RU2563354C2 - Adjuvant, based on low-molecular peptidoglycan of bacterial cell wall - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of pharmaceutics and deals with adjuvant, its application for obtaining composition, composition itself and set. Characterised adjuvant represents mixture of three active compounds, contained in amount, constituting respectively 20-50%, 20-50% and 40-60% of the entire adjuvant weight, with the following structure: compound 1: β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl_D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid (GMtri); compound 2: β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine (GMtetra) and compound 3: dimer of GMtetra (doGMtetra) Bond between monomer residues of GMtetra is realised due to carboxyl group of terminal D-alanine of one GMtetra residue and ω-aminogroup of meso-diaminopimelic acid of other GMtetra residue.

EFFECT: claimed invention provides activation of immune system of organism in response to introduction of antigen of viral or bacterial origin, where antigen can represent weak immunogen.

19 cl, 2 tbl, 4 ex

Description

FIELD OF THE INVENTION

The invention relates to medicine, more specifically to the field of pharmaceuticals, and, in particular, to an adjuvant based on fragments of peptidoglycans of the cell wall of gram-negative bacteria. The proposed adjuvant, consisting of three active components, provides activation of the body's immune response in response to the introduction of antigen (s) of viral or bacterial origin. In this regard, the invention relates to the use of the claimed adjuvant in the composition of immunogenic compositions and vaccines for animals and humans.

BACKGROUND

Adjuvants are substances that enhance the antigen-specific humoral and / or cellular response to antigen (s).

The body's response to various antigens goes in two ways: (1) through humoral-mediated immunity, which includes stimulation of B cells and production of antibodies or immunoglobulins, induction of antigen-presenting cells (APC) and helper T cells (Th1 and Th2), and (2) through cell-mediated immunity, which also involves T cells, including cytotoxic T lymphocytes, while other cells, such as Th1, Th2, and APC cells, can be involved in the body’s immune response.

In general, the body’s immune response can be simplified as a body reaction based on the specificity of B and T cells. These cells can acquire a specific reactivity after activation with the corresponding specific antigen and possess a memory mechanism in case of possible contact with this antigen in the future.

Therefore, vaccination against pathogens, aimed at significantly facilitating and accelerating the formation of long-lived T and B memory cells and, as a result, the induction of specific circulating protective antibodies, is an important preventive measure.

Moreover, to protect against pathogens, it is often sufficient to use as an antigen only an element of its structure, which can be recognized by the body as foreign. This approach to creating vaccines consisting of major components, such as (i) a specific antigen and (ii) an adjuvant (excluding excipients and excipients), is recognized as the most promising, and this approach is preferably implemented when creating modern vaccine preparations.

Adjuvants in general are compounds that, when administered with an antigen (or mixed with an antigen, or administered before the antigen is administered) at least enhance the body's immune response to this specific antigen, and providing the immune system with a “danger” signal, bring it into a state "Anxiety." Moreover, adjuvants are used to achieve at least two goals: (i) (stimulate the response of the immune system) to increase the level of a protective immune response and (ii) to reduce the time between infection and the onset of activation of the immune system. In recent years, there has been growing interest, including commercial, in vaccine preparations, which include purified protein or carbohydrate-containing biomolecules, i.e. there is a shift away from vaccine technology for whole microorganisms.

Promising biomolecules, on the basis of which an effective vaccine can be developed, include biomolecules that are localized on the outer surface of pathogens. Such biomolecules having a unique primary structure and architectonics, and specific to a particular pathogen or group of pathogens, should ensure the activation of the body's immune system (including the synthesis of immunoglobulins), which is generally directed against pathogen (s) carrying such biomolecules on their surface. These biomolecules can belong to different classes, but, based on their functional properties when immunity is activated, they are collectively called antigens.

However, when developing vaccines based on purified antigens isolated, for example, from viruses or bacteria, almost always there are difficulties associated with the fact that the immune response in the form of the production of specific protective antibodies to such antigens is extremely low, and often completely absent (i.e., this response is below the sensitivity threshold of control methods). Such antigens are traditionally called "weak antigens" or "low immunogenic antigens."

It is the above antigens, collectively called “weak antigens” or “weak immunogens,” that seem to be promising for the creation of new vaccines, since in this case the body's immune response will be directed exclusively to pathogens that carry such an antigen.

To create vaccines (the traditional term for protective drugs that induce the body's immune response to the pathogen (s)), including a weak immunogen (weak antigen) as an active agent, including antigen in minimized amounts, adjuvants are used, i.e. substances that, when introduced with such an antigen (together or separately) enhance the body's immune response to this antigen.

The first effective adjuvant created was Freund's complete adjuvant (PFA), which contains a water-in-oil emulsion mixed with mycobacterial components. The presence of two types of active ingredients provides this adjuvant with properties of stimulating the immune response and creating a “depot” from which antigens are released for a considerable time, providing the required level of activation of the immune system. This adjuvant is traditionally considered one of the best in laboratory practice in studies on animal models, however, PFA is not applicable in medical practice due to adverse reactions.

Currently, almost the only commercial adjuvant widely used in the manufacture of vaccines is aluminum hydroxide (and some of its derivatives), the use of which provides intensive production of antibodies, almost independent of the type of antigen. However, studies conducted in recent years have shown that the use of adjuvants based on aluminum compounds with which a weak antigen is associated leads to the induction of immunity according to the Th2 type, and as a result, immunity is directed primarily against extracellular pathogens. Given the fact that in the case of viral and many microbial infections, the Th1-type immune response is most effective, there is an obvious need to create new adjuvants or adjuvant systems for weak antigens.

The exact mechanism of action of adjuvants is currently unknown in detail. Research into the development of new adjuvants is partly empirical, and often based on unexpected observations. Each adjuvant described in the prior art has its positive and negative properties.

However, in the case of the provision of adjuvant (s), new, known or commercially available, the selection of the adjuvant-antigen pair and their ratio is, in fact, the trivial task of "enumerating pairs of variants" when the choice is made by evaluating the effectiveness of specific adjuvant combinations -antigen. The problem is to have a pharmaceutically acceptable, functionally active adjuvant for such experiments, since the adjuvant properties of the compound or adjuvant system, as a rule, extend to a large group and a wide type of compounds with antigenic (immunogenic) activity. An example is vaccine immunogenic preparations based on Freund’s adjuvant, for which, in essence, the species, type and class of compound with potential antigenic activity or a cell against which immunity is induced does not play a role.

