External cow lipopolysaccharide epitope h. pylori

FIELD: chemistry.

SUBSTANCE: present invention relates to biotechnology and provides a α1,6-glucan-containing compound of Helicobacter pylori. The present invention also discloses a conjugate for inducing immune response against H.pylori, which contains said compound conjugated with a carrier protein. The present invention also discloses an immunogenic composition, use of said composition and a method of inducing immune response against H.pylori using said composition. The present invention also discloses immune serum for neutralising H.pylori in mammals, which is obtained by immunising said mammal with an immunogenic composition containing said immunogenic composition. The present invention discloses an antibody which recognises said α1,6-glucan-containing compound of H.pylori, use of said antibody and a method of inducing complement-mediated bacteriolysis of H.pylori strains which express α1,6-glucan using said antibody.

EFFECT: invention improves the effectiveness of immunogenic compositions against Hpylori.

27 cl, 8 dwg, 21 tbl, 11 ex

 

INFORMATION ABOUT the PREVIOUS REQUEST

According to this application claims priority of the provisional application for U.S. patent 61/230315, filed July 31, 2009, which is incorporated into this description by reference in full.

The technical field TO WHICH the INVENTION RELATES

The present invention relates to a new external korouoma the LPS epitopeHelicobacter pylori. More specifically, the present invention relates to a new external korovou epitope ofH. pylorito its synthesis, and conjugation.

The prior art INVENTIONS

Helicobacter pyloriit is considered the most common bacterial infection associated with chronic gastritis, peptic ulcer and carcinoma of the stomach in humans. The prevalence ofH. pyloriis estimated as approximately half the population of the entire world, reaching up to 70% in developing countries and 20-30% in industrialized countries (Dunn et al., 1997). The vast majority of individuals becomesH. pyloriin childhood, the prevalence of infection among children in developing countries is associated with low socioeconomic status and poor sanitary conditions (Castillo-Rojas et al., 2004).H. pyloriis defined by the world Health Organization (WHO) as a class I carcinogen because it increases the relative risk of stomach cancer at minicamera six times. Stomach cancer is the second most frequent cause of death worldwide and accounts for 700,000 deaths each year (Parkin et al., 2002).

Modern strategy of destructionH. pyloribased on the use of proton pump inhibitors and antibiotics. However, the effectiveness of chemotherapeutic limited impact of antibiotic resistance that is often found among isolates ofH. pyloriand failure of the immune response during re-infection. Thus, the need for new types of therapeutic agents, which provides the global strategy for the prevention and elimination of infectionsH. pylori.

Despite the fact that in the most recent studies focused on the role of protein components in the pathogenesis ofH. pyloriand their role in protective immunity (Ruggiero et al., 2003; Rossi et al., 2004), relatively few studies have been conducted to assess the possibility of including antigens that are different from the proteins in the vaccine formulations (Angelakopoulos and Hohmann, 2000). For example, it is known that conjugate vaccines based on polysaccharides prevent the development of systemic infection and inhibit the colonization of the host (Anderson et al., 1986; Chu et al., 1991; Pon et al., 1997; Passwell et al., 2001; Passwell et al., 2003). In recent studies of intestinal pathogens were studied approaches based on the LPS conjugates as vaccine candidates (Gu et al., 1996 Mieszala et al., 2003; Cox et al., 2005; Yu and Gu, 2007).

Lipopolysaccharide (LPS) is a major component of the cell surface ofH. pylori. Studies of the structure, carried out in a number of isolates ofH. pylori(Monteiro, 2001), has led to the creation of the structural model of LPS consisting of O-chain and crustal oligosaccharide that is attached to the molecule of lipid A. the structure of the polysaccharide backbone of the O-chain of most strains ofH. pyloriisunique and reflects the determinants of blood group Lewis (Le) type 2 and/or type 1, which simulate the determinants that are present on the surface of stomach cells and human tumor cells (Wirth et al., 1996); these determinants may be involved in unwanted autoimmune reactions leading to atrophic gastritis (Appelmelk et al., 1996). In addition, external boundary Kora LPSH. pyloricontains two unusual polymer component: DD-heptahelical and α1,6-glucan (Monteiro, 2001). Polymer α1,6-glucan external crustal plot LPS isolates ofH. pyloriis synthesized by the gene product HP0159 with an open reading frame. The presence and expression of the gene HP0159 inH. pyloriare normal.

Describes several biosynthetic genes LPSH. pyloriand defines their role in the pathogenesis and colonization (Logan et al., 2000; Logan et al., 2005; Hiratsuka et al., 2005; Chandan et al., 2007; Altman et al., 2008). Were created mutants HP0826H Pyloriand it was shown that this mutation Pref�leads to the formation of truncated LPS, deprived of antigen Le (Logan et al., 2000). However, was not carried out a complete characterization of the structure of LPS.

Despite advances in this area, immunogenic epitopes, are effective in many types ofH. Pyloriremain unclear.

Summary of the INVENTION

The present invention relates to a novel epitope of outer area of the cortex LPSHelicobacter pylori. In more detail, the present invention relates to a novel epitope of outer area of the cortex LPSH. pylorihis synthesis, description, and conjugation.

The present invention relates to α1,6-glucan-containing compoundHelicobacter pylori,containing the structure of formula I:

R represents trisaharid α-DDHep-3-α-L-Fuc-3-β-GlcNAc substituted α1,6-glucan associated with α1,3-DD-heptane, and the last remnant of DD-Hep α1,3-DD-heptane Kairouan the remnant β-GlcNAc. In the described connection of α1,6-glucan may contain from about 3 to 12 α1,6-linked glucose residues, and α1,3-DD-heptane may contain from about 2 to 6 α1,3-linked residues, heptose.

The group R of the compounds described above, can be a

where is the rest of L β-linked GlcNAc to O-2 of Hep G. In connection, which is described above, the residues Q and Z glucans represent α1,6-linked glucose residues, and n can have any value from 1 to 11; residues T, Y and X represent the FDS�nd α1,3-linked residues of heptose, and m can have any value from about 0 to 4.

The compound described above may be isolated or purified from strain HP0826::KanH. pylori.

In the compounds described above, the structure of formula I may also contain a molecule of A lipid, covalently associated with the Kdo residue C. the Molecule of lipid A can be O-diallylamine or can be split off by hydrolysis ketose communication from the Kdo residue.

The present invention also relates to a conjugate containing essentially linear α1,6-glucan-containing compound, anywhereman with the linker molecule, the carrier protein or a combination of both. Essentially linear α1,6-glucan-containing compound may be a compound described in the present application, characterized in that the structure of formula I anywhereman with the linker molecule, the carrier protein or a combination of both. Essentially linear α1,6-glucan-containing compound may alternatively be a Dextran, such as Dextran T5. Protein carrier can be a tetanus toxoid or bovine serum albumin.

The present invention also includes a composition comprising one or more compounds or conjugates described above.

The present invention further relates to an antibody directed against compounds contained�Amigo α1,6-glucan epitope, described in the present description. The antibody may be a monoclonal antibody 1C4F9. The invention furthermore relates to hybrid cell lines 1C4F9, which produces a monoclonal antibody 1C4F9.

A monoclonal antibody, described above, can be used for the induction of complement-mediated bacteriolysis α1,6-glucan-expressing strains ofH. pyloriin the individual, if necessary.

The present invention also relates to the application of an effective amount of the composition described above, for inducing an immune response againstH. pyloriin the individual. The compound(I) in the composition may be anywhereman with a suitable carrier protein; in addition, compound(I) in the composition may be anywhereman with a suitable carrier protein via 2-keto-3-deoxy-octulosonic acid (Kdo) of lipopolysaccharide.

The present invention furthermore relates to the immune antiserum obtained by immunization of a mammal the immunogenic composition described in the present application. Immune antiserum may contain IgG that recognizes α1,6-linked glucan epitope in homologous and heterologous interviewee and failed to be typed strains ofH. pylorimutant and wild type. IgG can induce complement-mediated bacterials strains ofH. pylorimutant and wild-type,expressing the α1,6-�Lucan.

Additional aspects and advantages of the present invention will be apparent from the following description. Detailed description and examples, in which are shown preferred embodiments of the invention, shown only as an illustration, since various changes and modifications within the invention will be obvious to specialists in this field in light of the description of the present invention.

BRIEF description of the DRAWINGS

These and other features of the invention will be described in the present application in the form of an example with reference to the accompanying drawings, where:

FIGURE 1 shows the analysis of CE-MS LPS glycoforms of fraction 1 delipidation LPS of strain 26695 HP0826::KanH. pyloriin the regime of positive ions. Figure 1A shows the selected mass spectrum (m/z 1000-1500), while figure 1 B shows the spectrum of the product ions of the ions at m/z 1266,3.

FIGURE 2 shows the analysis of CE-MS glycoforms of LPS O-decelerating LPS of strain 26695 HP0826::KanH. pyloriin the regime of negative ions. Figure 2A shows the selected mass spectrum (m/z 600-2000), whereas figure 2B shows the spectrum of the product ions of the ions at m/z 1597,7. Charged fragments corresponding to the group of lipid A that are marked with an asterisk.

FIGURE 3A shows the structure of a main glycoform LPS of strain 26695 HP0826::KanH. pylori; acylation is not shown. Products�you KOH-diallylamine LPS figure 3A is shown in figure 3B (compounds 1-4). The products of deamination of compound 4 is shown in figure 3C (compounds 5 and 6). The products of periodate oxidation of compound 6 is shown in figure 3D (compounds 7 and 8). Structures previously proposed in this area for LPSH. pyloristrain 26695 (figure 3E) and LPSH. pylorimutant strain 26695 HP0826::Kan (figure 3F), are also shown (adapted from Logan et al., 2000). PEtn = phosphoethanolamine; Glc = D-glucopyranose; Gal = D-galactopyranose; Kdo = 2-keto-3-deoxy-octulosonic acid; LDHep = L-glycero-D-mannoheptose; DDHep = D-glycero-D-mannoheptose; GlcNAc - 2-atsetamido-2-deoxy-D-glucose; GlcN=2-amino-2-deoxy-D-glucose; Fuc=L-fucose; P=phosphate; and Gro=glycerin.

FIGURE 4 shows a reaction scheme of obtaining conjugates on the basis of the LPS of the present invention.

FIGURE 5 shows the analysis of CE-MS basic fraction delipidation LPSH. pyloriO:3HP0826::Kan in the regime of negative ions. ●: glycoform LPS containing one Hep residue in the side chain, R=Hex5-13Hep, HexNAc, Fuc; ■: glycoform LPS containing two Hep residue in the side chain, R=Hex8-13Hep, Hep, HexNAc, Fuc.

FIGURE 6 shows the graphs of determining the specificity of rabbit antibodies induced by conjugate orastream-TT, using inhibition ELISA using LPSH. pylori26695 HP0479::Kan (figure 6A) and 26695 HP0826::Kan (figure 6B). The application of LPS is depicted as follows: filled squares - 26695; shaded diamonds - 26695 HP0826::Kan; shaded to�ug - 26695 HP0159::Kan; filled triangles - 26695 HP0479::Kan; empty circles - SS1; empty squares - SS1 HP0826::Kan; unfilled triangles - SS1 HP0159::Kan; empty inverted triangles - SS1 HP0479::Kan.

FIGURE 7 presents graphs illustrating the formation ofH. pylori-specific antibodies in mice CD-1. Mice were vaccinated four times with weekly intervals 25 μg/mouse conjugate orastream-TT with added adjuvants 1 μg/mouse of cholera toxin (shaded bars), the acellular sonicate PJ2 added as an adjuvant of 1 μg/mouse of cholera toxin (hatched bars) or saline (open bars). Four weeks after the first immunization, collected samples of serum, feces and vaginal swabs and tested forH. pylori-specific IgG and IgA. Readings of individual mice plotted with a horizontal line indicating the average value in the group (n=5/group).*p<0.05, and**p<0.01 according to the one-sided Mann-Whitney test.

FIGURE 8 is a bar graph depicting loadH. pyloriin the stomachs of mice CD-1. Mice were vaccinated four times with weekly intervals 25 μg/mouse conjugate orastream-TT with added adjuvants 1 μg/mouse of cholera toxin (hatched bars), the acellular sonicate PJ2 added�th adjuvants 1 μg/mouse of cholera toxin (hatched bars) or saline (open bars). Five weeks after the first immunization, the mice were administered orally through the probe three times a day for -108SOMEH. pyloristrain PJ2. After four weeks the mice were sacrificed and counted the number of live bacteria in the stomachs. The columns represent groups of 4-5 mice ±SEM. *p<0,05, one-sided Mann-Whitney test.

