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The comparative study of 4 constructed protective antigen producing strains of the classical biovar and V. cholerae strains 569 B Inaba and M41 Ogawa, used in manufacturing the cholera chemical vaccine "cholerogen-toxoid", was carried out. The study revealed that V. cholerae plasmid strains 2414 Ogawa, 2415 Inaba and nonplasmid strains 2416 Ogawa, 2417 Inaba had a higher level of production of the main protective antrigens in comparison with producer strains. They also synthesized much more (4-5 fold) cholera toxin, toxin co-regulated adhesion pili, contained protein OmpU in their outer membrane, exceeded 2- to 3-fold in the synthesis of pathogenicity enzymes (proteases, phospholipases) and synthesized the same amounts of 01 antigen, serovars Inaba and Ogawa. The use of the newly created protective-antigen producing strains in vaccine manufacturing could facilitate the preparation of a more effective cholera chemical vaccine "cholerogen-toxoid".

The influence of the newly developed complex vaccine Pyopol, containing the antigens of opportunistic microorganisms and polyoxydonium used as immunomodulator, on the oxygen-dependent metabolism of neutrophils was studied. The study revealed that the main components of the vaccine, both individually and in association, did not change cellular activity in the range of concentrations used in this study. The inhibition of the oxygen-dependent metabolism of neutrophil granulocytes in the presence of native or weakly diluted vaccine occurred due to the cytotoxic effect of thimerosal used as preservative.

Ribosomes, sufficiently purified by physicochemical criteria (with the RNA/protein ratio equal to 1.6 - 1.7), were isolated from disintegrated C.perfringens cells, type A, strain BP6K, by ultracentrifugation or by fractionation with polyethylene glycol. The purified conjugates of ribosomes and C.perfringens toxoid, absorbed on aluminum hydroxide, were obtained. The ribosomal preparations had low toxicity and high protective activity: when used in a dose of 6 binding units, they protected guinea pigs and mice inoculated with 8 - 9 LD50 of C.perfringens culture.

The reaction of the bacteriosorption of immune complexes (RBIC), based of on the avidity of horse IgG (T) to protein A in interaction with complement antigen, is proposed. The possibility of using RBIC for the evaluation of the titer of native antitoxic sera and the degree of the enzymolysis of specific antitoxins with pepsin has been shown.

The reactor technology for obtaining suspensions of Staphylococcus aureus (strain Cowan 1) with active protein A was developed. On the basis of the sensitization of bacterial suspensions with antitoxic rabbit sera staphylococcal reagents to diphtherial, gas gangrene (perfringens, oedematiens), botulinic toxins-toxoids were obtained and for the first time the coagglutination test with toxoids was carried out. More sensitive "specifically directed" reagents for the coagglutination test were obtained; for this purpose bacterial suspensions were sensitized with pure antibodies isolated from rabbit immune sera by immunosorption. The possibility of using the coagglutination test with specifically directed staphylococcal reagents for the rapid evaluation of the dynamics of toxin formation in reactors for the titration of purified toxoids, for the control of the purification of toxoids by ultrafiltration, as well as for the evaluation of the sorption-desorption of toxoids in the production of the corresponding preparations, was shown.

The effectiveness, both immunological (by an increase in the titers of antitoxins) and protective (by resistance to the inoculation of the absolute lethal dose of infective agents), of the regional (wound) revaccination with tetratoxoid (Clostridium perfringens, C. oedematiens, C. septicum, C. histolyticum) was demonstrated on the experimental model of wound infection (gas gangrene) of guinea pigs. The schedule of rapid immunization with tetratoxoid was developed, which made it possible to create good immunological preparedness (basic immunity) for subsequent revaccination in case of traumas within 6 days. The effectiveness of rapid immunization by the application of tetratoxoid on the wound was shown. This immunization ensured a considered increase in the titers of antitoxins within the first 6 days, which increased the protection of the animals from infection with each of the four causative agents of gas gangrene.

The safety, reactogenicity and immunogenic activity of P. aeruginosa toxoid were studied in 46 volunteer donors. Systemic reactions to the injection of the preparation were absent in all vaccinees, in 2 subjects (4.3%) insignificant reactions at the site of injection in the form of hyperemia, sized 13-14 mm and disappearing within 48 hours, were registered. The preparation was found stimulate humoral immunity, which was manifested by an increase in the number of B-rosette-forming lymphocytes and the level of antitoxic IgG in the blood of the vaccinees. Besides, immune sera obtained from the blood of the volunteers were found to possess protective properties.

