Immunogenic conjugate of beta-propionamide-bound polysaccharide with protein applied as vaccine

FIELD: immunology.

SUBSTANCE: the innovation deals with new immunogenic conjugates of beta-propionamide-bound polysaccharide and N-propionamide-bound oligosaccharide with protein, and the method to obtain these conjugates has been suggested, as well. Conjugates should be applied to obtain vaccines against infectious diseases and cancer that enables to broaden the number of preparations applied in treating the above-mentioned diseases.

EFFECT: higher efficiency.

1 dwg, 2 ex, 8 tbl

 

The present invention relates to the immunogenic conjugates β-propionamides of polysaccharide with protein and to methods of producing these conjugates from bacteria, yeast or cancer cells. These conjugates are used as vaccines.

Bacterial infections caused by gram-positive bacteria, such as Streptococcus, Staphylococcus, Enterococcus, Bacillus, Corynebacterium, Listeria, Erysipelothrix, and Clostridium, and gram-negative bacteria such as Haemophilus, Shigella, Vibrio cholerae, Neisseria and some types of Esherichia coli, cause significant morbidity worldwide. This fact, coupled with emerging resistance, which are bacteria against antibiotics, determines the need for the development of bacterial vaccines. For example, streptococci represent a large and diverse genus of gram-positive bacteria, which was subdivided into several groups on the basis of antigenicity and structure of polysaccharides in their cell walls (26, 27). Two of these groups are associated with severe human infections. Streptococci group And cause a number of infectious disorders, including strep throat, rheumatic fever, streptococcal impetigo and sepsis. Streptococci group are important perinatal pathogens in the United States and in developing countries (37).

Gram-negative bacteria are also a significant cause of disease. To RA is the development and application of recently polysaccharide-protein vaccines directed against the bacterium Haemophilus influenzae type b (Hib), a bacterial infection Hib was the cause of numerous cases of mental retardation in children. Infection of N. menigitidis and E. coli K1 responsible for neonatal meningitis. Strains of gram-negative bacteria, E. coli, associated with severe disease, including death of the body, coming through the consumption of meat infected with strains of E. coli.

Conjugates of polysaccharides with other immunogenic molecule such as a polypeptide or protein, was used to initiate humoral immune responses to a number of gram-negative and gram-positive bacteria. Conjugation of polysaccharide or oligosaccharide with the polypeptide converts the immune response to the polysaccharide or oligosaccharide, which is generally T-independent in the T-dependent response.

In previous prior art reveals both direct and indirect connection of polysaccharides to proteins with the formation of conjugates (result announced in the link (11) and U.S. patent No. 5 306 492). Methods of conjugating include diazoketone, thioester linkage, amidation, reductive amination and thiocarbamoylation for connection to a protein carrier.

Gever strategy et al., Med. Environ. Immunol., 165: 171-288 (1979) described the obtaining of some conjugates of capsular polysaccharide fragments of Klebsiella pneumoniae with nitrate ylethylamine linker by reductive amination and join sugar derived using the result.

In U.S. patent No. 4 057 685, McIntire, described the lipopolysaccharide of Escherichia coli with reduced toxicity, covalently associated with a protein antigen as a result of interaction with galogenangidridy.

In U.S. patent No. 4 356 170, Jennings et al., describes how to obtain a polysaccharide-protein conjugates by the method of reductive amination.

In U.S. patent No. 4 673 574, 4 761 283 and 4 808 700, Anderson described obtaining immunogenic conjugates comprising the product of the reductive amination immunogenic capsular polysaccharide fragment derived from the capsular polymer of Streptococcus pneumoniae or N. influenzae containing regenerating the end, obtained using methods such as oxidative decomposition of periodate or by hydrolysis of glycosidic bonds, and a bacterial toxin or toxoid as the protein carrier.

In U.S. patent No. 4 459 286, Hillman et al., describes how to obtain a polysaccharide-protein conjugate by activating the polysaccharide of N. influenzae type b labronico, deriving activated polysaccharide with spacer elements molecule, 6-aminocaproic acid and joining the main protein of the outer membrane of Neisseria meningitidis using water-soluble carbodiimide with amide bond formation type of protein through a complex series of links from the spacer 6-aminocaproic acid to the polysaccharide.

In U.S. patent No. 4 965 338, Gordon describes the obtaining of a water-soluble covalent conjugate of a polysaccharide with diphtheria toxoid, where pure polysaccharide of N. influenzae type b activate labronica and immediately mixed with diphtheria toxoid modified ADH-spacer.

In U.S. patent No. 4 663 160, Tsay et al., described detoxified enduring polysaccharide gram-negative bacteria covalently associated with detoxificating protein derived from the same species of gram-negative bacteria, by means of a fragment having 4-12 carbon atoms.

In U.S. patent No. 4 619 828, Gordon et al., described conjugates of molecules of polysaccharides of pathogenic bacteria, such as Haemophilus influenzae type b, Streptococcus pneumoniae, Neisseria meningitidis and Escherichia coli, with T-dependent antigens such as diphtheria and tetanus toxoid.

In U.S. patent No. 4 711 779, Porro et al., described vaccines based on conjugates of glycoproteins having trivalent immunogenic activity, containing antigenic determinants of the capsular polysaccharides of gram-positive bacteria, as well as any CRM197, the tetanus toxoid, or pertussis toxin.

In U.S. patent No. 5 306 492, Porro, described oligosaccharide conjugate with protein carrier, obtained by the interaction of the oligosaccharide having a terminal after-care group, with diaminoethane in the presence of pyridinoline, this reaction is vosstanovit the Noah amination, interaction laminirovannogo oligosaccharide product with a molecule having two functional groups, and then the interaction of the activated oligosaccharide product with protein carrier.

In U.S. patent No. 5 192 540, Kuo et al., described oligosaccharide conjugate protein comprising the product of the reductive amination of oxidized polyribosyl-revitalift polysaccharide fragment derived from the capsular polysaccharide of Haemophilus influenzae type b, and protein of the outer membrane of Haemophilus influenzae type Century.

In the publication of a European patent application no EP 0747063 A2 describes the modified capsular polysaccharide containing various derivatives of sialic acid, and heterobifunctional linker molecule that is associated with media molecule. Linkers are used for N-alkylation of about 5 residues of sialic acid polysaccharide. Then the remaining amino acelerou propionic or acetic anhydride.

There is a need for more efficient purified immunogenic polysaccharide-protein conjugates, which can be obtained in a simple way and with a higher yield for large-scale gain-based vaccines immunogenic polysaccharide-protein conjugates.

This invention relates to immunogenic conjugate β-propionamide polysaccharide and ; -propionamide oligosaccharide with protein.

The purpose of this invention is the provision of a method of producing immunogenic conjugates β-propionamides of polysaccharide with protein, which has advantages compared to commonly used at present methodologies. A further purpose of this invention is the provision of pharmaceutical compositions, vaccines and other immunological reagents derived from immunogenic conjugates β-propionamides of polysaccharide with protein.

Provided is a method of obtaining immunogenic polysaccharide-protein conjugates, which includes de-N-acetylation of polysaccharide or of oligosaccharide by primary or enzymatic hydrolysis with subsequent N-akrilovye N-deacetylating polysaccharide. N-acryloylcholine polysaccharide directly attached to the carrier protein with the formation of immunogenic conjugate β-propionamides of polysaccharide with protein.

The polysaccharide capsule and cell surface can be extracted in accordance with this invention or from a cell supernatants bacteria, yeast or mammals, or directly from bacterial cells, yeast or mammalian, by hydrolysis sensitive to the effects of communication base, which connects the floor of the saccharide with other cellular components, or by enzymatic hydrolysis. Part of the N-acetyl groups removed by hydrolysis of the polysaccharide are substituted with N-acryloyl group, which, in turn, are directly connected with protein, with the formation of a conjugate according to the invention.

In one aspect of the present invention are oligosaccharides and polysaccharides, which are directly attached to the protein (proteins) in several provisions.

In another aspect the present invention provides a method of immunization of a mammal against bacterial or yeast infections, or cancer, which comprises the administration to a mammal an effective amount of the vaccine according to the invention to prevent the infection from a pathogenic organism or a cancer.

An aspect of the present invention is a method of inducing the production of antibodies in mammals using conjugates β-propionamides of polysaccharide with protein that protects a mammal against infection or disease.