DESCRIPTION of PREFERRED embodiments

The present invention relates to a novel epitope of outer area of the cortex LPSHelicobacter pylori. In more detail, the present invention relates to a novel epitope of outer area of the cortex LPSH. pylorihis synthesis, and conjugation.

Unless otherwise indicated, all technical and scientific terms used in this application have the meaning understood by the person skilled in the art to which the invention relates. Although any methods and materials similar or equivalent described in the present application methods and materials that can be used in practice or testing of the present invention, the following describes the preferred methods and materials. All mentioned in the present application publications incorporated by reference.

In this context, a “purified” does not necessarily mean absolute purity, but rather is intended as a relative definition. Similarly, in this context, “selected” relative�designed to remove something from its natural environment.

Helicobacter pyloriis a bacterial pathogen associated with human chronic gastritis, peptic ulcer and gastric cancer; the result is that the risk of gastric cancer increases with the presence of infectionH. pylorithis pathogen was classified as a carcinogen of class I. Lipopolysaccharide (LPS) is a major component of the cell surface ofH. pylori. Existing to date publications related to the studies of the structure of LPSH Pyloriled to the creation of a model in which the polysaccharide O-chain covalently linked to the oligosaccharide bark, which, in turn, attached to a molecule of lipid A. the Polysaccharide skeleton of the O-chain of most strains ofH. pyloriis unique and can be determinants of blood group Lewis (Le) type 2 and/or type 1; specified polysaccharide component is antigenic. "Interviewee's" strains ofH. pylorihave the Lewis epitopes (antigens Le X and/or Le (Y), which can be recognized by the antibodies anti-Lewis (anti-Le); such antibodies are commercially available and which helps in typing. "Failed to be typed" strains do not contain structures Lewis.

Other studies of the structure showed that the external plot Cora LPSH. pyloricontains two unusual polymer component: DD-heptahelical and α1,6-glucan side chains (Monteiro, 2001). Logan et al. (2000) also gave a presentation about �the LPS structure. In particular, the proposed structure (see Fig. 3E and 3F) provided that DD-heptose (DD-Hep) the side chain attached to the DD-Hep main chain, whereas the α1,6-glucan is attached to the specified DD-Hep side chain and forms another branch. In particular, Logan et al. (2000) determined the length of glycanova chain LPS mutant 0826, which ranges from one to three molecules of glucose, based on the spectra, FAB-MS (mass spectrometry with the bombardment of accelerated atoms). The presence of α1,6-linked glucose in LPSstrain 26695H. pyloriit was also described based on data analysis of methylation, but the length of the glucan was not installed. In addition, it has been suggested that in the LPS of strain 26695, 3-substituted Hep forms the link between the element of GlcNAc O-chain and core. It was also found the presence of 3-linked heptose in LPSH. pyloriHP0826::Kan, but the presence of α1,3-heptane or its length has not been described. Other information about the structure or length heptane or glucan side chains do not exist. The structure of LPSH. pyloriit is currently installed.

The present invention relates to a novel α1,6-glucan-containing compoundHelicobacter pyloricontaining the structure of formula I:

in which R represents a trisaharid α-DDHep-3-α-L-Fuc-3-β-GlcNAc substituted α1,6-glucan followed by α1,3-DD-heptane, where the last remnant of DD-Hep α1,3-DD-heptane� Kairouan the remnant β-GlcNAc.

In the structure described above, the β-GlcNAc of trisaccharide (α-DDHep-3-α-L-Fuc-3-β-GlcNAc), is associated with α-DDHep G. α-DDHep of trisaccharide is associated with α1,6-glucan, which in turn is associated with α1,3-DD-heptane. Then α1,3-DD-heptane is connected to the remainder of the β-GlcNAc; the latter can provide a point of attachment for the polysaccharide O-chain. The term "associated" or "substituted" means that the two groups are connected by a covalent bond.

The term "α1,6-glucan" as used in this application, may also be used interchangeably as a "glycan", "α1,6-glucan side chain", "glucan side chain", "α1,6-glucan fragment" and/or "glucan fragment". α1,6-Glucan is a linear polysaccharide chain composed of glucose subunits linked α1,6 O-glycosidic bonds. In one non-limiting example, the α1,6-glucan may be a linear polysaccharide. α1,6-Glucan may contain any suitable number of α1,6-glucose residues. For example, the glucan may include without limitation from about 3 to 12 α1,6-linked glucose residues; in particular, glucan fragment can contain approximately 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 α1,6-linked glucose residues, or the number of residues can be in any range defined by any two of the listed values. In non-limiting example, the α1,6-glucan may contain 9 to 12 ostatka� α1,6-glucose; in another non-limiting example, the α1,6-glucan may contain 10 α1,6-glucose residues.

The term "glycoform", as used in the present description, refers to the various forms or types of connections with the same LPS structure, but a different number of residues of the α1,6-glucose or α1,3-heptose. For example, and without regard to any theory, each glycoform may contain glucan and/or heptane fragment of a specific length, or a combination thereof.

The term "α1,3-heptane used in the present application, can also interchangeably be referred to as "α1,3-DD-heptane", "heptane", "α1,3-heptane side chain", "side chain heptane", "α1,3-heptane fragment", "heptane fragment" and/or "DD-heptahelical". α1,3-Heptane represents a polysaccharide chain consisting of the monomers heptose linked α1,3-O-glycoside bonds. In one of the examples that are not intended to limit in any way, α1,3-heptane can be a linear polysaccharide. α1,3-Heptane may contain any suitable number of residues of the α1,3-heptose. For example, heptane may include, without limitation, approximately 2-6 α1,3-linked residues, heptose; in particular, heptane fragment may contain approximately 2, 3, 4, 5 or 6 α1,3-linked residues, heptose or the number of residues of heptose may be in the range, asked Liu�bubbled two of the above values.

The last remnant of DD-Hep α1,3-DD-heptane, as described above, Kairouan the remnant β-GlcNAc. The term "teperovannyj" means that the residue of the β-GlcNAc is the last residue side chain; can also be used the term "limit". β-GlcNAc may be associated with DD-Hep through the position O-2 heptose. Out of touch with any theory, the remnant β-GlcNAc can provide a point of attachment for the polysaccharide O-chain.

In non-limiting example, R may be a

where is the rest of L β-linked GlcNAc to O-2 of Hep residue G. In this example, the remainder of W β-GlcNAc can provide a point of attachment for the polysaccharide O-chain. In connection, which describes the remnants of Q and Z glucans represent α1,6-linked glucose residues, and n can have any value from 1 to 11, so glucan contains approximately 3 to 12 glucose residues in α1,6-connection; in one specific non-limiting example, the main glycoform contains 10 consecutive α1,6-linked glucose residues (n=9). In connection, which describes the remnants of T, Y and X represent α1,3-linked residues of heptose, and m can have any value from 0 to 4, so that the heptane contains from about 2 to 6 residues of heptose in α1,3-joint; in one specific non-limiting example, the main glycoform contains 4 consecutive α1,3-linked residues, heptose (m=2.

The structure may be isolated and/or purified from any suitable strain ofH. pylori; as an example, and without intent to limit in any way, truncated LPS moleculeH. pylorican be isolated from a strain failed to be typedH. pylori(i.e., strain, devoid of Lewis antigens), this, but without limitation, as a strain ofH Pylorihaving a mutation in the gene HP0826, resulting in the isogenic mutant, devoid of the polysaccharide O-chain. In non-limiting example, the compound described in the present description, can be isolated and/or purified from strain 26695 HP0826::Kan or strain PJ2H. pylori.

The structure of formula I, also referred to in the present description as the "molecule of the inner portion of the cortex, may also enable the molecule, A lipid, covalently attached to the Kdo residue, for example, to the Kdo residue C. In other embodiments, the molecule of lipid A can be O-diallylamine or can be completely diallylamine. In other embodiments, the molecule of lipid A are split off by hydrolysis ketose communication from the Kdo residue. Out of touch with any theory, the cleavage of lipid A can be done to eliminate the toxicity of LPS and avoid possible aggregation and insolubility of the conjugate. Specialists in this area known methods of O-diallylamine, diallylamine, or hydrolysis ketose bond of the molecule of lipid A (see,for example, Hoist et al., 1991; Altman et al., 2003).

The present invention also relates to a conjugate containing essentially linear α1,6-glucan-containing compound, anywhereman with carrier protein. Essentially linear α1,6-glucan-containing compound may be any suitable essentially linear polysaccharide consisting of α1,6-linked glucose residues. The term "substantially linear" means that α1,6-glucan contains several branches; for example, the α1,6-glucan may without limitation as to contain from about 0 to 5% branching in α1,6-glucane. In particular, the α1,6-glucan may include approximately 0, 0,5, 1, 1,5, 2, 2,5, 3, 3,5, 4, 4,5 or 5% branching, or any number between the specified values; the connection can also be a mixture, in which the degree of branching in the mixture varies from one connection to another. In non-limiting example, the α1,6-glucan-containing compound may be a structure described above, in the present description in another example, the α1,6-glucan-containing compound may be a Dextran. Dextran may be of any suitable Dextran, which meets the requirements described above and has a molecular weight of from 1 to 10 kDa; for example, Dextran may be without limitation a molecular weight of 1, 3,5, 5, 6,5, 8, or 10 kDa, or any molecular weight mezhdugorodnymi values. In specific non-limiting example, the Dextran can be a Dextran T5. In yet another example, the α1,6-glucan-containing compound may be a linear chain of 5-8 α1,6-linked glucose residues; for example, the α1,6-glucan-containing compound may be without limitation a linear chain of 5, 6, 7, or 8 of α1,6-linked glucose residues.

α1,6-Glucan-containing compound kongugiruut with the linker molecule and/or carrier protein; the person skilled in the art it is obvious that the structure described in the present description, can be konjugierte directly with the carrier protein or can be konjugierte with a linker molecule (also referred to in this application as "linker"), which, in turn, conjugated with carrier protein. The term "conjugated" means that the structure of the covalently attached or linked to the linker molecule and/or carrier protein. Methods of covalent attach the linker and/or a carrier protein are well known to specialists in this field; and as will be clear to the person skilled in the art, the method of covalent joining (and regardless of whether the linker) can vary with the applied a carrier protein. Without intent to limit in any way, figure 4 shows one such method, which was adapted from Fernandez-Satana et al. (1998); in this method, easilyonly or delipidation LPS activated covalent joining of a carboxyl group of the Kdo residue to a linker molecule, followed by the introduction maleimide functional groups. Activated LPS mixed with etiolirovannykh carrier protein to obtain a conjugate structure. As will be appreciated by specialists in this field, the method described in the present application, is General and therefore can be used any other suitable method (e.g., but not limited to the methods described by Chu et al., 1991; Cox et al., 2005; Gu et al., 1996; Mieszala et al., 2003; Yu and Gu, 2007). α1,6-Glucan-containing compound can be attached to the linker/squirrel-a carrier with a carboxyl group of any suitable carbohydrate residue patterns. In specific non-limiting example, the α1,6-glucan-containing compound may have the structure of the present invention and may be attached by means of balance, Kdo inner portion of the bark.

Protein carrier can be any suitable carrier known in this area, including immunogenic carriers. For example, the protein carrier may be, but not limited to, tetanus toxoid, bovine serum albumin (BSA), diphtheria toxoid mutant diphtheria toxoid, CRM, protein CRM197protein AndPseudomonas, �trees cholera toxin (CT), protein cholera toxin mutant CT-E29H, and other carrier proteins, known in this field, for example, but without limitation parts of flagella, pili and other toxins.

As previously stated, the conjugates can be obtained directly by coupling a carrier protein and the structure of the present invention through a natural group, or connection with the introduction of spacer or linker molecules, including, but without limitation simplest amides, hydrazides, thiols, carboxyl groups, and others.

The present invention furthermore relates to compositions containing one or more compounds described above, one or more conjugates as described above, or a combination of both. In one embodiments, the composition may contain a mixture of glycoforms of the compounds described above; as an example, and without intent to limit in any way, the composition may contain a main glycoform, including 10 consecutive α1,6-linked glucose residues (n=9) in the fragment glucan. Similarly, the composition may contain, without limitation conjugates derived from more than one glycoform described in the present application. As shown in the examples, the structure of the LPS produced by a strain ofH. pylori26695 HP0826::Kan, which was used in obtaining conjugates, was a mixture of three Glyco�ORM: oligosaccharide main circuit I, the core oligosaccharide chain I, teperovannyj GlcNAc [GlcNAc, Fuc, Hep], and oligosaccharide containing the α1,6-linked glucan, with the longest glycanova chain of approximately twelve α-1,6-linked residues, as determined by analysis of CE-MS (table 2, Fig. 2).

