KR20050021357A - Method of isolating biologically active fraction containing clinically acceptable native s-lipopolysaccharides obtained from bacteria producing endotoxic lipopolysaccharides - Google Patents

Abstract

A biologically active fraction comprising S-lipopolysaccharide (LPS) from Gram-negative bacteria, which produces endotoxin LPS as a main component, is disclosed. This fraction is selected from the group consisting of D-glucosamine and β-hydroxydecanoic acid, β-hydroxydodecanoic acid, β-hydroxytetradecanoic acid and β-hydroxyhexadecanoic acid in lipid A of S-LPS. The molar ratio of β-hydroxy acid is about 2: 1-4, and the molar ratio of D-glucosamine and higher fatty acids linked by amide and ester bonds in lipid A of S-LPS is about 2: 3-7. Also disclosed is a method of isolating BAF comprising predominantly Gram-negative S-LPS producing endotoxin S-LPS.

The obtained BAF has the pyrogenicity, low toxicity, and high immunogenicity of commercial polysaccharide vaccine levels, and can be used as a vaccine for mammals including humans. They may be considered as inducers of cytokines and as prophylactic resistant antishock agents.

Description

METHODO OF ISOLATING BIOLOGICALLY ACTIVE FRACTION CONTAINING CLINICALLY ACCEPTABLE NATIVE S -LIPOPOLYSACCHARIDES OBTAINED FROM BACTERIA PRODUCING ENDOTOXIC LIPOPOLYSACCHARIDES}

The present invention relates to biologically active fractions comprising predominantly natural low toxicity S-lipopolysaccharides (S-LPS) obtained from the pharmaceutical field, in particular from bacteria producing endotoxin lipopolysaccharides (LPSs). , BAF), for clinical or experimental drug use for the prevention and treatment of diseases.

LPSs are the major polysaccharide antigens of Gram-negative bacteria. They are located on the outer surface of the outer membrane of the cell wall and play an important role in the pathogenesis of many infections (1). LPSs are actively involved in the formation and functioning of microbial physiological membranes and are critical to their growth and viability (Chapter 1, 2). LPS, on the other hand, is the primary target of interaction with antimicrobials and elements of the host's immune system.

LPS molecules are formed by repeating oligosaccharide units and basically include hydrophilic heteropolysaccharides (O-specific polysaccharides) that determine the immune specificity of the bacterial cells. O-specific polysaccharides covalently bond with side chain “core” oligosaccharides containing 10 to 12 monosaccharide residues, which in turn pair with hydrophobic lipid fragment-lipid A and support LPS molecules in the outer membrane of microbial cells. do. The three residues of 2-keto-deoxyoctulose acid (KDO), whose ketosidic bonds are extremely acid labile, are "core" and lipid A fractions. Located at the edge between. LPS consists of three parts, O-specific polysaccharides, “cores” and lipid A, which are Gram-negative microbial strains in the form of smooth colonies cultured in solid culture grown media. Is separated from the so-called S-LPS. LPS, which loses O-specific polysaccharide chains, so-called R-LPS, is usually isolated from strains that grow in the form of rough colonies.

When distinguished from O-specific polysaccharides, it is immunologically inactive in itself, and the lipid component of the LPS determines the overall complexity of the pathophysiological properties of the LPS, in particular its high endotoxin and pyrogenicity. Decide The lipid A portion also characterizes the adjuvant of LPS and consequently their high immunogenicity.

LPS has a wide range of "beneficial" biological characteristics. It has been intensively studied in humans and animals as well as in vitro experiments. It is generally believed that a broad range of "positive" biological properties means that LPS represents itself as a potent protective antigen, exerts antimicrobial and antiviral abilities and also acts as an adjuvant and adjuvant (1).

Immunization of animals with μg of LPS induces several kinds of specific antigens with high titers, which determine their vaccine potential. On the other hand, when macrophages recognize LPS, many α-necrosis factors (α-TNF), interleukins 1, 6 and 12 (IL-1, IL-6, IL12), interferon (IFN) α and β, chemokines (chemokines) ), It induces many proinflammatory mediators, including lipid mediators (1). Moreover, macrophages can be activated by LPS to act as antigen-presenting cells for CD4 ⁺ T cells. In cooperation with IFN- [gamma], LPS stimulates macrophages to transcription of genes encoding NO-synthetase that triggers the synthesis of toxic oxidant-NO. Stimulation of macrophages with the help of LPS and IFN-γ results in a highly active phenotype characterized by strong bactericidal and anticancer capacity. This stimulation of macrophages with LPS improves local immunity against Gram-negative bacteria and causes several distant and systemic effects due to the production of IL-1, IL-6 and α-TNF, which not only fight tumors, but also tumors. It also plays an important role in the fight against.

On the other hand, natural traditional LPS is a biologically active substance, which is very dangerous for humans. Incorporation of small amounts of LPS in bacterial products or contaminants into the body can cause endotoxin shock with severe hemodynamic disorders (Chapter 1, 55). However, the useful pharmacological effects of LPS in a number of pathological conditions (infection and tumor progression) are of great importance, and attempts have been made to use LPS or its derivatives in various directions in clinical medicine (Chapter 1, 63). .

Current research for the manufacture of clinically applicable LPS involves some modification of its structure, ie modification of some biological properties.

One such study is the chemical modification of LPS, such as altering the structure of lipid A with weak alkali or enzymatic treatment. Subsequent removal of fatty acids and phosphate-containing substituents reduces the endotoxin of LPS on the one hand, but on the other hand, greatly reduces the level of immune response resulting in complete loss of immunobiological function of the modified LPS.

Prior to developing the group of compounds according to the invention, it was not possible to use natural LPS directly as a vaccine because of its high toxicity. Thus, several conjugated vaccines have now been developed that contain conjugates and protein carriers of OPS-specific polysaccharide components of LPS against infections caused by Samonella enterica, S. paratyphi A, Shigella sonnei and Shigella flexneri. And were tested by volunteers (2, 3, 4). The immunogenic potential of LPS is lost due to the removal of lipid A, which is partially recovered by the use of protein carriers that induce a secondary immune response and by optimization of the immune design.

Another use of clinical applications of denatured LPS is anti-shock vaccines that cause resistance. The concept of using LPS as a resistance-induced anti-shock vaccine to produce organism resistance to endotoxins that will later be introduced in large quantities is not new (Chapter 1, 50). At the same time, this approach did not lose reality in the sense that control of the clinical situation resulting in endotoxin shock is extremely important. Recently clinical and experimental findings of inducing "early" or "late" resistance to subsequent endotoxin administration, through the administration of synthetic analogs of LPS as well as its derivatives, monophosphoryl-lipid A (MPL) and lipid A. The study was conducted (Chapter 1, 50). MPL is also well known as an immunomodulator and also as an adjuvant with a broad domain of action.

Other methods of reducing endotoxins in LPS are functional detoxification of LPS with cyclic peptides (chapters 1, 23), specific proteins (chapters 1, 19, and 21), or polycationic compounds (chapters 1, 11). detoxification, each of which has the ability to specifically bind to the lipid A portion of LPS. This trend is believed to ensure the purification of the blood or plasma of the patient in a system of extracorporeal treatment, using a solid matrix comprising ligands specific for lipid A. However, there is currently insufficient research on the processes that can occur when LPS complexes with lipid A ligands enter animal organs. Thus, the question of their immunogenicity, biodegradability, toxicity and the possibility of accumulation in other tissues is uncertain.

Trials that use LPS in the treatment of cancer require individual consideration. Clinical studies on the application of LPS to the treatment of cancer are well known (Chapter 1, 63). Currently all possible variants of modified LPS (chemical derivatives, synthetic analogs, functional detoxification products), as well as natural high-toxic endotoxins, are used in the treatment of tumors. Recent studies in this application suggest the possibility of combined LPS and γ-interferon treatment. Because of the high endotoxin, it was only possible to use very small amounts of LPS, such as intravenous administration of 1 to 4 ng per kg body weight.

Distinguished from traditional LPS, the groups of low-toxic native BAFs of LPS disclosed herein differ in extremely wide dose intervals for clinical use. This creates new possibilities for physicians in making effective schemes of treatment.

The product obtained by the present invention was subjected to NMR-spectroscopy and chromato-mass specrometry analysis.

1 shows Sh. ¹³ C-NMR spectrum of sonnei's O-specific polysaccharide.

2 shows Sh. ¹ H-NMR spectrum of sonnei's O-specific polysaccharide.

3 shows Sh. Chromatogram of methanolysis of lipid A isolated from BAF of sonnei.

Figure 4 is a Sh. Separated by the Westphal method. Sonnei is a chromatogram of methanolytes of lipid A of BAF.

The original study we propose to obtain BAF of low-toxic natural S-LPS is a combination of modern and effective methods, on the one hand, which is likely to yield high purity LPS (no impurities exceeding 4%). On the other hand, the possibility of separating low endotoxin fractions from total "parent" LPS. In addition, the conditions under which all stages of separation and purification are performed are believed to maintain the primary LPS structure to the maximum.

Isolation and purification of the claimed BAFs includes extraction, enzymatic treatment of extracts to destroy foreign nucleic acids and proteins, and subsequent fractionation of raw LPS by various methods.

The main method of the extraction step is to treat with phenol in 45% hot aqueous solution according to the Westphal method (5). Currently this method is particularly used for separating LPS, which on the one hand provides a high yield of LPS and on the other hand glycosidic and ketoside linkages of monosaccharide residues and polysaccharides of LPS and Estimate the stability of the linkage of non-carbohydrate substituents of the lipid moiety.

Enzymatic treatment of the extract is carried out for the degradation of ribo- and deoxyribonucleic acids and also for the degradation of protein components of bacterial cells. Enzymatic hydrolysis of nucleic acids is performed by the simultaneous action of ribonuclease-A and deoxyribonuclease in a buffer containing calcium and magnesium ions. The selection of the nuclease is not essential for characterization: it is necessary to increase its concentration and reaction time when enzymes with reduced activity are used. Furthermore, the disodium salt of ethylenediaminetetraacetic acid (EDTA) is added to the reaction mixture and paired with a divalent metal and a proteinase, usually proteinase K.

