Experimental study of the preventive properties of immune milk containing Shigella antibodies

The protective properties of the mammary gland secretions of cows immunized with Shigella sonnei into the udder were studied. As a model for this study the intranasal and intraperitoneal infection of white mice was used. Immune milk was found to have pronounced protective properties against S. sonnei. When introduced intraperitoneally, this milk protected the animals infected with S. sonnei from death. When introduced intranasally, it not only protected the animals from death, but perceptibly inhibited the development of the pathological process in the pulmonary tissue, preventing the multiplication of shigellae and accelerating the elimination of the infective agents from the lungs of the infected animals. As a rule, the degree of protective action was determined by the level of antibodies to shigellae in the substrate under test.     

Protective role of O antigen in Salmonella typhi-murium infection

Mice were actively immunized with preparations produced from different Salmonella strains. They were challenged with 2 LD50 of a virulent S. typhi-murium strain and examined for viable cell counts in the liver 4, 7 and 11 days postinfection. Whole cell vaccines, ribosomal extracts and endotoxin preparations of the O antigen-deficient variant of S. typhi-murium strain LT2-MI failed to protect the mice or did so in a much lower degree than preparations of the corresponding O antigen-bearing variant. Preparations from other salmonellae exerted a protective action only if the strain had an O-antigen identical with that of S. typhi-murium. The results pointed to a considerable protective role of the O antigen.     

O-specific L-hapten in the composition of Shigella sonnei

Along with classical lipopolysaccharide (LPS), O-specific material not precipitated by ultracentrifugation has been isolated from the water-phenol extract of S. sonnei avirulent strain 9090 possessing complete antigenic properties. The purification of O-antigen contained in the supernatant fluid has been carried out by the gel filtration of the fluid, previously treated with ribonuclease, in a column packed with Sephadex G-100. The polysaccharide nature of O-antigen thus obtained, the absence of lipid A and KDO and the low content of hexoses, or core-specific saccharides of S. sonnei LPS, in this antigen make it possible to classify this material with O-components of microbial cells, described by different authors as "native protoplasmic polysaccharide" or "L-hapten" and formed by polymers of LPS O-side chains. The content of this component in S. sonnei strains under study is, on the average, 2.5% of the weight of dry microbial substance. L-hapten preparations obtained in the course of our investigations have been found to contain two O-specific antigens detected by immunoelectrophoresis and immunodiffusion, as well as by sedimentation in saccharose gradient, where they form peaks corresponding to 4.3 S and 10.8 S. This polysaccharide O-antigen is supposed to be capable of interaction with ribosomal particles and suitable for use as a component of ribosomal dysentery vaccines.     

The protective properties of the endotoxin protein

The isolation and properties of endotoxin protein, or lipid A-associated protein (LAP), from Shigella sonnei were described earlier (Zh. mikrobiol. epidemiol. immunobiol., 1991, No. 4, pp. 11-17, and No. 7). In this report the data on its protective activity are presented. In experiments on mice one nanogram of LAP injected i. v. protected 50% of the animals against i. p. challenge with 40 LD50 of virulent S. sonnei. Guinea pigs injected s. c. with 10 micrograms of LAP were protected against local (keratoconjunctival) challenge with S. sonnei, the efficiency of immunization being 58%. LAP preparations containing no detectable amounts of O-antigen (less than 0.003%) were found to have a protective effect. Hyperimmune anti-LAP rabbit serum prevented local infection when incubated with S. sonnei challenge inoculum before injection into guinea pigs. Both active and passive protection induced by LAP was specific since no effect was observed in animals challenged with Shigella flexneri. In the homologous system the protective effect of anti-LAP serum was abolished by the addition of protein-free LPS. These results are compatible with the hypothesis that the protective activity of LAP depends on the presence of minute amounts of O-antigen whose immunogenic effect is greatly amplified by the protein component of the natural endotoxin complex.     