The composition described above, can be immunogenic. The term "immunogenic" means that the composition can induce an immune response againstH. pyloriwild and/or mutant strains. The immune response can provide a broad immunogenic response against the subject and failed to be typed strains ofH. pylori.

The present invention also relates to the application of an effective amount of the composition as described in the present application, for the induction of an immune response againstH. pyloriin the individual. As described previously, the composition may contain one or more than one compound of the present invention. One or more than one connection can be anywhereman with the linker and/or a suitable molecule-carrier.

The present invention furthermore relates to the immune antiserum obtained by immunization of a mammal the immunogenic composition as described above. Immune antiserum may contain or provide palimony serum IgG that recognizes α1,6-linked glucan epitope in homologous and heterologous t�piramyd and failed to be typed mutant and wild strains of H. pylori. IgG can induce complement-mediated bacterials mutant and wild α1,6-glucan-expressing strains ofH. pylori.

The present invention furthermore relates to anti-α1,6-glycanova antibodies. Antibodies can be induced against compounds of the invention, which is described in the present application, or can be induced against other α1,6-glucan-containing molecules, such as Dextran or Dextran conjugates, for example, but without limitation conjugates BSA-Dextran). In one non-limiting examples, the antibody may be a monoclonal antibody obtained by the method known in the art (see Example 10; Altman et al., 2005). For example, the antibody may be without limitation a monoclonal antibody induced against α1,6-glucan epitope presented in the outer crust area LPS mutant HP0826H. pylori; in more detail, the antibody may be induced against the connection presented on figure 5. In a more particular example, the monoclonal antibody may be an IgM 1C4F9 produced by hybrid cell line 1C4F9. Specified the cell line is registered with the International Depository authority of Canada (National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, Manitoba, CANADA) on 30 July 2009 with reference number 300709-01. As shown in the examples, the antibody 1C4F9 recognize� α1,6-glucan epitope in LPS and whole cells of the subject and failed to be typed strains of H. pylorithat was easily accessible on the surface of live bacteria and reacted equally well with the conjugates LPS-OH-TT and orastream-BSA or orastream-TT.

The present invention relates to the use of antibodies 1C4F9, for the implementation of complement-mediated bacteriolysis mutant and wild interviewee failed to be typed and α1,6-glucan-expressing strains ofH. pyloriin the individual, if necessary. As described earlier, the interviewee strains ofH. pylorihave Lewis antigens recognized by antibodies anti-Le, whereas strains failed to be typed don't have them. However, as interviewee and failed to be typed strains contain α1,6-glucan, both types of strains will be recognized by the antibody 1C4F9.

Currently in this area, the strains ofH. pylorican be typed using commercially available antibodies against antigens Lewis; since, however, failed to be typed strains do not contain Lewis structures, they cannot be classified with the use of this approach. Since most of the interviewee and failed to be typed strains ofH. pyloriI have the α1,6-glucan epitopes, anti-α1,6-glucan antibodies such as mAb 1C4F9) can provide an additional way of screening and descriptions of isolates ofH. pylori.

LPS of strain 26695 HP0826::KanH. pyloriwas purified and its chemical structure was identified by composition, methylation, in-depth analysis of nuclear MAG�itogo resonance (NMR) data and analysis CE-MS. The presence of α1,6-linked glucan in the outer crust area LPS mutant HP0826H. pyloriit was also shown; this structure is recognized by monoclonal antibody 1C4F9 specific to α1,6-glucan. The latter antibody was obtained using the formalin-fixed cells of a mutant strain OF:3 HP0826::KanH. pylori. These antibodies have been available on the cell surface and was bactericidal. It was previously thought that only Lewis structures are antigenic; thus, antibodies against α1,6-glucan was unexpected.

To examine the vaccine potential of LPSH. pylorimodified LPS mutant 26695 HP0826::KanH. pyloriconjugated with tetanus toxoid (TT) or bovine serum albumin (BSA). Applied two approaches for obtaining partially delipidation or delipidation LPS: O-diallylamine LPS with mild hydrazinolysis (LPS-OH) or delipidization LPS mild acid treatment (orastream). Additional ways of delipidization and/or partial delipidization well known in this field, and such suitable methods can be used in the scope of the present invention. LPS-OH and orastream covalently linked through the residue of 2-keto-3-deoxy-octulosonic acid (Kdo) with a spacer containing diaminopropan, followed by an introduction maleimide functional groups and con�garavanian with etiolirovannykh TT or BSA to obtain conjugates LPS-OH-TT, orastream-BSA and orastream-TT, respectively. In a separate experiment, LPS failed to be typed strain PJ2 was delipidation and used for conjugation with obtaining conjugate orastream(PJ2)-TT.

Conjugates LPS-OH-TT, orastream-BSA, orastream-TT and orastream(PJ2)-TT retained the antigenicity of the surface available α1,6-glycanova determinants, which was evaluated in an indirect ELISA test with IgM 1C4F9. The antibody has been shown to possess high selectivity for α1,6-glycanova the determinants and characteristics of binding was determined in the test of inhibition ELISA oligosaccharides from isomalto-series and purified LPS interviewee and failed to be typed strains ofH. pylori.These studies confirmed that the antibody 1C4F9 required 5-6 consecutive α1,6-linked glucose residues, the model is consistent with the size of the plots of the binding of anti-α-(1→6)dextran, postulated Kabat (1993).

Conjugates LPS-OH-TT, orastream-BSA, orastream-TT or orastream(PJ2)-TT was immunogenum in mice and rabbits and caused a statistically significant IgG antibodies in response to LPS homologous and heterologous wild-type strains ofH. pylori. In mice and rabbits, which received orastream-containing conjugate, developed ten times more strong IgG immune response to immunization with an antigen. Pastamania serum of rabbits immunized with either LPS-OH-TT, orastream-BSA, orastream-TT or orastream(PJ2)-TT showed bactericidal activity against mutant 26695 HP0826::Kan and dickoh� 26695 strains of H. pylori.

Thus, these results show that protein conjugates on the basis of delipidation or partially delipidation-based LPSH. pyloridevoid of antigen Le and carrying long α1,6-glucan chain are immunogenic in mice and rabbits and induce the formation of bactericidal antibodies. It is important to note that the epitope was identified as immunogenic. This fact has not been previously installed, because it was known that only Lewis structures are antigenic and induce the formation of specific antibodies.

The present invention will be further illustrated in the following examples. However, it should be understood that these examples are merely illustrative and should not be used to limit the scope of the present invention in any way.

Example 1. Isolation and structural analysis of LPSH. pylori26696 HP0826::Kan

Strain 26695Helicobacter pyloriwas obtained from Dr. R. Aim (Astra Zeneca, Boston, MA), an isolate ofH. pyloriAbout:3 was obtained from Dr. J. Penner, J99 was obtained from Dr. D. Taylor (University of Alberta, Edmonton, Canada), SS1 was obtained from Dr. A. Lee (The University of New South Wales, Sydney, Australia), clinical isolates of PJ1 and PJ2 was a fresh clinical isolates from Dr. W. Conlan (IBS, NRC) and the M6 was obtained from Dr. K. Eaton (Michigan State University, MI).

Cultivation of bacterial strains was performed as described by Hiratsuka et al (2005). Briefly, cells were grown at 37°C on plates with Columbia blood agar (Difco) with added antibiotics) containing 7% horse blood in a microaerophilic conditions for 48 hours (Kan 20 μg/ml) as previously described (Hiratsuka et al., 2005). For growth in suspension culture with the addition of antibiotics liquid Brucella medium containing 10% fetal bovine serum, inoculable cellsH pyloricollected after 48 hours with a layer of nutrient medium Columbia blood agar/horse blood, and incubated for 48 hours in a shaker in microaerophilic conditions (85% N2, 10% CO2, 5% O2as described previously (Hiratsuka et al., 2005).

The strains ofH. pyloriwere cultured in liquid culture, as described above, and the wet cell mass obtained by centrifugation of the bacterial culture, twice successively washed with ethanol, acetone and light petroleum ether and air dried. LPS was extracted from air-dried cell mass by extraction procedure with a mixture of hot phenol-water, described by Westphal and Jann (1965). LPS was obtained from the aqueous phase after extensive dialysis and lyophilization. Then LPSH. pyloriwas purified by ultracentrifugation (105000 x g, 4°C, 12 hours), and the precipitate was suspended in distilled water and liofilizirovanny.

Analysis of the composition of sugars conducted aldit-acetate method (Sawardeker et al., 1967). Hydrolysis in�terminal facilities in 4 M trifluoroacetic acid at 100°C for 4 hours or in 2 M trifluoroacetic acid at 100°C for 16 hours followed by reduction in H 2O NaBH4and subsequent acetylation with acetic anhydride/pyridine. Aldit-acetate derivatives were analyzed as previously described (Altman et al., 2003). Analysis of methylation was performed according to the method of Ciucanu &Kerek (1984) and describing dermatillomania aldit-acetate derivatives using gas-liquid chromatography - mass spectrometry (GLC-MS) as described previously (Altman et al., 2003).

Analysis of sugars purified LPS from strain 26695 HP0826::KanH. pyloriin aldit-acetates revealed the presence of L-fucose (L-Fuc), D-glucose (D-Glc), D-galactose (D-Gal), N-acetyl-D-glucosamine (D-GlcNAc), D-glycero-D-manno-heptose (DD-Hep) and L-glycero-D-manno-heptose (LD-Hep) in approximately molar ratio 0,4:5,0:1,5:4,3:6,4:1,4, pointing to the presence of the structure, devoid of O-chain (Logan et al., 2000). The methylation analysis performed on intact LPS of strain 26695 HP0826::Kan, was consistent with these results and showed the presence of terminal L-Fuc, 3-substituted L-Fuc, terminal D-Glc, terminal D-Gal, 3-substituted glucose, 4-substituted D-Gal, 6-substituted glucose, terminal DD-Hep, 2-substituted DD-Hep, 6-substituted DD-Hep, 3-substituted DD-Hep, 7-substituted DD-Hep, 2,7-substituted DD-Hep, 2-substituted LD-Hep, 3-substituted LD-Hep, terminal D-GlcNAc and 3-substituted D-GlcNAc in the approximate molar ratio 0,1:1,0:1,1:0,1:1,2:1,0:6,0:0,4:1,2:1,1:3,1:0,5:1,6:1,2:0,1:0,3:0,4. Not found a 3,4-substituted D-GlcNAc and 2-linked D-Gal, characteristic of the O-chain containing �ntigen Le. The purity of LPS was confirmed by the absence of Rabita derived from RNA.

The analyses using the composition and methylation LPS of strain 26695 is described elsewhere (Logan et al., 2005). All sugars are present in the pyranose form.

Example 2. Description delipidation LPSH. pylori26695 HP0826::Kan through a system of capillary electrophoresis - mass spectrometry(CE-MS)

Purified LPS 26695 HP0826::Kan (20 mg) obtained in Example 1 hydrolyzable in 0.1 M sodium-acetate buffer, pH of 4.2, for 2 hours at 100°C and fractionally using gel filtration on a column of Bio-Gel P-2 as described previously (Altman et al., 2003) to obtain delipidation LPS (orastream). Collected three fractions (fractions 1-3) and analyzed using a combination of capillary electrophoresis and mass spectrometry (CE-MS; table 1).

For CE-MS system Prince CE (Prince Technologies, The Netherlands) connected to a mass spectrometer 4000 QTRAP (Applied Biosystems/MDS Sciex, Canada). The flow-through solution (isopropanol-methanol, 2:1) was passed at a flow rate of 1.0 µl/min.

The division received approximately unmodified silica capillary length 90 cm with application of 15 mm ammonium acetate in deionized water, pH of 9.0. Used the ionization voltage of elektrorazpredelenie 5 kV for determination mode of positive ions. Tandem mass spectra were obtained using enhanced mode, scanning the product ion (EPI) with the velocity�computer scan 4000 Da/s. Nitrogen was used as a “gas curtain” (at size 12) and for gas collisions (set to a scale of "high").

Analysis of CE-MS basic fraction 1 in the definition of the positive ions confirmed the presence of a series of three ions at m/z 1212,3, m/z 1266,3 and m/z 1320,4 in accordance with the sequential addition of residues Hex, the longest glucan chain represented approximately eleven α-1,6-linked residues. Based on the signal intensity of the ions, the most common glycoform at m/z 1266,3 contained ten α-1,6-linked Hex (Fig. 1). Range ion product ions at m/z 1266,3 in the mode of determination of positive ions confirmed the presence of diagnostic ions at m/z 1244,5 and m/z 1447,6 in accordance with the previously observed fragments of the main chain Cora Hex2Hep3(PEtn)Kdo and HexNAcHex2Hep3(PEtn)Kdo, respectively (Fig. 1). Fractions 1 and 2, bearing α1,6-glucan in the cortex of LPS were put together and used for conjugation.