Another aspect of the present invention are immunoglobulin and the selected antibody-containing substances that occur in response to immunization with conjugates β-propionamides of polysaccharide with protein. Such immunoglobulin and the selected antibody used as drugs and diagnostic reagents.

BRIEF ABOUT ISANA DRAWING

Drawing 1. Schematic illustration of the method of producing immunogenic conjugates β-propionamides of polysaccharide with protein.

DETAILED description of the INVENTION

This invention presents a new polysaccharide-protein conjugate and oligosaccharide-protein conjugates used as immunogens and vaccines against bacterial infections, yeast infections, and also as anti-cancer drugs. Polysaccharides or oligosaccharides that are used for the formation of immunogenic conjugates β-propionamides of polysaccharide with protein, derived from sources of polysaccharides or oligosaccharides, which include, but are not limited to, gram(+) or gram(-) bacteria, yeast, cancer cells or cancer tissue and the like, in which the polysaccharide or oligosaccharide is used as a virulent factor of the cells during the formation of the protective mechanisms of the host. Polysaccharide-protein conjugates of the present invention are formed as a result of the direct connection of N-grilliravintola polysaccharide to a protein by attaching the nucleophilic sites of proteins by Michael.

Polysaccharides or oligosaccharides can be obtained from a number of sources, including gram-negative, gram-positive bacteria, yeast, cancer cells or recombinant forms of each of them, by basic or enzymatic hydrolysis of connection, which connects the polysaccharide or oligosaccharide with cellular components. The polysaccharide or oligosaccharide can be extracted from an organism or cell by bringing into contact of an organism or cell, or a solution containing the fragments of the organism or cells, with a base or an enzyme. Then, after the main or enzymatic hydrolysis of the polysaccharide or oligosaccharide can be distinguished using a variety of methods. Non-limiting examples of gram-positive bacteria and recombinant strains suitable for use in accordance with this invention, are Streptococci, Susceptible, Enterococci, Bacillus, Corynebacterium, Listeria, Erysipelothrix, and Clostridium. In particular, the use of Streptococci is more preferred, and the use of types Ia, Ib, II, III, IV, V and VIII Streptococci group is most preferred. Non-limiting examples of gram-negative bacteria and their recombinant strains suitable for use in accordance with this invention, include Haemophilus influenzae, Neisseria meningitides, Escherichia coli, Salmonella typhi, Klebsiella pneumoniae Pseudomonas aeruginosa. In particular, the use of N. influenzae type b, N. meningitides types b, C, Y and W135, E. coli E. coli K1 and K92 is preferable. Examples of yeast suitable for use in the present invention include, but are not limited to, Cryptococcus neoformans. When the minimum level of cancer cells or cancer tissues, suitable for use in the present invention include, but are not limited to, small cell lung carcinoma, neuroblastoma, breast cancer, carcinoma of the colon, etc.

For hydrolysis of the polysaccharide or oligosaccharide in water or an organic solvent in accordance with this invention using methods known in this field can be used in a wide range of conditions. The degree of hydrolysis of N-acetyl relations carbohydrates can be controlled by the reaction conditions. In one embodiment at least about 50% N-acetyl groups are removed by hydrolysis, preferably removed from approximately 50% to 100%, more preferably remove approximately 90% or more of the native N-acetyl groups. In a particular embodiment, about 95% or more N-acetyl groups of the polysaccharide is subjected to hydrolysis by treating gidrolizuut reagent.

Capsular polysaccharides that are extracted in the presence of a base, are polysaccharides, which have no sensitive to the effects of the Foundation of the Deputy, which cannot be substituted, such as O-acetyl groups required for demonstration of immunogenicity. Other capsular polysaccharides that are extracted in the presence of a base, represent the policy is Aridi, have no fosfomifira communication and uronic acid residues attached at the 4 position.

In the preferred embodiment of the basic hydrolysis of CPS (capsular polysaccharides) are extracted from group b Streptococci (GBS). In the most preferred embodiment of the CPS extracted from GBS type Ia, Ib, II, III, V and VIII.

In another preferred embodiment of the basic hydrolysis CPS extracted from S.pneumoniae. In a more preferred embodiment of the basic hydrolysis CPS extracted from S.pneumoniae types III, IV and XIV.

In another preferred embodiment of the basic hydrolysis CPS extracted from the bacteria Neisseria or Escherichia. In a more preferred embodiment of the basic extraction CPS extracted from Neisseria meningitidis type b, C, Y or W135, Escherichia coli K1 or Escherichia coli K92.

Polysaccharides, which are subject to enzymatic deacetylation, are polysaccharides that do not have any unstable to the enzyme Deputy needed for demonstration of immunogenicity in which the Deputy may not be replaced or substituted immunogenic fragment, these polysaccharides include, but are not limited to, GBS, etc.

A. Obtaining N-acryloylmorpholine polysaccharides

1. The deacetylation of polysaccharides

(a) the Original substance

The polysaccharide or oligosaccharide can be obtained using alkaline and enzymatic hydrolysis of concentrated bacterial cells, yeast cells or mammalian cells, or recombinant forms of these cells, or from supernatants homogenised cells, or air-conditioned environment, using standard methods known in this field. The polysaccharide or oligosaccharide may be isolated and purified using standard methods known in this field. As starting substances can also be used polysaccharide or oligosaccharide from commercial sources.

Methods of extraction of polysaccharide used depend on the specific polysaccharide. A common method is the use of ionic detergent for the formation of a complex with a charged polysaccharide. This complex is precipitated and isolated. Then the complex is dissolved in the solution with high ionic strength, such as calcium chloride, and then the polysaccharide precipitated with ethanol.

Isolated and purified polysaccharides and oligosaccharides obtained for use in this invention, intended for application to humans, preferably contain less than 1% impurities of nucleic acids and protein. Due to the presence of inorganic salts after cleaning is often observed purity, the degree of which is 80-100% carbohydrates.

b) Basic hydrolysis

To remove the N-acetyl groups of the purified polysaccharides or oligosaccharides can processing is to be grounds. Non-limiting examples of bases that can be used in accordance with this invention, are NaOH, KOH, LiOH, Panso3, Na2CO3To2CO3, KCN, Et3N, NH3H2N2H2, NaH, NaOMe, NaOEt or KOtBu. Such grounds as NaOH, KOH, LiOH, NaH, NaOMe or KOtBu most effectively used in the range of 0.5 N to 5.0 n Such grounds as NaHCO3, Na2CO3To2CO3and KCN can be used in such high concentrations, which only allows their solubility. Organic base such as Et3N can be used in an environment at a high (50-100%) concentrations, as there is such an agent, such as water or alcohol, to effect hydrolysis. Such grounds as NH3or H2N2H2can be used in almost any concentration, including 100%. You can use such solvents as water, alcohols (preferably C1-C4), dimethylsulfoxide, dimethylformamide or mixtures of these and other organic solvents. Solutions of bases, including water, are the most preferred.

The range of pH that is most effective for the removal of N-acetyl groups of the polysaccharide or oligosaccharide is from about 9 to 14, and the optimal pH value of approximately 12. Then N-deacetylating olished clear of the remaining reagents by ultracide using membrane or dialysis using standard methods, known in this field.

(C) Enzymatic hydrolysis

For enzymatic removal of N-acetyl groups of the polysaccharide or oligosaccharide is possible to use the enzyme N-deacetylase. In one embodiments the enzyme N-deacetylase used to remove N-acetyl residues of polysaccharides or oligosaccharides, as described in references 47, 48 and 49. Enzymatic hydrolysis of the polysaccharide or oligosaccharide and the deacetylase enzyme is mixed with the appropriate enzyme buffer system under appropriate pH and temperature and allowed to interact for a period sufficient to remove the N-acetyl groups. To obtain the N-deacetylating polysaccharide in one of the embodiments of the polysaccharide and enzyme N-deacetylase mixed with the appropriate enzyme buffer system, for example 50 mm MES, 10 mm MnCl2, pH 6.3 at 37°C, for 60 minutes. The reaction is stopped by using the appropriate stop solution, for example 1M monochloracetic acid, 0.5m NaOH, 2M NaCl, or by dilution of the appropriate buffer solution.