The effect of Shigella sonnei strains opposite in their virulence on different links in the immune system]

For the first time different action of S. sonnei strains, opposite in their virulence, on hematopoiesis and the functional activity of T- and B-lymphocytes has been shown. The hematopoiesis-disturbing action of virulent shigellae is manifested by their capacity, more pronounced than similar capacity of an avirulent (vaccine) strain, for stimulating the processes of endo- and exocolony formation, the proliferation of hematopoietic stem cells and their migration to the blood. The effect produced by shigellae on T-cell-mediated immune response is manifested by the suppression of macrophage migration and its subsequent activation, whose manifestations and duration depend on the virulence of S. sonnei strains under study. The modulating effect of S. sonnei on B-cell-mediated immune reactions is manifested by the inhibiting action of S. sonnei virulent strain and the stimulating action of S. sonnei vaccine strain on the formation of antibody-producing cells synthesizing S. sonnei lipopolysaccharide antibodies shortly after the injection of shigellae. The results of this study indicate that S. sonnei virulent and avirulent (vaccine) produce multifunctional and differing effects on cell-mediated immune reactions, these processes being dependent on the virulence of shigellae and their individual specific antigens.     

The isolation of the ribosomal fraction of Clostridium perfringens type A and an evaluation of its protective activity

A method for isolation of the ribosomal fraction (RF) from the cytoplasm of type-A C. perfringens strain BP6K was developed and its chemical and antigenic properties characterized. RF has been found to possess protective properties: two subcutaneous immunizations of mice with RF preparations adsorbed on Al(OH)3 in doses of 250 and 500 micrograms (dry weight) has ensured, on the average, the protection of 41.9% of the immunized animals from 1 DCL of type-A C. perfringens strain BP6K culture.     

Immunological interrelations between Shigella sonnei differing in enzymatic activity and colicinogenicity

For the first time immunological interrelations between S. sonnei differing in enzymatic activity and colicinogenicity were studied. Specific postinfectious immunity against Shigella pneumonia in intranasally infected mice was used as a model for testing S. sonnei strains, biovars II and III, with colicinogenic markers designated S5 and IE2, respectively. The development of homologous and heterologous immunity was shown to occur in the animals; immunity to the more virulent strain II S5 proved to be significantly more intense than in comparison with immunity to the less virulent strain III IE2. The unequal effectiveness of immunity to these strains (the more virulent were the strains, the more effective immunity they produced) was due to the fact that their populations contained different amounts of organisms in phase I responsible for infective action and immunity, and also, which was heretofore unknown, for the sensitivity of these bacteria to immunological action: the greater their virulence, the greater their sensitivity.     

Comparative study of the immunogenicity of neurotoxin preparations and their separate fractions obtained from Shigella sonnei, phases I and II

The immunogenicity of the preparations of phase I S. sonnei neurotoxin, determined by the keratoconjunctival test on guinea pigs, is linked mainly with the presence of a high-molecular component other than endotoxin in these preparations. The preparations of phase II S. sonnei neurotoxin do not contain the endotoxin and high-molecular antigens of S. sonnei, phase I, which protect guinea pigs from the development of experimental keratoconjunctivitis; these preparations are toxic for mice and possess low immunogenicity.     

Neutralization of toxic action of endotoxins of gram-negative bacteria by unithiol and magnesium sulphate

The experiments were carried out on mice of CBA strain. The animals received lysate of S. typhimurium in the dose of LD50. The injection of lysate was followed by an increase in the level of malonic dialdehyde (MDA) suggesting the lipid peroxidation (LPO) activation in the liver. At the same time the concentration of cyclic adenosine monophosphate (cAMP) in the liver and the lungs was decreasing, which was indicative of the decrease in the activity of prostaglandins. The injections of unithiol prevented both the activation of LPO and the decrease in the concentration of cAMP, that effect having been caused by the antioxidant action of unithiol. The repeated injection of unithiol protected the animals from death in intoxication by lysate of S. typhimurium and Shigella sonnei; the protection manifested itself in the 4.2 and 3.7-fold increase in LD50 respectively. Magnesium sulphate enhanced the protective action of unithiol.     