Table 1
The results of CE-MS in the determination mode of positive ions and the estimated composition of LPS glycoforms in delipidation LPS of strain 26695 HP0826::KanH. pylori
Combined fractionsObserved ion (m/z) Molecular weight (Da)The estimated composition ofb
observableCalculateda
Faction 1[M+H+2NH4]3+
1212,33599,93599,1Hex12Hep5PEtn1HexNAc1Fuc1anhydroKDO1
1218,13617,33617,1Hex12Hep5PEtn1HexNAc1Fuc1KDO1
1266,33761,93761,2Hex13Hep5PEtn1HexNAc1Fuc1anhydroKDO1
1272,03779,03779,2Hex13Hep5PEtn1HexNAc1Fuc1KDO1
1320,43924,2 3923,4Hex14Hep5PEtn1HexNAc1Fuc1anhydroKDO1
1326,43942,23941,4Hex14Hep5PEtn1HexNAc1Fuc1KDO1
Faction 2[M+N+NH4]2+
902,41785,81785,5Hex2Hep4PEtn1HexNAc1Fuc1anhydroKDO1
911,41803,81803,5Hex2Hep4PEtn1HexNAc1Fuc1KDO1
983,51948,01947,7Hex3Hep4PEtn1HexNAc1Fuc1anhydroKDO1

992,51966,01965,7 Hex3Hep4PEtn1HexNAc1Fuc1KDO1
1064,62110,22109,8Hex4Hep4PEtn1HexNAc1Fuc1anhydroKDO1
1073,62128,22127,8Hex4Hep4PEtn1HexNAc1Fuc1KDO1
1145,52272,02271,9Hex5Hep4PEtn1HexNAc1Fuc1anhydroKDO1
1154,52290,02290,0Hex5Hep4PEtn1HexNAc1Fuc1KDO1
Fraction 3[M+N+NH4]2+
Inhabited by 631.61244,21244,0Hex2Hep3PEtn1anhydroKDO1
�� 640,61262,21262,0Hex2Hep3PEtn1HexNAc1KDO1
733,21447,41447,2Hex2Hep3PEtn1HexNAc1anhydroKDO1
742,11465,21465,2Hex2Hep3PEtn1HexNAc1KDO1
aBased on the average value of the atomic mass
bHex, HexNAc, Hep, KDO, PEtn and Fuc represent a hexose, hexosamine, heptose, 3-keto deoxy-D-manno-2-octulosonic acid, phosphoethanolamine and fucose are presented, respectively.

Example 3. Description O-decelerating 26695 LPS HP0826::Kan using CE-MS

O-diallylamine 26695 LPS HP0826::Kan (Example 1) was carried out according to Holst et al. (1991) with some modifications. Briefly, LPS (4 mg) was stirred in anhydrous hydrazine (0.2 ml) at 37°C for 4 hours. Cooled the reaction mixture and slowly added to cold acetone (2 ml) to decompose the excess of hydrazine. After 30 min, the precipitated O-easilyonly LPS (LPS-OH) was collected by centrifugation (4°C, 9300 x g, 10 min). The precipitate guidetravel cold acetone, was dissolved in water and liofilizirovanny with getting LPS-OH (3.5 mg).

Analysis of CE-MS O-decelerating 26695 LPS HP0826::Kan (LPS-OH) in the definition of the positive ions was performed as described in Example 2. The results of CE-MS match MS data obtained for delipidation LPS and presented the three main double-charged ion at m/z 1137,2, m/z 1239,2 and m/z 1408,0 corresponding to the presence of oligosaccharide main chain and main chain oligosaccharide, caerbannog and HexNAc [HexAc, Fuc, Hep], respectively (table 2), whereas three times charged ions at m/z 1597,7, m/z 1651,4 and m/z 1705,5 consistent with the presence of glucan, the longest glucan chain represented approximately twelve α-1,6-linked residues (table 2, Fig. 2). Spectrum MS/MS at m/z 1597,7 showed the presence of diagnostic ions at m/z 1447,6 and m/z 1244,5, which is consistent with continuous removal [Fuc,Hep] and [Fuc,Hep,HexNAc], respectively (Fig. 2) and, additionally, gave weakly charged ion at m/z 1786,8 corresponding korovou fragment FucHex2HexNAcHep4(PEtn)Kdo connected through the Kdo residue with O-diallylamine the lipid A (lipid A-OH), while ions at m/z 444,4 and m/z 887,8 consistent with the molecule of lipid A-OH, consisting of dglucosamine main chain substituted by two linked amide coupling circuits 3-hydroxyoctadecadienoic [C18:0(3-OH)] fatty acids (Fig. 2).

Table 2
The results of CE-MS in the determination mode of positive ions and the estimated composition of LPS glycoforms in O-diallylamine LPS of strain 26695 HP0826::KanH. pylori
Observed ion (m/z)Molecular weightThe estimated composition ofb
observableCalculateda
[M+2H]2+
1137,22272,42272,1Hex2Hep3PEtn2HexN2(3-OH C18:0)2KDO1
1239,22476,62475,3Hex2Hep3HexNAc1PEtn2HexN2(3-OH C18:0)2KDO1
14082814,42813,5Fuc1Hex2Hep4HexNAc1PEtn2HexN2(3-OH C18:0)2KDO1
[M+3H]3+
1597,74790,14789,2Fuc1Hex13Hep5HexNAc1PEtn2HexN2(3-OH C18:0)2KDO1
1651,44951,24951,3Fuc1Hex14Hep5HexNAc1PEtn2HexN2(3-OH C18:0)2KDO1
1705,55113,55113,5Fuc1Hex15Hep5HexNAc1PEtn2HexN2(3-OH C18:0)2KDO1
aOn the basis of the average values of the atomic masses
bHex, HexN, Hep, KDO, PEtn and Fuc represent a hexose, hexosamine, heptose, 3-keto deoxy-D-manno-2-octulosonic acid, phosphoethanolamine and fucose are presented, respectively.

Example 4. The NMR analysis of the LPS of strain 26695 HP0826::KanH. pylori

The degradation of the LPS of strain 26695 HP0826::Kan (Example 1)H. pyloristarted with full diallylamine 4 M KOH in the presence of NaBH4for quick recovery GlcN lipid A, since alkaline hydrolysis of the Deputy PEtN leaves regenerating hair end without GlcN aglycone (Holst et al., 1991). The separation of the products by gel chromatography gave two fractions, - eluted in the region oligos�reed chromatograms. Further analysis showed that these fractions contained similar compounds, obviously differing in the length of glycanova chain. Fraction with lower molecular weight compounds contained 1-3 identified by mass spectrometry, and both fractions contained the compound 4 with glycanova part of a different length (Fig. 3).

The NMR spectra (DQCOSY, TOCSY, NOESY,1H-13C HSQC and HMBC) were recorded on a spectrometer Varian 500 or 600 MHz using standard software, as described previously (Brisson et al., 2002). All NMR experiments were performed at 25°C using acetone as an internal standard δ 2,225 M. D.1H spectra and 31,45 M. D.13C spectra.

The NMR spectra of both fractions was consistent with the compound 4, although the spectra could not be fully interpreted because of their complexity. The protons H-1 terminal residues of α-1,6-glucan and DD-heptane does not overlap with the other H-1 glucan and heptane, which allows to identify the position of these homopolymers within a structure, as shown in Fig. 3. However, the connection position of the reducing end residue Glc Z α-1,6-glycanova chain is not installed. He showed a strong NOE correlation with unidentified proton (later attributed to H-6 N Hep). The results of NMR of compound 4 showed clearly that the α-1,6-glucan is between DD-heptane and internal�them with a section of the bark, since the first Hep residue fragment of heptane (X) is associated with O-3 nevosstanovlenie terminal residue Glc V belonging to the α-1,6-glucan. Nevosstanovlenie end DD-heptane substituted at O-3 of β-GlcN W. Connection, identical to the small oligosaccharides 1-3 previously detected as the major components of the LPSH. pylorimutant, devoid of α-1,6-glucan, HP0159::Kan, and the published NMR results (Altman et al., 2008). Some of the results of NMR of compound 4 is presented in table 3. It is clearly seen that previously proposed a sequence of four consecutive residues of heptose related to Kdo, not represented in any of the considered compounds (Fig. 3). The residue is L β-GlcN was directly linked to O-2 of Hep G, and GlcN was not connected to O-7 Hep in any part of the structure of LPS.

For further analysis of the LPS structure of compounds 1-4 was dezaminirovanie. Briefly, the samples were dissolved in 10% AcOH was added an excess of NaNO2; after 3 hours the product was isolated by gel chromatography on a column of Sephadex G-15. Some compounds are then recovered NaBD4and were desalted. The final products consisted of compounds 5 and 6. The NMR spectra of compounds 5 (performed as described above) showed two isomers with the phosphate at position 6 or 7 of the Hep E (due to the rearrangement of phosphate in alkaline conditions). And in this case, were represented �ri serial Hep residue, but not four, as one would expect if the earlier proposed structure was correct. The removal of the entire side chain using the method of deamination (described above) indicated that GlcN L forms the link between the sugars 5OS and the remainder of the molecule.

Compound 6 contained DD-heptane, α-1,6-glucan and trisaharid DDHep-Fuc-anh-Man-ol (N-M-L, anh-Man L obtained from L GlcN) on revitalising the end. Spectra of this product were less congested and it became possible to determine the point of attachment of α-1,6-glucan to O-6 DDHep N (TOCSY between H-1 and H-6 DDHep N). The structure was linear, which was confirmed by methylation analysis, which showed branched sugars; all expected products have been identified in accordance with the proposed structure (table 4).

In addition, the conformation of the structure in the field of DD-heptane-α-1,6-glucan was obtained from the results periodate oxidation of compound 6. Periodate oxidation was carried out with an excess of 0.1 M NaIO4within 24 hours; ethylene glycol was added and the product was isolated by gel-chromatography on a column of Sephadex G-15. The product was restored NaBD4, were desalted and hydrolizable 2% AcOH for 3 hours at 100°C. Received two major product 7 and 8, and was isolated by gel-chromatography on a column of Sephadex G-15.

Voss�yavlenie oxidized oligosaccharide NaBD 4allowed to identify the oxidized carbons in the NMR spectra (inverted phase signals CHDOH in comparison with the signals of CH2OH in APT-HSQC). The structure of compounds 7 and 8 were determined using NMR spectroscopy and methylation analysis. The formation of the compound 7 proved that DDHep N was not substituted in position 3. Since methylation of compound 6 showed only limit, 3 - and 6-substituted DD-Hep, DD-Hep N has been substituted in position 6, as would be expected, based on the results of NMR of compound 6. The formation of the compound 8 with glycerol (Gro) on revitalising the end confirmed that Glc V 6 OS was glycosylamine the following residue Glc in position 6, as there were no other components of the oligosaccharide 6, which could form the glycerin after the oxidation-recovery. This fact also proves the joining of fragment DD-heptane to nevosstanovlenie the end of α-1,6-glucan in position 3 terminal glucose balance.

You can estimate the size of the DD-heptane domain: compound 8 contained four mannose residue obtained from DD-heptose. Spectra of compound 8 had a good resolution and integration of the signals of H-1 gave almost equimolar ratio and indicated the presence of four mannose residues. Thus, an intact DD-heptane domain in the connection 6 consisted mainly of 5 links DD-heptose, one of which (concavo�) was removed periodate oxidation. The conclusion is confirmed by the results of mass spectrometry. The structure of the main glycoform LPS, produced by the strain 26695 HP0826::KanH. Pyloriis illustrated in Figure 3.

Example 5. Receipt and description of conjugates LPS-OH-TT, orastream-BSA, orastream-TT and orastream(PJ2)-TT

Received Kdo-linked conjugates LPS-OH (see Example 3) and orastream (see Example 2) with bovine serum albumin (BSA) and/or tetanus toxoid (TT).