In recent years, proteosomes, synthetic unmethylated guanidine-cytosine sequences (CpG oligodeoxynucleotides), as well as medications approved for medical use based on monophosphoryl lipid A (AS04), squalene (MF59), saponin (ISCOMATRIX), and others have been used as adjuvants for research purposes. similar, using which the immune response profile has a mixed Th1 / Th2 type. When using such adjuvants, problems often arise due to their multicomponent nature, difficulty in unification, and low stability, which necessitates the development of new highly effective adjuvants.

As promising adjuvants, products of bacterial origin are often studied, such as lipopolysaccharides (LPS) of gram-negative microorganisms, fragments of the cell wall peptidoglycan (PCS) - muramyl peptides, B-subunit of cholera toxoid, etc. However, specific examples of effective adjuvants that are acceptable in medical practice and supplied on medical practice market for these purposes is unknown.

The first mention of the adjuvant activity of PKC fragments (Adam A, Ciorubaru R, Ellous F, Petit JF, Lederer E. Adjuvant activity jf monomeric bacterial cell wall peptidoglycanes. Biochem Biophys Res Commun 1974; 56. 561-6) was associated with an attempt to replace mycobacteria cells in Freud's adjuvant for soluble hydrolysis products of PKC lysozyme from Mycobacteria and E. coli. This observation served as an impetus for further research, but this area of research was not implemented due to the impossibility of using Freud's adjuvant in clinical practice. Thus, this assumption has not been properly developed in the field of creating new adjuvant systems.

In particular, the prior art describes muramyl dipeptide N-acetylmuramyl-L-alanyl-D-glutamic acid (Ellouz AF, Ciorubaru R, Lederer E. Minimal structural requirements for adjuvant activity of bacterial peptidoglycan subunits. Biochem Biophys Res Commun 1974; 59: 1317 -25). This muramyl dipeptide, having a powerful immunostimulating and adjuvant action, was unsuitable for clinical use due to pronounced pyrogenicity.

The experience of the authors of the present invention with LPS and PKC bacteria has revealed a wide group of compounds that are active against the warm-blooded immune system. In general, the types of activities that exhibit the components of the bacterial cell wall are quite diverse - from general toxic effects on the body to stimulation of nonspecific immunity. The type of activity depends, of course, on the particular compound. However, since all potentially active compounds are part of the cell wall polymers or are products that are obtained from them, it is necessary to isolate either a specific compound or a complex of compounds that will have the desired type of activity.

In particular, it was found that preparations of a mixture of PKC fragments of an indeterminate composition, isolated from the cell wall by rough cleaning, showed adjuvant properties when used to immunize mice (Pathological Physiology and Experimental Therapy, 2011, No. 3, pp. 40-42. ) The results obtained were a prerequisite for the search for potentially active substances from the number of fragments of PCS for use as effective adjuvants.

On the other hand, as mentioned above, quite a lot of weak immunogens are currently described, which can be considered as the basis for modern subunit vaccines, but, unfortunately, due to the low molecular weight that is characteristic of synthetic, genetic engineering, many natural peptides are not in themselves effective inducers of the immune response. Their use as vaccines is possible only with an effective adjuvant or adjuvant system. This situation also applies to some bacterial peptides of the bacterial wall, which, due to the structural features of the lipid component of LPS - lipid A, are not effective inducers of the immune response. Similarly, detoxified LPS (d-LPS) and natural low-endotoxic LPS (ne-LPS) are also not pronounced immunogens. All of the above examples of weak immunogens require, if used as inducers of the immune response, the presence of a potent or highly active adjuvant or adjuvant system.

In this regard, it should be noted that the problem of creating an effective adjuvant, i.e. a substance capable of at least enhancing and accelerating the body's immune response to a weak antigen, and provided that this adjuvant is acceptable in medical practice, remains an urgent task requiring new technical solutions.

Therefore, the present invention was the creation of a new adjuvant suitable for use in medical practice, and capable of providing the body's immune response to antigens with weak immunogenicity. The use of such an adjuvant allows the creation of new products, including vaccines, for delivery to the market and their use in medical or veterinary practice.

BACKGROUND OF THE INVENTION

It is an object of the present invention to provide an adjuvant that would, upon administration of an antigen, induce T and B cells in the animal's body with a Th1 / Th2 type immune response profile, while the antigen is a “weak antigen”, i.e. . the antigen does not cause a significant immune response in the body. Moreover, the concept of “does not cause a significant immune response” implies a response that cannot be objectively registered by modern methods and means of immunological diagnostics.

Preliminary studies of the authors of the present invention showed that the peptidoglycan fraction obtained from gram-negative microorganisms has adjuvant properties and can induce the desired immune response of the body. This has been shown using low molecular weight peptides and detoxified lipopolysaccharides.

Specific fragments of PKC, which are part of the general fraction and are responsible for the adjuvant effect, were not previously identified. In addition, the total fraction of PKC fragments, including a large number of compounds with different molecular weights, has toxicity, limiting the use of such a fraction for introduction into a living organism.

In this regard, studies were aimed at searching for specific compounds in the total fraction of bacterial PKC fragments responsible for adjuvant activity. The requirement for the level of toxicity of the compounds was also taken into account, since such adjuvants are intended for use in vaccines for the prevention and treatment of diseases of mammals, including humans, as well as birds and animals.

In addition, the economic aspects of their preparation were also taken into account when choosing the active ingredients of the fraction of PCB fragments.

In particular, products derived from PKC bacteria were investigated. So, for example, from the PCB of the gram-negative microorganism Salmonella typhi, due to partial hydrolysis by lysozyme, a substance was obtained which is a polymer fragment of PCS, where the number of repeating units of GlcNAc-MurNAc ranged from 5 to 20. The method of preparation and some properties of this substance are described in patent RU 2412197.

As a result of further studies and screening tests, it was found that the fraction of products of enzymatic hydrolysis of PKC bacteria, containing mainly a mixture of three active components (muramyl peptides), meets the above requirements for adjuvants. This fraction mainly included active ingredients with the following structure:

compound 1: β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid (GMtri);

compound 2: β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine (GM),

Compound 3: GMetter dimer (diHMtetra), in which the bond between the monomer residues of the GMetter is due to the carboxyl group of the terminal D-alanine of one GMetetra residue and the ω-amino group of the meso-diaminopimelic acid of the other GMetter.