2. N-acryloylmorpholine polysaccharide

Alkaline or enzymatic hydrolysis of the polysaccharide or oligosaccharide leads to the destruction of N-acetyl groups from sialic acid and aminosugar residues of polysaccharides or oligosaccharides. After hydrolysis of the polysaccharide or oligos harid subjected to N-acrylonitril to the required extent with the help of a number of acryloyloxy agents.

In one embodiment of this method involves adding aceloularoer reagent for N-acryloylmorpholine N-deacetylating polysaccharide or oligosaccharide. Examples acryloyloxy reagents include, but are not limited to, akriloilkhlorida, acrylamide, acrylic acid and dehydrating agent such as DCC, CH2CHCOCN etc. used in excess in the concentration component of approximately 1M. In the method of N-acryloylmorpholine N-deacetylating polysaccharide pH adjusted to values in the range from about 9 to 11, preferably equal to approximately 10, and maintains this value during the reaction. The temperature during the reaction is from about 2°to 8°C, preferably about 4°C. the Reaction is carried out over a period of approximately 1 hour. The N-acryloylcholine polysaccharide or N-acryloylcholine oligosaccharide is allorerung at least 95% or more.

C. Obtaining conjugates β-propionamides of polysaccharide with protein

The polysaccharide or oligosaccharide of the present invention in the formation of a conjugate with other immunogenic molecule such as a polypeptide or protein, can be used for induzione in the body of the humoral response to a number of gram-negative and g is analogically bacteria, yeast and cancer. Conjugation of polysaccharide or oligosaccharide with the polypeptide converts the immune response to the polysaccharide or oligosaccharide, which is generally T-independent in the T-dependent response. Accordingly, the preferred amount of the polypeptide is an amount that is sufficient to ensure conversion of the response of T-independent in T-dependent. For the purpose of providing secondary immunogen may be useful to use smaller polypeptides. The molecular mass of the protein carrier is usually from about 50 000 to 500 000.

Preferred carrier proteins include, but are not limited to, the tetanus toxoid, diphtheria toxoid, the subunit In the cholera toxin proteins of the outer membrane of Neisseria meningitidis,-β a protein of Streptococcus group b, C-β a protein of Streptococcus group B, non-binding IgA, toxoid Pseudomonas aeruginosa, pertussis toxoid, synthetic protein containing residues of lysine or cysteine, etc. Protein carrier may be native protein, chemically modified protein, detoxified enduring protein or a recombinant protein. Molecule conjugates obtained in accordance with this invention, as for the protein component can be monomers, dimers, trimers and molecules with a higher degree of crosslinking.

This image is eenie provides the opportunity to gain molecule conjugates, in which the protein is associated with the polysaccharide or oligosaccharide through one or more of the provisions on the polysaccharide or oligosaccharide. The size of the polysaccharide or oligosaccharide may widely vary. One or more polysaccharides or oligosaccharides can be cross-linked with one or more proteins. The conjugates of the present invention preferably represent a lattice structure. The attachment points are located between lysine residues or cysteine protein and N-akrilovymi groups of the polysaccharide or oligosaccharide.

One of the ways to obtain immunogenic polysaccharide-protein conjugate polysaccharide selected (glycosaminoglycan), in which the remains of the sugar component of the repeating units contain a free amino group or N-acyl group (for example, N-acetyl group), is first subjected to hydrolysis using a base or enzyme to remove part or all of its N-acyl groups. Free amino groups are then subjected to N-acylation with N-acridoidea reagent with the formation of N-grilliravintola polysaccharide described above. Then N-acryloylcholine polysaccharide directly attached to the protein in terms of optimum pH, temperature and time for formation of immunogenic conjugate β-propionamide polysaccharide is a protein.

In one of the embodiments for optimal reactivity of the free ε-amino lysine residues in the protein conjugation is carried out at a pH above to 9.0, preferably at pH of about from 9.0 to 10.0. In another embodiment for optimal reactivity of the thiol (SH) groups of cysteine residues in the protein conjugation is carried out at a neutral pH of approximately 7,0. The choice of pH for implementing the method of conjugation can be based on the number of reactive groups in a specific protein carrier. For example, the way in which use protein composed of more reactive lysine residues compared to cysteine residues, preferably carried out at basic pH values. The method of conjugation, which use protein composed of more reactive cysteine residues compared to lysine residues, preferably carried out at neutral pH values.

The conjugation reaction can be carried out in sautereau reagents, including but not limited to buffer reagent comprising carbonate/bicarbonate, borate, phosphate buffer and the like, the reaction Temperature conjugation is at least about 25°C, preferably about 37°during the periods of the a, preferably approximately 24 hours. Key reactions include 1,4-conjugate connection (Michael) nucleophilic cysteine thiol groups or lysine ε-NH2groups of proteins with N-acryloylmorpholine remnants of sugars, as described Romanowska et al. (46), which are present in the repeating units of the polysaccharide, as shown in the drawing. In the resulting conjugate β-propionamides of polysaccharide to protein ratio of polysaccharide to protein is approximately from 0.1 to 0.6.

Glucosamine residues of the polysaccharide having N-acyl group, responsible for direct conjugation with the remnants of cysteine and/or lysine protein include, but are not limited to, glucosamine, galactosamine, mannosamine, fucosamine, sialic acid, etc. Polysaccharide can be obtained from such natural sources as bacterial, yeast or cancer cells, or from synthetic sources. Synthetic sources include chemical synthesis, enzymatic synthesis and homofermentative synthesis. The synthesis may be a de novo synthesis or modification of natural carbohydrates. Selected natural carbohydrates can be modified by changing the functional groups of the carbohydrate residue, or by addition or removal of carbohydrate residues.

The size of polishuri the and or oligosaccharide, intended for use in obtaining conjugates β-propionamide polysaccharide and β-propionamides of the oligosaccharide to the protein of the present invention, may be varied in conjugation with the protein carrier. As defined in this document, oligosaccharide, designed for use in the present invention, includes at least 10 residues of sugars and preferably from 10 to 50 residues of sugars. The polysaccharide as defined herein, includes more than 50 residues of sugars and may include about 600 or more residues. In some cases, to enhance immunogenicity requires conjugates large sizes. The methods of this invention provide the use of polysaccharides are very large, as in one polysaccharide, you can enter many reactive sites. Another advantage of this method compared with the prior art is that no changes occur in the charged functional groups of the polysaccharide or oligosaccharide, which often interact with the epitope responsible for immunity, or form part of such epitope.

C. Vaccines

This invention is also directed to the production of vaccines. In accordance with this invention the selected conjugates β-propionamide policy is arid with protein, described above, can be used as an antigen that causes the formation of antibodies active against polysaccharide or oligosaccharide and therefore active against the organism or cells, which was dedicated this polysaccharide or oligosaccharide. Vaccines of the present invention can provide a Raman or multicomponent vaccine, further comprising, along with the conjugate β-propionamides of polysaccharide with protein, other components, including, but not limited to, diphtheria-tetanus-pertussis (DTP), tetanus-diphtheria (TD), DTaP, DTap-Hib vaccine, DTap-IPV-Hib vaccine and the like, and combinations thereof, to provide a multifunctional vaccine used for immunization against a number of organisms or cells, causing disease.

Vaccines of the present invention can provide an active or passive immunity. Vaccines are designed to provide active immunity include isolated and purified N-acryloylcholine polysaccharide or oligosaccharide conjugated to at least one antigenic peptide.

D. Pharmaceutical compositions

The pharmaceutical compositions of the present invention may include at least one polysaccharide-protein conjugate and pharmacologically acceptable carriers, such the AK physiological saline solution, dextrose, glycerol, ethanol or the like In another embodiment the pharmaceutical composition includes other immunogenic fragment such as a peptide or composition comprising the antibody, the formation of which is called one of the CPS of the present invention. The composition can also include adjuvants to enhance the immune response in the recipient. Such adjuvants can be adjuvants on the basis of aluminum, such as alum, or long-chain alkyl adjuvants, such as sterilisation (see U.S. patent serial No. 583 372, filed 9/17/90; European patent, EP 0 549 617 B1; Moloney et al., U.S. patent No. 4 029 258), muramyldipeptide (MDP) or its derivative, monophosphorylated A (MPL), saponin (Quil-A), etc. Cm. also Jennings, et al., U.S. patent No. 5 683 699 and Paoletti et al., J. Infectious Diseases 1997; 175:1237-9. The pharmaceutical composition may also include one or more additional immunogenic, including, but not limited to, diphtheria-tetanus-pertussis (DTP), tetanus-diphtheria (Td), DTaP, DTaP-Hib, DTaP-IPV-Hib and the like, and combinations thereof. Especially, these pharmaceutical compositions can be used as vaccines.