Separation of BAF of S-LPS from the unpurified LPS obtained after enzymatic treatment is carried out by chloroform-methanol-0.2M HCl extraction mixture [6]. However, when a mixture with the proportion of extractants indicated in the paper [6] is used for extraction, the isolated product is the reference for the polysaccharide vaccine given in Pharmacopedae (0.05 µg / kg of rabbit body weight). It has a pyrogenicity exceeding 8 to 16 times.

In this respect, the solvent ratio of the extraction mixture was changed from that described in the paper [6] to increase its polarity and hydrophilicity, and became 1: 1: 0.4-0.5. Wherein the obtaining of nonpyrogenic BAF from S-LPS did not exceed 1% of the unpurified LPS weight.

The ultracentrifugation was carried out in an aqueous solution which may have been buffered and may contain chaotropic agents, in which the concentration of LPS is not more than 1%. The conditions of ultracentrifugation, including speed and time, were selected specifically for LPS isolated from each of the solidified microorganisms. Thus, when the rate and / or time of ultracentrifugation is shortened, low-molecular LPS is seen to increase in the final product, and when this parameter increases-there is a sharp decrease in yield. The criterion for optimal selection in conditions of ultrafiltration can be the level of endotoxin of BAF isolated from the supernatant.

It is emphasized that the ultracentrifugation and also all the methods for separating nonpyrogenic BAF from S-LPS, described above, can be used for the product obtained during the extraction by a mixture of the product of enzymatic treatment and chloroform-methanol-0.2M HCl. It is important to do.

Fractionation of BAF using preliminary electrophoresis in polyamide gels seems relatively promising because of separating sufficiently narrow LPS fractions and varying the number of cross-links in the gel (7,5 to 5). 12.5%) is likely. However, there are many disadvantages instead. Thus, the separation of gram-level quantities of initial LPS is obviously difficult. There is also a difficulty associated with the possibility that the required product is contaminated with fragments of degraded (mechanically or chemically) polyamide gels.

The disadvantages presented above do not appear when classifying LPS using the "Milipore" ultrafiltration membrane. However, the pores of these membranes vary considerably in size, so that only moderately "wide" fractions of S-LPS are separated as a result of classification. It divides so that the space | interval of molecular weight value may reach 20-30 kDa. That is, for example, using a 50 kDa "blocking" membrane, S-LPS molecules of molecular weight 60 or even 70 kDa can also pass through the pores. Furthermore, in some cases, it may be necessary to use detergents to improve the quality of the separation, such as Triton X-100, which removes LPS micelles. In such cases, removing the detergent from the final product is a particular problem. For this purpose, lower alcohols, usually ethanol, are often added to the LPS solution during fractionation up to 30% concentration. In addition, the use of this approach is most convenient for removing low-molecular weight fractions in unclassified LPS.

Ion exchange gel chromatography is a fairly convenient method only when O-specific polysaccharides contain residues filled with S-LPS, for example LPS Sh. sonnei, phase 1 (if the O-polysaccharide has a carboxyl group and an amino group).

The main difficulty of both when using ion exchange and gel permeation chromatography is the apparent tendency of LPS to aggregate. In order to eliminate the influence of this undesirable factor, it is possible to include an organic solvent (alcohol, acetonitrile), a chaotropic drug or a detergent (nonionic in the case of ion exchange chromatography) in the eluent. Despite the wide selection of commercially available gels, DEAE-Sefadex is commonly used for ion exchange chromatography and Sepharose 2B, 4B or 6B-CL are commonly used for gel permeation chromatography.

One of the most convenient ways to separate amphiphilic polymeric compounds is hydrophobic chromatography using butyl-, octyl- or phenyl-sepharose. We found that electrodialysis and subsequent formation of triethylammonium salt of LPS could prepare a sample preparation of preparative hydrophobic chromatography [7] and could be successfully replaced by chelating ion exchange resin treatment. It should be noted that the total hydrophobicity of the LPS molecules confirms not only the lipid component, but to a considerable extent, the properties of the monosaccharides from which the O-specific polysaccharides are made (therefore O- and N-acetyl- And also the presence of deoxysaccharides). On the other hand, the wide selection of matrices and eluents for the separation of components makes it possible in many cases to optimize the process for successfully separating the desired fractions of S-LPS.

Thus, in a methodology for isolating claimed BAFs from different Gram-negative bacteria, an essential step involves extraction, followed by enzymatic digestion of foreign proteins and nucleic acids, furthermore the method described above to obtain the desired product. We propose a method that includes classification performed using one or a combination thereof. Here, in each specific case, several factors influence the choice of method of classification, in particular the species of the microorganism, the desired set of immunobiological characteristics (eg pyrogenicity, toxicity), the yield, cost and product characteristics of BAF and Other factors are like this.

"Nativity" (distinguishable from the preparation of modified LPS) by the design of the above-mentioned S-LPS-based claimed BAF, which is purified and purified. mature)) and "non-degeneration" of the LPS primary structure.

When analyzing the composition and structure of the claimed BAF, based on the NMR spectral data, in all cases the structure of the 0-specific polysaccharide component of LPS included in the claimed BAF composition is described in the literature. It should be noted that it is not different from that.

Regarding the oligosaccharide "core" located at the edge between the 0-specific polysaccharide and lipid A of the LPS, studies to determine if it is present in the composition of the claimed BAF include two "essential" components for the "core" component. Was performed with the identification of -heptose (using bound gas-liquid chromatography-mass-spectrometry (GLC MS)) and KDO ( Using a colorimetric reaction of thiobarbituric acid).

As a result of comparative analysis of lipid A fatty acid composition by the current GLC-MS method, only structural differences between the “parent” LPS and the proposed BAF formulation were detected.

Thus, particular attention has been paid to the lipid composition of the claimed BAF, particularly within the framework of the present work. Because the components, as noted above, determine the total complexity of the pathophysiological properties of LPS. Some specificity of the lipid component of the claimed BAF will be considered after a brief review of the structure of lipid A from LPS with Enterobacteriaceae.

At the base of lipid A is disaccharide β- (1'-6) -D-glucosamyl-D-glucosamine, which has two phosphate groups, one at the 4 'position of the shortened residue of D-glucosamine and the other. Carries (at an α-bond) one position of the shortened residue of D-glucosamine. Both amino groups of glycoprotein residues in disaccharides are substituted with residues of (R) -3-hydroxytetradecanoic acid [14: 0 (3-OH)]. While the other two residues of this acid are linked by ester bonds to the 3 and 3 ′ positions of the two residues of D-glucosamine of the disaccharide fragment. The two residues [14: 0 (3-OH)] located at the unreduced residues of D-glucosamine are in turn substituted by residues of tetratradecanoic and dodecanoic acid. This so-called "traditional" structure of lipid A is a feature that accounts for the overwhelming majority of LPS with Enterobacteriaceae (along with some minor modifications to the properties of substituents in the case of phosphates).

Many publications in the past have established theories and also the separation of the primary structure of lipid A of LPS, their synthesis, and their synthesis of many analogs, separated from microorganisms of its family, as well as other families and species of Gram-negative bacteria. And comparative studies on the biological properties of lipids synthesized and synthesized (chapters 1, 14, 46 and 47). An analysis of these findings has been made in the conclusion that any serious departure from the "traditional" structure of lipid A results in obvious and in some cases dramatic endotoxin reduction (Chapter 1, 46 and 47). The qualitative and quantitative composition of fatty acids is the most important property here. Thus, lipid A, which does not contain a "traditional" set of six fatty acids and has seven or five residues in its composition, exhibits significantly lower endotoxin activity in vivo compared to "traditional" lipid A [ Pyrogenic (rabbit) and lethal toxicity (rat)] (Chapter 1, 46 and 47). Furthermore, distinguished from "traditional" lipid A, the properties of lipid A described above are double-stranded with methylene linkages to C-2 (not C-3), HO groups, oxo groups, and adjacent methyl groups. The presence of fatty acids with longer or shorter hydrocarbon chains, which can include binding, cyclopropane fragments. Thus, it was known that the low LPS toxicity of Bacteroides fragilis is associated with the fact that lipid A of this LPS has a unique set of fatty acids substituted with fatty acids with 16 carbon atoms in which some of the fatty acids with tetradecanoic acid skeletons are known. 1, Chapter 7).

Lipid A of BAF claimed within the framework of the current study, along with "essential" fatty acids (particularly [14: 0 (3-OH)], tetradecanoic acid and dodecanoic acid), are also rare fatty acids such as hexa- and octa Decanone, where a significant decrease in the contents of [14: 0 (3-OH)] was observed. As a result, the structure of lipid A of claimed BAF based S-LPS is quite different from the "traditional" one, which will obviously affect their endotoxin properties.

In this regard, the high endotoxin LPS of Gram-negative microorganisms can now include fractions of S-LPS with reduced levels of endotoxins and pyrogenicity of S-LPS, which could be used for clinical practice. It became possible.

Some evidence has been obtained for the "nativity" of the claimed formulation in this study. First, comparing the NMR spectra of polysaccharide fragments of BAF separated by fatty acid composition of lipid A and by other methods (e.g., by the action of aqueous phenol and trichloroacetic acid), their characteristics are exactly the same. Showed. Secondly, a clear serological response between BAF and the corresponding anti-O-serum, as well as the serum of the patients (bacterial confirmed), was found in all cases. Third, in the case of the method of separating the widest range of LPS—extracted with hot aqueous phenol solution—no alteration of the fatty acid composition nor modification of the spectral characteristics of the isolated preparation occurred in the iterative process of the isolated preparation under the extraction conditions. Fourth, all BAFs showed high immunobiological activity.