Since the other components of the isolated fraction did not have a pronounced adjuvant effect, a mixture (purity not lower than 95%) of the indicated compounds was isolated from this fraction, and also by the additional separation of this mixture, micro amounts of each of the compounds (1) - (3) were isolated.

This mixture in its composition generally corresponds to the previously studied mixture described in patent RU 2441906, and the method for its preparation was disclosed in application RU 2008144644. However, on the priority date of these documents, only immunostimulating functions were detected in the above mixture of muramyl peptides, in particular - the type of activity of this mixture was defined as non-specific protection against pathogens, i.e., in fact, they act as activators of innate immunity.

Since the purpose of the adjuvant is to activate the antigen of the presenting cells belonging to the innate immunity system, it could be assumed that the mixture (1) - (3) will have adjuvant properties. However, there was no direct experimental confirmation of this at the priority date. In addition, activators of natural immunity, as a rule, suppress the activity of third-party immunogens introduced into the body.

Screening studies of the microquantities of compounds (1) - (3) isolated in an individual state confirmed that each of them has adjuvant activity at approximately the same level. Since the separation of a mixture of muramyl peptides into specific substances is a rather complex and highly costly procedure, a mixture of the three compounds, without separation, was chosen as an adjuvant of the present invention.

This adjuvant, consisting of three active compounds, which are muramileptides, with the structure of the above compounds (1), (2) and (3), provides an increase in the immune response in the body in response to the antigen administered.

Given that each of the compounds (1) to (3) has an adjuvant activity at approximately the same level, their ratio in the mixture is not critical, however, the present invention assumes that all three compounds are necessarily present in the mixture.

In one of the embodiments of the invention, the ratio of the compounds (1), (2) and (3) in the mixture is based on the typical content of muramyl peptides, which are respectively 20-50%, 20-50% and 40-60% by weight of the total adjuvant.

The adjuvant of the invention enhances the body's immune response to an administered antigen. Moreover, the antigen is a weakly immunogenic antigen, or the amount of antigen administered without the indicated adjuvant is insufficient to induce the body's immune response. Thus, the adjuvant of the invention is intended not only to perform an adjuvant function for traditional antigens, but also allows you to use weakly immunogenic antigens with it, or significantly reduce the amount of antigen administered.

The adjuvant of the invention can be used as part of immunogenic (vaccine) compositions intended for administration to mammals, including humans, as well as animals and birds. Such compositions (vaccines) are intended for the treatment or prophylaxis of diseases and conditions caused by a pathogen, the prophylaxis or treatment of which requires activation of immunity against the specified pathogen.

The compositions contain, as active substances, the antigen and adjuvant of the invention. The antigen that is part of the composition is presented in an effective amount, i.e. the amount necessary and sufficient to activate immunity in the subject, which drive this composition. This amount of adjuvant, with a single injection, is usually from 0.1 to 100 μg per kilogram of body weight. In this case, the typical ratio of antigen / adjuvant in the composition is from about 1: 1 to 10: 1 by weight.

The composition according to the invention is intended for subcutaneous administration, intramuscular administration, intradermal administration, intraperitoneal administration, nasal administration, intrapulmonary administration, percutaneous administration, oral administration and mucosal administration. The specific form of administration of the composition is determined based on the properties of the pathogen and / or its antigen used in the composition.

In principle, the list of antigens that can be used in conjunction with the adjuvant of the invention is not limited, since any antigen acts by similar mechanisms of immunity induction.

In this regard, the present invention is also directed to the use of the adjuvant of the invention for the production of vaccines based on antigens that are weak immunogens, or vaccines in which the amount of antigen used, administered without any adjuvant, is insufficient to induce immunity.

The present invention also provides a kit used to treat or prevent diseases and conditions caused by a pathogen, the prevention or treatment of which requires activation of immunity against the pathogen. This kit contains a package in which an effective amount of an adjuvant of the invention and an antigen are placed in an antigen / adjuvant ratio of 1: 1 to 10: 1 by weight. The kit also, as a rule, contains instructions for its use.

In this case, the adjuvant and antigen may optionally be in the kit in the same closure in the form of a mixture (composition).

The above essence of the invention will be presented more clearly in the detailed description, and will also be illustrated by examples and experimental data confirming the effectiveness of the adjuvant and the technical result achieved in terms of the potential expansion of the arsenal of drugs for the treatment and prevention of diseases and conditions caused by various pathogens.

DETAILED DESCRIPTION OF THE INVENTION

As part of a study of the chemical and immunological characteristics of a mixture of three active components, which are soluble fragments of PKC of gram-negative bacteria, it was confirmed that the primary structure of the cell wall peptidoglycan is almost identical for many microorganisms. PCB is a spatially cross-linked heteropolymer built of polysaccharide chains, the repeating unit of which is β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramic acid disaccharide attached to the β- (polysaccharide chain 1-4) bonds, with the carboxyl group of the muramic acid residue having a tetrapeptide residue: N- (L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine), and the adjacent tetrapeptide fragments located in adjacent polysaccharide chains in in turn, due to the peptide bond between the carboxyl group of the terminal D-alanine residue of the tetrapeptide fragment from one polysaccharide chain and the ω-amino group of the meso-diaminopimelic acid residue of the tetrapeptide fragment from the other polysaccharide chain form octapeptide "bridges" between adjacent polysaccharide chains, thus creating a heterogeneous formation.

It is known that during the processing of water-insoluble PCB gram-negative microorganisms with lysozyme, an enzyme that hydrolyzes glycosidic bonds between the residues of muramic acid and glucosamine in polysaccharide chains, a mixture of water-soluble low molecular weight fragments of PCB is formed, which are conventionally called muramyl peptides. Mass spectrometry analysis of all products of the enzymatic cleavage of the cell wall peptidoglycans of many gram-negative microorganisms isolated by high-performance chromatography showed that the products of enzymatic hydrolysis in all cases contain a mixture of murapeptides that are presented in the present invention as compounds (1), (2) and (3 ) The ratio of these compounds in the hydrolysis products depends mainly on the type of gram-negative bacterium from which PKC is isolated, and also, to some extent, on the conditions of cell cultivation.