To induce passive immunity pharmaceutical composition may include polyclonal or monoclonal antibodies, derivatives thereof or fragments and their recombinant forms. The amount of antibody fragment or a derivative thereof is therapeutically or prophylactically effective amount, which is determined using standard clinical methods.

Drugs of this invention can be administered to the subject using methods that are known in this area are effective. These include intradermal, intraperitoneally, intravenous, subcutaneous, intramuscular, oral, and intranasal routes of administration, but they are not alone.

The compositions of this invention may include suitable for standard vaccines carriers, buffers or preservatives, known to specialists in this field, including, without limitation, any suitable pharmaceutically acceptable carrier such as physiological saline solution, or other suitable injection fluid. The vaccines can also be a normal extension agents, for example stabilizers such as lactose or sorbitol, and adjuvants to enhance the immune response, such as phosphate or aluminum hydroxide, or sulfate and sterilisation. Vaccines received

in accordance with this invention, can also be used as components of multivalent vaccines that induce the formation of an immune response against a variety of infectious agents.

Vaccines of the present invention is administered in amounts sufficient to induce the production of antibodies is AK integral part of the immune response. To induce the production of antibodies IgG and IgM, the vaccine can be given parenterally or can be delivered to the mucous membrane to induce the production of antibodies IgA on the surface of the tissue. The dosage may be established on the basis of size, weight or age of the subject receiving the vaccine. Humoral response in the subject can be monitored by analysis of antibody titer or bactericidal activity and increase it if necessary amplification of the response. Usually a single dose for a child is approximately 10 µg of the vaccine comprising the conjugate per dose, or about 0.5 μg-20 μg/kg. Adult dose comprising about 0.5-20 mcg/kg vaccine comprising the conjugate. For vaccine comprising the conjugate of the CRP protein, the usual dose is approximately 25 µg of each individual CPS per dose. Then there is a vaccine against streptococcus group may include 25 μg of each of the CPS from each of the nine serotypes.

E. Antibodies

The formation of antibodies against the polysaccharide may induction using any well-known in this area of the method. In accordance with one approach, the antibodies can coil by introducing a selected immunogenic conjugate β-propionamides of polysaccharide with protein W the animal host. Animal host can be a rat, mouse, non-human primates or people, but this list is not limiting. Preferably the host is a human. In one of the embodiments of the immunological responses can be enhanced by applying known in the field of adjuvants.

By using any of the well-known in the field of methods can be also obtained monoclonal antibodies against the polysaccharide. In accordance with one of the methods used culture hybridoma cell lines (Kohler and Milstein (1975) Nature 256: 495-497). Monoclonal antibodies against the polysaccharide may be human monoclonal antibodies, chimeric monoclonal antibodies or humanized monoclonal antibodies obtained using any of the well-known in the field of methods. In accordance with one approach can be obtained chimeric monoclonal antibodies, which contain the nonhuman (e.g., mouse) antigennegative domain, combined with a human constant region (Takeda et al. (1985) Nature 314: 452). Humanized antibodies can be obtained in accordance with methods of the Queen et al., U.S. patent No. 5 089 585 and U.S. patent No. 5 530 101. Using known in the field of methods can be constructed single-chain antibodies (U.S. patent No. 4 946 778; Davis, G.. et al., 1991 Biotechnology 9: 165 to 169; Pluckthun, A 1990 Nature 347: 497-498). The domains of the constant regions of the antibodies can be modified using known in the field of methods (WO 89/07142).

Antibodies against the polysaccharide or oligosaccharide may be purified by any of well-known in the field of methods, including, not agrarias them, immunoabsorption or immunoaffinity chromatography or other chromatographic methods (e.g., HPLC). Antibodies can also be isolated in the form of immunoglobulin fractions from the serum, plasma or cell culture medium.

The antibody molecules of the present invention may be intact molecules of immunoglobulins, essentially intact molecules of immunoglobulins or fragments of molecules of immunoglobulins, such as Fab fragments, which contain antigennegative plot. The antibody molecules may belong to any class, including IgG, IgM and IgA.

Fragments of antibodies against CRP can be obtained using any of the known in the field of methods (Campbell (1985) Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Burdon, et al. (eds.), Elsevier Science Publishers, Amsterdam).

The antibody or antigennegative fragment can be used as a therapeutic agent for providing passive protection against diseases caused by gram(+)gram(-) bacteria or yeast. The antibody or antigennegative fragment also can is t be used as a diagnostic reagent in standard immunological analysis for registracii and/or identification of bacteria yeast or cancer cells. The antibody can be delivered as only set or together with standard reagents for immunological analysis.

In another embodiment of this invention antibodies against the polysaccharide or oligosaccharide of the present invention can be used as a drug in therapeutic or prophylactic use for the transmission of passive immunity from the subject the owner to another entity (i.e. to enhance the immune response of a subject against gram-positive or gram-negative bacteria or yeast, or to provide response in patients with the risk of immune diseases, or in immunocompromised patients, including patients with AIDS). Passive transfer of antibodies is known in the field and can be carried out using any of the known methods. In accordance with one method of antibodies against conjugates of this invention are formed in immunocompetent animal host ("the donor"), they are collected from an animal host and introduce the subject to the recipient. For example, to generate antibodies that are active against polysaccharide-protein conjugate of the present invention, it is possible to use human donor. Then the antibodies can be introduced into therapeutically or prophylactically effective amounts to a human recipient, needs l the treatment, whereby the recipient is transmitted resistance to bacteria that are bound by antibodies, the formation of which is induced by using the polysaccharide component (see Grossman, M. and Cohen, S.N., in "Basic and Clinical Immunology", 7thEd., (Stites, D.P. and Terr, A. T. eds., Appleton & Lange 1991) Chapter 58 "Immunization").

In some cases, the polysaccharide used in connection with this invention, can cause the formation of antibodies that cross-react with other pathogenic organisms and, thus, have the ability to protect the body from infection of these other bacteria.

F. Diagnostic kits

In another embodiment of the PCC of the present invention, or derivatives thereof, or fragments may be provided in the diagnostic kits designed to determine the presence of antibodies against bacteria, yeast or cancer cells. The presence of these antibodies can be determined before exposure to the pathogen and thus it is possible to predict which subjects will be resistant to infection. Diagnostic kit may include at least one of the CPS of the present invention, or its derivatives, or fragments, alone or in conjugate with protein, and suitable reagents for determination of antibody response, when modified KPS, or their derivatives, or fragments mixed with a sample containing antibodies against the gram-negative, gram-positive bacteria, yeast or cancer cells or cancer tissue. The reaction of antibodies can be determined using any of the methods described in this area, including, but not limited to, ELISA. Knowledge of this is important and can help to avoid vaccination, which is not necessary.

Alternative diagnostic kit may further include a solid substrate, or magnetic beads, or plastic matrix and at least one of the CPS of the present invention, or its derivatives, or fragments.

In some cases it may be preferred that the CPS or their derivatives, or fragments are labeled. Aiming agents are well known in this field. For example, aiming agents include, but are not limited to, radioactive, chemiluminescent, bioluminescent, fluorescent or other identification markers suitable for analysis. Body fluid or tissue samples (e.g., blood, serum, saliva) can be collected, cleaned and analyzed using a diagnostic kit. KPS, derivatives or fragments can be purified or not purified and can consist of a mixture of molecules.

The solid matrix is known in this field and are available, they include, but are not limited to, polystyrene, polyethylene, poly is roelen, polycarbonate or any solid plastic material in the form of tubes, beads, microparticles, sticks for dipping, tablets or other Advanced matrix include, but are not limited to, membranes, 96-well microtiter plates, test tubes and Eppendorf tubes. Basically, these matrices include any surface to which can be attached legendbase.ui agent, or surface, which itself provides a site of attachment of the ligand.

Everything mentioned here publications, patents and articles specifically incorporated herein fully by reference. The following examples are provided to illustrate the present invention, but they in no case should not be construed as limiting the scope of the present invention. Professionals in this field should be clear that many changes and substitutions can be made without deviating from the spirit and scope of this invention.