The BAFs protected in the present invention differ significantly from the LPS formulations obtained by conventional methods in terms of the level of safety. They meet the requirements of the Technical Committee of Experts of the WHO in safety criteria for meningococcus and typhoid Vi-antigen polysaccharide vaccines (8).

In the framework of clinical trials of dysentery vaccines under the supervision of the National Control Authorities of Russia (NCA), the applicants were advised to Sh. After parenteral administration of sonnei-derived BAF at doses of 25, 50, 70 μg, there was no severe systemic or local endotoxin response as well as endotoxin shock. Thus, during individual clinical studies, the dose of clinical use of the claimed BAF is comparable to that of capsule vaccine polysaccharides and is significantly higher (up to 100-1000 fold) than the dose of conventional LPS administered to humans (9). Many subsequent clinical trials of this formulation confirmed its high degree of safety. There was no overall response, such as chills and headaches, only a slight increase in body temperature (<37.6 ° C) occurred in people who did not exceed 10% of the vaccine doses, and very rare local reactions occurred in the form of redness or pain at the site of administration. .

Sh. Sonnei-derived BAF was also safe in subcutaneous administration at doses of 25-50 μg to children of different age groups of 3-6 years, 7-10 years, 10-14 years. The levels of reactogenicity measured in systemic and local reactions were not significantly different in children and adults.

The safety of BAF, i.e. the absence of a pyrogenic response when administered subcutaneously with a dose of the agent in clinical and laboratory studies, is due to the relatively low activation of α-TNF (one of the important mediators of endotoxin responses) by BAF. It can be described as. Sh. When subcutaneous administration of sonnei-derived BAF at 50, 75 μg doses did not result in a significant increase in the α-TNF content in the serum of the volunteers after the first or second immunization.

It was also found that the BAF claimed in the laboratory and clinical studies performed was a potent immunogen and could be used as a vaccine. They induce O-specific protective antibodies in animals, including humans, where humoral immune responses were seen by all major types of antibodies IgG, IgA, IgM. These antibodies have bactericidal effects against the microorganisms they target. It provides defense of macroorganisms against homotypic strains of typhoid or heterologous pathogens in an anti-infective immune model (10). When immunizing volunteers with each BAF agent, a clear increase in the concentration of serum O-specific antibodies and seroconversion (more than fourfold increase in antibody levels) were observed in 80-90% of the immunized humans. Sustained high concentrations of specific antibodies protect people against Sonne shigellosis infection.

The characteristic nature of the immunogenicity of low-endotoxin BAF is the strong systemic and multisystemic activation of the local IgA immune response. Guinea Pig Sh. Immunization with sonnei-derived BAF induces local immunity and protects the mucous membranes of their eye conjunctiva from infections of lethal, homologous strains. O-specific antibodies produced locally and detected with MATs for α chain and secretory components were measured in the saliva of immunized volunteers.

In a field vaccine experiment against Sonne shigellosis performed in a pandemic region of Saratov region by the NCA of Russia, the vaccine was a BAF-based pharmaceutical composition, which showed a high preventive efficacy of the formulation. The formulation protected the citizens in the most unpleasant summer-autumn period when Sonne shigellosis development occurred. The mean index of vaccine efficacy exceeded 90% (12).

The use of low toxicity BAF as a resistance-induced anti-shock vaccine is very effective and may eliminate the toxicity issues of similar formulations. This is very important for patients in surgical hospitals. BAF from LPS of Shigella sonnei, Salmonella enterica and typhi, Escherichia coli 12, 12 hours before the lethal dose of posterior endotoxin was injected, and the complete survival of the experimental animals demonstrated a clear immunity to bacterial endotoxin. do.

Low endotoxin BAF acts on many aspects of the immune system and is an immunomodulator that affects the organ's resistance to tumor growth. The immunomodulatory action of BAF specific for vaccine action is evident especially at high doses of 50 to 150 μg and repeated administration of the agent.

The group of agents claimed has an anticancerogenic effect that was established in vivo experiments with inoculated cells of mastocytoma P855. Two doses of BAF preparations from Samonella enterica and typhi were administered to Balb / c mice, and then inoculated with mastocytoma cells to increase animal viability. Similar effects were obtained when the formulations were administered early (up to 24 hours) after inoculation.

LPS-derived BAF significantly increases resistance to Salmonella enterica and typhimurium natural infections with essential intracellular parasitic bacterial pathologies in mice.

The main immunomodulatory effect of the claimed group of agents is the rational activation of cytokines that mediate anti-infective immune mechanisms. Of the key immunity mediators that play an important role in viral and bacterial infections in the cell, the most important is γ-interferon. BAF derived from LPS of Shigella sonnei is a potent inducer of γ-interferon in vivo when administered at a dose of 100 μg.

The BAF proposed in the present invention can be used not only as a vaccine but also as a vaccine matrix for making a conjugated vaccine for mammals including humans. Conjugates of capsular polysaccharides of S. typhi (Vi-antigen) vaccine properties are described in Sh. It is selected as a carrier with sonnei-derived BAF, obtained using a conjugating agent, in particular carbodimide. Conjugates linked by strong amide bonds are stable under dissociation conditions. BAF can also be used as a matrix for protein antigens, which is particularly important because in the vaccine field almost such a matrix is completely absent.

The possibility of vaccine and immunogen formation based on the proposed BAF without forming covalent compound bonds between polymer molecules should be noted. We have established that the composition of polymer micelles formed during BAF self-assembly contains model protein antigen-human serum albumin. This "quasi-conjugated vaccine" is a polymer complex of 1000 to 5000 kDa, which contains an immunogenic BAF carrier which forms the required antigen and micelles.

The problem of the structural theory of the supermolecular structure of LPS, which is present on the outer wall of Gram-negative bacteria and is clearly destroyed during extraction, is recovered after removal of polymers and low molecular weight bacterial components in the purification of LPS. LPS isolated in aqueous solution tends to self-organize into a structure that remembers one fragment of a bacterial membrane, as seen by electron microscopy (Chapter 1, 11).

Thus, the group of agents proposed is a clinically applicable highly immunogenic BAF comprising S-LPS having primarily low levels of endotoxins and pyrogenicity, and the fatty acid composition of the initial total LPS of the corresponding microbe. Includes lipid A of different fatty acid composition.

The following examples are intended to illustrate and explain the invention and do not have the purpose of limiting the claims of the invention.

Example 1

1. Extract

Sh. Dried in acetone with 45% aqueous phenol solution (700 ml) with strong mechanical agitation at 68-72 ° C. for 15 minutes in a thermostat vessel. Extraction of sonnei, phase 1 bacterial cells (20 g) was performed and the suspension was cooled to 10-15 ° C. and centrifuged at 5000 g for 40 min. The upper aqueous layer was separated and dialyzed against distilled water for 5 days, undissolved residues were removed by centrifugation at 13000 g and the supernatant was lyophilized. The yield of the extract was 12% of the dry cell weight. The contents of the nucleic acid (UV-absorption) and the protein (Laury method) were 35-40% and 10-15%, respectively. The resulting extract was obtained with an initial concentration of 100 µl / ml. The reaction was performed in a reaction of inhibiting passive hemagglutination (RIPHA) using rabbit serum obtained by immunization with a dead culture of sonnei, phase 1 (diluted 1: 128).

2. Enzymatic Hydrolysis

2 g of crude extract containing predominantly LPS, nucleic acid, and soluble protein are dissolved in 100 ml of buffer, pH 7.2 to 7.6, containing 0.2 M NaCl, 0.05 M TRIS-HCl, 0.001 M MgCl ₂ and CaCl ₂ At 37 ° C. with ribonuclease A (activity 50-100 units / mg, 50 μg / ml of solution) and deoxyribonuclease (activity 400-800 units / mg, 5 μg / ml of solution) Stir for time. Proteinase K (activity 10-20 units / mg, 20 μg / ml of solution) was then added to the solution and stirred at 50 ° C. for 1 hour. The reaction mixture was dialyzed against distilled water for 72 hours and lyophilized.

3. ultracentrifugation

One gram of the product obtained in step 2 was dissolved in 120 ml of water, and the solution was ultracentrifuged using a Bekman ultracentrifuge (U.S.A.) for 8 hours at an acceleration of 80,000 g at 5 ° C. The obtained supernatant was lyophilized.

4. Separation of BAF by Extraction

If necessary, instead of step 3, extraction was carried out with a mixture of chloroform-methanol-0.2M HCl aqueous solution. Sh. In order to obtain sonnei-derived BAF, the above-mentioned mixture had a component ratio of 14: 13: 5 and a solid phase content of 10 mg / ml.

5. Fractionation in porous matrices

If necessary, instead of steps 3 and / or 4, a 10% polyacrylamide gel (1.5 mm thick gel, mobility of BAF to bromine phenol blue 0.1-0.3, Whole Gel Eluter from Bio-Rad) Preparative electrophoresis or hydrophobic chromatography (octyl-Sepharose column in buffer at pH8.1 containing 10% propanol and 0.05M ammonium bicarbonate) or Sephadex Ion exchange chromatography on superfiltration or DEAE cellulose (ammonium bicarbonate 0.01-0.75M) column using GLC column chromatography (1.5 M aqueous ammonium bicarbonate solution) or Pellicon system (Milipore) (membrane blocking 10 kD) Photography was used.

6. Purification of BAF

If necessary, desalination of BAF after fractionation was carried out at 4 ° C using gel chromatography of Sepharose 4B-Cl column in water or in 0.05M pyridine-acetate buffer. Fractions containing BAF were separated, bound and lyophilized around the void volume of the column.