The total fraction containing a mixture of compounds (1), (2) and (3) was present in all samples isolated from gram-negative microorganisms, and the content in the isolated mixture of compounds (1), (2) and (3) was approximately 20-50, respectively %, 20-50% and 40-60% by weight, based on the total weight of their mixture. The specific values included in this interval varied for different samples obtained from different microorganisms.

The analysis of a large body of literature data showed that a wider variation in the ratio of compounds (1), (2) and (3) in their mixture is possible for different types and strains of gram-negative bacteria. However, based on the general principle of constructing the cell wall of such microorganisms, there is every reason to believe that all of the above compounds (1) - (3) will always be present in the products of enzymatic hydrolysis of PKC gram-negative bacteria.

Isolation of individual substances turned out to be a rather difficult and expensive procedure, which had to be abandoned when it was experimentally established that each of compounds (1) - (3) has adjuvant activity at approximately the same level. This was confirmed by isolating micro amounts of individual compounds and screening for their adjuvant activity using low molecular weight peptides and bacterial polysaccharides.

On this basis, a choice was made in favor of a mixture of compounds (1), (2) and (3), without their separation into individual components.

This choice was also justified by the fact that there is a well-established methodology for isolating such a mixture (see patent application RU RU 2008144644, according to which a patent was issued at the end of 2012), which can be implemented on a semi-industrial scale, and its technology can be created on the basis of industrial scale.

This mixture was previously quite well characterized as a nonspecific immunoactivator, and it was experimentally confirmed that this mixture of compounds does not have a pyrogenic effect, as well as other side effects, when administered to a person in a dose of up to 10 μg / kg / day, and can be administered once at a maximum dose of up to 100 μg / kg (see patent for invention RU 2441906).

Thus, a mixture of substances was obtained that possesses potential adjuvant activity, and which is safe for administration to humans and, therefore, other animals and birds, in which the tolerance to polymuramyls (otherwise called muramyl peptides, muropeptides) is usually significantly higher.

To use the mixture of compounds (1) to (3) as an adjuvant, this mixture can be used as part of a vaccine composition that contains the adjuvant of the invention and an antigen to which it is desirable to induce an immune response of the body.

Considering the high adjuvant effectiveness of the resulting mixture of compounds, substances that have low immunogenicity, i.e. if such substances are introduced into the body without any adjuvant, the immune response is essentially not induced. The latter suggests that objective immunological control methods do not confirm the presence of immunity.

To illustrate the possible uses of the adjuvant of the invention, compositions based on it were created and tested, which included both traditional antigens and antigens with weak immunogenicity, belonging to different classes of natural biopolymers, as well as, in some cases, their modified derivatives . Some of these antigens are already used as components of licensed conjugate vaccines.

As immunogens, an unlimited list of such compounds, including mixtures thereof, can be used.

The general concept of antigen selection for a new highly effective adjuvant is to use known, previously obtained, isolated and studied antigens to induce an immune response that require the presence of adjuvant and on the basis of which it is desirable to create vaccines for the prevention and treatment of common epidemic and non-epidemic diseases . Typical antigens are described in various sources. In particular, the international patent application WO 2012/026508 provides a list of typical antigens of greatest interest for the creation of preventive and therapeutic drugs.

The type of antigen included in the adjuvant composition is not limited, and it may be a peptide, protein, glycoprotein, glycolipid, lipid, carbohydrate, nucleic acid, or a poly- or oligosaccharide-containing polymer; or a virus, bacterial cell, allergenic substance, tissue, or cell including them. Specific examples of the antigen include pollen derived antigens, hepatitis A virus antigens, hepatitis B virus antigens, hepatitis C virus antigens, hepatitis D virus antigens, hepatitis C virus antigens, antigens derived from hepatitis E virus, antigens derived from hepatitis F virus, antigens derived from HIV virus, antigens derived from influenza virus, antigens derived from herpes virus (HSV-1, HSV-2), antigens derived from causative agent of anthrax, antigens s derived from chlamydia, antigens derived from pneumococcus, antigens derived from Japanese encephalitis virus, antigens derived from measles virus, rubella, antigens derived from Clostridium tetani, antigens derived from chickenpox virus, antigens derived from SARS (SARS), antigens derived from Epstein-Barr virus (EB), antigens derived from papilloma virus, antigens derived from Helicobacter pylori, antigens derived from rabies virus, antigens derived from West Nile virus, antigens derived from hantavirus a, antigens derived from Streptococcus, antigens derived from Staphylococcus, antigens derived from Bordetella pertussis, antigens derived from Neisseria meningitidis groups A and C, antigens derived from Haemophilus influenzae, antigens derived from Escherichia coli, antigens derived from Escherichia coli, antigens derived from Salmonella, antigens derived from Shigella, antigens derived from Mycobacterium tuberculosis, antigens derived from Plasmodium, antigens derived from poliovirus, antigens derived from various zoonotic infections, cancer antigens and antigens derived from various food allergens.

For an antigen in the adjuvant composition, there is no requirement to be the only antigen. In light of the application of the present invention, immune responses can be induced against cancer cells, bacteria, viruses or the like, which are composed of many elements. In such cases, the antigen may be a plurality of types of proteins or the like that may elicit immune responses, or may be a mixture of substances whose types cannot be identified. In addition, the inclusion of many types of antigens for the successful induction of immune responses against many types of antigens is one form of use of the immunogenic composition of the present invention. Preferably, more than 3 types of antigens can be encapsulated in the adjuvant microparticle, more preferably one type of antigen.

In the experimental evaluation of the adjuvant according to the invention and vaccines based on it, a mixture of the adjuvant according to the invention and CPS from Neisseria meningitidis of groups A and C, as well as detoxified LPS (d-LPS) from Escherichia coli O111: B4 and d-LPS were used as typical compositions. from Salmonella enterica, typhimurium serovar, ne-LPS from Pseudomonas aeruginosa and ne-LPS from Brucella abortus, a specific protein of influenza virus (hemagglutinin) and hepatitis A.

The level of antibody IgG response after the second immunization with immunogenic compositions containing the adjuvant of the invention was selected as the main criterion for evaluating the results. This criterion is considered universally recognized, since a high level of rapid antibody IgG response and immune memory provide effective protection against pathogens during vaccination.