LPS-OH (4 mg, 0.8 μmol, based on the assumed average molecular weight 4789 Yes) or orastream (4 mg, 1 mmol, based on assumed average molecular weight Yes 3779) was dissolved in 0.1 M buffer 2-(N-morpholine)econsultancy acid (MES; Sigma-Aldrich, St. Louis, Mo), pH of 4.8, containing 0.1 M NaCl (0.4 ml) was added 1-ethyl-3-dimethylaminopropyl of carbodiimide (EDC; 34,38 mg, molar ratio 100:1; Sigma-Aldrich) followed by the addition of 1,8-diamino-3,6-dioxaoctyl (15 μl, 103 μmol; Sigma-Aldrich), and the reaction was carried out at a pH of 4.8 for 4 hours at 22°C. the Solution was adjusted to pH 7.0 and were dialyzed against distilled water, or were desalted with the use of centrifugal devices Microsep, the threshold value of 1000 Da (Pall Life Sciences, Ann Arbor, MI) and liofilizirovanny.

The conjugation procedure was carried out mainly as described for oligosaccharide Fernandez-Santana et al. (1998). Briefly, the reaction was performed containing the spacer LPS-OH (2 mg, 0.4 μmol) or orastream (2 mg, 0.5 mol) with N-hydroxysuccinimidyl ether 3-Malek�dipropionovoi acid (BMPS; 2 mg, 7.5 μmol; Sigma-Aldrich) in dry DMSO for 24 hours at 22°C. the Solution were dialyzed against distilled water and liofilizirovanny.

To activate BSA was added 3,3-dithiodipropionic acid di(N-hydroxysuccinimidyl ester) (DTSP; 0,63 mg, 1.6 μmol; Sigma-Aldrich) in dry DMSO, in the atmosphere of N2to a solution of bovine serum albumin (BSA) (molecular weight 66320 Yes) (8 mg, 0.12 mmol) previously dissolved in 10 mm PBS buffer, pH 8,0, containing 6 mm EDTA (final concentration 4 mg/ml), and the mixture was stirred for 2 hours at 22°C. was Then added to dithiothreitol (DTT; 7,12 mg, 46 μmol; Sigma-Aldrich) in an atmosphere of N2and the mixture was stirred for 1 hour at 4°C. the resulting solution deliberately against 10 mm PBS buffer, pH of 7.2, containing 5 mm EDTA in the cell for ultrafiltration (Millipore, Billerica, MA), using N2as the source pressure on the membrane from regenerated cellulose, a threshold value of 30,000 Da (YM30, Millipore) at 4°C. the protein Content and SH were determined using analytical set with bicinchoninic acid (BCA; Pierce, Rockford, IL) and by the method of Ellman (1959), respectively. Reached the molar substitution 20-22 SH-groups.

For activation of tetanus toxoid (TT) (molecular weight, 150000 Yes), DTSP (0,316 mg, 0.8 μmol) in dry DMSO (25 ml) was added, in an atmosphere of N2to a solution of TT (4 mg, 0,03 mmol), in 10 mm PBS buffer, pH 8,0, containing 6 mm EDTA (horse�Naya concentration of 4 mg/ml), and the reaction was carried out as described for BSA. Then added dithiothreitol (DTT; 3,56 mg, 23 μmol; Sigma-Aldrich) in an atmosphere of N2and the mixture was stirred for 1 hour at 4°C. the Activated TT was transferred into a cell for ultrafiltration (Millipore) and were dialyzed against 10 mm PBS buffer, pH of 7.2, containing 5 mm EDTA on the membrane of regenerated cellulose, the threshold value 100000 Yes (YM100, Millipore) at 4°C.

To a solution of BSA-SH21-22or TT-SH21-22in 10 mm PBS buffer, pH of 7.2, containing 5 mm EDTA, was added a solution derived maleimido-functionalized LPS-OH or orastream in 10 mm PBS buffer, pH of 7.2, containing 5 mm EDTA (final concentration 4 mg/ml) (molar ratio 3:1) in the atmosphere of N2. The reaction mixture was stirred for 24 hours at 4°C. Then was added N-ethylmaleimide (1 mg; Sigma-Aldrich). The reaction was performed for 30 min at 22°C, and the resulting conjugate were dialyzed against 10 mm PBS buffer, pH of 7.2, for 4 days at 4°C and sterilized by filtration using 0.22 μm membrane polyvinylidene fluoride (PVDF) (Millex-GV, Millipore, Cork, Ireland). Conjugates were tested for the content of carbohydrate and protein by the method of determination of neutral sugars by phenol and sulfuric acid (Dubois et al., 1956) and using a standard kit for protein determination using BCA (Pierce), respectively, with LPS-OH or orastream and BSA as standards. The efficiency of conjugation was confirmed using Vysokova�tive liquid chromatography (HPLC; Agilent 1200 series, Agilent Technologies, Waldbronn, Germany) using the column Superose 12 10/300 GL (Amersham Biosciences, Uppsala, Sweden), equilibrated 10 mm PBS buffer pH of 7.2. Chromatography was carried out at room temperature and a flow rate of 0.5 ml/min, the Elution was monitored at 210 nm and 280 nm using the detector diode matrix (Agilent Technologies).

The presence of the spacer was confirmed by1H-NMR spectroscopy by the appearance of new proton resonance at 3,22 M. D., corresponding to the group of CH2NH2. Then received a derivative of the amino group of the spacer molecule by reaction with N-hydroxysuccinimidyl ether 3-multimediaphoto acid for the formation of maleimide-funkzionirovanija LPS-OH or orastream, which confirmed the presence of proton resonances at 2.55 m. D. and 6.9 M. D., of the respective groups CH2α and CH=CH β-multimediaphoto, respectively (Fig. 4). Derived phosphoethanolamine inner portion of the bark of LD-Hep maintained at a minimum level when using stoichiometric amounts of reagent. Glycoconjugate received by etiolirovaniya a carrier protein and add tarirovannogo protein to maleimido-functionalized LPS-OH or orastream (Fig. 4). The efficiency of conjugation was monitored using HPLC. The resulting conjugate was tested for the content of carbohydrate and protein.

The molar ratio of LPS-OH to TT in the three conjugates of�analog from 10:1 to 20:1, and the output is changed from 13% to 22%, based on the carbohydrate content (table 5). As a result of conjugation with orastream BSA or TT received conjugates orastream-BSA-2, orastream-TT or orastream(PJ2)-TT with a significantly higher content of carbohydrates (table 5). Conjugates LPS-OH-TT and orastream-BSA or orastream-TT reacted equally well with α1,6-glucan-specific mAb in conducting ELISA, suggesting that the conformation glucan epitope has not been changed.

Table 5
Composition and yield of conjugates used in this study
ConjugateAmt (ug/ml)The mole ratio CHO3to proteinOutput (%)
CHO1Protein2
LPS-OH-TT-112026015:122
LPS-OH-TT-211034010:122
LPS-OH-TT-38012020:1 13
LPS-OH-TT-414031014:118
orastream-BSA-17510013:118
orastream-BSA-2850120013:129
orastream-TT49096020:116
orastream(PJ2)-TT39076024:132
1The amount of carbohydrate (CHO) were determined according to Dubois et al. (1956); LPS-OH or orastream was used as standard.
2The amount of protein was determined using a BCA test; BSA was used as standard.
3The mole ratio of CHO to protein was determined using the values of the average molecular weight, based on the average length of glycanova chain: 4789 Yes for LPS-OH and Yes 3779 for orastream (n=10). For orastream(PJ2) the value of the average molecular weight was 3261 Yes based on the average length glycanova chain (n=8) (Atman et al., 2003).

Example 6. Immunogenic conjugates LPS-OH-TT, orastream-BSA, orastream-TT and conjugates orastream(PJ2)-TT in mice and rabbits

Immunogenic conjugates (LPS-OH-TT, orastream-BSA, orastream-TT, and orastream(PJ2)-TT) of Example 5 was tested in mice and rabbits.

Five female mice BALB/c aged 6-8 weeks were immunized intraperitoneally relevant conjugates. Each mouse received 2 μg or 10 μg of carbohydrate in 0.2 MLM Ribi adjuvant per injection. The mice were injected booster injections on the 21st and 42nd day and received serum after terminal cardiac puncture on the 51st day.

Three white New Zealand rabbits were immunized subcutaneously relevant conjugates. Each rabbit received 10 μg or 50 μg of carbohydrate in 0.5 ml of incomplete adjuvant of franda. The mice were injected booster injections on the 28th and 56th day and received the serum after the bloodletting on the 65th day.

Antibody level of anti-LPS serum was measured using ELISA test, in which the purified LPS was used as antigen, covering the hole (1 µg/well). After washing with PBS the plates were blocked with 1% (wt/V) solution of bovine serum albumin (BSA) in PBS or diluent/blocking solution containing milk (MDB) (KPL, Gaithersburg, MD) for 1 hour at 37°C. was Added diluted mouse or rabbit pre - or postmania serum and the plates were incubated for 2 hours at 37°C. For inhibition ELISA serial is� " s inhibitory LPS of strain 26695 HP0479::Kan or LPS of strain 26695 HP0826::Kan) was mixed with a predetermined dilution of the rabbit sera, which corresponded to the value of the OD450=0.6 and 0.8. This mixture is incubated for 15 min at 22°C and then transferred to the original microtiter tablet, blocked by adsorbed antigen LPS, where the mixture is incubated for 2 hours at 37°C. After this stage was performed by indirect ELISA test. Briefly, the plates were washed with PBS and the second antibody conjugate with horseradish peroxidase goat antimachine IgG+IgM (Caltag, So. San Francisco, CA) was added for 1 hour at room temperature. After the final wash add substrate 3,3',5,5'-tetramethylbenzidine (TMB) (KPL, Gaithersburg, MD) and the reaction was stopped with 1 M phosphoric acid. The absorbance was determined at 450 nm using a spectrophotometer for microtiter plates (Dynatech, Chantilly, VA).

The percentage of inhibition was calculated by the following formula:

% inhibition = 100×[(OD with inhibitor-OD without inhibitor)/OD without inhibitor]

Built curves of inhibition versus log concentration for each inhibitor, and determined the concentration required for premaxillae inhibitory concentration (IC50) as a result of extrapolation of the curves.

All conjugates caused the IgG response against homologous LPS (26695 HP0826::Kan) and corresponding wild (26695) strains in rabbits and in mice after three injections (table 6-9), although the response was generally weaker in mice and rabbits, immunis�aligned LPS-OH-TT, than in animals immunized orastream-BSA, orastream-TT or orastream(PJ2)-TT. Control rabbits immunized with a mixture of LPS-OH, orastream or orastream(PJ2) and a carrier protein (with adjuvant) did not show or showed a low level of specific response to LPS homologous strain (mutant 26695 HP0826::Kan) or the corresponding wild strain 26695H. pyloriafter three immunization.

Table 6
The formation of antibodies in mice and rabbits in response to LPSH. pylori26695 HP0826::Kan LPS and 26695 induced by conjugates LPS-OH-TT
aEach mouse received 10 μg of carbohydrate per injection.
bEach rabbit received 50 μg of carbohydrate per injection.
cEach rabbit received 10 μg of carbohydrate per injection.
Table 7
The formation of antibodies in mice and rabbits in response to LPS of strain 26695 HP0826::KanH. pyloriand LPS of strain 26695 induced by conjugates orastream-BSA

aEach mouse received 10 μg of carbohydrate per injection.
bEach mouse received 2 μg of carbohydrate per injection
cEach rabbit received 10 μg of carbohydrate per injection.
dAdjuvant (IFA) only.

Table 8
The formation of antibodies in mice and rabbits in response to LPS of strain 26695 HP0826::Kan LPS and strain 26695H. pyloricaused by conjugate orastream-TT

aEach mouse received 2 μg of carbohydrate per injection.
bEach rabbit received 10 μg of carbohydrate per injection.
cAdjuvant (IFA) only.

Table 9
The formation of antibodies in mice and rabbits in response to LPS of strain 26695 HP0826::Kan LPS and strain 26695H Pyloricaused by conjugate orastream(PJ2)-TT
aEach mouse received 2 μg of carbohydrate per injection.
bEach rabbit received 10 μg of carbohydrate per injection.
cAdjuvant (IA) only.

Studies of cross-reactivity was performed with posthumanity sera of rabbits immunisierung LPS-OH-TT-2, LPS-OH-TT-3, LPS-OH-TT-4, orastream-BSA-2, orastream-TT, or orastream(PJ2) against LPS from strains ofH Pylorirepresenting various glycolipid LPS (Monteiro, 2001) and selected mutant strains. The results are presented in tables 10, 11 and 12.