Unpurified Sh. Isolation of BAF in sonnei LPS. step Way Yield of non-pyrogenic (when dose of 0.05 μg / kg of rabbit body weight) of% by weight of raw LPS 3 Ultracentrifugation 3-5% 4 Extraction of LPS with a mixture of chloroform-methanol-0.2M HCl solution <One% 5 Preparative Gel-electrophoresis on Polyacrylamide Gel (PAGE) 1-3% * 5 Ultrafiltration at the "Milipore" membrane 5-7% * 5 Ion exchange chromatography <One% 5 Hydrophobic chromatography 3-5% 5 Gel chromatography 3-5%

*-Higher dose of product is pyrogenic, reducing dose to 0.0125-0.0250 μg / kg of rabbit body weight

Example 2

Sh. Using NMR spectrometer and chromatographic mass spectrometry. Confirmation of the structure of O-specific polysaccharide and lipid A isolated from sonnei-derived BAF

As a result of the weak acid hydrolysis followed by precipitation of insoluble lipid A, Sh. O-specific polysaccharide and lipid A were isolated from sonnei-derived BAF.

The ¹³ C-NMR and ¹ H-NMR spectra of O-specific polysaccharides (FIGS. 1 and 2, respectively) show the Brucker WM-250 device at a temperature of 297 ° K or 303 ° K in heavy water (D ₂ O) solution. Were recorded (samples were lyophilized from heavy water before recording ¹ H-NMR spectra).

Analysis of the spectral data obtained according to the COZY, TOCSY and ROESY methods yielded clear properties of all signals in the ¹³ C-NMR and ¹ H-NMR spectra, with the repeat unit of PS being β- (1-3) -linked. Isosaccharide 2-acetamido-2-deoxy-4-O- (2-acetamido-4-amino-2,4,6-trideoxy-β-D-galactopyranosyl) -L- It was confirmed that it is altopyranosyluronic acid.

Sh. The fatty acid composition of lipid A of sonnei-derived BAF was verified by GLC MS method using methyl ether of fatty acid formed from lipid A after methanolysis (1M HCl in methanol, 100 ° C., 6 hours). Sh. Chromatograms of LPS-derived lipid A isolated from the methanol decomposition product of sonnei-derived BAF lipid A and the Bestpal method are shown in FIGS. 3 and 4, respectively.

Example 3

Preparation of conjugates of Vi antigen of Shigella sonnei-derived BAF with Salmonella enterica and S. typhi.

20 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide was added to a solution in which 20 g of Vi antigen was dissolved in 2 ml of water, and 0.1 M HCl was added to maintain pH at about 5.0 for 10 minutes at room temperature. Stirred. After stirring for 30 minutes at pH 5.0, the Sh. A solution of 5 mg BAF from sonnei was added to the reaction mixture. The reaction mixture was stirred at 10-12 ° C. for 16 hours and lyophilized by dialysis in distilled water for 72 hours. The product obtained was gel chromatographed on a sephacryl S1000 (exclusion limit> 8 × 10 ⁸ D) column (1 × 100) in 0.2 M NaCl. Fractions isolated around the pores of the column (calculated after 7 mg dialysis) were obtained by Sh. There was activity in serum RTPGA against sonnei, phase 1-derived LPS and S. typhi Vi antigens. Initial antigens were separated from the column for a longer duration. The data presented in the formation of the aforementioned polymeric conjugates include both antigens linked by amide bonds, which allows the use of BAF as a matrix to bind antigens of various origins. Therefore, the possibility of modifying BAF with different spacer groups and the possibility of creating "leg" structures linked to the macromolecules of antigen are within the scope of the present invention.

Example 4

From human serum albumin Sh. Preparation of sonnei-derived BAF associations

As a protein, Sh. The likelihood of forming a conjugate of sonnei-derived BAF was determined by gel chromatography.

Sh. 0.5 mg of sonnei-derived BAF was dissolved in 1 ml of 0.9% NaCl, and the resulting solution was heated at 60 ° C. for 15 minutes, gel chromatographed on a Sepharose 4B-CL column and equilibrated with 0.9% NaCl. Sh. sonnei-derived BAF was isolated near the void. Under these conditions, human serum albumin (HSA) was isolated with Kd = 0.67 and cholera anatoxin with Kd = 0.85. To produce this combination, Sh. A solution of sonnei-derived BAF (10 mg / ml concentration) was mixed with an equal volume of HSA solution (1 mg / ml concentration) and incubated at 60 ° C. for 30 minutes. The resulting mixture was applied to a Sepharose 4B-CL column (1 × 100 cm) and balanced with 0.9% NaCl. The formation of the bond is judged by the complete absence of the peak in the region Kd = 0.67 and the presence of a single peak separated in the pores of the column. Similar results were obtained by Sh. It was also shown in the analysis of the combination of sonnei-derived BAF and cholera anatoxin.

Thus, the ability to form a covalent bond between the macromolecule of the lipopolysaccharide antigen and the protein opens up the utility of BAF as a carrier for the protein antigen.

Example 5

Confirmation of pyrogenicity of BAF formulation in rabbit experiment

The pyrogenicity of the BAF formulations derived from Shigella sonnei, phase I, Shigella flexneri 2a, Shigella dysentereae type I, Salmonella enterica, S. typhi, Escherichia coli 055 compared with LPS samples obtained by the BestPal method from the same strain of endotoxin microorganisms. It was confirmed. Experiments were conducted with rabbits in accordance with the requirements of State Pharmacopoeia 7th edition, European Pharmacopoeia, USP. Pyrogenicity experiments were performed with three rabbits of the Shinshill species weighing 2.8-3.05 kg. After administration of the formulation, the rectal body temperature of rabbits was measured three times at 1 hour intervals. The formulation was determined to be nonpyrogenic if the total body temperature rise (in three rabbits) did not exceed 1.15 ° C. The results of body temperature measurement are shown in Table 2.

According to the data in Table 2, all LPSs isolated by BestPal have a high level of pyrogenicity, whereas BAF proposed in the present invention had no exothermic effect in rabbits when administered at a dose of 0.05 μg / kg rabbit body weight. . At the level of pyrogenicity, it satisfies the requirements of the Technical Committee of Experts of WHO for polysaccharide vaccines.

Pyrogenicity of BAF Preparation Formulation Temperature measurement data Pyrogenicity Level Check Result Vaccine VIANVAC ¹⁾ (0., 0.3, 0.3) Σ0.7 Non-pyrogen Vaccine TYPHIM Vi ²⁾ (0.2, 0.5, 0.2) Σ0.9 Non-pyrogen BAF Shigella flexneri 2a (0.5, 0.2, 0.4) Σ1.1 Non-pyrogen BAF Shigella sonnei, phase I (0.5, 0.2, 0.3) Σ1.0 Non-pyrogen BAF Shigella dysenteriae Type I (0.2, 0.5, 0.3) Σ1.0 Non-pyrogen BAF Salmonella enterica and S. typhi (0.3, 0.3, 0.5) Σ1.1 Non-pyrogen BAF Escherichia coli (0.2, 0.4, 0.3) Σ0.9 Non-pyrogen LPS Shigella flexneri 2a (0.8, 0.6, 0.8) Σ2.2 High-pyrogen LPS Shigella dysenteriae Type I (1.3, 1.2, 1.0) Σ3.6 High-pyrogen LPS Escherichia coli (Sigma) (1.5, 1.0, 1.0) Σ3.5 High-pyrogen LPS Shigella sonnei, phase I (1.4, 1.0, 0.9) Σ3.3 High-pyrogen LPS Salmonella enterica and S. typhi (Sigma) (1.6, 1.1, 0.9) Σ3.6 High-pyrogen

^{1) A} Vi-antigen polysaccharide typhoid vaccine from the commercially available sample "Gritvak" of Russia (International Trademark ATV-D-TEAM).

²⁾ Vi-antigen polysaccharide typhoid vaccine from the commercially available sample "Pasteur-Merier" of France.

Example 6

Confirmation of Endotoxin of BAF in D-galactosamine-sensitized Rat Experiment

Measurement of endotoxins was carried out using a special model of cross-bred mice sensitized with D-galactosamine (D-GalN), a hepatotoxic drug that rapidly increases rat sensitivity to produce endogenous tumor necrosis factor (TNF) induced by LPS. In vivo experiments were used. The individual's sensitivity to the toxic products of macrophages increases without modification of the endogenous toxin, ie the actual cause of the macrophage's reactivity to respond to LPS. 15 mg of D-GalN was intraperitoneally injected into rats weighing 18-20 g along with LPS samples obtained by BAF or BestPal method. Survival rates of mice in a group of 5 individuals were confirmed at the 48 hour course (Table 3). The complete survival of the rats was recorded when BAF was administered in the following dose ranges: Shigella sonnei, BAF-100 μg from phase I, BAF-1 μg from Shigella flexneri, BAF-10 from Shigella dysenteriae type I Μg, Salmonella enterica and typhi derived BAF-1 μg. The survival rate of the rats was noted upon injection of LPS preparations obtained by BestPal method at a dose 1000 times less than BAF.

Confirmation of LPS Endotoxin When Administered to D-GalN-sensitized Cross-White Rats Formulation Survival rats after (μg) dose of D-GalN 15 mg + preparation 0.001 0.01 0.1 1.0 10 100 400 BAF Shigella flexneri 2a ND ND 100% 100% 0% 0% 0% BAF Shigella sonnei ND ND 100% 100% 100% 100% 60% BAF Shigella dysenteriae Type I ND ND 100% 100% 100% 0% 0% BAF Salmonella enterica and typhi ND 100% 100% 100% 0% 0% 0% LPS Shigella sonnei 0% 0% 0% 0% 0% ND ND LPS Salmonella typhimurium (Sigma, U.S.A.) 0% 0% 0% 0% 0% ND ND

Example 7

Side effects of clinically applying BAF to adult volunteers

In accordance with the provisions of the National Control Authorities of the Ministry of Health of the Russia Federation, Sh. Sonnei-derived BAF as a heterologous candidate vaccine with doses of 25, 50, 75 μg in phenol-phosphate buffer as a solvent 1, 2, 3 times 3-4 weeks at 1765 adult volunteers Subcutaneously administered to the site. The appearance of toxic reactions included three groups: endotoxin shock symptoms, systemic (general) side effects, and local side effects were recorded for 72 hours.