A general immunization schedule may include both a single immunization and a booster schedule. A typical immunization schedule is presented below.

Vaccination of mice is carried out subcutaneously, with the second immunization followed 14 days after the first. To facilitate comparison of results, all immunizations use the same dose of the adjuvant composition — 50 μg and the same antigen – adjuvant ratio of 1: 9. Immune sera are collected 7 days after the second injection.

It should also be borne in mind that systems based on effective adjuvants provide a faster immune response of the body to a pathogen antigen, compared with the pathological process caused by this pathogen entering the body. This well-known pattern is the basis for the use of adjuvant / antigen compositions in the treatment of diseases and disorders that arise after infection of the body with a pathogen. Those. a composition comprising an antigen and an adjuvant may be administered when the first symptoms of a disease caused by a pathogen appear, or if infection is suspected of being caused by a pathogen that causes the disease.

Most preferred in the framework of this invention are the "weak" immunogens proposed below, which are of undoubted interest from the epidemic point of view in terms of developing vaccine preparations. In the experimental evaluation of the adjuvant according to the invention and vaccines based on it, a mixture of adjuvant and CPS from Neisseria meningitidis of groups A and C, as well as Haemophilus influenzae, type B, were used as typical compositions. These polysaccharides, having a low molecular weight (15-20 kDa) and a linear structure without pronounced structural epitopes, have low immunological activity and are therefore used in commercial vaccine preparations in the form of conjugates with bacterial toxoids.

From the group of detoxified due to alkaline treatment of LPS, d-LPS from Escherichia coli O111: B4 (Sigma) and Salmonella enterica, typhimurium serotype (Sigma), were used as model ones. This choice is due to the fact that, despite the relevance of vaccines for the prevention of Escherichiosis and salmonella, commercial protective drugs based on their main virulence factor - highly endotoxic LPS have not yet been created. d-LPS, in contrast to native bacterial LPS, has a low level of endotoxicity, but detoxification of LPS is accompanied by a sharp decrease in immunogenicity, therefore, their use without adjuvant is impossible.

The choice of vaccine compositions containing native ne-LPS from Pseudomonas aeruginosa (a typical pathogen for nosocomial Pseudomonas infection) and from Brucella abortus (the causative agent of a difficult to diagnose and not always treatable human disease that is transmitted through home and wild animals) was made on the basis that, on the one hand, the corresponding microorganisms cause dangerous diseases, and, on the other hand, the fact that there are no antigenic subunit vaccines for their prevention due to the extremely low immunogenicity of the polysaccharide-containing main somatic antigen - LPS. And this, in turn, is due to the unique structure of lipid A - the endotoxic onset of LPS of these pathogens. The “classical” enterobacteria lipid A contains basically bisphosphorylated at positions 1 and 4′ disaccharide, constructed from two residues of D-glucosamine and acylated with (R) -3-hydroxymyristic acid or (R) -3-acyloxymyristic acid (where acyl is characterized by length chains of 12, 14 or 16 carbon atoms) at positions 2, 3, 2 'and 3'. In contrast, lipid A from Pseudomonas aeruginosa does not carry phosphate substituents and the acylating fatty acid residue is represented by decanoic acid (acyl-C10), and 2,3-diamino-2 was identified in lipid A from Brucella abortus instead of one of the D-glucosamine residues , 3-dideoxy-D-glucopyranose. LPS and other microorganisms with such deviations in the structure of lipid A from the “classical” one are also characterized by extremely low immunogenicity and, in particular, inability to induce specific protective anti-LPS antibodies. Activation of the immune system of this kind of LPS is possible when vaccinated with compositions containing an effective adjuvant.

In addition, the immunological characteristics of adjuvant compositions containing a specific protein of the influenza virus (hemagglutinin) and hepatitis A virus as an immunogen were evaluated. The basis of commercial vaccines against these infectious diseases are conjugates of specific viral proteins with adjuvant proteins, the production of which is of high cost, and their effectiveness is poor. Therefore, the development of adjuvant compositions based on the proposed muramyl peptide adjuvant, the use of which does not require the creation of a chemical bond with a "weak" antigen, is preferable.

Evaluation of effectiveness was carried out by the level of induction of specific antibodies of class G.

Since adjuvants essentially do not have selective activity, to confirm its properties it was enough to prove its activity using examples of typical antigenic compounds belonging to different classes. This evidence-based approach is generally accepted in research practice, in the presentation of candidate adjuvants, and in the patent, in the protection of new adjuvants and adjuvant systems. The data and results of experimental evaluations are given in the Examples section.

Based on the data obtained, it was concluded that the adjuvant according to the invention has all the claimed properties and functions, namely, it is able to provide activation of the body's immune response for weakly immunogenic compounds of the composition.

Based on typical antigen / adjuvant ratios ranging from 1: 1 to 10: 1, selective experiments with the adjuvant of the invention were carried out. The obtained data confirmed that within the specified interval the adjuvant activity of the mixture of compounds (1) - (3) is preserved, but it was found that there are optima for the quantitative ratio of antigen / adjuvant for a particular antigen. The selection of the ratio of antigen / adjuvant for each specific formulation of the composition is a trivial task, and the optimal ratio can be easily selected by a person skilled in the art by routine experimentation.

The immunogenic composition contains an effective amount of antigen. The term "effective amount of antigen" refers to the amount of antigen that, when administered to a subject in combination with an adjuvant, causes the induction of a specific immune response to this antigen. The subject may be a mammal, including a human, animal or bird. This statement is based on the principle of generality of the mechanism of induction of the immune response.

The present invention also provides an immunogenic (vaccine) composition that contains an effective amount of an antigen as well as an adjuvant of the invention.

Since the adjuvant of the invention has extremely high activity in terms of inducing immunity, the weakest immunogenic antigen is the most preferred antigen used in conjunction with said adjuvant, i.e. such that, when administered without the antigen of the invention, a substantially immune response of the organism, i.e. it does not cause a sufficiently high response or does not cause any response recorded by objective methods of immunological control. In another embodiment, the amount of antigen may be that which is insufficient to induce an immune response of the body, with the introduction of only one antigen.

Moreover, as indicated above, bacterial KPS, d-LPS, ne-LPS or peptide can be used as antigen, each of which, in the most preferred embodiment, does not cause an immune response due to its chemical nature, being a weak immunogen .