EXAMPLE 1

Obtaining conjugates β-propionamides of polysaccharide with protein carrier

The following non-limiting examples describe a number of clinically significant polysaccharide-protein conjugates for vaccines against Streptococcus pneumoniae (type 14), Streptococcus group B (GBS)type III and type II, and E. coli K1. All of the above polysaccharides used in this example, represents the t of a glycosaminoglycan, which contain N-acetyl group in one or more of helicoiling balances representing the part of their recurring structural units.

A. Depolymerization of pneumococcal polysaccharide type 14

To increase the solubility of the polysaccharide is first subjected to partial depolymerization using ultrasonic treatment. 200 mg of pneumococcal polysaccharide type 14 (lot No. 2020510, American type culture collection) was dissolved in 20 ml of PBS and treated with ultrasound for 4 hours at 0°using ultrasonic generator Branson model 450. The obtained polysaccharide dialist, lyophilizer and then fractionary on column superdex 200, balanced saline solution with phosphate buffer (PBS). Peak fractions are combined and then cialiswhat against di water using Spectra/Por® Membrane MWCO:3500. The yield after drying is 157,5 mg solids. Processed ultrasound polysaccharide has an average molecular weight of approximately 50 000, as determined using SEC-MALLS with miniDAWN (Wyatt Technology Corp., Santa Barbara, CA).

V. De-N-acetylation of pneumococcal polysaccharide type 14

100 mg fractionated pneumococcal polysaccharide type 14 was dissolved in 10 ml of 2N NaOH and then to the reaction mixture add 10 mg of NaBH4. This mixture is heated at 100°With whom for one hour and then cooled to room temperature. N-deacetylating component cialiswhat against di (deionized) water using Spectra/Por® Membrane MWCO:3500 and lyophilizers getting 84 mg solid white. As discovered through the analysis of N-deacetylating polysaccharide with N1NMR at 500 MHz, it contains less than 5% of residual N-acetyl groups.

C. N-acryloylmorpholine N-deacetylating pneumococcal polysaccharide type 14

84 mg of N-deacetylating pneumococcal polysaccharide type 14 was dissolved in 4.2 ml of D. I. water. In a water bath, the pH of the solution was adjusted to 10 with 2N NaOH. Then add 420 μl mixture akriloilkhlorida:dioxane 1:1 vol./about. and bring the pH to 11 with 2N NaOH. The reaction mixture is allowed to stand for another hour at pH 11, in order to ensure complete hydrolysis of esters, which can be caused by O-acylation. This solution cialiswhat and lyophilizers, receiving 42 mg of the dry powder. After analysis with N1NMR at 500 MHz, it was found that the polysaccharide is N-acryloylcholine more than 95%.

D. Attaching N-grilliravintola pneumococcal polysaccharide type 14 to the monomer tetanus toxoid

22 mg of N-grilliravintola pneumococcal polysaccharide type 14 was dissolved in 1.1 ml of carbonate/bicarbonate buffer, pH of 9.5. To the reaction mixture are added 22 mg of monomer of stabn knogo the toxoid. The reaction mixture was incubated over night at 37°S. the Course of conjugation analyzed by Biologic system (Bio-Rad)equipped with a column with supersoil 12. Conjugation of polysaccharide with tetanus toxin track by a progressive increase in peak, which is recorded by measuring UV absorption at 280 nm, elution occurs in the free volume. After conjugation, the solution is neutralized to pH 7 with 0,1N HCl and then cialiswhat against PBS. The conjugate is purified by passing through a column of 1.6×60 cm with superdex 200 PG (Pharmacia) and elwira with PBS containing 0.01% thimerosal. Fractions corresponding to the peak of free volume, unite. The content of carbohydrates and protein in the conjugate assessed by staining vinylsulfonic acid by Dubois et al. (51) and staining of Kumasi by Bradford (9).

Similar methods are used for polysaccharides GBS type II, type III, and E. coli K1 and meningococcus C. the reaction Conditions for each of these polysaccharides are shown in the tables below.

TABLE 1
E. De-N-acetylation of GBS polysaccharide of type II and type III
 Size* PS (kDa)PS (mg)NaOH NaBH4TemperatureThe reaction timeOutput
GBSP II25063 mg6 ml12 mg110°6 h63 mg
GBSP III11050 mg5 ml10 mg110°6 h55 mg
*Determined using SEC-MALLS
TABLE 2
E. N-acryloylmorpholine polysaccharide GBS type II and type III
 The quantity PS (mg)Di waterAkriloilkhlorida:dioxane 1:1 vol./about.Output
GBSP II603 ml300 ml60 mg
GBSP III552,75 ml275 ml55 mg
TABLE is CA 3
That is, the Adherence of GBS polysaccharide II and III GBS to the monomer tetanus toxoid
 PS (mg)ST* (mg)The carb./bicarb. buffer rn,5TemperatureThe incubation period
GBSP II10100.5 ml37°during the night
GBSP III10,529,520.5 ml37°during the night
*ST - tetanus toxoid

F. De-N-acetylation of the K1 polysaccharide

300 mg of PS K1 dissolved in 15 ml of 2, ON NaOH solution and add 150 mg of sodium borohydride. The solution is heated at 110°C for 6 hours, cooled to room temperature and diluted 20-fold volume of deionized water. After diafiltration through the membrane Amicon YM3 in a stream of deionized water, the solution lyophilizer getting 255 mg of N-deacetylating PS K1. H1NMR at 500 MHz confirms that M-deacetylation was complete.

G. M-acryloylmorpholine poly is Farida K1

To 10 ml of a solution of 250 mg of de-N-acetylated PS K1 in deionized water, cooled in a bath with ice, add dropwise a solution of akriloilkhlorida (Aldrich, Milwaukee, WI), obtained by combining 1 ml of akriloilkhlorida with 1 ml of dioxane. the pH of the solution support in the range of 7.0 to 10.5 by adding 2N sodium hydroxide solution. After complete addition, the pH increased to 13 and is supported in the interval from 12.9 to 13.1 for one hour at room temperature. the pH of the solution was adjusted to 9.5 by adding dropwise IN HCl. The solution is subjected to diafiltration using membrane Amicon YM3 in the cell with stirring in a stream of deionized water. Withheld the remainder lyophilizer to dryness and the resulting material (N-acryloyl SS K1) stored in a desiccator in a freezer at -20°C. N.1NMR at 500 MHz shows that the reaction is complete N-acryloylmorpholine.

N. β-Propionamides K1-rPorB conjugate (K1-rPorB I)

A solution containing an 8.4 mg N-acryloyl SS K1 and 4.0 mg of recombinant PorB of Neisseria meningitidis in 0.3 ml of 0.2m borate, 0.05% of Zwittergen™ 3,14 (Boehringer Mannhein) pH of 9.5, and incubated at 37°C for 3 days. The conjugate is purified using chromatography with the exception of size on the preparative column with superdex 200, elwira PBS containing 0.01% thimerosal. Fractions with active UV signal at 280 nm, eluruumina in St. the free volume of the column, or close to it, combine and store in the refrigerator. Conjugate analyze the content of sialic acid and protein by staining resorcinol, Kumasi respectively.

I. Receiving tarirovannogo rPorB

To one ml rPorB porina with a concentration of 10 mg/ml in 0,25 M HEPES buffer pH 8.5, containing 0,25 M sodium chloride and 0.05 twitterget 3-14 add 0.2 ml 0,05 m solution of N-Succinimidyl 3-[2-pyridyldithio]propionate. The solution is thoroughly mixed and allowed to stand at room temperature for one hour. To this solution was added 0.06 ml of 1M solution of dithiothreitol in the same buffer. The solution is again thoroughly mixed and left to stand at room temperature for another two hours. The solution is diluted with 1.3 ml of 0,25M buffer HEPES pH 7.0, containing 0,25 M sodium chloride and 0.05 twitterget 3-14, and loaded onto a column for desalting Pharmacia PD-10, which was pre-equilibrated with the same buffer. The elution is conducted with the same buffer, the eluate is collected and concentrated using an Amicon concentrator Method 30 at 5 000 rpm for one hour. The residue is collected, and determine the protein concentration.