Local immunity in the parenteral immunization of guinea pigs with a ribosomal dysentery vaccine

After immunization of guinea pigs with Shigella sonnei ribosomal vaccine O-antibodies appeared not only in the blood serum of the animals, but also in their lacrimal fluid. Since no correlation between the levels of serum and secretory antibodies was detected and since the time course of changes in these antibody levels was quite different (serum antibodies reached their peak on day 7 while secretory antibodies, on day 14 after vaccination), antibodies in lacrimal fluid were supposed to reflect local immune response induced by parenteral administration of ribosomal vaccine, irrespective of systemic immune response. The peak of secretory O-antibodies coincided in time with the period of the highest protection of guinea pigs from Shigella keratoconjunctivitis. The animals with a high level of secretory antibodies were better protected from Shigella infection than those with a low level of secretory antibodies. These data suggest that locally produced O-antibodies play an important role in protective immunity induced by parenteral administration of the ribosomal vaccine.     

Lipopolysaccharide changes in smooth-type avirulent Shigella in comparison with initial virulent strains]

The comparative study of the lipopolysaccharides (LPS) of virulent and avirulent strains of S. sonnei, phase I (smooth colonies), has been made. Electrophoresis of LPS and subsequent densitometry of electrophoregrams have revealed the increase of the fraction of long 0-chains with a considerable number of recurring elements in 2 out of 3 LPS preparations obtained from avirulent shigellae. In mice immunized with these LPS preparations a considerably greater number of antibody-producing cells can be detected in Jerne's test on sheep red blood cells (SRBC) sensitized with the LPS of a virulent strain than on those sensitized with the above LPS preparations. Long 0-specific chains supposedly inhibit the fixation of individual complement components on the corresponding sensitized SRBC. The LPS of the third avirulent strain of S. sonnei, phase I, with transposon integrated into its genome, which has led to the formation of the avirulent variant of a previously virulent strain, seems to contain fine structural differences from the initial virulent strain. The immunogenicity of the LPS of this avirulent strain is greatly (3-4 times) decreased, which is manifested by the number of antibody-producing cells detected in Jerne's test on SRBC sensitized with LPS preparations obtained from these two strains.     

Citrate method of extracting antigens from Shigella sonnei. I. Comparison with other methods

The comparative characteristic of extracts obtained from Sh. sonnei by the method of Raynaud and Digeon and neurotoxin obtained by sedimentation with trichloroacetic acid from chloroform autolysates by the method of Mesrobeanu et al. is presented. Both preparations were shown to be similar in their antigenic structure and immunogenicity for mice and guinea-pigs. Nevertheless the extracts obtained by the citrate method contained considerably less protein and endotoxin and had no enterotoxic activity. This allows to recommend the citrate method for extracting protective antigens from Sh. sonnei.     

Mechanism of the nonspecific protective action of Shigella antigens

The mechanism promoting the nonspecific action of antigens obtained from S. flexneri and S. sonnei by a sparing method has been studied. These antigens stimulate the T- and B- systems of immunity, that is followed by activation of myelopoiesis and the humoral protective factors of the body, which seems to underlie the formation of resistance to infection caused by nonspecific microorganisms.     

Comparative analysis of the protein composition of the external membrane of a virulent strain of Shigella sonnei and of genetically related avirulent bacteria

The outer membrane proteins of intact S. sonnei cells of a strain virulent in the keratoconjunctival test contain up to 24 polypeptides; one of them (with a molecular weight of about 70 KD) is absent in similar preparations of avirulent nonpenetrating shigellae in phase I, genetically related to the above strain. Study of the composition of the outer membrane proteins of S. sonnei cells in phase II has revealed that these cells, when compared with smooth bacteria, show qualitative and quantitative differences in their polypeptide fractions; of these, three fractions are characteristic of rough bacteria.     