The protective activity of the liposomal form of P. aeruginosa toxoid and intact phosphatidylethanolamine liposomes has been evaluated in mice with experimental P. aeruginosa infection. It has been revealed that the liposomal form of P. aeruginosa toxoid, introduced in a single injection, ensures the formation of more intense and prolonged immunity in comparison with that induced by the toxoid adsorbed on aluminum hydroxide. This investigation has revealed that intact phosphatidylethanolamine liposomes also possess protective properties, protecting 10-36.7% of the animals from infection with P. aeruginosa toxigenic strain.

One of the stages of a new technological scheme for obtaining C1. oedematiens toxoid (detoxication with formaldehyde of a purified concentrated toxin) was elaborated under experimental conditions. It was found that under the effect of increased formalin concentrations (0.4--0.2%) or of prolonged action of heat there developed in the toxin a reduction of antigenic activity, expressed on immunoelectrophoregrams in the form of a change of surface potential of one of the electrophoretic fractions. Detoxication of the purified concentrated C1. oedematients toxin occurred in the course of 3 days without any losses of its antigenic activity under the following conditions: formalin concentration 0.1%, temperature 37-38 degrees C, pH L.7--6.9. No reversion of toxicity of the preparations detoxified under the mentioned conditions was revealed under the action of formalin-binding agent (sodium sulfite) on them.

Antisera obtained as a result of immunization of rabbits with concentrated E. coli enterotoxin, its active fraction or toxoid, had a pronounced toxin-neutralizing activity. At the same time the cytotoxic activity of antisera to the purified fraction was considerably higher than that of the antisera to the concentrated enterotoxin. Such antisera had also a neutralizing effect on thermolabile enterotoxin. The neutralizing activity of antisera to E. coli enterotoxin was clearly manifested in the paw edema test.

A possibility was shown of purification of the concentrated toxoids of the Cl. perfringens and Cl. oedematiens species in examination of KSK-2, KSK-2,5; MCA-2 silicagels. The specific activity of the preparations obtained and the yield approached the analogous indices obtained in stratification of Sephadex G-75. The effect of preliminary silicagel treatment and also of the nature of buffer solutions on the efficacy of the process of purification of the gangrenous toxoids was established.

The studies described in this work have indicated that cholera enterotoxin and its components (cholerogenoid and subunit B) can be detected in amounts of 0.25, 0.28 and 0.6 microgram of protein per 1 ml, respectively, by means of erythrocytes sensitized with gangliozide-containing complex. The conditions for erythrocyte sensitization have been established. The methods of cholerogen titration in the passive hemagglutination test by means of the erythrocytic gangliozide diagnostic reagent and in Craig's skin test have been shown to correlate. Similarly, the passive hemagglutination test is supposed to be suitable for detecting other bacterial toxins interacting with gangliozides.

The method for the titration of Shigella dysenteriae I neurotoxin, toxoid prepared from this toxin and different antitoxic preparations in the mouse paw edema test has been developed. The quantitative determination of antitoxin is based on the neutralization test. The conditions of titration (the dilutions of the neurotoxin and the neutralization doses of the antitoxin), as well as criteria for the evaluation of the positive or negative results, have been established.

Three fractions isolated from the culture fluid of the 569B cholera vibrio strain were studied. The fractions differed by the extent of purification, and the content of the toxid and the O-antigen. In intraintestinal application to rabbits all of them caused formation of antitoxins and vibriocidal antibodies in the blood serum. The immunizing dose of the preparations in intraintestinal administration exceeded the dose required for subcutaneous application. Fraction I should be used for Producing tablet form of cholera vaccine because it was least toxic and provided the greatest yield.

The levels of antitoxic and vibriocidal antibodies in the sera of suckling rabbits after their parenteral immunization with cholera vaccine, cholera toxoid and a combination of cholera vaccine and toxoid were examined. Cholera vaccine induces intensive production of vibriocidal antibodies, and cholera toxoid, of antitoxic antibodies. The parenteral administration of the serum of rabbits immunized with cholera toxoid neutralized the action of cholera toxin in the small intestine of suckling rabbits. The complex preparation combines the properties of the corpuscular vaccine and the toxoid, inducing the production of both vibriocidal and antitoxic antibodies.