In particular embodiments, the bacterial polysaccharide is a capsular polysaccharide from microorganisms of the genus Salmonella, Ecsherichia, Shigella, Neisseria meningitidis and Haemophilus enfluenzae or Streptococcus.

In particular embodiments, LPS is LPS isolated from the microorganisms Pseudomonas aeruginosa, Yersinia pestis, Helicobacter pylori, and Brucella. LPS can also be d-LPS derived from the highly endotoxic LPS of the microorganisms Salmonella and Ecsherichia coli, Shigella, Klebsiella, Neisseria, Bordetella or Vibriocholerae.

The peptide used in conjunction with the claimed adjuvant may be peptides of the HIV virus, herpes virus, specific proteins of the influenza virus (hemagglutinin) and hepatitis virus.

An immunogenic composition according to the invention may optionally contain an auxiliary substance or substances providing the utilitarian properties of the composition, such as storage, stability, ease of use, ease of administration to a patient, reduction of side effects, and the like. Such substances include traditional auxiliary ingredients known from the prior art, which are a surfactant, a buffer, amino acids, a preservative, an antioxidant, a stabilizer, means of delivery of the composition (for example, propellants for intranasal use, penetrants for percutaneous use, an oil-wax base for suppositories, etc.), anesthetic, antibiotic, etc., as well as combinations thereof.

The composition for can be presented both in dry (sublimated) and liquid (ready for use) form.

Methods of sublimation of both the ingredients and their mixtures are known in the art.

The ability to obtain a liquid composition follows from the ability of the adjuvant of the invention to maintain its properties in solution during prolonged storage. In particular, the mixture of compounds (1) to (3) can be stored in the form of a solution for 6 months without changing its properties (see patent RU 2441906). As a rule, antigens in the form of dilute solutions are also stored for a long time. Given the fact that the antigen and adjuvant are inert compounds, chemical interaction between them is not expected. This makes it possible to obtain, store and use the finished liquid vaccine compositions containing the adjuvant of the invention.

Both liquid and dry compositions may contain additional ingredients that do not affect the immunogenic properties of the composition of the invention, but improve its other, additional properties. For example, adjuvants known in the art from the prior art that are conventionally used in vaccine compositions can be added. These can be substances that improve the solubility and stability of the composition (surfactant, buffer, amino acids, preservative, antioxidant, stabilizer, etc.), substances useful in the use of the composition, such as antibiotics, anesthetics, means of improving the delivery of the composition for example, penetrants, in the case of transdermal administration, etc.

An immunogenic composition may be administered to a subject in various ways. An adjuvant is a mixture of muramyl peptides, which, in addition to the traditional parenteral, intraperitoneal, subcutaneous, intradermal administration, can be administered by other methods, such as oral, oral, buccal, sublingual, intranasal, pulmonary, rectal or vaginal routes. The listed additional methods are based on the property of muramylpetids to penetrate through the epithelial barriers into the bloodstream (Andronova TM, Pinegin BV Likopid (GMDP) - a modern domestic highly effective immunomodulator. Russia, 2005). This determines the possibility of release in the form of pharmaceutical compositions for transmucosal and transdermal use. Thus, the vaccine composition according to the invention, in addition to traditional injectable forms, can be produced in the form of compositions for oral and oral administration, in particular in the form of tablets, capsules, oral solutions, troches and buccal forms, as well as in the form of suppositories for rectal and vaginal use. For pulmonary and intranasal routes of administration, the composition may be used in the form of an aerosol or spray.

The route of administration of the vaccine composition of the invention is determined solely by the properties of the antigen contained in the composition. Pharmaceutical forms and methods for their preparation using traditional techniques and auxiliary ingredients are known in the art. Specialist in this field, guided by general knowledge in the field of pharmacy, and taking into account the disclosure of the present invention, as well as the pharmacokinetic properties of the specific antigen used, are able to prepare pharmaceutical forms suitable for the above routes of administration.

The active ingredients that make up the composition, or a mixture thereof, may be in a dry state, in particular, freeze-dried. In this case, to obtain a composition, the starting components are reduced in an acceptable solvent, for example, physiological saline or other liquid, taking into account the route of administration to the patient.

As an embodiment of the invention, since at least one active agent of the vaccine composition can be stored in the form of a solution, one of the components can be presented in dry form and the other in the form of a solution, and both forms are mixed before use. Those. one ready-made solution is reducing for the other component in the kit in a dry, for example, freeze-dried, form.

Finished forms of pharmaceutical preparations, compositions or individual ingredients for obtaining on-site pharmaceutical form, ready for administration to a patient, are packaged in ampoules, vials, boxes, bags, blisters, tubes, etc. depending on the traditions and needs of the market.

The package should also contain instructions regarding the name of the drug, its labeling and, if necessary, instructions for its use. Various packaging options are known in the art and do not require further description.

Vaccination with the antigen / adjuvant composition can be carried out in advance, before potential contact with the pathogen (prophylactic use), or after contact with the pathogen, at a time when the clinical manifestations of the pathology have not yet fully manifested (i.e., use in treatment). Vaccination regimes and dosages, as a rule, are determined by the attending physician on the basis of their general instructions for the use of the drug, clinical manifestations of the pathology, the patient’s condition, etc. As a rule, the prophylactic use of vaccines includes a single vaccination, which can be supplemented by booster immunization, which is traditionally used in case of weakly immunogenic antigens. Control of the level of immunity is carried out using conventional forms of immune control. The choice of control methods is not fundamental. The only requirement for it is that this method should be an objective and evidence-based method for monitoring the patient's condition.

EXAMPLES

The following examples, given in order to detail and illustrate the invention, are not intended to limit the claims of the present invention, and demonstrate the industrial applicability of the present invention, as well as the effectiveness of the proposed adjuvant.

Example 1

Obtaining the adjuvant of the invention.

The adjuvant of the invention was obtained by the method described in the application RU 2008144644, without its fundamental modifications.