Table 10
Education rabbit antibodies in response to purified LPSH. pyloricaused by conjugates LPS-OH-TT and orastream-BSA
aPostmonday serum, the titer of 1:100
bPostmonday serum, titer 1:1000
COD450±10%

Table 11
Education rabbit antibodiesain response to purified LPSH. pyloricaused by conjugateborastream-TT
aPostmonday serum, titer 1:1000
bOD450±10%

Table 12
Education rabbit antibodiesain response to purified LPSH. pyloricaused by conjugateborastream(PJ2)-TT

aPostmonday serum, titer 1:1000
bOD450±10%

Reactivity Bestimmung sera obtained from the rabbits, which were immunized by LPS-OH-TT-2, LPS-OH-TT-3 or LPS-OH-TT-4 showed the need for the presence of α1,6-glucan, because it received only weak cross-reactivity with LPS strains SS1 and SS1 HP0826::Kan, who are representatives of strains ofH. pyloriunable to attach α1,6-glucan (Logan et al., 2005) (table 10). Surprisingly, the serum obtained from the rabbits, which were immunized by orastream-TT, also recognize the crust LPS epitopes that did not contain α1,6-glucan, namely LPS strains SS1, SS1 HP0826::Kan, M6 and J99 (table 11), which shows a wide korovou recognition and suggests that conjugate orastream-TT was more immunogenic, possibly due to the presence of carrier protein TT (table 11). Alternatively, since the conjugates were obtained from a mixture of 3 glycoforms, the immune response could be generated to other minor components of LPS; mailobject the observed cross-reactions.

To examine the specificity of binding of rabbit sera induced by conjugate orastream-TT, conducted inhibition test, ELISA, purified LPS of strain 26695 HP0479::Kan, consisting of two glycoforms (approximate 1:1 ratio), linear structure of the core oligosaccharide chains and a linear main chain oligosaccharide, caerbannog [GlcNAc, Fuc] (Hiratsuka et al., 2005), and LPS of strain 26695 HP0826::Kan. Binding of rabbit sera with glucan-negative LPS strains SS1, SS1 HP0826::Kan HP0159::Kan and SS1 HP0479::Kan (Fig. 6) significantly ingibirovalos when 26695 LPS HP0479::Kan was used as the inhibitor, while 26695 LPS HP0826::Kan was the most effective inhibitor, if 26695 LPS was used as a covering of the antigen (Fig. 6).

The ability of rabbit Bestimmung sera to recognize heterologous interviewee and failed to be typed strains were also tested in an indirect ELISA test with whole cell (WCE) against selected clinical isolates ofH. Pylorirepresentatives interviewee and failed to be typed strains ofH. pylori. Indirect ELISA test with whole cell (WCE) was performed as described previously (Altman et al., 2008). Briefly, the wells of a microtiter plate were coated with 100 µl of bacterial suspension, 108cells/ml and left overnight at 4°C. Then the wells were fixed with methanol and blocked with 200 μl diluent/blocking solution containing milk, (MDB) (KPL, Gaithersburg, MD) in tech�of 2 hours at 37°C. Then, the wells were incubated for 2 hours at 37°C with 100 µl of ascitic fluids 1C4F9, diluted 1:500 in MDB, followed by incubation with a conjugate anti-mouse IgG+IgM with horseradish peroxidase (Caltag) diluted 1:1000 in MDB, for 1 hour at room temperature. Added substrate, as described for indirect ELISA test. Nonspecific background values were determined as OD450the negative control wells containing bacterial cells conjugate secondary antibodies and substrate. The values OD450was ≤0,2. The values of optical density at OD450<0,2 classified as negative reactions and the values of the OD450≥0,2 classified as positive reaction. To ensure comparability between tablets cells of strain 26695H. pyloriwas used as a positive control. The test results differed by no more than 10%.

Serum obtained from rabbits that were immunized with a conjugate orastream-TT, showed the strongest cross-reactivity with all tested strains (table 13) use of whole cells in an indirect ELISA test.

Table 13
The cross-reactivity of rabbit Bestimmung serumaagainst clinical isolates ofH. pyloriin indirect ELISA testb with whole cells
aPostmonday serum, the titer of 1:100.
bOD450≥ 0,2±10%.

Example 7. The bactericidal activity of rabbit antisera

Antibacterial test was performed using sera of pre - or postvaccination rabbits (Example 6).

Grown on plates of the cellH. pyloricollected and washed with 5 ml PBS per Cup. After centrifugation, suspended sediments in 25 ml of PBS. The final bacterial suspension was diluted in saline solution, phosphate buffered, Dulbecco (DPBS) (Invitrogen, Grand Island, NY) at a concentration of 4×106CFU/ml. Antibacterial test was performed in titration the microplate with flat bottom (ICN). Tenfold serial dilution decompencirovannah pre - or Bestimmung sera (50 µl) was added to each well. Then added to the bacterial suspension (25 μl) and pre-incubated for 15 min at 37°C. the Preparation of the complement of small rabbit (Cedarlane Laboratories, Hornby, ON) was diluted 1:50 and 25 μl was added to appropriate wells. The plate was incubated for 45 min at 37°C and then placed on ice. An aliquot of 10 µl was applied in triplicate on Columbia blood agar, grown for 4 days, and then produced� - Odile counting on the Cup to measure the number of colony forming units (CFU). Control Cup seeding with bacteria and complement, but without serum were used to calculate percentage of lysis. Bactericidal activity was defined as the highest dilution of serum that caused 50% lysis.

Blood serum taken before immunization or serum obtained from rabbits immunized with a mixture of a carrier protein and either LPS-OH, orastream, or orastream(PJ2) (with adjuvant) did not show or showed low levels of bactericidal activity against the homologous strain ofH. pylori26695 HP0826::Kan or wild-type strain 26695. Pastamania serum obtained from rabbits immunized with LPS-OH-TT-4, orastream-BSA-2, orastream-TT and orastream(PJ2)-TT, showed statistically significant functional activity against mutant 26695 HP0826::Kan and the corresponding wild-type strains (table 14), and serum after vaccination, obtained from rabbits immunized with a conjugate orastream-TT, showed the highest functional activity against the wild strain 26695 (table 14).

Table 14
Bactericidal activity against strains ofH. pylori26695 HP0826::Kan and 26695 rabbit antisera induced by conjugates
a Each rabbit received 10 μg of carbohydrate per injection.
bCorresponding rabbit preimmune serum.

In addition, the bactericidal activity of rabbit sera was also tested against selected clinical isolates ofH. pylorinamely strains 002CL, 0153CL and 058CL (Altman et al., 2008). These isolates were selected as representatives of strong, moderate and weak binding agents based on their values OD450in tests WCE with mAb anti-α1,6-glucan: strain 002CL - OD4501,361, strong binding agent; strain 153CL - OD4500,29, moderate binding agent; and strain 058CL - OD4500,162, weak linking agent. It is important to note that only pastamania serum of rabbits immunized with conjugate orastream-TT or orastream(PJ2)-TT, showed functional activity with all three tested clinical isolates (table 15).

153CL
Table 15
Bactericidal activity against clinical isolates ofH. pylorirabbit antisera induced by conjugates
The immunogenSerum bactericidal titer (50% cytolysis) againstH. Pylori1:
002CL058CL
LPS-OH-TT-4 controla1131
107
407
79
0
25
orastream-BSA-2
controla
864
93
1259
75
66
53
orastream-TT
controla
509
28
1445
36
311
21
orastream(PJ2)
controla
374
18
1738
20
215
21
aCorresponding rabbit preimmune serum.

Example 8. Defence research using conjugate orastream-TT in mice

Potential conjugate orastream-TT as a vaccine candidate was evaluated in outbred mice CD-1.

Groups of 5 mice CD-1 was vaccinated four times intranasally with weekly intervals 25 μg/mouse conjugate orastream-TT, with added adjuvants 1 μg/mouse recently isolated toxin (CT; Sigma), to 31.5 μg/mouse the acellular sonicator PJ2 added as an adjuvant of 1 μg/mouse recently isolated toxin (control), or saline (control). A week after the last immunization were collected on�the serum samples, the feces and vaginal swabs and tested forH. pylori-specific IgG and IgA. The levels of serum IgG antibodies and serum, fecal and vaginal IgA were determined using standard ELISA test. The tablets were coated with 1 μg/well LPS of strain 26695 HP0826::KanH pylorias described in the present application. Serum samples were diluted 1:100 test for IgG and 1:50 for the test with IgA. Samples of feces were diluted 1:2 and vaginal samples were diluted 1:20. Determined the levels of antibodies specific to H.pyloriand the data were analyzed using the Mann-Whitney test with the use of GraphPad software, version 5.0.

Five weeks after the first immunization, the mice oral gavage was administered three times a day ~108SOMEH. pyloristrain PJ2 (Altman et al., 2003). Four weeks later mice were killed, counting live bacteria isolated from their stomachs, and the data were analyzed using the Mann-Whitney test using GraphPad software, version 5.0.

Intranasal immunization with a conjugate orastream-TT plus CT created a very strong IgG response specific to LPSH. pylori26695 HP0826::Kan, which was measured using ELISA, and was also found to moderate the response of serum IgA specific toH. pylori26695 HP0826::Kan (tables 16 and 17, Fig. 7). In addition, the formation of IgA specific for LPSH. pylori26695 HP0826::Kan, was also found in the vagi�social or fecal samples (p< 0,01) (table 17, Fig. 7).

Table 16
Levels ofH. pyloriLPS-specific serum IgG against LPS of strain 26695 HP0826::Kan measured using ELISA. Samples of sera were collected one week after the last immunization and before the bacterial infection with a strain PJ2H. pylori
TimeOD405(Mean±SEM) IgG
orastream-TT+CTconicet PJ2+CTsaline
Original value3,004±0,3151,467±0,2950,089±0,017

Table 17
Levels ofH. pyloriLPS-specific serum, fecal and vaginal IgA against LPS of strain 26695 HP0826::Kan measured using ELISA. The samples were collected one week after the last immunization and before the bacterial infection with a strain PJ2H. pylori
SampleOD405(Mean±SEM) IgA
orastream-TT+CTconicet PJ2+CT saline
Serum0,473±0,1001,099±0,3970,036±0,004
Excrement0,689±0,5050,674±0,3210,017±0,003
Vaginal wash0,927±0,6740,269±0,168-0,008±0,002

All groups of mice then urogastrone was inoculable failed to be typed colonizing mice with the strain PJ2H. pylorithat is, as shown above, contains a long α1,6-glucan (Altman et al., 2003). Protective action is defined as a statistically significant decrease in bacterial burden in vaccinated groups compared to control groups. The results of colonisation for groups of mice immunized intranasally conjugate orastream-TT plus CT was statistically significant (p <0,05) (Fig. 8). In addition, these results suggest that education only IgA serum antibodies specific to LPS 26695 HP0826::KanH. pylorimay not be sufficient to protect against bacterial contamination and that the education of serum IgG antibodies specific to LPS, may also contribute to enhanced protection.

Example 9. Accessed� and description of the conjugate Dextran-BSA and studies of immunogenicity and functional activity

Conjugate Dextran-BSA was prepared by dissolving 10 mg of Dextran T5 (MW 5 KDa, Pharmacosmos A/S, Holbaek, Denmark) in 250 ál of 0.2 M borate buffer, pH 9,0; the specified solution was added to a solution (250 µl) containing 1.8-diamino-3,6-dioxaoctyl (25 µl) and cyanoborohydride sodium (10 mg) in 0.2 M borate buffer, pH 9,0 (as described by Roy et al., 1984). The reaction was performed for 5 days at 55°C. the reaction Product was purified as described above, to obtain conjugates on the basis of LPS.

This reaction has led to the introduction of the spacer. Then received a derivative of the amino group in the molecule of the spacer by reaction with N-hydroxysuccinimidyl ether 3-multimediaphoto acid, as described for conjugates on the basis of LPS (see Example 5). Glycoconjugate received by milirovanie a carrier protein and the addition of tarirovannogo protein to maleimidopropionamide the Dextran T5. The mole ratio of Dextran to BSA in the conjugate was 20:1, and the yield was 32%, based on the carbohydrate content.

Studies of cross-reactivity was performed with posthumanity sera of rabbits immunized with Dextran-BSA against LPS of strains ofH Pyloricharacteristic of different glycation LPS, LPS and selected mutant strains (table 18).

Table 18
Educationbrabbitandantibodies in response to PTS�by LPS H Pyloricaused by conjugate Dextran-BSAc
andEach rabbit received 10 μg of carbohydrate per injection.
bCaption postmonday serum 1:100.
cOD450± 10%

Reactivity bastianich of sera obtained from rabbits that were immunized with Dextran-BSA, testified about the need for the presence of α1,6-glucan, as I have not received cross-reactivity with glucan-negative strains SS1 and SS1 HP0826::Kan.