Symptoms of endotoxin shock: tachycardia, lowering blood pressure, and a sharp increase in body temperature were not seen in any of 1765 volunteers in the 17-65 year old age group who received single or multiple doses.

Systemic Side Effects: Fatigue, headache, nausea, diarrhea, rhinitis, stomach pain, chills, and dizziness were recorded in 129 patients in two clinical control groups using placebo, 1 or 2 at doses of 25, 50, even 75 μg. It was not recorded in those who received one dose.

Non-severe local side effects: Pain and redness were found in the vaccine and placebo groups at 7.47% and 8.3%, respectively. No infiltrate, abscess, lymphangitis, pain or enlargement of regional lymph nodes were found.

Therefore, the complications of side effects were judged to be mild.

Example 8

Temperature response when clinically applying BAF to adult volunteers

Sh. Sonnei-derived BAF as a heterologous candidate vaccine with doses of 25, 50 and 75 ㎍ in phenol-phosphate buffer as a solvent. Subcutaneously administered to the site. For 72 hours, the incidence of the three groups—about <37.5 ° C., medium 37.6-38.5 ° C., heavy> 38.6 ° C.—temperature reaction was recorded. Strong and medium temperature responses were not recorded in volunteers of all planned and immune doses used. Only a slight rise in body temperature to 37.5 ° C. was observed relatively rarely (not more than 5-10% of donors), and this indicator was not very different from the number of temperature responses in the placebo group.

Example 9

Side effects and temperature response when clinically applying BAF to children

In accordance with the provisions of the National Control Authorities of the Ministry of Health of the Russia Federation, Sh. Sonnei-derived BAF is a heterologous candidate vaccine with a dose of 25 and 50 µg in phenol-phosphate buffer as a solvent, 35 children aged 2.8-6 years and 21 children aged 7-10 , 18 children aged 10-14 years were given subcutaneously in the upper third of the shoulder.

Symptoms of endotoxin shock: Acute tachycardia, lowering blood pressure, and a sharp increase in body temperature were not seen in 74 volunteers in the 2.8-14 year old age group who received one or two doses.

Systemic Side Effects: Fatigue, headache, nausea, diarrhea, rhinitis, stomach pain, chills and dizziness were also not recorded (Table 4).

Mild topical side effects (pain) were only seen in 2.7%.

Strong and moderate temperature responses were not recorded in immune children of all schemes and doses used. A slight increase in body temperature up to 37.5 ° C. was observed in 5.4% of recipients.

Sh. Systemic and local side effects after nausea avoided with sonnei-derived BAF Side Effect Sh. Frequency of adverse events recording in children immunized with sonnei-derived BAF (74 patients) temperature Body temperature rise to 37.1-37.5 degrees Celsius Absolute Number (Abs.) 4 % 5.4% Body temperature rise to 37.6-38.5 ℃ Absolute 0 % 0 38.6 ℃ or higher body temperature Absolute 0 % 0 Topical Redness 0 Induration 0 Pain at the site of administration Absolute 2 % 2.7% whole body nausea 0 diarrhea 0 headache 0 Rhinitis 0 dizziness 0 tired 0 chills 0

The complications of the side effects were evaluated as "weak".

Example 10

Induction of Tumor Necrosis Factor in Voluntary Subcutaneous Administration of Vaccine Dose of BAF

Sh. Sonnei-derived BAF 50 and 75 μg doses were compared with baseline levels for the production of tumor necrosis factor (α-TNF) 3 hours after the first and second subcutaneous immunizations. The contents of α-TNF, expressed in pg / ml, were investigated with the "Pro Cont-TNF" experimental system.

There was no statistically significant increase in the main mediator-concentration of the α-TNF-endotoxin response, even at the maximum dose of 75 μg (Table 5).

Low induction of α-TNF after immunization of volunteers with BAF Sh. sonnei-derived BAF dose, μg N * α-TNF, pg Before vaccination 3 hours after vaccination Before re-vaccine 3 hours after re-vaccine 50 10 208.42 ± 53.82 150.76 ± 35.98 118.82 ± 23.99 143.49 ± 16.66 75 12 172.41 ± 49.44 244.31 ± 74.17 79.13 ± 12.58 140.02 ± 38.03

N *-Number of experimental serum pairs of volunteers.

Example 11

Serological analysis of O-antigenic determinants in BAF preparations

Sh. sonnei, Sh. flexneri 2a, Sh. Serological activities of dysenteriae type 1, Salmonella enterica and typhi, Escherichia coli 055 BAF preparations were compared with the corresponding commercial ERYTHROCYTIC DIAGNOSTICUM (MSRIEM, Russia) and Sh. sonnei, Sh. flexneri 2a, Sh. Passive hemagglutination inhibition (IHA) was measured using monoreceptors (SPbNIVS, Russia, MSRIEM; Diagnostic Pasteur, France) of dysenteriae type 1, Salmonella enterica and typhi, Escherichia coli 055. Samples of 50 μg / ml concentrations of LPS or BAF formulations prepared by the BestPal method were placed sequentially in cups. The corresponding antiserum concentration was 4SU (serumological unit). The minimum concentration that inhibits reaction after addition of ERYTHROCYTIC DIAGNOSTICUM was expressed in μg / ml.

The inhibition points of each sample of BAF and LPS are shown in Table 6. The serological activity of BAF was not different from that of the corresponding LPS. Therefore, during the preparation of BAF described in Example 1 of the present invention, the structure of the O-specific polysaccharide chain was not changed and their antigen activity was maintained high.

Sh. sonnei, Sh. flexneri 2a, Sh. concentration of IHA in each sample of dysenteriae type 1, Salmonella enterica and typhi, Escherichia coli 055 BAF Sample Inhibitory concentration, µg / ml BAF Salmonella enterica and typhi 1.56 LPS Salmonella enterica and typhi 1.56 O-PS Salmonella enterica and typhi 12.5 BAF Escherichia coli 055 3.12 LPS Escherichia coli 055 1.56 BAF Sh. flexneri 2a 1.56 LPS Sh. flexneri 2a 0.78 BAF Sh. dysenteriae Type 1 0.37 LPS Sh. dysenteriae Type 1 1.56 BAF Sh. sonnei 3.125 O-PS Sh. sonnei 25 LPS Sh. sonnei 3.125

Example 12

Activation of Humoral Immune Responses of BAF Agents in Laboratory Animal Experiments

To confirm the induction of the humoral immune response, rats (CBAXC57B1 / 6) FI Sh. sonnei, Sh. flexneri 2a, Sh. dysenteriae type 1 BAF preparations were immunized intraperitoneally to each rat at doses of 400, 100, 10, 1 and 0.1 μg. In another experiment, Salmonella enterica, typhi, and Escherichia coli 055 BAF were administered at 1, 10 and 50 μg per rat. Peripheral blood serum was taken from animals after 15 and / or 30 days to determine the level of O-specific antigen by ELISA experiments and IHA. After a single dose BAF stimulated the immune response and the titer of anti-O-antibody was confirmed on day 15, where a maximum immune effect was observed at a dose range of 10-50 μg per horse. Significant amounts of IgG, IgM, IgA, the major antibody classes against homologous LPS, have been identified in peripheral blood.

Example 13

Induction of adaptive immunity to typhoid fever by immunizing experimental animals with BAF of Salmonella enterica and typhi

The rat (CBAXC57B1 / 6) FI was intraperitoneally injected with Salmonella enterica and typhi-derived BAF and Salmonella enterica and typhi-derived LPS at 25, 5, 1, 0.2, 0.04, 0.008 and 0.0016 µg per horse. Immunized. Physiological solutions were administered to the animals of the control group. After 12 days, pathogenic typhoid strain Ty2 No. 4446 was infected with each group at a dose of 1 × 10 ³ cells (50LD ₅₀ ) using sterile physiological solution containing 5% mucin. Survival of the animal group was recorded for 7 days. Both high endotoxin LPS and low endotoxin BAF induce adaptive immunity to typhoid fever and show high levels of protection (80-100%) in animals when infected at a dose of 25-5 μg (Table 7). All animals in the control group died. ED ₅₀ BAF-0.037 μg, ED ₅₀ LPS-0.081 μg.

Induction of adaptive immunity to typhoid fever after immunization of experimental animals with BAF of Salmonella enterica and typhi Immune dose (μg) Survival of rats after immunization with Salmonella enterica and typhi BAF and infection with 1000 cells of Salmonella enterica and typhi Ty2 strains Survival of rats after immunization with Salmonella enterica and typhi LPS followed by infection with 1000 cells of Salmonella enterica and typhi Ty2 strains 25 10/10 8/10 5 8/10 7/10 One 5/10 6/10 0.2 8/10 6/10 0.04 6/10 4/10 0.008 5/10 6/10 0.0016 2/10 4/10 - 0/10 0/10

Example 14

Sh. Sh. after the whole body immunization of experimental animals with sonnei-derived BAF. Induction of mucosal immunity to sonnei infection (Sereny test)

Sh. To guinea pigs weighing 200-250 g. 25, 50 μg doses of sonnei-derived BAF were immunized subcutaneously in the back region twice daily at 10 day intervals. The control group was injected with a physiological solution instead of the preparation. Ten days after the last immunization, the pathogenic strain Sh. A suspension of 30 μl of sterile physiological solution of sonnei 5063 cells was inoculated into the conjunctiva to a dose close to ID ₁₀₀ (2 × 10 ⁹ cells) and to a dose close to 2ID ₁₀₀ (4 × 10 ⁹ cells). Shigella keratoconjunctivitis progressed in all animals of control 5 infected with 4 × 10 ⁹ cells and 75% of animals 6 of control 6 infected with 2 × 10 ⁹ cells (Table 8). Animals immunized twice with 50 μg dose protected 55% of eyes when infected with 4 × 10 ⁹ cell dose, and 75% of animals when infected with 2 × 10 ⁹ cell dose. There was a protective effect. Animals immunized twice at 25 μg dose had protective effect on 75% of animal eyes when infected with 4 × 10 ⁹ cell dose and 80% of animals when infected with 2 × 10 ⁹ cell dose. There was a protective effect. Sh. Obvious local anti-infective immunity was confirmed when reversed with sonnei-derived BAF preparations.