PCS isolation

20 g of S. typhi dried acetone and ether (or, respectively, 120-180 g wet) bacterial cells were suspended with vigorous stirring in 350 ml of distilled water. The resulting suspension was heated to a temperature of 68-70 ° C and 350 ml of 90% aqueous phenol heated to the same temperature was added. The mixture was stirred for 20-30 minutes at a temperature of 65-68 ° C, and then cooled to a temperature of 10-12 ° C and centrifuged for 40 minutes at 5000 rpm. The precipitate after decantation of the aqueous and phenolic layers was re-extracted according to the same scheme. The precipitate obtained by extraction was washed with 0.2 M ammonium bicarbonate buffer until absorbance at 260-280 nm in the UV spectrum of the supernatant (the region of specific absorption of nucleic acids and proteins), and then 2-3 times with water.

The output of the washed and lyophilized dried pellet, which is a crude PCS, amounted to 20-40% by weight of dry cells. Amino acid analysis showed that the resulting product also contains a significant amount of bound protein, which must be removed from the drug.

Enzymatic hydrolysis of PKC with lysozyme

5 g of PCB was suspended in 100 ml of 0.2 M triethylammonium acetate buffer pH 7.2. To a suspension stirred at a temperature (hereinafter) of 10 ° C, 300 mg of lysozyme was added and after an hour of stirring, the reaction mixture was transferred to a dialysis tube, which was placed in a vessel with 300 ml of the above buffer. Dialysis was carried out for 3 days, periodically (1-3 times a day) removing the dialysate and adding a fresh buffer solution to the external vessel. The resulting combined dialysates were evaporated in vacuo to a volume of 200 ml at a temperature of not more than 40 ° C, the residue was lyophilized, and then dissolved in 200 ml of deionized water, and then re-lyophilized. The yield of the target mixture was 320 mg.

Isolation and purification of target compounds and their mixtures

Preparative isolation of the mixture of substances was carried out using gel chromatography on a column of Sephadex G-50 in 0.05 M ammonium bicarbonate buffer pH 8.3. A mixture of substances elutes in the form of two peaks close in terms of area, with Kd 0.42 and 0.53, with a shorter time eluting a diHMetter, and with a longer time a mixture of GMtri and GMtetra. The combined fractions were lyophilized and then re-lyophilized from deionized water. Alternatively, the combined fractions may be desalted on a Sephadex G-10 column (eluent is deionized water).

From the data of mass spectrometry (MALDI-TOF), it follows that the resulting mixture of substances contains three components - GMtri, GMtetra and diHMtetra with molecular masses (calculated values in brackets) 868.4 (868.8), 939.4 (939.8 ) and 1860.8 (1861.7), respectively.

An analysis of the 13C-NMR spectrum confirmed the absence of significant amounts of impurities.

Example 2

Assessment of potential adjuvant toxicity

Testing the resulting preparation in rabbits showed that the resulting preparation at a dose of 0.1 μg / ml does not cause a pyrogenic effect.

When testing the resulting preparation in the LAL test, it was found that the content of bacterial endotoxins in the drug solution with a concentration of 100 μg / ml does not exceed 1.5 EU / ml.

A single intramuscular and intraperitoneal administration of the drug to BALB / C mice and Wistar rats in the dose range of 1-500 mg / kg does not cause any signs of intoxication, and does not lead to the death of animals or to their abnormal behavior or pathological symptoms.

The highest dose tested in mice and rats at a dose of 500 mg / kg is more than 300,000 times higher than the highest therapeutic dose (1.5 μg / kg) taken for humans.

Since toxicity tests were carried out according to the generally accepted methodology and recognized systems for evaluating and extrapolating the results, the resulting adjuvant was found to be safe for administration to humans or other mammals, as well as birds.

Example 3

In accordance with International requirements (Guideline on Adjuvants in vaccines), the adjuvant effect of the proposed adjuvant has been studied. For this, 5 μg of ovalbumin (Sigma) in a volume of 0.25 ml of 0.9% NaCl is added to 50 μg of adjuvant in a plastic ampoule. In accordance with the procedure, a finely divided, slowly settling suspension is prepared, which is immunized subcutaneously with 6 mice of the line (CBAxC57BL) F1 in a volume of 0.25 ml. 6 control mice (group 1) were injected subcutaneously with 50 μg of ovalbumin. Control mice of the 2nd group are injected with 0.9% NaCl solution. On the 12th day, serum is obtained in which the titers of IgG antibodies to ovalbumin are determined by enzyme-linked immunosorbent assay.

Table 1
The adjuvant effect of the drug with a single administration
simultaneously with ovalbumin at a dose of 5 mcg / mouse
(optical density, serum dilution 1:50) Ovalbumin +
adjuvant Ovalbumin
1st group Saline
2nd group one 0.688 0.119 0,084 2 0.648 0.380 0,060 3 0.443 0.226 0,064 four 0.242 0.122 0.058 5 0.582 0.338 0,090 6 0.582 0.106 Average 0.531 0.215 0,071 Standard deviation 0.164 0,120 0.015 ** - p <0.005 (significance of differences between groups 1 and 2)

As can be seen from the table, the administration of ovalbumin to mice causes the development of a humoral immune response, which the adjuvant preparation reliably enhances by more than 2 times. In accordance with the Guideline on adjuvants in vaccines for human use, prepared by the European Medical Agency, an adjuvant is considered to be a substance that gives a statistically significant increase in antibody titer 2 times or more compared to control.

In this case, an adjuvant effect is observed in relation to ovalbumin, which is traditionally considered a non-immunogenic substance.

These data prove the presence of an adjuvant effect against "weak" or non-immunogenic compounds.

Example 4

The ability to induce specific IgG antibodies during immunization with “weak” polysaccharide and protein antigens was also studied. To do this, 50 μg of the “weak” antigen and 450 μg of the adjuvant were dissolved in 5 ml of phosphate-buffered saline (PBS), and 0.5 ml of the resulting solution was used to immunize mice (each group of 10 animals). The comparison group consisted of animals to which the antigen was administered in Freund's complete adjuvant (PAF) and to which pure FSB was administered in the same doses. Animals were immunized twice, with an interval of two weeks, subcutaneously. On the 7th day after the second immunization, serum was obtained. Antibody titers were determined using enzyme immunoassay.

table 2
The adjuvant effect of immunizing mice with “weak” antigens in the presence of the adjuvant of the invention. The results are presented in the form of a dilution of pooled sera Bacterial or viral
antigen Breeding Serum Antigen Antigen + Adjuvant Antigen + PAF Capsule Polysaccharide Neisseria meningitides Group A twenty 80 40 Capsule polysaccharide Neisseria meningitidis, group C 10 80 10 Haemophilus enfluenzae capsule polysaccharide type B. 0 40 10 Degraded LPS
Escherichia coli O111: B4 twenty 160 40 Degraded LPS
Salmonella enterica, typhimurium serotype twenty 160 40 Low Edotoxic LPS Pseudomonas aeruginosa 0 40 0 Low endotoxic LPS of Brucella abortus 0 80 10

Influenza virus hemagglutinin 40 160 80 Hepatitis A virus specific protein 40 320 160

As can be seen from the table, the use of adjuvant allows you to strengthen the humoral immune response to "weak" antigens of various origin. In addition, the adjuvant has a much more pronounced immunogenic effect compared to Freund's complete adjuvant (PAF).