N. Obtaining N-grilliravintola K1-S-rPorB conjugate (K1-S-rPorB)

To 0,17 ml of the solution obtained previously tarirovannogo of grow with a concentration of 25 mg/ml added 9 mg of N-grilliravintola K1 polysaccharide. The solution thoroughly the mix and incubated in a thermostat at 37° C for 18 hours. The solution is purified by passing through a column superdex 200 (Pharmacia)using PBS as eluent. UV active at 280 nm of the fractions, eluruumina in the free volume of the column or close to it, unite. The analyses show that the conjugate contains 25 μg/ml of polysaccharide and 188 μg/ml of protein.

I. Obtaining N-grilliravintola GCMP-S-rPorB conjugate (GCMP-S-rPorB)

Similarly N-acryloylcholine GCMP-S-rPorB get in the way, comparable to that described above, to obtain the N-grilliravintola K1-S-rPorB conjugate, it was discovered that the resulting conjugate contains 43 μg/ml of polysaccharide and 200 μg/ml protein.

TABLE 4
Analytical data for the above-described conjugates
 Protein concentration, mg/mlThe PS concentration, µg/mlThe percentage of PS in the conjugate
Pn14-ARTICLE (3)547293 (1)35
GBSII-CT (3)377160 (2)30
GBSIII-CT (3)365115 (2)24
K1-rPorB I (3)14717 (2)10
K1-rPorB II (4)40641 (2)9
K1-S-rPorB (3)18825 (2)12
GCMP-S-rPorB (3)20043 (2)18
(1) Total carbohydrate analysis by Dubois

(2) Resorcinol analysis of sialic acids

(3) is produced by the direct connection of N-grilliravintola polysaccharide to the corresponding protein-media. (4) the Control conjugate obtained by reductive amination of periodate-oxidized N-grilliravintola PS K1 with rPorB

EXAMPLE 2

Immunogenicity and efficacy of conjugates β-propionamides of polysaccharide with protein carrier

Preclinical evaluation of conjugates in mice

Immunological tests: Serum antibodies to each polysaccharide conjugate is measured by ELISA. Conjugates of human serum albumin (CSA) (Sigma, St Louis, MO)used for ELISA assays, obtained by reductive amination. The oxidized polysaccharide is added to CSA and then subjected to reductive aminating with NaCNBH3. Conjugates emit using gel-filtration chromatography and stored dried from the frozen state at -70°C. the Titles of PS-specific antibodies was determined by ELISA as follows. Polystyrene 96-well flat-bottomed Mick is attrvalue plates (NUNC Polysorb) (Nunc, Naperville, IL) cover the conjugates PS-CSA in PBS (0,01M phosphate sodium, 0,15M NaCl, pH 7.5) in an amount of 0.25 μg/well (100 μl/well) by incubation for 1 hour at 37°C, then 5 times washed with PBS-Tween (0.05 per cent.about. Tween 20 in PBS). All subsequent incubation was performed at room temperature. For all necessary washes using PBS-Tween. Coated tablets are then blocked with PBS-BSA (0.5 percent wt./about. bovine serum albumin in PBS) for IgG ELISA, or 0.1% of the mass/about, nonfat dry milk Carnation for IgM ELISA, in the amount of 0.15 ml/well for 1 hour, then washed. Diluted 2 times with serum, in repeats, making the tablet in the amount of 100 μl/well and incubated for 1 hour, then washed. Add conjugate antibodies (antimachine goat antibody labeled with peroxidase) (Kirkegaard &Perry Lab, Gaithersburg, MD) in an amount of 100 μl/well and incubated for 30 minutes, then washed. Add the dye and the substrate solution (Kirkegaard &Perry TMB) 1:1 and peroxide in 0.05 ml/well and incubated for 10 minutes. Peroxidase-catalyzed reaction is stopped with 1M H3RHO4, 0.05 ml/well, and the plate is read on a reading device for microtiter plate reader Molecular Devices Emax (Molecular Devices, Menio Park, CA) at a wavelength of 450 nm, using 650 nm as the wavelength of the comparison. The background absorption is determined in several not containing serum counter is selected wells and average for each tablet. For each dilution of serum subtract the average background absorption, and then get the average value of the absorption of whey repetitions. For subsequent data analysis used a modified schedule of Scatchard, where absorbance (Y-axis) was laid against the corresponding absorption reverse dilution (X axis). The conditions for equilibrium and excess antibody, for each series of dilutions of serum get a straight line; this line to extrapolate the X-axis to determine the antibody titer. Positive serum control with pre-defined antibody titer is used on each plate to ensure the inclusion, in relation to which standardizes all serum, minimizing the scatter of the data that exists for different tablets and different days. The results of these data are shown in tables 5, 6 and 7.

Opsonophagocytosis analysis (PA): the Ability to opsonization mouse antisera against streptococcal (GBS) and pneumococcal conjugates was determined in opsonophagocytosis lyse test in vitro using human promyelocytic leukemia cell line HL-60 (ATS (American type culture collection) No. CCL 240). Namely 200 colony forming units (CFU) cell strain M GBS type III, or pneumococcal strain type 14, mixed in equal about is yamah with serum antibodies, and incubated with shaking for 15 minutes at 35° C in an incubator with 5% CO2. To this mixture add the complement from rabbits and cells HL-60 (5×105), cultured for 5 days in the presence of 90 mm in DMF) and incubated 1 hour at 37°when shaken. To quantify the growth of the culture selected aliquots. Titers determined by extrapolating the cultivation of antibodies, corresponding to fifty percent of surviving bacteria. The results of these analyses are given in table 5 for pneumococcal conjugates of type 14 and in table 6 for the conjugates of GBS type III.

Serum bactericidal assay (SBA): antibody-dependent complementarian bactericidal activity measured in units of bactericidal titer, or reverse breeding, which provides a 50% destruction of bacteria targets. Complement all sera initially incubated at 56°C for 30 minutes Then a series of 2-fold dilutions were prepared for each serum GBSS in sterile 96-well tablets with a U-shaped bottom (Sigma), receiving the final volume of 50 µl/well. Serum complement rabbits (Pel-Freez, Brown Deer, WI) diluted 1:1 working concentration of bacteria GBM (serotype 15 strain 44/76) or meningococcal C11 group With strain comparison and 50 μl added to each well containing the diluted serum, getting the reaction mixture with a final volume of 100 µl/well. This reaction with the offer, containing 50% serum (V / V heat inactivated and diluted), 25% rabbit serum complement and 25% bacteria (working concentration), incubated for 60 minutes in a humid incubator at 37°C and 5% CO2on the shaker for microtiter plates (LKB-Wallac; Pharmacia Biotech) at high speed.

The contents of all wells then placed onto chocolate agar by spraying 30 µl/plate. Also put the initial (zero time point of incubation) bacterial samples. All plates are incubated overnight as before. Then think of colony forming units (CFU) using an automatic counter colonies from Imaging Products International (Chantilly, VA), taking the mean of the three readings die. Reverse breeding, or titer, which corresponds to 50% of the deaths directly read from the graph, where the X-axis presents the values of log10 corresponding reverse breeding, and on the Y-axis shows the percentage of survival. The results of this analysis are shown in table 7.

The control vaccine is a conjugate polysaccharide type 14-tetanus toxoid obtained by reductive amination. Pn14 conjugate-ST is the product of the direct connection of N-grilliravintola pneumococcal polysaccharide type 14 to the tetanus toxoid.

For this research group, consisting of 10 CD1 mice (Charles River Laboratory) age 6-8 weeks, were injected subcutaneously at 0, 28 and 38 day 2.0 µg conjugated polysaccharide. The animals took blood at 0, 28, and 38 days, and on day 59 of them were driven away. ELISA performed using conjugate Pn14 polysaccharide-CSA obtained by reductive amination. Titers determined using ELISA, are shown in table 5 represent the total IgGs. Shows the titers of antibodies against pneumococcal strain type 14 was determined using the EOS.

The control conjugate vaccine is a conjugate polysaccharide GBS type III-tetanus toxoid obtained by reductive amination periodicocitara of GBS polysaccharide of type III and tetanus toxoid. Conjugate GBS III-CT is the product of the direct connection of N-grilliravintola polysaccharide of type III to the tetanus toxoid.