Sh. Sh. after the whole body immunization of experimental animals with sonnei-derived BAF. Induction of mucosal immunity to sonnei infection (Sereny test) No. Vaccine Dose Infection dose Infected animals Infected eyes Number of eyes with keratitis conjunctivitis Number of eyes defended against keratitis Percent of eye defended One 50 µg 50 µg 0.4 × 10 ¹⁰ cells / eye 10 20 9 11 55% 2 50 µg 50 µg 0.2 × 10 ¹⁰ cells / eye 10 20 5 15 75% 3 25 µg 25 µg 0.4 × 10 ¹⁰ cells / eye 10 20 5 15 75% 4 25 µg 25 µg 0.2 × 10 ¹⁰ cells / eye 10 20 4 16 80% 5 Control 0.4 × 10 ¹⁰ cells / eye 10 20 20 0 0% 6 Control 0.2 × 10 ¹⁰ cells / eye 10 20 15 5 25%

Example 15

Activity of Systemic Immune Responses by BAF Agents During Immunization of Adult Volunteers

Sh. As a sonnei candidate vaccine, Sh. A study of sonnei-derived BAFs conducted an engineered experiment to immunize 18-22 year old adults according to the regulations of the National Control Authorities of the Ministry of Health of the Russia Federation. Sh. The systemic immune response to sonnei-derived BAF was studied by examining pairs of venous blood serum taken from patients before and 28-30 days after immunization. Specific indirect haemagglutination-IHA reactions were performed using a commercially available kit (Series 3 K 60) of the Gabrichevsky Enterprise Institute (Moscow) for serological diagnosis of Sonnei dysentery. To identify IgG, IgA, and IgM types of serum antibodies, Sh. ELISA was performed using an ELISA experimental system based on sonnei derived LPS. Sh. A total of 380 sera from sonnei-derived BAF and placebo were tested.

Specific anti-LPS Sh. Showed a 18.5-fold increase in GM titer, 5.9-fold IgG, 2,6-fold IgM, and 19.3-fold increase in IgA. An increase in sonnei antibodies was observed in Sh. It appeared in people vaccinated with sonnei-derived BAF (Table 9). There was no statistically significant change in GM titers of placebo-treated people before or after the vaccine.

Sh. Percentage of humans with seroconversion (4 fold or more increase in antibody) a month after receiving sonnei-derived BAF, 94.7% for anti-O-antibody, 71.6 for IgG %, 51% for IgM and 77% for IgA.

Combining clinical immunological data from volunteers, Sh. Systemic humoral immune response to sonnei-derived BAF, particularly high levels of IgA.

Evaluation of systemic humoral immune response to BAF after one month of immunization of volunteers Multiple of increase in IgA anti-O-antibody 19.3 - Geometric mean titers of IgA anti-O-antibodies (number of experimental pairs of serum studied) 4 weeks after vaccination 1235 ± 168 (75) 58 ± 9 (80) Before vaccine 64 ± 7.5 (75) 83 ± 17 (80) Multiple of increase in IgM anti-O-antibody 2.62 1.45 Geometric mean titers of IgM anti-O-antibodies (number of experimental pairs of serum studied) 4 weeks after vaccination 249 ± 72 (78) 153 ± 28 (81) Before vaccine 95 ± 29 (78) 105 ± 33 (81) Multiple of increase in IgG anti-O-antibody 5.9 - Geometric mean titers of IgG anti-O-antibodies (number of experimental pairs of serum studied) 4 weeks after vaccination 940 ± 150 (78) 131 ± 16 (81) Before vaccine 181 ± 40 (78) 138 ± 22 (81) Multiple of increase in aggregated antibody 18.5 - Geometric mean titer (GMTA) of IgA anti-O-antibody (number of experimental pairs of serum studied) After vaccine 724 (95) 42 (80) Before vaccine 39 (95) 34 (80) Formulation name Sh. sonnei-derived BAF Placebo

Example 16

Mucosal Immunity in Immunized Adult Volunteers with BAF

Sh. The presence of specific secretory antibodies to LPS was studied with pairs of saliva samples from volunteers (30) before and after immunization with sonnei-derived BAF. All saliva samples and coprofiltrate were coded and bound into blocks and stored at minus 18-20 ° C. before the experiment.

Antibodies were identified by IHA and ELISA using monoclonal antibodies against IgA (clone GA) of secretory components.

In the IHA test, there was a statistically significant increase in the antibody titer in saliva after vaccination where there was a 4-fold change in antibody in 72% of human saliva, with a multiple of 4.21. In the ELISA test, a statistically significant increase in geometric mean titer for O-specific IgG, IgA antibody, and IgA (sIgA) with secretory components were observed in saliva. The folds of increase in antibody titers were 5.47, 4.37 and 4.0, respectively (Table 10).

Sh. GM titers detected in the saliva of volunteers before and after immunization with sonnei-derived BAF Antibodies Number of test saliva samples GM titers of anti-LPS antibodies Multiple of increase Before vaccination 4 weeks after vaccination Cohesive 30 2.4 10.1 4.21 IgG 30 9.2 50.3 5.47 IgA 30 358 1563 4.37 sIgA 30 18.2 72.8 4.0

Example 17

Activation of Systemic Immune Responses after Immunizing Children with BAF

Three groups of children were made by random selection (selection unit-one child): the first group consisted of 35 children aged 2.8 to 6 years; The second group consisted of 21 children aged 7 to 10 years; The third group consisted of 18 children aged 11 to 14. Sh. Sonnei-derived BAF preparations were immunized subcutaneously with doses of 50 μg in 69 children and 25 μg in 5 children aged 2.8 to 4 years. Experimental pairs of pre- and post-immune venous blood serum using commercially available kits (Series 3K 60), a Gabrichevsky Enterprise Institute (Moscow) ELISA test system for the diagnosis of Sonne Shigellosis, to identify systemic O-LPS specific immune responses The pair of sera of venous blood taken before and after 14 days after immunization from 41 patients receiving the formulation was examined by IHA assay and ELISA.

Sh. Children of three age groups. Activation of systemic immune response 14 days after immunization with sonnei-derived BAF Vaccinated group Number of test serum GM titers of aggregated anti-LPS antibodies GM titers of IgG anti-LPS antibodies GM titer of IgM anti-LPS antibody GM titers of IgA anti-LPS antibodies Before vaccine 2 weeks after vaccine Before vaccine 2 weeks after vaccine Before vaccine 2 weeks after vaccine Before vaccine 2 weeks after vaccine 2.86-6 year olds 14 141 ± 33 1187 ± 326 44 ± 8.5 168 ± 88 138 ± 42 476 ± 93 42 ± 3 411 ± 109 Children 7-10 years old 9 323 ± 93 2389 ± 519 74 ± 18 435 ± 262 217 ± 72 507 ± 103 44 ± 4 1164 ± 304 Children aged 11-14 18 299 ± 108 2580 ± 332 71 ± 16 593 ± 144 186 ± 26 419 ± 61 58.7 ± 12 1280 ± 397 2.8-14 year olds 41 235 ± 55 1947 ± 240 61 ± 9 360 ± 95 174 ± 24 456 ± 47 48.9 ± 6 673 ± 216

Sh. In all groups of children receiving sonnei-derived BAF preparations, the percentages of 4-fold seroconversion of cohesive, IgG, IgM, and IgA antibodies were 90.2%, 73%, 48.7%, and 92.7%, respectively, 14 days after vaccination. In the first group of vaccinated children (aged 2.8-6 years), the percentage of fourfold seroconversions of cohesive, IgG, IgM, and IgA antibodies was 100%, 57%, 71%, and 85.7%, respectively, and the second group (7-10). 71.4%, 66.7%, 44.4%, and 85.7%, respectively, and 89.5%, 88.7%, 33.3%, and 100% in the third group (11-14 years old), respectively.

The geometric mean titer (GM titer) of the anti-O aggregated antibody was determined by Sh. In the group of children receiving sonnei-derived BAF, the vaccine was significantly higher than the baseline level (Table 11). The GMTAs of IgG antibodies before vaccination in the vaccinated group were 44, 74, and 71 in the first, second, and third groups, respectively, and 168, 435, 593 after 14 days of vaccination, respectively. The multiplicity of antibody titers was 3.8, 5.9 and 8.9, respectively. But Sh. In people immunized with sonnei-derived BAF agents, the increase in IgM anti-O-antibodies was statistically significant in all groups immunized. Sh. Note the apparent induction of anti-O IgA antibodies that play an important role in immunity to dysentery by sonnei-derived BAF preparations. More significant increases in IgG antibodies were observed, specifically GMTA before immunization were 42, 44 and 58.7, respectively, afterwards 411, 1164 and 1280, and the folds of increase in antibody titers were 6.6, 17.3 and 21.7 times, respectively (Table 11). .

Example 18

Induced resistance to endotoxin with BAF

To confirm the induction of resistance to lipopolysaccharide, rats (CBAXC57B1 / 6) FI (8 per group) received Salmonella enterica, S. typhi, Shigella sonnei, Escherichia coli 055 BAF formulations from 0.1, 1.0 per horse. And immunization by intraperitoneal injection at a dose of 10.0 μg. The same dose of Shigella sonnei-derived LPS and nonpyrogenic physiological solution was injected into the control group. After 12 hours, 0.1 μg of LPS preparation and 15 μg of D-galactosamine per horse were intraperitoneally injected to model the endotoxin shock. It has already been found that this dose of LPS is absolutely lethal (100LD ₅₀ ). In the group of animals immunized with Salmonella enterica, S. typhi, Shigella sonnei, Escherichia coli 055-derived BAF and Shigella sonnei-derived LPS, the survival rate of the animals was 100% as a result of induction of resistance. In the control group, all animals died within 24 hours. Thus, low endotoxin BAF derived from Salmonella enterica, S. typhi, Shigella sonnei, Escherichia coli 055 has a definite resistance-inducing effect in the range of 0.1 to 10 µg, like LPS derived from traditional Shigella sonnei.