Ribosomal dysentery vaccine. II. Biological trials of active protection for guinea pigs and mice]

Ribosomal vaccine from Sh. sonnei injected subcutaneously once or twice in physiological saline or in Freund's complete adjuvant produces a marked protective effect against experimental keratoconjunctivitis in guinea pigs. Inhibition of the protective effect by high doses (above 100 microgram) of ribosomal vaccine is evident after a single, but not repeated injections. Protective effect in mice is achieved by immunization with very low doses of ribosomal vaccine: ED50 is 1.2 ng after challenge with 5.6 LD50. The nature of immunogenic factor responsible for the biological activity of the ribosome vaccine is still obscure. In contrast to Boivin's antigen, ribosomal preparations, even in high doses (1000--2000 microgram), have no toxic effect on mice and guinea pigs.     

Stimulation of the secretory IgA system in monkeys by parenteral immunization with a ribosomal Sonne dysentery vaccine

The experiment was made on 16 monkeys (rhesus macaques). Only 1 out of 12 monkeys immunized with S. sonnei ribosomal vaccine and all 4 control monkeys fell ill as the result of oral challenge with S. sonnei virulent strain. The immunized monkeys stopped excreting Shigellae earlier than the control monkeys. Antibody to lipopolysaccharide (LPS) in the serum and saliva of the monkeys were studied in the enzyme immunoassay with monospecific antibodies to human IgA, IgG and IgM. A single injection of the ribosomal vaccine in a dose of 600 micrograms was shown to lead to a considerable increase in the levels of IgA, IgG and IgM antibodies to LPS in saliva. In parenteral immunization with the ribosomal vaccine the stimulation of secretory IgA system is similar to that resulting from oral challenge with Shigella virulent strain introduced in a dose of 50 X 10(9) microbial cells. No difference in the response of monkeys to primary and booster immunization was noted.     

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

The molecular heterogeneity of Shigella sonnei lipopolysaccharide based on polyacrylamide gel electrophoretic data

The molecular heterogeneity of S. sonnei lipopolysaccharide (LPS), reflecting the size of lateral O-specific polysaccharide chains, has been established by the method of electrophoresis in acrylamide gel in the presence of sodium dodecyl sulfate and urea. The dominating components fall into three types, viz. those with 0-3, 10-16 and 35-40 repeating structures, the remaining components being minor ones. The electrophoretic profile of S. sonnei LPS considerably differs from the profiles of Escherichia coli and S. flexneri LPS, but coincides with the LPS profiles of other strains with different virulence. The preparations of LPS obtained by extraction with trichloroacetic acid have the same electrophoretic profiles as LPS obtained by the method of aqueous phenol extraction. The domination of certain molecular variants reflects, seemingly, specific features of the biosynthesis of LPS, characteristic of a given strain. The mechanisms of the preferable synthesis of lateral O-specific chains of the definite size and the importance of the molecular parameters of lateral chains for the biological properties of LPS require further study.     

A protective factor of ribosomal shigellosis vaccine

Shigella ribosomal vaccine was shown previously to possess protective properties in the keratoconjunctival test on guinea pigs and to be capable of preventing experimental infection in 90% of challenged monkeys. The presence of the O-specific component (OSC) constituting about 0.5% of the ribosomal preparation by serological activity suggested its importance for the protective effect. This was studied in experiments with two O-specific immunosorbents prepared by coupling anti-O rabbit antibodies with Staphylococcus aureus cells or with CNBr-Sepharose. Ribosomes treated with immunosorbents proved to be lacking the serologically active OSC and lost their ability to induce O-antibody response in rabbits and mice. After the removal of this component ribosomal preparations were incapable of ensuring protection from Shigella kerato-conjunctival infection. The isolated OSC was also inactive in this test. The data obtained in this investigation confirm the hypothesis stating that the protective activity of Shigella ribosomal vaccine is based on the combined action of ribosomes and O-specific factor whose nature and properties require further study.