Pastamania serum obtained from rabbits immunized with Dextran-BSA, exhibit functional activity against mutant 26695 HP0826::Kan and the corresponding wild-type strain (table 19).

Table 19
Bactericidal activity against strains ofH. pylori26695 HP0826::Kan and 26695 rabbit antisera induced by conjugate Dextran-BSA
The immunogenImmune serumSerum bactericidal titer againstH. pylori(50% destruction) 1:
26695 26695 HP0826::Kan
rabbits
Dextran-BSAa1259203
2374149
Controlb1513
aEach rabbit received 10 μg of carbohydrate per injection.
bCorresponding rabbit preimmune serum.

The ability of rabbit Bestimmung sera to recognize heterologiczne interviewee and failed to be typed strains and induce bactericidal antibodies reflects the possibility that a conjugate consisting of Dextran, a polymer containing a linear main chain of α1,6-linked repeating glucose residues, or conjugate containing optimized linear oligosaccharides consisting of a sequence α1,6-linked glucose residues, and a suitable protein carrier, can be sufficient to provide protection against infection withH. pylori.

Example 10. Studies of the specificity of anti-glycan�'s mAb

Received monoclonal antibodies that are specific to the strain O:3 HP0826::KanH. pyloriand studied their specificity.

Created hybridomas as previously described (Altman et al., 2005). Six mice BALB/c (Charles River Laboratories, St Constant, QC) were injected intraperitoneally (i.p.) 108(200 μl) fixed in formalin cells of strain O:3 HP0826::KanH. pylori5 times for 82 days to reach a significant titer. Entered a final intravenous (i.v.) injection and 3 days later the merged cells. Spleen cells of two mice connected with cells plasmacytoma line Sp2/O by Kohler and Milstein (1975). Organized the screening of the supernatants of the original merged cells from 368 holes using indirect enzyme-linked immunosorbent assay (ELISA). For screening hybridomas with an ELISA titration microplates (ICN, Costa Mesa, CA) were coated with 10 μg/ml of the corresponding LPSH. pyloriO:3 HP0826::Kan in 50 mm carbonate buffer, pH of 9.8, for 3 hours at 37°C. After washing with PBS the plates were blocked with 5% (wt/V) solution of bovine serum albumin (BSA) in PBS. Added depleted supernatants, and the plates were incubated for 3 hours at room temperature. The plates were washed with PBS and added a second antibody conjugate with horseradish peroxidase goat antimachine IgG+IgM (Caitag, So. San Francisco, CA) for 1 hour at room temperature. After the final wash add substrate 3,3',5,5'-tetramethylene�siden (TMB) (KPL, Gaithersburg, MD) and the reaction was stopped with 1 M phosphoric acid. The absorbance was determined at 450 nm with a spectrophotometer for titration microplates (Dynatech, Chantilly, VA). After this stage was performed indirect ELISA test as described in Example 6.

Two suitable hybridomas were obtained by cloning by serial dilutions. One clone, 1C4F9, IgM, was selected for further description. Ascitic fluid was obtained in mice BALB/c, and a monoclonal antibody 1C4F9 (mAb) was isolated from ascitic fluid with the use of IgM-specific columns for affinity chromatography (Pierce, Rockford, IL) according to the manufacturer's instructions.

Studies of the specificity of glycan-specific antibodies was performed with the use of a series of linear oligosaccharides consisting of a sequence α1,6-linked glucose residues Glc→[α(1→6)Glc]n=1-6and selectively purified LPSH. pylori. For inhibition ELISA, preparing serial dilutions of inhibitors, linear α1,6-linked gluconeogenesis oligosaccharide isomalto-series [Glcα1→(6Glcα1→)n=1-6] (USBiologicals, Swampscott, MS) or LPSH. pylori26695, O:3 and PJ2, and mixed with the prepared by dilutions of purified antibodies 1C4F9 that gave an OD value450=1. After incubation (22°C, 15 min) the mixture was transferred into a source blocked microtiter tablet with adsorbed antigen LP, where the mixture is incubated for 2 hours at 37°C. After this stage was followed by indirect ELISA test as described in Example 6. The percentage of inhibition was calculated by the following formula:

% inhibition = 100×[(OD with inhibitor - OD without inhibitor)/ OD without inhibitor]

For each inhibitor were building the curve of inhibition as a function of log concentration and determined the concentration required for premaxillae inhibitory concentration (IC50from extrapolation of the curves.

Inhibitory properties had optimal values when using isomaltulose (n=5) and smaltimento (n=6) (table 20). LPS strain PJ2H. Pylorias shown previously, with an average of six to eight residues in glycanova chain (Altman et al., 2003), was the most effective inhibitor (table 20).

Table 20
Inhibition ELISA series linear α1,6-linked gluconeogenesis oligosaccharide and the LPS of strains ofH. pylori26695, A:3 and PJ2
InhibitorIC50b
Isomaltose Glcα1→(6Glcα1→)n=10
Isomaltulose Glcα1→(6Glcα1→)n=2a10000
1600
Isomaltose Glcα1→(6Glcα1→)n=4460
Isomaltulose Glcα1→(6Glcα1→)n=5290
Isomaltulose Glcα1→(6Glcα1→)n=6210

26695 LPS (n=3-4)170
O:3 LPS (n=5-6)52
PJ2 LPS (n=6-8)0,29
aThe α1,6-linked glucose residues
bThe concentration of the oligosaccharide or LPS (µg/ml) required for 50% inhibition.

Example 11. Research availability and bactericidal tests using antiglobulin mAb

Antiglutamate mAb were easily accessible on the surface of live bacteria of representative strains ofH. Pylorias shown by studies of indirect if microscopy (table 21). α1,6-glucan and CagA can be simultaneously seen on the bacterial surface.

To determine the bactericidal activity of a monoclonal antibody, a tenfold serial dilution of mAb 1C4F9 from lisanna� complement ascitic fluid (50 µl) was added to each well followed by the addition of bacterial suspension (25 µl) and preincubated for 15 min at 37°C. After this stage the test was performed as described in Example 7. Binding of cell surface 1C4F9 correlated with functional activity, which was determined using antibacterial tests against wild and mutant strains ofH. pylori(Table 21).

Table 21
Characterization of binding of mAb 1C4F9 with α1,6-glucan strains ofH. pyloriwith WCE, IF-adhesion and bactericidal tests
aNotation:++++ strongly positive; - , negative.
bBactericidal (BC50) titers represented as the inverse of the dilution of antiserum which reduces the number of living cells by 50%.
cMonteiro, 2001.
dLoganet al., 2000.
eAltmanet al., 2003.
dNot specified.

Although preferred embodiments of the invention have been described above, consideration should be given to understand that it can be made various modifications and it is intended that the appended claims cover all such modifications that may correspond to the nature and scope of the invention.

LINKS

In�e patents, patent applications, and publications referred to in this application and throughout the application that are included, therefore, as a reference.

1. Dedicated α1,6-glucan-containing compound Helicobacter pylori, containing the structure of formula I:

in which R represents a trisaharid α-DDHep-3-α-L-Fuc-3-β-GlcNAc substituted α1,6-glucan containing from 4 to 12 α1,6-linked glucose residues, linked to the α1,3-DD-heptane, in which the last remnant of DD-Hep α1,3-DD-heptane Kairouan the remnant β-GlcNAc.

2. The compound according to claim 1, wherein the glucan contains 3-12 α1,6-linked glucose residues.

3. The compound according to claim 1, wherein the fragment contains heptane 2-6 α1,3-linked residues, heptose.

4. The compound according to claim 1 in which R contains

where is the rest of L β-GlcNAc is connected with 0-2 Hep G, n = 1-11, and m is 0-4.

5. The compound according to claim 4, in which n is equal to 9.

6. The compound according to claim 4, in which m is equal to 2.

7. The compound according to claim 5, in which m is equal to 2.

8. The compound according to any one of claims.1-7, in which the structure of formula I further comprises a lipid molecule And covalently attached to the Kdo residue C.

9. The compound according to claim 8, in which the molecule of lipid A O-decelerate or cleaved by hydrolysis to�tosignal connection Kdo residue.

10. The compound according to claim 8, which is isolated or purified from a strain of H. pylori NR::Kan.

11. Conjugate to induce an immune response against H. pilory containing essentially linear α1,6-glucan-containing compound according to any one of claims.1-10, anywhereman with the linker molecule, the carrier protein or a combination of both by means of a Kdo residue of the inner portion of the bark.

12. The conjugate according to claim 11, characterized in that the compound containing essentially linear α1,6-glucan, is a dextran.

13. Conjugate according to any of claims.11 and 12, characterized in that the protein carrier is a tetanus toxoid, bovine serum albumin, CRM or CRM197.

14. Composition for induction of an immune response against H. pilory, containing one or more compound according to any one of claims.1-10 and/or conjugate according to any of claims.11-13, and one or more pharmaceutically acceptable carriers.

15. An antibody that recognizes the compound containing an α1,6-glucan epitope according to claim 1, obtained with this connection.

16. The antibody according to claim 15, where the antibody is a monoclonal antibody which is produced by hybridoma 1C4F9, which is deposited in National Microbiology Laboratory, Public Health Agency of Canada, July 30, 2009 under the serial number 300709-01.

17. The use of monoclonal antibodies according to claim 15 for the induction of complement-mediated bacteriolysis strains of N. pylori, Express�tion of α1,6-glucan, in the individual, if necessary.

18. A method of inducing complement-mediated bacteriolysis strains of N. pylori expressing α1,6-glucan, comprising administering a monoclonal antibody according to claim 15 to the individual, if necessary.

19. The use of an effective amount of a composition according to claim 14 for inducing an immune response against N. pylori infection in the individual.

20. The use according to claim 19, characterized in that the connection anywhereman with a suitable carrier protein.

21. The use according to claim 19, characterized in that the composition anywhereman with a suitable carrier protein via 2-keto-3-deoxy-octulosonic acid (Kdo) of lipopolysaccharide.

22. A method of inducing an immune response to N. pylori infection in the individual, comprising the introduction of a specified individual of an effective amount of a composition according to claim 14.

23. A method according to claim 22, characterized in that the connection anywhereman with a suitable carrier protein.

24. A method according to claim 20, characterized in that the composition anywhereman with a suitable carrier protein via 2-keto-3-deoxy-octulosonic acid (Kdo) of lipopolysaccharide.

25. Immune antiserum to neutralize N. pylori infection in the mammal, obtained by immunization of a specified mammal the immunogenic composition of claim 14.

26. Immune antiserum according to claim 25, antiserum contains IgG that recognizes α1,6-linked glucan epitope in gomologichnyi heterologous interviewee and failed to be typed strains of N. pylori mutant and wild-type.

27. Immune antiserum according to claim 26, characterized in that the IgG induces complement - mediated bacterials strains of H. pylori that produce α1,6-glucan mutant and wild type.



 

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22 cl, 20 dwg, 4 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology and biotechnology. There are presented versions of nucleic acids each of which codes a heavy-chain amino acid sequence of immunoglobulin IgG1. The above chain contains glycine-lysine dipeptide coded by ggaaaa, ggcaaa or gggaaa codon at the C terminal of the CH3 domain. There are described: a plasmid coding a heavy chain of immunoglobulin; version cells providing immunoglobulin IgG1 expression; a method for producing immunoglobulin in mammalian cells; a method for improving immunoglobulin expression in the mammalian cells; - using the versions of a nucleic acid.

EFFECT: using the invention provides preventing the by-product expression of weight 80 kDa that can find application in producing immunoglubulins.

18 cl, 7 dwg, 3 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly a polypeptide bearing a human BNP(1-32) epitope, for producing ligands that are directed against the human BNP(1-32) or human proBNP(1-108), where said polypeptide has the formula α1-R1-X1-FGRKMDR-X2-R22. Disclosed is use of said polypeptide to produce ligands directed against the human BNP(1-32) or human proBNP(1-108) and for producing a hybridoma which secretes a monoclonal antibody directed against the human BNP(1-32) or human proBNP(1-108). Disclosed is a method of producing a hybridoma which secretes a monoclonal antibody directed against the human BNP(1-32) or human proBNP(1-108), as well as the obtained hybridoma. Disclosed is a ligand which is specific to an epitope with the sequence FGRKMDR, as well as use thereof to detect human BNP(1-32) or human proBNP(1-108) in a biological sample. Disclosed are methods of detecting human BNP(1-32) or a human proBNP(1-108) derivative in a biological sample, a method for in vitro diagnosis, prediction, risk stratification or subsequent observation of long-term results of cardiac and/or vascular pathology in an individual, as well as a method for in vitro diagnosis of stroke in an individual using said ligand. Disclosed is a multi-epitope calibrator designed to obtain calibration curves for analysis of BNP(1-32), proBNP(1-108), as well as a kit for detecting human BNP(1-32) or human proBNP(1-108).