Example 19

Induction of early γ-interferon in vivo by BAF preparation

To study the production of γ-interferon, rats (CBAXC57B1 / 6) were immunized with 10 and 100 μg intraperitoneal injections of Shigella sonnei-derived BAF and Shigella sonnei-derived LPS. The initial peak was measured in the serum of peripheral blood of the animals after 7.5 hours. The concentration of gamma interferon was measured using the OptEIA ^™ mouse interferongamma measurement system (Pharmagen). As evident in Table 12, BAF formulations result in an increase in the concentration of gamma interferon in serum. For BAF formulations derived from Shigella sonnei, an increase in gamma interferon production with dose was seen.

Controlled Induction of Early γ-Interferon In Vivo by BAF Agents Formulation Concentration of gamma interferon, pg / ml 10 μg / horse 100 μg / horse Shigella sonnei-derived LPS 541 ± 158 1039 ± 728 BAF from Shigella sonnei 55 ± 50 407 ± 253 Control 25 ± 25 50 ± 48

Example 20

Induction of resistance to rats against Staphylococcus aureus infection by BAF preparations derived from Salmonella enterica, S. typhi, Shigella sonnei, Escherichia coli 055

To confirm the induction of resistance to Staphylococcus aureus, strain 209, white mice were immunized with intraperitoneal administration of Shigella sonnei-derived BAF formulation at a dose of 100 μg per horse (equivalent to human dose), and 7 days later, Staphylococcus aureus, Infected with 5 × 10 ⁶ cells of strain 209. Mice from the experimental and control groups (untreated) were dissected on Day 1, Day 2, Day 3, Day 4 and Day 7 (5 in each group on each day mentioned above). Blood, liver, spleen, mesentery and kidneys were planted in solid nutrient media followed by colonies sorted. The index of dissemination—positive seeding ratio of the total number of samples—was measured. Dynamics of seeding index was used as an index of nonspecific resistance of rats to Staphylococcus aureus (Table 13).

Dynamics of the Index of Sowing of Internal Organs of Rats Date range Seeding index 100 µg experimental group of BAF derived from Shigella sonnei Control One 0.91 1.0 2 0.48 0.93 3 0.20 0.75 6 0 0.12 10 0 0.05

As such, Shigella sonnei-derived BAF preparations increased nonspecific resistance to Staphylococcus infection at 100 μg.

Example 21

Induction of rat resistance to influenza A virus PRA-8 strain infection by BAF preparations derived from Salmonella enterica, S. typhi, Shigella sonnei, Escherichia coli 055

To study the significance of resistance to viral infection by the action of BAF, mice (CBA line, males of 18-20 g, 10 per group) were uninfected with influenza A virus PRA-8 strain 1DCL under mild ether anesthesia. Two days before infection, 100 μg of Salmonella enterica, S. typhi, Shigella sonnei, and Escherichia coli 055-derived BAF were injected subcutaneously. The survival rate (observed for 10 days) of rats by BAF formulation was 80-90%, whereas the control group showed 85-90% mortality. In this model mentioned above the BAF showed a definite protective action.

Example 22

Anticancer Activity of BAF Agents on Infection of DBA / 2 Mice by Mast Cell P815 Transplantation Cells

Anticancer activity was measured with DBA / 2 mice. Ten rats per group were immunized with intraperitoneal injections of S. typhi-derived BAF preparations 24, 72 hours and 12 and 24 hours, respectively, at 10 and 100 μg doses of mast cell tumor P815 cells. The mast cell tumor P815 cells were injected into the hind limbs at a dose of 1 × 10 ⁵ cells, and the same doses of cancer cells were injected into the site using mice in the same line as a control group. The percentage of survival of the animals for 31 days was identified.

Group II injected twice with 100 μg dose of S. typhi-derived BAF preparation 24 and 72 hours before tumor cell injection or group IV injected 12,24 hours after injection of tumor cells were BAF derived from S. typhi. Survival was significantly increased compared to group I, which did not receive the formulation. At the same time, the group receiving S. typhi-derived BAF at a dose of 10 μg did not activate anticancer immunity and the survival index decreased regardless of the time of tumor cell administration (Group III, Group V). As such, the S. typhi-derived BAF formulation had a protective effect against tumor growth when administered twice at 100 μg per horse (Table 14).

Anticancer Activity of BAF Agents on Infection of DBA / 2 Mice by Mast Cell P815 Transplantation Cells Work Animal survival rate,% I * II * III * IV * V * 0 100 100 100 100 100 7 60 100 60 100 60 11 - 80 0 80 0 19 0 60 0 60 - 31 0 20 0 60 0

I * -control. Mastocytoma P815 cell administration.

Administration of S. typhi-derived BAF twice a dose of 100 μg 24 and 72 hours prior to II * -mastomas P815 cells.

Administration of S. typhi-derived BAF at 10 μg 24 hours prior to administration of III *-mastocytoma P815 cells.

Twelve and 24 hours after IV *-mastocytoma P815 cells were administered two doses of S. typhi-derived BAF at 100 μg.

12 and 24 hours after administration of V *-mastocytoma P815 cells, two doses of S. typhi-derived BAF were administered at 10 μg.

Example 23

Effectiveness as a vaccine against Sonne shigellosis of Shigella sonnei-derived BAF-based pharmaceutical compositions

A field trial of a vaccine against Sonne shigellosis, a BAF-based pharmaceutical composition, was conducted in Romanovsky County, Saratov, where the disease is prevalent. Immunized 3068 people. Among them, 1802 volunteers received the vaccine subcutaneously and 1266 were placed on the placebo. The infection rate among the vaccinated people was 0.55 per 1000 people and 7.9 per 1000 people in the placebo group. The average value of vaccine efficiency after 9 months of observation was 92.9%.

During the most unpleasant summer-autumn period when Sonne shigellosis infection occurs, the formulation provides effective prevention for the citizens.

Example 24

BAF drug type

Vaccine formulations were prepared by mixing the following components in a volume of 0.5 ml liquid.

Active Ingredient: BAF 0.050mg from Shigella sonnei

Dihydroorthophosphate Sodium 0.052mg

(Sodium dihydroortophosphate)

Hydroorthophosphate Sodium 0.017mg

(Sodium hydroortophosphate)

Sodium chloride 4.150mg

Phenolic 0.750mg

0.5 ml of distilled water.

The resulting formulation can be stored at 2-8 ° C. for 36 months.

[references]

Helmut Brade et al., Eds. Endotoxin in Health and Disease, New York: Marcel DEKKER, 1999.

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Ashkenazi S, Passwell JH, Harlev E, Miron D, Dagan R, Farzan N, Ramon R, Majadly F, Bryla DA, Karpas AB, Robbins JB, Schneerson R. Safety and immunologenicity of Shigella sonnei and Shigella flexneri 2a O- specific polysaccharide conjugates in children. J Infect Dis. 1999 Jun; 179 (6): 1565-8.

Cohen D, Ashkenazi S, Green MS, Gdalevich M, Robin G, Slepon R, Yavzori M, Orr N, Block C, Ashkenazi I, Shemer J, Taylor DN, Hale TL, Sadoff JC, Pavliakova D, Schneerson R, Robbins JB. Double-blind vaccine-controlled randomised efficacy trial of an investigational Shigella sonnei conjugate vaccine in young adults. Lancet, 1997 Jan 18; 349 (9046): 155-9.

Westphal O., Lueritz O., and Bister F. Uber die Extraktion von Bakterien mit Phenol / Wasser. Z. Naturforch. 7: 148-155. 1952.

6. Galano C, Jiao BH, Komuro T, Freudenberg MA, Luderitz O. Large-scale fractionation of S-form lipopolysaccharide from Salmonella abortus equi. Chemical and serological characterization of the fractions. J Chromatogr. 1998 May 25; 440: 397-404.

Mikes O et al., Eds., Laboratory handbook of chromatographic and allied methods, J. Wiley, Chichester, New York, Brisbane, Toronto, 1979.

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9.Aparin P.G., Golovina M.E., Yolkina S.I., Gantcho T.V., Ershov V.I., L'vov V.L. First CLINICAL INVESTIGATION OF NEW LIPOPOLYSACCHARIDES: PHASE I-II CLINICAL TRIALS OF Shigellae sonnei LPS VACCINE. Proceedings of The Third Annual Conference on Vaccine Research, Bethesda, USA, 1-2 May, 2000. Abstract S18.

10.L'vov V.L., Verner I.C., Golovina M.E., Aparin P.G. Immunobiology of low toxic & pyrogenic lipopolysaccharide from Salmonella Typhy. Medical Journal of Indonesia, Oct., 1998, V. 7, Supp. 1: 247-251.

11.Aparin PG, Golovina ME, Ershov VI, Gancho TV, Shmigol VL, Pavlova LI, Chuprynina RP, Yolkina SI, Rachmanov RS, and L'vov VL, Systemic and mucosal (local) immune response after immunization with new type low- endotoxic LPS vaccine Shigella sonnei. Proceedings of The Fourth Annual Conference on Vaccine Research, Arlington, USA, April 23-25, 2001. Abstract S6.

12.Aparin P.G., Pavlova L.I., Gorbunov M.A., Golovina M.E., Gantcho T.V., Chuprinina R.P., Nemirovskaya T.I., Rachmanov R.S., Ershov V.I., Kurenkova E.B., Prochnitskaya I.A., Anvovol and Ivovol and Ivovol. Phase III efficacy trials of LPS Shigella sonnei vaccine: seasonal protection against shigellosis Sonnei in mid-Volga region. Proceedings of The Fifth Annual Conference on Vaccine Research, Baltimore, USA, May 6-8, 2002. Abstract.