For this study, groups of 10 mice CD1 (Charles River Laboratory) age 6-8 weeks, were injected subcutaneously at 0, 21 and 42 day 2 μg of conjugated polysaccharide. In mice took blood at 0, 21 and day 42, and 52 day they were driven away. Titers were measured by ELISA using the GBS polysaccharide of type III, attached to human serum albumin. The titles given in table 6 represent the total IgGs against polysaccharide of type III.

The control vaccine (K1-rPorB II) is the product of the reductive amination periodicocitara N-grilliravintola K1 polysaccharide and tetanus toxoid. Vaccine K1-rPorB I is the product of the direct connection of N-grilliravintola K1 polysaccharide to the tetanus toxoid. K1-S-rPorB is the product of the direct connection tarirovannogo of grow porina to N-grilliravintola the K1 polysaccharide.

For this study the group consisting of 10 mice CD1 (age 4-6 weeks), obtained from Charles River Laboratory, was administered intraperitoneally immunized at 0, 28 and 42 day. In mice took blood at 0, 28 and 42 day, and then they were bled on day 52. Titers were measured by ELISA, using N-propenylidene the K1 polysaccharide attached to human serum albumin. The titles given in table 7 represent the total IgGs against the modified N-propikirovana K1 polysaccharide. Table 7 also shows the antibody titers against strain N/76 N. meningitidis group b serotype 15, defined by serum bactericidal test (SBA) for blood collected on day 52.

TABLE 8
Immunogenicity of conjugates GCMP
The vaccine/adjuvantELISA day 38SBA day 38
GCMP-S-grow/alum lot440002100
GCMP-S-rPorB/alum lot430002800
S/alum<50<50

GCMP-S-rPorB is the product of the direct connection of N-grilliravintola polysaccharide meningococcal disease group (GCMP) and tarirovannogo of grow. These animal studies to groups of 10 outbreeding female mice Swiss Webster (age 6-8 weeks) from HSD, was subcutaneously injected at 0 and 28 day 2 μg of conjugated polysaccharide per dose. Animals were bled on day 38. The titers of antibodies against polysaccharide of group C was measured using ELISA using GCMP attached to human serum albumin. Serum bactericidal titers were obtained using strain comparison meningococcal strain C11.

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47. Orellana. A. and C.B.Hirshberg. 1994. Molecular Cloning and Expression of a Glycosaminoglycan N-Acetylglucosaminyl N-Deacetylase/N-Sulfotransferase from a Heparin-producing Cell Line. J.Biol. Chem. Vol 269, No. 3 pp.2270-2276.

48. Cheung, W-F., I.Eriksson, M.Kusche-Gullberg, U.Lindahl, and L.Kjéllén, 1996. Expression of the Mouse Mastocytoma Glucosaminyl N-Deacetylase/N-sulfotransferase in Human Kidney 293 Cells Results in Increased N-Sulfation of Heparan Sulfate. Biochem 35: 5250-5256.

49. Kusche-Gullberg, M., I.Eriksson, D.Sandback Pikas, and L.Kjellen 1998. Identification and Expression in Mouse of Two Heparan Sulfate Glucosaminyl N-Deacetylase/N-Sulfotransferase Genes. J. Biol. Chem. Vol 273, No. 19: 11902-11907.

50. Finke, A., D.Bonner, A.V.Nikolaev, .Jaim and K. Jann. 1991. Biosynthesis of the Escherichia coli K5 Polysaccharide, a Representative of Group II Capsular Polysaccharides: Polymerization In Vitro and Characterization of the Product. J. Bacteriology 173, No. 13: 4088-4094.

51. Dubois, M. et al 1965. Colormetric Method for the Determination of Sugars and Related Substance. Analytic. Chem. Vol. 28: 350-366.

1. Immunogenic polysaccharide-protein conjugate or oligosaccharide-protein conjugate containing N-promieniowania polysaccharide or N-promieniowania oligosaccharide, is directly attached to the protein through sites β-positions of one or more propionate fragments N-promieniowania polysaccharide or N-promieniowania oligosaccharide; N-promieniowania polysaccharide or N-promieniowania oligosaccharide, directly connected with protein, it causes the production of protective antibodies reactive against N-promieniowania polysaccharide or N-promieniowania oligosaccharide; N-promieniowania polysaccharide or N-promieniowania oligosaccharide derived from a polysaccharide or oligosaccharide containing N-acetyl group, and where at least 50% of the N-acetyl groups substituted N-akrilovymi groups obtained in N-promieniowania polysaccharide or N-promieniowania the oligosaccharide, which is subject to the connection with the specified protein.

2. The conjugate according to claim 1, where the polysaccharide or oligosaccharide derived from a polysaccharide derived from bacteria, yeast, cancer cells, or synthetic the new chemical means.

3. The conjugate according to claim 1, where the polysaccharide or oligosaccharide derived from a polysaccharide derived from Escherichia coli, Meningococcus, Pneumococcus, Streptococcus, Haemophilus, Neisseria, Salmonella, Klebsiella, or Pseudomonas.

4. The conjugate according to claim 1, where the polysaccharide or oligosaccharide derived from a polysaccharide derived from Streptococcus group selected from the group consisting of type Ia, type Ib, type II, type III, type V, type VIII and their combinations.

5. The conjugate according to paragraph 3, where the polysaccharide or oligosaccharide derived from a polysaccharide derived from Meningococcus group selected from the group consisting of group b, group C, group Y, group W135 and their combinations.

6. The conjugate according to claim 3, where the polysaccharide or oligosaccharide derived from a polysaccharide derived from E. coli K1, E. coli K92, Pneumococcus type 4, Pneumococcus type 14, Streptococcus group A, Streptococcus group or their combinations.

7. The conjugate according to claim 1, where the protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid, protein outer membrane of Neisseria meningitidis, pneumolysoid,-β-protein of group b Streptococcus, and does not bind IgA C-β protein from Streptococcus group C.

8. Polysaccharide-protein conjugate or oligosaccharide-protein conjugate according to claim 7, where the protein obtained using recombinant methods.

9. Polysaccharide-protein conjugate or oligosaccharide-protein conjugate of claim 8, where the protein is a recombinant protein of the outer membrane of N. meningitidis.

10. The floor of the saccharide-protein conjugate or oligosaccharide-protein conjugate according to claim 1, where the polysaccharide or oligosaccharide contains glycosaminoglycan.

11. Polysaccharide-protein conjugate or oligosaccharide-protein conjugate according to claim 1, where the polysaccharide or oligosaccharide contains glucosamine remnants of the structural repeating unit having at least one free amino group or N-acyl group.

12. Polysaccharide-protein conjugate or oligosaccharide-protein conjugate according to claim 11, where Picatinny residue selected from the group consisting of glucosamine, galactosamine, mannosamine, fucosamine and sialic acid.

13. Polysaccharide-protein conjugate or oligosaccharide-protein conjugate according to claim 1, where N is promieniowania polysaccharide or N-promieniowania oligosaccharide is directly connected with free ε-amino group of a lysine residue or a thiol group of the cysteine residue of the protein.

14. The conjugate according to claim 1, where the conjugate is selected from the group consisting of conjugate polysaccharide, propionamido-group Streptococcus pneumoniae type 14 with tetanus by toxoid, conjugate polysaccharide, propionamido-group b Streptococcus type III with tetanus by toxoid, conjugate polysaccharide, propionamido-group b Streptococcus type II and tetanus by toxoid, conjugate polysaccharide, propionamido group E. coli K1 with tetanus by toxodon and conjugate meningococcal polysaccharide of propionamido group With tetanus by toxodon where indicated the p conjugate is used as a vaccine.

15. The conjugate according to claim 1 for use in the method, characterized in that causes the mammal the production or antibody-based test response to polysaccharide or oligosaccharide component of the polysaccharide-protein conjugate or oligosaccharide-protein conjugate, comprising the introduction of this conjugate in an amount sufficient to provide protective immunity.