Research work has been carried out with the aim of obtaining hyperimmune antitoxic plasma against Pseudomonas aeruginosa. Hyperimmune plasma active against P. aeruginosa toxin has been obtained from donors immunized with P. aeruginosa toxoid. The preparation is highly specific and active, which is revealed by in vivo and in vitro tests. The clinical study of the preparation indicates its high efficacy in the treatment of diseases caused by different P. aeruginosa serotypes.

The protective properties of formulated toxoid obtained from the highly purified preparation of P. aeruginosa exotoxin A have been studied in the test of the active immunization of mice. The study has revealed that the preparation when introduced in 1 or 2 injections in a dose of 15 micrograms, shows faint protective potency with respect to P. aeruginosa strains differing in virulence. Immunization with this toxoid in 3 and 4 injections has been found to ensure 60-100% and 50-60% protection of mice infected with P. aeruginosa toxigenic and proteolytic strains respectively. Immunization with toxoid has been found to induce the appearance of short-term antibacterial immunity which loses its capacity to protect the immunized animals, challenged with both toxigenic and proteolytic P. aeruginosa strains, as early as on day 28. The immunization of mice with toxoid in 4 injections has been shown to induce the development of antitoxic immunity capable of neutralizing up to 150 LD50 of purified exotoxin A.

The authors discussed the results of study of the titre of vibration antibodies and antitoxins one, three and five months after the vaccination of adults with an optimal vaccination dose of cholerogen toxoid (24OO BU). Comparison of the immunological information obtained at the above mentioned periods showed the expediency of revaccination three months after the vaccination. Immunogenicity of the three doses of the cholerogen-toxoid tested (1200, 2400, 3600 BU) displayed no statistically significant difference in revaccination. Taking into consideration that the least reactogenic dose was 1200 BU this dose is recommended as the optimal revaccination dose for adults.

Guinea-pigs were immunized with anatheta-hemolysin preparations adsorbed on aluminum hydroxide, as well as with the mixture of anatheta-hemolysin and type A Cl. perfringens toxoid, purified and concentrated. Anatheta hemolysin preparations were obtained with the use of homogeneous theta hemolysin, as well theta hemolysin of various purification degrees. As a result, antatheta hemolytic guinea-pig sera capable of neutralizing 2,000-8,000 HU of theta-hemolysin were obtained. Tests made to establish the degree of protection in the immunized guinea-pigs did not show that the animals immunized with the mixture of anatheta-hemolysin and type A Cl. perfringens toxoid, purified and concentrated, had any advantages in the degree of protection over the animals immunized with the toxoid alone. But there is no doubt that this component plays a positive role under the conditions of natural gas gangrene when the hemolytic action of Cl. perfringens toxin becomes considerably pronounced.

Comparative experiments were conducted on rabbits and guinea pigs; a study was made of the immunological efficacy of the toxoids with a different content of the immunizing units (IU/ml). There was revealed a regular quantitative interrelationship between the content of the IU/ml in the toxoids, the level of the antitoxin formation and the extent of protection against the experimental gas gangrene in the immunized animals.

The nontoxic (on the model of raw edema in white mice) anaform of E. coli enterotoxin was obtained under the action of heat and formalin. In the process of detoxication the charge of the enterotoxin molecules was changed. The toxoid thus obtained preserved the antigenic activity of the initial preparation and, when used for the immunization of rabbits, stimulated the production of active antitoxic sera.

The authors present a method of obtaining relatively homogeneous preparations of alpha-toxoid of Cl. perfringens, type A, including the primary conception of the alpha-toxin proteins, their chromatography on DEAE-cellulose, fractionation with (NH4)2SO4, detoxication, with the subsequent gel-filtration through sephadex and isoelectric focussing. Sedimentation coefficient of the preparation proved to be 3.8 S, isoelectric point-4.83 +/- 0.07. In studying the immunogenic properties of alpha-toxoid in experiments on guinea pigs and rabbits their high immunogenicity, exceeding that of the industrial toxoid 8- and 6-fold, respectively, was established. Homogeneous preparations of alpha-toxoid provided intense anti-microbial immunity. Interlinear differences in the levels of the immune response of inbred mice, highly-reactive (BALB/c) and low-reactive (C57BL/6) to alpha-toxoid, reached 20-fold; in combination with the high immunogenicity of this antigen for mice this permits to recommend it for immunogenic studies.