Claims (43)

In a method for separating a biologically active fraction (BAF) mainly comprising S-LPS of Gram-negative bacteria producing endotoxin S-LPS (LIPOPOLYSACCHARIDES), (I) followed by extraction, separation of insoluble material and lyophilization with subsequent dialysis; (II) treatment with nuclease and proteinase K to purify the extract obtained in step (I) from protein and nucleic acid impurities, followed by dialysis to separate LPS and lyophilize; And (III) optionally, dividing the LPSs obtained in step (II) into fractions by ultracentrifugation; And (IV) optionally, extracting the LPS obtained in step (II) or (III) into a mixture of chloroform-methanol-aqueous hydrochloric acid (aqueous HCl) and dividing the fraction into fractions; And (V) Optionally, the LPSs obtained in (II), (III) or (IV) are selected from the group consisting of methods including gel electrophoresis, column chromatography, ultrafiltration on preliminary polyacrylamide gels. Dividing the LPSs into fractions on porous matrixes using; (VI) separating the final product by dialysis and / or gel-penetrating chromatography. The method of claim 1, wherein the method of extracting the natural bacterial LPS that provides the invariance of the primary structure is used to obtain the extract of step (I). 3. The method of claim 2, wherein extraction with Bestphal is used to obtain the extract. The method of claim 3, wherein the intermediate obtained according to claim 6 is treated with nuclease and protease for 0.2 to 60 hours at a temperature of 4 to 60 ℃. The separation of BAF according to claim 4, wherein the intermediate is treated with a deoxyribonuclease having an activity of 600 to 4000 units / mg and a ribonuclease having an activity of 50 to 140 units / mg. Way. 5. The method of claim 4, wherein the intermediate is treated with a protease having an activity of 1 to 20 units / mg. The method of claim 1, wherein the extract of step (I) is ultracentrifuged at an acceleration of 50000 to 150000g at a temperature of 4 to 50 ° C. for 0.5 to 24 hours. The method of claim 1, wherein column chromatography selected from the group of methods including ion exchange chromatography, hydrophobic chromatography and gel permeation chromatography is used in step (V). The method of claim 1, wherein the dividing into fractions of step (V) is performed by gel permeation chromatography on a porous gel using water-soluble buffered eluents, which may include chaotropic drugs or detergents. Separating method of BAF, characterized in that carried out. The method according to claim 8, wherein the gel permeation chromatography of BAF characterized in that it is performed with a Sephadex column eluting with a buffer containing 0.05 to 4.0 moles of the component forming the lyophilizing buffer. Separation Method. The method of claim 1, wherein the dialysis of steps (I) and (II) is performed for 3 to 5 days. The method for separating BAF according to claim 1, wherein the vacuum freeze drying of steps (I) and (II) is performed for 16 to 24 hours. For BAF containing predominantly S-LPS from Gram-negative bacteria producing endotoxin LPSs, BAF is β-hydroxydecanoic, β-hydroxydodecanoic acid in lipid A of S-LPS. ), the molar ratio of D-glucosamine and β-hydroxy acid selected from the group comprising β-hydroxytetradecanoic and β-hydroxyhexadecanoic acid is about 2: 1 to 2: 4. And the molar ratio of D-glucosamine and higher fatty acids linked by amide bonds and ester bonds in lipid A of S-LPS is about 2: 3 to 2: 7. The BAF of claim 13, wherein the BAF is obtained from an endotoxin microorganism having a synthetic ability of S-LPS. The BAF of claim 13, wherein the BAF is isolated from endotoxin strains of Salmonella enterica, S. typhi, Shigella sonnei phase I, Shigella flexneri 2a, Shigella dysentereae type I (Shiga), Escherichia coli 055. The degree of pyrogenicity of BAF, according to any one of claims 13 to 15, in which the total body temperature rise does not exceed 1.15 ° C in an exothermic experiment conducted at a dose not exceeding 25 ng / kg of rabbit body weight. BAF, characterized in that. 16. The clinical trial of any of claims 13-15, wherein the exothermic response in a clinical trial to determine the degree of pyrogenicity by subcutaneously administering a dose of up to 75 μg (<37.6 ° C.) to an adult volunteer does not exceed 20% of cases. BAF, characterized by appearing. The method according to any one of claims 13 to 15, wherein in an experiment for determining the degree of pyrogenicity by subcutaneously administering a dose of up to 50 μg to a group of children aged 3 to 6 years, 7 to 10 years, and 10 to 14 years old. Weak (up to 37.5 ° C.) exothermic reactions are observed in BAF. 16. BAF according to any one of claims 13 to 15, wherein the maximum tolerated dose when administered once subcutaneously to an adult volunteer is no more than 150 μg. 16. The BAF of any of claims 13-15, wherein the BAF is a vaccine for a mammal, including humans. The method of claim 13, wherein the BAF is selected from Sh. Obtained from sonnei culture, it activates specific immune responses that produce systemic and secretory IgA antibodies when parenterally administered to volunteers at doses of 1-100 μg, and seroconversion. ) (4 fold or more increase in anti-O antibody) BAF. 21. The parenteral administration of any one of claims 13-20, wherein the parenteral administration is carried out at a dose of 1-50 μg to a group of children and teenagers aged 3 to 6 years, 7 to 10 years and 11 to 14 years of age. Activating specific immune responses to produce systemic and secretory IgA antibodies, and providing more than 80% of serum changes (four-fold or more increase in anti-O antibodies) BAF. 20. The method of any one of claims 13-19, wherein the BAF comprises heterologous protective natural antigens and synthetic antigens nontoxic complexes or humans of proteins and natural polysaccharides. BAF characterized in that it is a vaccine carrier which can be made together with a conjugated compound for the purpose of enhancing the immunogenicity of such antigens in mammals. 21. The method of any one of claims 13-20, wherein the BAF is a safe tolerogenic anti-shock vaccine, which reduces the sensitivity of mammalian bacteria to endotoxins upon parenteral administration. BAF, characterized in that. 20. The method according to any one of claims 13 to 19, wherein the BAF is an immunostimulator and has a therapeutic effect in the case of a disease or condition that needs to enhance the immunity of a mammal, including a human. BAF. 20. The BAF of any one of claims 13 to 19, wherein the BAF is an immunostimulant and has a therapeutic effect in the case of a viral infection that needs to enhance the immunity of a mammal, including humans. 20. The BAF according to any one of claims 13 to 19, wherein the BAF is an immunostimulant and has a therapeutic effect in the case of bacterial infections that need to enhance the immunity of mammals including humans. 20. The BAF according to any one of claims 13 to 19, wherein the BAF is an adjuvant and has a therapeutic effect in the case of a neoplastic disease that needs to enhance the immunity of a mammal including a human. 21. BAF according to any one of claims 13 to 20, characterized in that administration to humans at a dose of 100 μg does not induce endotoxin shock or serious reactions. 20. BAF according to any one of claims 13 to 19, which activates the production of cytokines, in particular γ-interferon in vivo . A pharmaceutical composition comprising BAF according to any one of claims 13 to 19 and a pharmaceutically acceptable additive. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition is a vaccine against Sh.sonnei and is administered parenterally to adult volunteers at a dose of 1-100 μg. O-specific humoral immune response It activates the production of systemic and secretory IgA antibodies, activates serum changes (four-fold and higher anti-O antibody concentrations) by more than 80%, and in situ conditions for shigellosis Sonne (in field). condition) A pharmaceutical composition characterized by activating mucosal immunity and defense. 32. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition is a vaccine for children against Sh.sonnei and is used in a group of children and teenagers aged 3 to 6 years, 7 to 10 years, and 11 to 14 years of age. Dosing parenterally at doses activates the production of O-specific humoral immune responses, systemic and secretory IgA antibodies, and increases serum changes (four-fold and higher anti-O antibody concentrations). A pharmaceutical composition, which activates more than% and activates mucosal immunity and defense in field conditions against shigellosis Sonne. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition is a vaccine against Sh.flexneri 2a infection comprising BAF isolated from Sh.flexneri 2a and is administered parenterally when administered to a mammal at a dose of 1-100 μg. A pharmaceutical composition characterized by activating the production of specific humoral immune responses, systemic and secretory IgA antibodies, and activating serum changes (four-fold and higher anti-O antibody concentrations) by 80% or more. . The pharmaceutical composition of claim 31, wherein the pharmaceutical composition is a vaccine against Sh.dysenteriae, type1 infection comprising BAF isolated from Sh.dysenteriae, type1 and is administered parenterally to a mammal at a dose of 1-100 μg. Pharmaceutical production characterized by activating the production of O-specific humoral immune responses, systemic and secretory IgA antibodies, and activating serum changes (4 times or more increase in anti-O antibodies) by 80% or more Composition. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition is a vaccine against S.enterica and typhi infection comprising BAF isolated from S.enterica and typhi and when administered parenterally to a mammal at a dose of 1-100 μg. Activates the production of O-specific humoral immune responses, systemic and secretory IgA antibodies, activates seroconversion (four-fold and higher levels of anti-O antibodies) by more than 80% and protects against typhoid infection Pharmaceutical composition, characterized in that to activate the defense against. 32. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition is a resistant to anti-shock vaccine and reduces the sensitivity of the response to bacterial endotoxins in mammals, including humans, upon parenteral administration. . A pharmaceutical composition according to claim 31 is administered to a mammal, including human, once or more, subcutaneously, orally, intravenously, intramuscularly, intraperitoneally, sublingually, intravaginally, rectally, nasally and percutaneously. How to induce protective immunity. 32. A method for improving a condition of a diseased patient in need of an enhanced immune status, comprising administering the pharmaceutical composition of claim 31 to a patient in need thereof. 40. The method of claim 39, wherein said disease requiring enhancement of immunity is a neoplastic disease requiring enhancement of immune status. 40. The method of claim 39, wherein the disease requiring enhancement of immunity is a viral disease. 40. The method of claim 39, wherein said disease requiring enhanced immunity is a bacterial disease. 40. The method of claim 39, wherein the disease requiring enhancement of immunity is a fungal disease.