EFFECT: invention enables to efficiently detect cardiac and/or vascular pathologies in an individual.

15 cl, 17 dwg, 12 tbl, 18 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a lymphotoxin-alpha (LTα) antibody, including chimeric and humanised versions thereof. Where are disclosed compositions containing it, using it for preparing a medication for autoimmune disorders, a method of inhibiting ex vivo lymphotoxin-alpha activated cell proliferation with using the antibody under the invention, as well as a nucleic acid, an expression vector, a host cell and a method of producing the antibody that proceeds from using them.

EFFECT: present invention can find further application in a therapy of the autoimmune diseases.

51 cl, 21 ex, 27 dwg, 7 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used to obtain monoclonal antibodies against the Yersinia pestis V antigen. The strain of hybrid animal cells Mus musculus 2B8 is obtained by immunising BALB/c mice. The mice are immunised by four-time administration of a recombinant V antigen in a dose of 100 mcg/mouse. On the third day after the last immunication, splenocytes of immune mice (1×108 cells) are hybridised with mouse myeloma cells RZ-X63 Ag/8-653 (1×107 cells). The fusion agent used is polyethylene glycol (Sigma, USA). Hybridisation is followed by selection, screening, cloning and cryopreservation of the hybridoma. The strain is deposited in the state collection of pathogenic microorganisms and cell cultures (GKPM-Obolensk) under number N-20.

EFFECT: strain of hybrid cultured cells, which produces monoclonal antibodies which are specific to the Y pestis V antigen, is suitable for constructing test systems for detecting plague pathogens.

8 dwg, 3 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used to obtain monoclonal antibodies against the Yersinia pestis V antigen. The strain of hybrid animal cells Mus musculus 5G6 is obtained by immunising BALB/c mice. The mice are immunised by four-time administration of a recombinant V antigen in a dose of 100 mcg/mouse. On the third day after the last immunication, splenocytes of immune mice (1×108 cells) are hybridised with mouse myeloma cells RZ-X63 Ag/8-653 (1×107 cells). The fusion agent used is polyethylene glycol (Sigma, USA). Hybridisation is followed by selection, screening, cloning and cryopreservation of the hybridoma. The strain is deposited in the state collection of pathogenic microorganisms and cell cultures (GKPM-Obolensk) under number N-19.

EFFECT: strain of hybrid cultured cells, which produces monoclonal antibodies which are specific to the Y pestis V antigen, is suitable for constructing test systems for detecting plague pathogens.

8 dwg, 2 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: osteons and intermediate lamellas are delineated and framed as members of analysis; osteocyte lacunae are numbered in each formed image; total number is presented, as well as a number of lacunae containing minerals in an amorphous phase. A percentage of the latter is described as a coefficient of osteocyte involvement in bone matrix mineralisation. If the minerals in the amorphous phase are found in less than 30% of lacunae, mineralisation activity of osteocyte is considered to be low. The presence of the amorphous minerals in 30-80% of lacunae shows moderate activity, and if more than 80% of lacunae contain the minerals in the amorphous phase, high activity is stated.

EFFECT: improved assessment accuracy.

1 ex, 2 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to binding compositions for insulation articles based on mineral wool. Disclosed is a binding composition based on mineral felt or glass fibre, which includes at least one saccharide, at least one organic polycarboxylic acid containing 2-4 carboxylic functional groups and having molecular weight less than or equal to 1000, and at least one polyorganosiloxane containing at least one functional group capable of reacting with at least one of the components of the binding composition. The invention also discloses a sound- and/or heat-insulation article and coating made of mineral fibre, glued using the disclosed composition and a method of producing a sound- and/or heat-insulation article.

EFFECT: disclosed binding composition does not contain formaldehyde and endows insulation articles with low water absorption.

25 cl, 2 tbl, 8 ex

Edible film coating // 2532180

FIELD: chemistry.

SUBSTANCE: invention relates to compositions of edible films which contain xanthan and chitosan. The invention is used in pharmaceuticals, medicine, veterinary, food or cosmetic industry. An edible film coating made from a thickener, chitosan, water, a solvent and glycerol. The thickener used is a microbial polysaccharide - xanthan, the chitosan solvent used is 3% citric acid solution, with the following ratio of components, wt %: xanthan - 50-55; chitosan - 10-15; water - 5-10; 3% citric acid solution - 5-10; glycerol - 5-10; 3% methyl cellulose solution - the balance.

EFFECT: invention enables to obtain a homogeneous film based on xanthan and chitosan.

2 dwg

FIELD: chemistry.

SUBSTANCE: method of producing fibre involves mixing hydrosilicate filler, which is pre-dispersed in an aqueous medium with pH 5-7 in an ultrasonic field with frequency v=20-100 kHz for 5-60 minutes, with chitosan in an amount which corresponds to concentration thereof in the solution of 1-4 wt %. The amount of the filler is 0.05-2% of the mass of the chitosan. The obtained mixture is intensely stirred at temperature of 20-50°C for 20-60 minutes. Concentrated acetic acid is then added in an amount which enables to obtain aqueous acetic acid solution with concentration of 1-8 wt % in the mixture. The mixture is intensely stirred at temperature of 20-50°C for 20-250 minutes. The mixture is filtered and deaerated. The fibre is formed through a draw plate into an alcohol or alcohol-alkaline precipitation agent, wherein the shear velocity of the solution in the plane of the cross-section of the capillary when the solution passes through the draw plate is selected in the range of 1.0-10-3 s-1. The fibre is stretched by 10-120%, washed with water and dried at temperature of 20-50°C. The fibre contains chitosan and hydrosilicate filler - halloysite, chrysotile, montmorillonite - in form of nanoparticles in amount of 0.05-2% of the mass of chitosan. The fibre has a single-phase homogeneous structure which corresponds to the basic form of chitosan.

EFFECT: obtaining a biocompatible, biodegradable composite elastic and strong fibre based on chitosan for use in medicine and biotechnology, particularly in suture filaments and in cellular engineering.

2 cl, 3 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: biopolymer composition contains a polymer which swells in water, the polymer being water-soluble preparations based on natural biopolymers of collagen or chitosan, phospholipid, oiling substances - fat emulsion based on modified natural and synthetic fats and/or oil, a bactericidal preparation and water-soluble dimethicone.

EFFECT: invention enables to optimise makeup of the composition, increases its stability over time, widens the range of processing properties which enable to obtain a considerable effect when restoring and stabilising properties of a fur semi-product which has lost its aesthetic and operational properties.

2 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to fluorinated compounds, method of their obtaining, fire-extinguishing compositions which include fluorinated compounds, and methods of fire extinguishing, control or prevention by means of said compositions. Fire-extinguishing composition contains solvent and fluorinated compound, representing fluoralkenylpoly[1,6]glycoside and containing C1-C20 fluorocarbon group. Compounds and compositions, described in the document, are useful as intermediate compounds in obtaining compositions of aqueous film-generating foams or as additives to compositions of aqueous film-generating foams, applied in extinguishing burning fuel and solvents.

EFFECT: compounds enhance fire-extinguishing properties and improve foam stability to a considerable degree.

23 cl, 1 tbl, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a method for preparing sodium salt of hyaluronic acid modified by boron compounds with no fluid medium added. The method consists in the fact that powdered sodium salt of hyaluronic acid together with a modifying agent and mixed modifying agents is pre-homogenised in a mixer at temperature ranging within 20° to 50°C; thereafter the prepared homogenous powder mixture is simultaneously exposed to pressure and shearing deformation in a mechanochemical reactor at temperature ranging within 20° to 50°C and pressure 5-1000 MPa.

EFFECT: invention provides preparing boron-containing sodium salt of hyaluronic acid applied in boron neutron capture therapy in one-stage process parameters with no fluid medium added which requires low power, labour and water consumptions.

13 cl, 15 ex

FIELD: food industry.

SUBSTANCE: thickening composition includes xanthum gum powder and a potassium salt bound to the xanthum gum powder surface wherein 0.5 - 7 wt. parts of the potassium salt bind 100 wt. parts of xanthum gum.

EFFECT: thickening composition, when added in a small quantity, quickly imparts viscosity to the used water containing material and may significantly reduce work time expenditures.

4 cl, 1 dwg, 2 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a viscoelastic composition for thickening, forming gels and for use as a surfactant. The composition contains water in amount not less than approximately 7% of the total weight of the composition, at least one inorganic salt in amount ranging from approximately 0.5 to approximately less than 15% of the total weight of the composition, at least one cationic carboydrate ether for regulating viscoelasticity of the composition. The inorganic salt is selected from a group comprising sodium chloride, potassium chloride, ammonium chloride, calcium chloride, sodium bromide, calcium bromide, zinc bromide, calcium formate, caesium chloride, caesium bromide and combinations thereof. The cationic carbohydrate ether contains one or more cationic fragments and one or more carbohydrate fragments bonded to one or more linking fragments through an ether bond. The linking fragment is selected from a group comprising an oxygen atom which forms an ether bond between two carbohydrate fragments, hydrocarbyl groups, and substituted hydrocarbyl groups. The cationic fragment contains one or more quaternary ammonium groups. The cationic carbohydrate ether contains at least one hydrophilic fragment. The hydrophilic fragment is chemically bonded to the substitute on the cationic fragment or the linking fragment through an ester, carboxamide or carboxamidine functional group.

EFFECT: invention can be used in well fluids for extracting hydrocarbons, which increases efficiency, lowers loss of fluid and increases the return of expensive and sometimes toxic metal salts.

19 cl, 18 dwg, 1 tbl, 13 ex

FIELD: chemistry.

SUBSTANCE: invention relates to methods for synthesis of new cross-linked salts of hyaluronic acid (HA) which are modified with vitamins - natural polymer from the polysaccharide family. The method involves chemical reaction of a salt of hyaluronic acid, vitamins together with at least one cross-linking agent, simultaneously subjecting the initial reagents to pressure ranging from 5 to 1000 MPa and shear deformation in a mechanochemical reactor at temperature ranging from 20°C to 50°C. The reactor used is preferably a Bridgman anvil or an auger-type device, e.g. a double-screw extruder.

EFFECT: design of a universal environmentally safe method which enables synthesis of a range of new, cross-linked salts of hyaluronic acid which are modified with vitamins, in a single-step production cycle in the absence of a liquid medium, to obtain desired products with quantitative output; the method does not require large energy-, labour- and water inputs, enables use of the most diverse salts of hyaluronic acid as initial reagents.

19 cl, 19 ex

FIELD: chemistry.

SUBSTANCE: invention relates to methods for synthesis of cross-linked salts of hyaluronic acid (HA) which are modified with folic acid - a natural polymer from the polysaccharide family. The method involves chemical reaction of a salt of hyaluronic acid, folic acid together with at least one cross-linking agent, simultaneously subjecting the initial reagents to pressure ranging from 5 to 1000 MPa and shear deformation in a mechanochemical reactor at temperature ranging from 20°C to 50°C. The reactor used is preferably a Bridgman anvil or an auger-type device, e.g. a double-screw extruder.

EFFECT: design of a universal environmentally safe method which enables synthesis of a range of new, cross-linked salts of hyaluronic acid which are modified with folic acid, in a single-step production cycle in the absence of a liquid medium, to obtain desired products with quantitative output; the method does not require large energy-, labour- and water inputs, enables use of the most diverse salts of hyaluronic acid as initial reagents.

19 cl, 18 ex

FIELD: chemistry.

SUBSTANCE: invention relates to methods of obtaining and modification of a hyaluronan derivative, containing an aldehyde group in position (6) of a glucosamine polysaccharide fragment. Claimed is a hyaluronic acid derivative. The derivative is oxidised by position 6 of the glucosamine fragment to the aldehyde (formula X)

.

Its hydrated form is called geminal diol (formula Y)

.

A method of obtaining the said hyaluronic acid derivative includes the interaction of hyaluronic acid with Dess-Martin periodinane (DMP) in a polar aprotic solvent. As the polar aprotic solvent, dimethylsulphoxide is preferably used. A method of modification of the obtained hyaluronic acid derivative is also claimed. The oxidised hyaluronic acid derivative is subjected to interaction with an amine of the general formula H2N-R or with hyaluronan, substituted with the group -R-NH2, where R is an alkyl with a linear or branched C1-C30 chain and optionally contains aromatic and heteroaromatic groups.

EFFECT: invention makes it possible to obtain the hyaluronic acid derivative with different possibilities of further modification due to the aldehyde group.

16 cl, 14 ex

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