The animal model used confirmed the high activity of the adjuvant of the invention. Considering the previously discovered ability of the adjuvant of the invention for use in various routes of administration to the body, these data can be extrapolated to all forms of administration of vaccine compositions indicated in the description.

Additional studies on a limited sample of model animals and some immunogens showed that the adjuvant effect occurs with a wide variation in the antigen / adjuvant ratio. In particular, test intervals ranging from 1: 1 to 10: 1 (based on the mass of antigen and adjuvant) confirmed that the adjuvant effect occurs within the indicated ratios (data not shown). Perhaps the adjuvant effect will take place at other ratios than the above, however, the indicated range of ratios is generally accepted in the design of vaccine preparations.

It is obvious that a mixture of antigens can be used within the scope of the invention. In particular, such an embodiment of the invention can be used to produce polyvalent vaccine formulations.

The above experimental data and results clearly show new and unexpected properties of the mixture of compounds (1) - (3) as an adjuvant.

The use of this mixture expands the arsenal of drugs used in the prevention and treatment of diseases caused by pathogens of various nature.

Claims (19)

1. Adjuvant to enhance the immune response in the body to an antigen, which is a mixture of three active compounds, which are compounds with the following structure:
compound 1: β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid (GMtri);
Compound 2: β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine (GM), and
compound 3: GMetter dimer (diHMetter), in which the bond between the monomer residues of the GMetter is due to the carboxyl group of the terminal D-alanine of one GMetetra residue and the ω-amino group of the mesodiaminopimelic acid of the other GMetter,
where compounds (1) to (3) are contained in an amount of 20-50%, 20-50% and 40-60% by weight of the total adjuvant, respectively. 2. The adjuvant of claim 1, wherein the antigen is a weakly immunogenic antigen that does not elicit an immune response when administered without said adjuvant, or a mixture of antigens. 3. The use of the adjuvant according to claim 1 for the preparation of an immunogenic composition containing said adjuvant and antigen or mixture of antigens, to induce an immune response in the body to said antigen or antigens, wherein the composition is suitable for administration to a mammal, including a human, and a bird. 4. The use of claim 3, wherein the antigen is a weakly immunogenic antigen that does not elicit an immune response when administered without said adjuvant, or a mixture of such antigens. 5. The use according to claim 3, wherein the composition is in a form suitable for subcutaneous injection, intramuscular injection, intradermal injection, intraperitoneal administration, nasal administration, percutaneous administration or oral administration. 6. An immunogenic composition for the treatment or prophylaxis of diseases and conditions caused by a pathogen, the prophylaxis or treatment of which requires the induction of immunity against the specified pathogen, containing an effective amount of antigen or antigens of the specified pathogen or pathogens and adjuvant according to claim 1 in a ratio of 1: 1 to 10 : 1 by weight. 7. The immunogenic composition according to claim 6, wherein the antigen is a weakly immunogenic antigen that does not elicit an immune response when administered without said adjuvant, or a mixture of antigens. 8. The immunogenic composition according to claim 6, which optionally contains an excipient selected from the group consisting of a surfactant, a buffer, amino acids, a preservative, an antioxidant, a stabilizer, a delivery vehicle, an anesthetic, an antibiotic, and a combination thereof. 9. The immunogenic composition according to any one of paragraphs. 6-8, in which the antigen is a bacterial polysaccharide. 10. The immunogenic composition of claim 9, wherein the bacterial polysaccharide is a capsular polysaccharide from microorganisms of the genus Salmonella, Ecsherichia, Shigella, Neisseria meningitides, Haemophilus enfluenzae or Streptococcus, or a mixture of polysaccharides. 11. The immunogenic composition according to any one of paragraphs. 6-8, in which the antigen is a lipopolysaccharide. 12. The immunogenic composition of claim 11, wherein the lipopolysaccharide is a lipopolysaccharide from the microorganisms Pseudomonas aeruginosa, Yersinia pestis, Helicobacter pylori, Brucella, or a mixture of lipopolysaccharides. 13. The immunogenic composition according to any one of paragraphs. 6-8, in which the antigen is a detoxified lipopolysaccharide derived from the highly endotoxic lipopolysaccharide of the microorganisms Salmonella, Ecsherichia coli, Shigella, Klebsiella, Neisseria, Bordetella or Vibrio cholerae, or a mixture of detoxified lipopolysaccharides. 14. The immunogenic composition according to any one of paragraphs. 6-8, in which the antigen is a peptide. 15. The immunogenic composition of claim 14, wherein the peptide is a peptide from an HIV virus, herpes virus, a specific protein of an influenza virus such as neuraminidase, and a peptide from hepatitis A, B, or C virus, or a mixture thereof. 16. The immunogenic composition according to any one of paragraphs. 6-8, in which the antigen is a mixture of antigens selected from the group consisting of the compounds listed in paragraphs. 9-15, in any combination. 17. A kit used to treat or prevent diseases and conditions caused by a pathogen, the prevention or treatment of which requires the induction of immunity against the specified pathogen, containing:
(1) the packaging in which
(2) an effective amount of an adjuvant according to claim 1;
(3) an effective amount of an antigen or mixture of antigens isolated from said pathogen in an antigen / adjuvant ratio of from 1: 1 to 10: 1 by weight; and
(4) optionally, instructions for using the kit. 18. The kit of claim 17, wherein the adjuvant and antigen or antigens are in the form of a mixture in one closure. 19. The kit of claim 17 or 18, wherein the adjuvant and / or antigen (s) are in a dry state, or the adjuvant and / or antigen (s) are in the form of a solution.