16. The conjugate containing N-promieniowania polysaccharide or N-promieniowania oligosaccharide, directly associated with the protein through sites β-positions of one or more propionate fragments N-promieniowania polysaccharide or N-promieniowania oligosaccharide; where the conjugate causes the production of protective antibodies reactive against N-promieniowania polysaccharide or N-promieniowania oligosaccharide; where specified conjugate obtained by the method, which includes

A) de-N-acetylation of selected polysaccharide or oligosaccharide with de-N-acetylurea reagent with the formation of the de-N-acetylated polysaccharide or de-N-acetylated oligosaccharide, where at least 50% of N-promieniowania polysaccharide or N-promieniowania oligosaccharide is de-N-acetylated;

B) N-acryloylmorpholine de-N-acetylated polysaccharide or de-N-acetylated oligosaccharide using aceloularoer re the Gent with the formation of N-promieniowania polysaccharide or N-promieniowania oligosaccharide, and

C) direct connection through the site β-positions of one or more propionate fragments N-promieniowania polysaccharide or N-promieniowania oligosaccharide to a bacterial protein or a synthetic protein containing lysine or cysteine residues, with the formation of polysaccharide-protein conjugate or oligosaccharide-protein conjugate, where the specified polysaccharide-protein conjugate or oligosaccharide-protein conjugate is used as immunogen or vaccine.

17. The conjugate according to clause 16, where the polysaccharide or oligosaccharide derived from bacteria, yeast or cancer cells, or chemically synthesized.

18. The conjugates according to clause 16, where the linking is carried out at a pH value approximately equal to 7.0.

19. The conjugate according to clause 16, where the linking is carried out at a pH value above 9.

20. The conjugate according to clause 16, where the binding is carried out in a reagent selected from the group consisting of phosphate buffer, bicarbonate buffer and borate buffer.

21. The conjugate according to clause 16, where the de-N-acetylide reagent is a base or an enzyme and acryloyloxy reagent selected from the group consisting of N-akriloilkhlorida, acrylamide, acrylic acid and dehydrating agent.

22. The conjugate according to clause 16 for use in the method, characterized in that causes the mammal to produce antic the high response to the polysaccharide or oligosaccharide component of the polysaccharide-protein conjugate or oligosaccharide-protein conjugate, includes the introduction of this conjugate in an amount sufficient to provide protective immunity.

23. A pharmaceutical composition comprising a conjugate according to any one of claim 1 or 16, and a pharmaceutically acceptable carrier, and a pharmaceutical composition provides protective immunity against at least one of the members of the species of the organism from which the polysaccharide or oligosaccharide component of the polysaccharide-protein conjugate or oligosaccharide-protein conjugate.

24. The pharmaceutical composition according to item 23, further comprising adjuvant.

25. The pharmaceutical composition according to paragraph 24, where the adjutant selected from the group consisting of alum and sterilising.

26. The pharmaceutical composition according to item 23, further comprising the second component, and the aforementioned second component selected from the group of immunogens consisting of diphtheria-tetanus-pertussis (DTP), diphtheria-tetanus-acellular pertussis (DTaP), tetanus-diphtheria (Td), diphtheria-tetanus-acellular pertussis-Haemophilus influenzae type b (DTaP-Hib), diphtheria-tetanus-acellular pertussis-inactivated poliovirus-Haemophilus influenzae type b (DTaP-IPV-Hib) vaccines and their combinations.

27. A method of obtaining a conjugate β-propionamido-linked polysaccharide-protein conjugate or β-propionamide with the provided oligosaccharide-protein conjugate, where these conjugates induce the production of protective antibodies reactive against polysaccharide or oligosaccharide, where specified conjugate is produced by a method that includes

A) de-N-acetylation of the polysaccharide or oligosaccharide with de-N-acetylurea reagent with the formation of the de-N-acetylated polysaccharide or de-N-acetylated oligosaccharide, where at least 50% de-N-acetylated polysaccharide or N-acetylated oligosaccharide is de-N-acetylated;

B) N-acryloylmorpholine de-N-acetylated polysaccharide or de-N-acetylated oligosaccharide using aceloularoer reagent, with the formation of N-promieniowania polysaccharide or N-promieniowania oligosaccharide, and

C) direct connection through one or more sites β-provisions propionate fragments N-promieniowania polysaccharide or N-promieniowania oligosaccharide to a bacterial protein or a synthetic protein containing lysine or cysteine residues, with the formation of β-propionamido-linked polysaccharide-protein conjugate or β-propionamido-linked oligosaccharide-protein conjugate.

28. The method according to item 27, where the polysaccharide or oligosaccharide is derived from polysaccharide originating from bacteria, yeast or cancer cells, or the synth is serout by chemical means.

29. The method according to item 27, where the polysaccharide or oligosaccharide is derived from the polysaccharide or oligosaccharide derived from Escherichia coli, Meningococcus, Pneumococcus, Streptococcus, Neisseria, Salmonella, Klebsiella, or Pseudomonas.

30. A vaccine comprising the conjugate according to claim 1 or 16, and a pharmaceutically acceptable carrier, where this vaccine provides protective immunity against at least one of the members of the species of the organism from which the polysaccharide or oligosaccharide component of the polysaccharide-protein conjugate or oligosaccharide-protein conjugate.

31. The vaccine according to item 30, where the organism is selected from the group consisting of bacteria and yeast.

32. The vaccine p, where the bacterium is selected from the group consisting of Escherichia coli, Meningococcus, Pneumococcus, Streptococcus, Neisseria, Salmonella, Klebsiella and Pseudomonas.

33. The vaccine according to item 30, further comprising a second immunogen in combination with a polysaccharide-protein conjugate or oligosaccharide-protein conjugate, and the specified second immunogen selected from the group consisting of DTP, DTaP, Td, DtaP, Hib, DTaP-IPV-Hib and their combinations.

34. The vaccine according to item 30 for use in the method of immunization of a mammal, at least against a disease caused by the organism from which was obtained the protein component of the polysaccharide-protein conjugate or oligosaccharide-protein conjugate, comprising administration to the mammal of an amount in which ctiny, sufficient to provide protective immunity.

35. The vaccine according to item 30 for use in the method of immunization of a mammal against Streptococcus pneumoniae, comprising the administration to a mammal amount of vaccine sufficient to provide protective immunity.

36. The vaccine according to item 30, for use in the method of immunization of a mammal against Streptococcus group B, comprising the administration to a mammal amount of vaccine sufficient to provide protective immunity.

37. The vaccine according to item 30, for use in the method of immunization of a mammal against Neisseria meningitidis group B, comprising the administration to a mammal amount of vaccine sufficient to provide protective immunity.

38. The vaccine according to item 30 for use in the method of immunization of a mammal against Neisseria meningitidis group C, comprising the administration to a mammal amount of vaccine sufficient to provide protective immunity.

39. The vaccine according to item 30 for use in the method of immunization of a mammal against Haemophilus influenzae type b, comprising the administration to a mammal amount of vaccine sufficient to provide protective immunity.

40. The selected immunoglobulin or its antigennegative fragment containing substances that occur in response to the conjugate according to any one of claim 1 or 16, and the specified immunoglobulin or Antiga is binding fragment is specifically immunoreactive against N-promieniowania polysaccharide or N-promieniowania oligosaccharide and immunoreactive against native N-acetylated polysaccharide, from which was obtained the N-promieniowania polysaccharide or N-promieniowania oligosaccharide.

41. The immunoglobulin or its antigennegative fragment on p, where the immunoglobulin is a selected antibody IgG.

42. The immunoglobulin or its antigennegative fragment on p selected from the group consisting of IgG antibodies, IgM antibodies, IgA antibodies and their combinations.

43. The immunoglobulin or its antigennegative fragment on p, where native N-acetylated polysaccharide is a component of bacteria, yeast or cancer cells.

44. The immunoglobulin or its antigennegative fragment according to item 43, where the polysaccharide derived from Escherichia coli, Meningococcus, Pneumococcus, Streptococcus, Neisseria, Salmonella, Klebsiella, or Pseudomonas.

45. The immunoglobulin or its antigennegative fragment on p, where the antibody is produced using recombinant methods.

46. The immunoglobulin or its antigennegative fragment on p for use in the method of passive immunization of a mammal against a disease caused by the organism or disease caused by cells, where the method includes the introduction of a number of immunoglobulin or antigen-binding fragment, where the specified number is sufficient to cause inhibition or death of the organism that causes the disease, or cells that cause Zab is levania.

47. The immunoglobulin or its antigennegative fragment according to item 46, where the immunoglobulin is selected antibody IgG or antigennegative fragment.

48. The immunoglobulin or its antigennegative fragment according to item 46, where the immunoglobulin is selected antibody IgM or antigennegative fragment.

49. The immunoglobulin or its antigennegative fragment according to item 46, where the immunoglobulin is selected antibody IgA or antigennegative fragment.



 

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