As a result of the study of lacalization of the C1. botulinum toxoids of the A, B, and E types in cells of the regional lymph nodes of rabbits by the indirect Coons' method and by the smear-print method it was revealed that different types of lymphoid tissue cells took part in the ingestion and digestion of these antigens; the antigens of the A and B types were at first revealed in the cytoplasm of pseudoeosinophils, and then in the macrophages. The antigen of the E type was revealed in the course of the whole experiment in the macro phages of the regional lymph node, although the number of pseudoeosinophils also increased. Apparently there was a different activity of pseudoeosinophils against the C1. botulinum toxoids, the types A, B and E.

Experiments were conducted on guinea pigs, rabbits and mice (mongrel and inbred); immunogenic properties of Cl. perfringens toxoids of different purity were studied. Toxin neutralization and passive hemagglutination tests were used to determine the antitoxic immunity level. It appeared that in the immunization of guinea pigs and rabbits the degree of immunogenicity of the preparations increased with the elevation of their specific activity. Under the same conditions both the mongrel and the inbred mice displayed the maximum immune response in the immunization with the least purified preparations, and the minimum after the injection of a highly purified antigen.

The study of different subcultures of V. cholerae strain 569 B Pakistan, used as the source of choleragen in the production of cholera vaccine, has been made. The composition of the population formed by this strain has been found to be heterogeneous with respect to toxin formation. The control of changes occurring in the production culture by analyzing its population composition has been proposed.

The data on the study of the protective activity of theta hemolysin and Cl. perfringens lecithinase preparations and the corresponding antitoxic sera obtained by indirect immune affinity chromatography are presented. Experiments in mice and guinea pigs indicate that the injection of antihemolytic serum and immunization with anatheta hemolysin ensures the protection of the animals from theta toxin. The enrichment of analecithinase preparation with anatheta hemolysin has been found to increase its protective properties against Cl. perfringens culture and toxin.

The authors discuss the results of studies on the choice of highly-reproducible and sufficiently informative methods of 9-antigen standardization in a new chemical vaccine against cholera (cholerogen-toxoid); materials were collected under conditions of controlled epidemilogical trial. Titration of O-antigen in industrial batches of the preparation with the aid of precipitation in gel against standard O-cholera serum could be used for its standardization. Quantitative limits of O-antigen responsible for the formation of vibration (antimicrobial) antibodies in the persons vaccinated were found by studying the immunogenicity and reactogenicity of a number of cholerogen-toxoid batches contrast by O-antigen content described by means of the precipitation test.

Crude cholera exotoxin (filtrate toxin) was irradiated with increasing doses of gamma radiation. A significant drop in enterotoxicity, in the activity of the permeation factor and a decrease in toxicity were shown to occur as radiation doses increased. Radiation doses of 50-70 kGy were found to completely inactivate enterotoxicity in liquid toxic preparations. A higher radioresistance of dried preparations in comparison with liquid ones was registered: inactivation occurred at 150-200 kGy. Different batches of the initial filtrate toxin had varying radiosensitivity. The sterilizing effect of gamma radiation was achieved at doses of 20 kGy for liquid preparations and 30 kGy for dried preparations. During the prolonged storage of the irradiated preparations of crude toxin (the term of observation being 1.5 years) at different temperatures no reversion of toxicity was found to occur, while their immunogenic properties remained unchanged.

Cholerogen-toxoid served as a complex vaccine preparation composed of the main pathogenicity factors of cholera vibrio--cholerogen (toxoid), endotoxin and a number of exoenzymes. The preparation contains 65 +/- 7.5% of protein, 12 +/- 1.2% of reducing sugars, 7 +/- 1.2% of lipids, and 2 +/- 0.3% of nucleic acids. Analytic disc-electrophoresis in polyacrylamide gel and immune disc-electrophoresis revealed at least seven individual proteins with the serological activity in the preparation. About 70% of these constituted toxoid proper; the content of O-antigen was 22%. In the cholerogen-toxoid there were revealed seven exoenzymes of cholera vibrio; proteinase, lecithinase, lipase, DNA-ase, RNA-ase, hyaluronidase, amylase. Antibodies against proteinase, lecithinase, amylase and RNA-ase of cholera vibrio were found in the serum of rabbits immunized with cholerogen-toxoid.