (1), IgG3 (6), or IgM (1) isotype and were specific for the O-polysaccharide region of Salmonella LPS, reacting with LPS from smooth S. typhimurium SR-11 and LT-2, but not with LPS from rough S. minnesota R60 (Ra), R345 (Rb), or R595 (Re). The effectiveness of each monoclonal antibody in protecting C3H/HeN and C3H/HeJ mice against the lethal effects of Salmonella infection was evaluated by comparing the median length of survival of groups of mice given antibody by i.p. injection before i.p. challenge with virulent S. typhimurium SR-11 to that of animals that received no antibody. Three out of eight monoclonal anti-O-polysaccharide antibodies, ST-1 (IgM), 10-5-47 (IgG3), and 10-5-6 (IgG3), provided significant (p less than 0.01) protection to C3H/HeN mice challenged with approximately 10(4) LD100 of Salmonella. Only antibodies ST-1 and 10-5-6, however, extended the median length of survival of C3H/HeJ mice beyond that of infected controls. Mouse antiserum prepared against S. typhimurium SR-11 was equally protective in C3H/HeJ mice. In an attempt to understand the contribution of antibody specificity to the relative differences in the protective capacities of the monoclonal antibodies, their reactivities with several Salmonella reference strains of different classical serotypes were examined. Although some differences in reactivity against the different strains were apparent, this approach was not adequate for defining the fine specificity of these monoclonal antibodies. The results of this study provide evidence that monoclonal antibodies with specificity to the O-polysaccharide region of Salmonella LPS can protect C3H mice against challenge with the homologous bacterial strain.     

Interfering effect of diphtheria-tetanus-acellular pertussis combined (DTaP) vaccines on the bacterial endotoxin test.

We evaluated applicability of the endotoxin test to DTaP vaccines. Although no DTaP vaccine batches showed immediate interference with the activating activity of spiked endotoxin on the clotting of Limulus amaebocyte lysate (LAL), a gradually progressing interference depending on the time after spiking was seen for some of the batches. The interfering DTaP vaccine batches, however, showed no significant effect onin vitro TNF-alpha induction in RAW264.7 cells and pyrogenicity in rabbits of spiked endotoxin. Aluminium hydroxide gel in the vaccines was suggested to be one of the causes of the interference. Accordingly, a careful evaluation of the interfering effect was assumed crucial for the effective application of the endotoxin test to check residual biological activity of endotoxin in DTaP vaccines.